Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B and G

PubMed Central

Stewart-Jones, Guillaume B. E.; Soto, Cinque; Lemmin, Thomas; Chuang, Gwo-Yu; Druz, Aliaksandr; Kong, Rui; Thomas, Paul V.; Wagh, Kshitij; Zhou, Tongqing; Behrens, Anna-Janina; Bylund, Tatsiana; Choi, Chang W.; Davison, Jack R.; Georgiev, Ivelin S.; Joyce, M. Gordon; Do Kwon, Young; Pancera, Marie; Taft, Justin; Yang, Yongping; Zhang, Baoshan; Shivatare, Sachin S.; Shivatare, Vidya S.; Lee, Chang-Chun D.; Wu, Chung-Yi; Bewley, Carole A.; Burton, Dennis R.; Koff, Wayne C.; Connors, Mark; Crispin, Max; Baxa, Ulrich; Korber, Bette T.; Wong, Chi-Huey; Mascola, John R.; Kwong, Peter D.

2017-01-01

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ~90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, which encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans amongst known broadly neutralizing antibodies that target the glycan-shielded trimer. PMID:27114034

Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved.

PubMed

Long, Hannah K; King, Hamish W; Patient, Roger K; Odom, Duncan T; Klose, Robert J

2016-08-19

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets.

PubMed

Nelson, Christopher S; Fuller, Chris K; Fordyce, Polly M; Greninger, Alexander L; Li, Hao; DeRisi, Joseph L

2013-07-01

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein's DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2's-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved.

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets

PubMed Central

Nelson, Christopher S.; Fuller, Chris K.; Fordyce, Polly M.; Greninger, Alexander L.; Li, Hao; DeRisi, Joseph L.

2013-01-01

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein’s DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2’s-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved. PMID:23625967

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

PubMed Central

Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

2010-01-01

Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles. PMID:20805402

Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1*

PubMed Central

Awad, Wael; Adamczyk, Barbara; Örnros, Jessica; Karlsson, Niclas G.; Mani, Katrin; Logan, Derek T.

2015-01-01

Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation. PMID:26203194

Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition.

PubMed

Wangkanont, Kittikhun; Wesener, Darryl A; Vidani, Jack A; Kiessling, Laura L; Forest, Katrina T

2016-03-11

Intelectins (X-type lectins), broadly distributed throughout chordates, have been implicated in innate immunity. Xenopus laevis embryonic epidermal lectin (XEEL), an intelectin secreted into environmental water by the X. laevis embryo, is postulated to function as a defense against microbes. XEEL is homologous (64% identical) to human intelectin-1 (hIntL-1), which is also implicated in innate immune defense. We showed previously that hIntL-1 binds microbial glycans bearing exocyclic vicinal diol groups. It is unknown whether XEEL has the same ligand specificity. Also unclear is whether XEEL and hIntL-1 have similar quaternary structures, as XEEL lacks the corresponding cysteine residues in hIntL-1 that stabilize the disulfide-linked trimer. These observations prompted us to further characterize XEEL. We found that hIntL-1 and XEEL have similar structural features. Even without the corresponding intermolecular disulfide bonds present in hIntL-1, the carbohydrate recognition domain of XEEL (XEELCRD) forms a stable trimer in solution. The structure of XEELCRD in complex with d-glycerol-1-phosphate, a residue present in microbe-specific glycans, indicated that the exocyclic vicinal diol coordinates to a protein-bound calcium ion. This ligand-binding mode is conserved between XEEL and hIntL-1. The domain architecture of full-length XEEL is reminiscent of a barbell, with two sets of three glycan-binding sites oriented in opposite directions. This orientation is consistent with our observation that XEEL can promote the agglutination of specific serotypes of Streptococcus pneumoniae. These data support a role for XEEL in innate immunity, and they highlight structural and functional conservation of X-type lectins among chordates. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition*

PubMed Central

Wangkanont, Kittikhun; Wesener, Darryl A.; Vidani, Jack A.; Kiessling, Laura L.; Forest, Katrina T.

2016-01-01

Intelectins (X-type lectins), broadly distributed throughout chordates, have been implicated in innate immunity. Xenopus laevis embryonic epidermal lectin (XEEL), an intelectin secreted into environmental water by the X. laevis embryo, is postulated to function as a defense against microbes. XEEL is homologous (64% identical) to human intelectin-1 (hIntL-1), which is also implicated in innate immune defense. We showed previously that hIntL-1 binds microbial glycans bearing exocyclic vicinal diol groups. It is unknown whether XEEL has the same ligand specificity. Also unclear is whether XEEL and hIntL-1 have similar quaternary structures, as XEEL lacks the corresponding cysteine residues in hIntL-1 that stabilize the disulfide-linked trimer. These observations prompted us to further characterize XEEL. We found that hIntL-1 and XEEL have similar structural features. Even without the corresponding intermolecular disulfide bonds present in hIntL-1, the carbohydrate recognition domain of XEEL (XEELCRD) forms a stable trimer in solution. The structure of XEELCRD in complex with d-glycerol-1-phosphate, a residue present in microbe-specific glycans, indicated that the exocyclic vicinal diol coordinates to a protein-bound calcium ion. This ligand-binding mode is conserved between XEEL and hIntL-1. The domain architecture of full-length XEEL is reminiscent of a barbell, with two sets of three glycan-binding sites oriented in opposite directions. This orientation is consistent with our observation that XEEL can promote the agglutination of specific serotypes of Streptococcus pneumoniae. These data support a role for XEEL in innate immunity, and they highlight structural and functional conservation of X-type lectins among chordates. PMID:26755729

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B, and G.

PubMed

Stewart-Jones, Guillaume B E; Soto, Cinque; Lemmin, Thomas; Chuang, Gwo-Yu; Druz, Aliaksandr; Kong, Rui; Thomas, Paul V; Wagh, Kshitij; Zhou, Tongqing; Behrens, Anna-Janina; Bylund, Tatsiana; Choi, Chang W; Davison, Jack R; Georgiev, Ivelin S; Joyce, M Gordon; Kwon, Young Do; Pancera, Marie; Taft, Justin; Yang, Yongping; Zhang, Baoshan; Shivatare, Sachin S; Shivatare, Vidya S; Lee, Chang-Chun D; Wu, Chung-Yi; Bewley, Carole A; Burton, Dennis R; Koff, Wayne C; Connors, Mark; Crispin, Max; Baxa, Ulrich; Korber, Bette T; Wong, Chi-Huey; Mascola, John R; Kwong, Peter D

2016-05-05

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer. Copyright © 2016 Elsevier Inc. All rights reserved.

N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A

PubMed Central

Yao, Guorui; Zhang, Sicai; Mahrhold, Stefan; Lam, Kwok-ho; Stern, Daniel; Bagramyan, Karine; Perry, Kay; Kalkum, Markus; Rummel, Andreas; Dong, Min; Jin, Rongsheng

2016-01-01

Botulinum neurotoxin serotype A1 (BoNT/A1) is one of the most dangerous potential bioterrorism agents, and exerts its action by invading motoneurons. It is also a licensed drug widely used for medical and cosmetic applications. Here we report a 2.0 Å resolution crystal structure of BoNT/A1 receptor-binding domain in complex with its neuronal receptor, the glycosylated human SV2C. We find that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an anti-botulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications to achieve highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors. PMID:27294781

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

PubMed Central

Crooks, Ema T.; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S.; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O’Dell, Sijy; LaBranche, Celia; Robinson, James E.; Montefiori, David C.; McKee, Krisha; Du, Sean X.; Doria-Rose, Nicole; Kwong, Peter D.; Mascola, John R.; Zhu, Ping; Schief, William R.; Wyatt, Richard T.; Whalen, Robert G.; Binley, James M.

2015-01-01

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine. PMID:26023780

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site.

PubMed

Crooks, Ema T; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O'Dell, Sijy; LaBranche, Celia; Robinson, James E; Montefiori, David C; McKee, Krisha; Du, Sean X; Doria-Rose, Nicole; Kwong, Peter D; Mascola, John R; Zhu, Ping; Schief, William R; Wyatt, Richard T; Whalen, Robert G; Binley, James M

2015-05-01

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

The Emerging Importance of IgG Fab Glycosylation in Immunity.

PubMed

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses.

PubMed

Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Kurosawa, Yoshikazu; Yokoyama, Shigeyuki

2014-07-01

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

PubMed Central

Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Yokoyama, Shigeyuki

2014-01-01

ABSTRACT Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines. PMID:24719430

Repertoire of human natural anti-glycan immunoglobulins. Do we have auto-antibodies?

PubMed

Bovin, Nicolai; Obukhova, Polina; Shilova, Nadezhda; Rapoport, Evgenia; Popova, Inna; Navakouski, Maksim; Unverzagt, Carlo; Vuskovic, Marko; Huflejt, Margaret

2012-09-01

Profiling of donor's antibodies using glycan arrays demonstrated presence of antibodies capable of binding to >100 mammalian glycans or their fragments. For example, relatively high binding to Galα1-4Galβ1-4GlcNAc (P(1)), Galα1-4Galβ1-4Glc (P(k)), Galβ1-3GlcNAc (Le(c)), 4-O-SuGalβ1-4GlcNAc, and GalNAcα1-3GalNAc (Fs) was found in all tested individuals. Affinity isolation using hapten-specific chromatography in combination with epitope mapping revealed their glycotopes. Notably, a significant part of the antibodies was capable of recognizing a fragment of larger glycans, for example, -Galβ1-4Glc of glycolipids, or Fucα1-3GlcNAc motif of Le(X)/Le(Y) antigens. Their epitope specificity did not vary between different healthy individuals. Nominally, all the mentioned immunoglobulins could be classified as auto-antibodies. In this work we re-evaluated results published earlier and analyzed new data to address the question why autologous antibodies found in healthy individuals do not cause severe auto-immune reactions. In all cases the presumably "auto" antibodies were found to bind short fragments "subtracted" from larger glycans whereas recognition of the same fragment in the context of the whole natural chain was completely abolished. Thus, in spite of numerous formally positive signals observed on the printed glycan array, we are yet unable to identify in blood serum of healthy individuals true auto-antibodies capable of binding carbohydrate chains in their naturally occurring form. The identified natural anti-glycan antibodies were found to be specific, high-titer and population conservative immunoglobulins - all of this suggesting as yet unknown biological role(s) of the studied proteins. This article is part of a Special Issue entitled Glycoproteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale

PubMed Central

Cockburn, Darrell W.; Orlovsky, Nicole I.; Foley, Matthew H.; Kwiatkowski, Kurt J.; Bahr, Constance M.; Maynard, Mallory; Demeler, Borries; Koropatkin, Nicole M.

2015-01-01

Summary Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute-binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, while the membrane associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute-binding proteins display a range of glycan-binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6-branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch-degrading Clostridium cluster XIVa organisms in the human gut. PMID:25388295

MCAW-DB: A glycan profile database capturing the ambiguity of glycan recognition patterns.

PubMed

Hosoda, Masae; Takahashi, Yushi; Shiota, Masaaki; Shinmachi, Daisuke; Inomoto, Renji; Higashimoto, Shinichi; Aoki-Kinoshita, Kiyoko F

2018-05-11

Glycan-binding protein (GBP) interaction experiments, such as glycan microarrays, are often used to understand glycan recognition patterns. However, oftentimes the interpretation of glycan array experimental data makes it difficult to identify discrete GBP binding patterns due to their ambiguity. It is known that lectins, for example, are non-specific in their binding affinities; the same lectin can bind to different monosaccharides or even different glycan structures. In bioinformatics, several tools to mine the data generated from these sorts of experiments have been developed. These tools take a library of predefined motifs, which are commonly-found glycan patterns such as sialyl-Lewis X, and attempt to identify the motif(s) that are specific to the GBP being analyzed. In our previous work, as opposed to using predefined motifs, we developed the Multiple Carbohydrate Alignment with Weights (MCAW) tool to visualize the state of the glycans being recognized by the GBP under analysis. We previously reported on the effectiveness of our tool and algorithm by analyzing several glycan array datasets from the Consortium of Functional Glycomics (CFG). In this work, we report on our analysis of 1081 data sets which we collected from the CFG, the results of which we have made publicly and freely available as a database called MCAW-DB. We introduce this database, its usage and describe several analysis results. We show how MCAW-DB can be used to analyze glycan-binding patterns of GBPs amidst their ambiguity. For example, the visualization of glycan-binding patterns in MCAW-DB show how they correlate with the concentrations of the samples used in the array experiments. Using MCAW-DB, the patterns of glycans found to bind to various GBP-glycan binding proteins are visualized, indicating the binding "environment" of the glycans. Thus, the ambiguity of glycan recognition is numerically represented, along with the patterns of monosaccharides surrounding the binding region. The profiles in MCAW-DB could potentially be used as predictors of affinity of unknown or novel glycans to particular GBPs by comparing how well they match the existing profiles for those GBPs. Moreover, as the glycan profiles of diseased tissues become available, glycan alignments could also be used to identify glycan biomarkers unique to that tissue. Databases of these alignments may be of great use for drug discovery. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Glycobiology simplified: diverse roles of glycan recognition in inflammation

PubMed Central

Schnaar, Ronald L.

2016-01-01

Glycans and complementary glycan-binding proteins are essential components in the language of cell-cell interactions in immunity. The study of glycan function is the purview of glycobiology, which has often been presented as an unusually complex discipline. In fact, the human glycome, composed of all of its glycans, is built primarily from only 9 building blocks that are combined by enzymes (writers) with specific and limited biosynthetic capabilities into a tractable and increasingly accessible number of potential glycan patterns that are functionally read by several dozen human glycan-binding proteins (readers). Nowhere is the importance of glycan recognition better understood than in infection and immunity, and knowledge in this area has already led to glycan mimetic anti-infective and anti-inflammatory drugs. This review includes a brief tutorial on human glycobiology and a limited number of specific examples of glycan-binding protein-glycan interactions that initiate and regulate inflammation. Examples include representatives from different glycan-binding protein families, including the C-type lectins (E-selectin, P-selectin, dectin-1, and dectin-2), sialic acid-binding immunoglobulin-like lectins (sialic acid-binding immunoglobulin-like lectins 8 and 9), galectins (galectin-1, galectin-3, and galectin-9), as well as hyaluronic acid-binding proteins. As glycoscience technologies advance, opportunities for enhanced understanding of glycans and their roles in leukocyte cell biology provide increasing opportunities for discovery and therapeutic intervention. PMID:27004978

Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Rui; McBride, Ryan; Nycholat, Corwin M.

2012-02-13

Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only formore » {alpha}2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For {alpha}2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with {alpha}2-6- and {alpha}2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for {alpha}2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.« less

Galectins as self/non-self recognition receptors in innate and adaptive immunity: an unresolved paradox

PubMed Central

Vasta, Gerardo R.; Ahmed, Hafiz; Nita-Lazar, Mihai; Banerjee, Aditi; Pasek, Marta; Shridhar, Surekha; Guha, Prasun; Fernández-Robledo, José A.

2012-01-01

Galectins are characterized by their binding affinity for β-galactosides, a unique binding site sequence motif, and wide taxonomic distribution and structural conservation in vertebrates, invertebrates, protista, and fungi. Since their initial description, galectins were considered to bind endogenous (“self”) glycans and mediate developmental processes and cancer. In the past few years, however, numerous studies have described the diverse effects of galectins on cells involved in both innate and adaptive immune responses, and the mechanistic aspects of their regulatory roles in immune homeostasis. More recently, however, evidence has accumulated to suggest that galectins also bind exogenous (“non-self”) glycans on the surface of potentially pathogenic microbes, parasites, and fungi, suggesting that galectins can function as pattern recognition receptors (PRRs) in innate immunity. Thus, a perplexing paradox arises by the fact that galectins also recognize lactosamine-containing glycans on the host cell surface during developmental processes and regulation of immune responses. According to the currently accepted model for non-self recognition, PRRs recognize pathogens via highly conserved microbial surface molecules of wide distribution such as LPS or peptidoglycan (pathogen-associated molecular patterns; PAMPs), which are absent in the host. Hence, this would not apply to galectins, which apparently bind similar self/non-self molecular patterns on host and microbial cells. This paradox underscores first, an oversimplification in the use of the PRR/PAMP terminology. Second, and most importantly, it reveals significant gaps in our knowledge about the diversity of the host galectin repertoire, and the subcellular targeting, localization, and secretion. Furthermore, our knowledge about the structural and biophysical aspects of their interactions with the host and microbial carbohydrate moieties is fragmentary, and warrants further investigation. PMID:22811679

Development and application of an algorithm to compute weighted multiple glycan alignments.

PubMed

Hosoda, Masae; Akune, Yukie; Aoki-Kinoshita, Kiyoko F

2017-05-01

A glycan consists of monosaccharides linked by glycosidic bonds, has branches and forms complex molecular structures. Databases have been developed to store large amounts of glycan-binding experiments, including glycan arrays with glycan-binding proteins. However, there are few bioinformatics techniques to analyze large amounts of data for glycans because there are few tools that can handle the complexity of glycan structures. Thus, we have developed the MCAW (Multiple Carbohydrate Alignment with Weights) tool that can align multiple glycan structures, to aid in the understanding of their function as binding recognition molecules. We have described in detail the first algorithm to perform multiple glycan alignments by modeling glycans as trees. To test our tool, we prepared several data sets, and as a result, we found that the glycan motif could be successfully aligned without any prior knowledge applied to the tool, and the known recognition binding sites of glycans could be aligned at a high rate amongst all our datasets tested. We thus claim that our tool is able to find meaningful glycan recognition and binding patterns using data obtained by glycan-binding experiments. The development and availability of an effective multiple glycan alignment tool opens possibilities for many other glycoinformatics analysis, making this work a big step towards furthering glycomics analysis. http://www.rings.t.soka.ac.jp. kkiyoko@soka.ac.jp. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

The Glycan Microarray Story from Construction to Applications.

PubMed

Hyun, Ji Young; Pai, Jaeyoung; Shin, Injae

2017-04-18

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

In Silico Analysis of Gene Expression Network Components Underlying Pigmentation Phenotypes in the Python Identified Evolutionarily Conserved Clusters of Transcription Factor Binding Sites

PubMed Central

2016-01-01

Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1) that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus. Our results provide insight into pigment phenotypes in pythons. PMID:27698666

In Silico Analysis of Gene Expression Network Components Underlying Pigmentation Phenotypes in the Python Identified Evolutionarily Conserved Clusters of Transcription Factor Binding Sites.

PubMed

Irizarry, Kristopher J L; Bryden, Randall L

2016-01-01

Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1) that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus . Our results provide insight into pigment phenotypes in pythons.

Expanding the universe of cytokines and pattern recognition receptors: galectins and glycans in innate immunity.

PubMed

Cerliani, Juan P; Stowell, Sean R; Mascanfroni, Iván D; Arthur, Connie M; Cummings, Richard D; Rabinovich, Gabriel A

2011-02-01

Effective immunity relies on the recognition of pathogens and tumors by innate immune cells through diverse pattern recognition receptors (PRRs) that lead to initiation of signaling processes and secretion of pro- and anti-inflammatory cytokines. Galectins, a family of endogenous lectins widely expressed in infected and neoplastic tissues have emerged as part of the portfolio of soluble mediators and pattern recognition receptors responsible for eliciting and controlling innate immunity. These highly conserved glycan-binding proteins can control immune cell processes through binding to specific glycan structures on pathogens and tumors or by acting intracellularly via modulation of selective signaling pathways. Recent findings demonstrate that various galectin family members influence the fate and physiology of different innate immune cells including polymorphonuclear neutrophils, mast cells, macrophages, and dendritic cells. Moreover, several pathogens may actually utilize galectins as a mechanism of host invasion. In this review, we aim to highlight and integrate recent discoveries that have led to our current understanding of the role of galectins in host-pathogen interactions and innate immunity. Challenges for the future will embrace the rational manipulation of galectin-glycan interactions to instruct and shape innate immunity during microbial infections, inflammation, and cancer.

A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo

PubMed Central

Freund, Natalia T.; Horwitz, Joshua A.; Nogueira, Lilian; Sievers, Stuart A.; Scharf, Louise; Scheid, Johannes F.; Gazumyan, Anna; Liu, Cassie; Velinzon, Klara; Goldenthal, Ariel; Sanders, Rogier W.; Moore, John P.; Bjorkman, Pamela J.; Seaman, Michael S.; Walker, Bruce D.; Klein, Florian; Nussenzweig, Michel C.

2015-01-01

The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans. PMID:26516768

The Populus ARBORKNOX1 homeodomain transcription factor regulates woody growth through binding to evolutionarily conserved target genes of diverse function

Treesearch

Lijun Liu; Matthew S. Zinkgraf; H. Earl Petzold; Eric P. Beers; Vladimir Filkov; Andrew Groover

2014-01-01

The class I KNOX homeodomain transcription factor ARBORKNOX1 (ARK1) is a key regulator of vascular cambium maintenance and cell differentiation in Populus. Currently, basic information is lacking concerning the distribution, functional characteristics, and evolution of ARK1 binding in the Populus genome.

Exploiting fungal cell wall components in vaccines.

PubMed

Levitz, Stuart M; Huang, Haibin; Ostroff, Gary R; Specht, Charles A

2015-03-01

Innate recognition of fungi leads to strong adaptive immunity. Investigators are trying to exploit this observation in vaccine development by combining antigens with evolutionarily conserved fungal cell wall carbohydrates to induce protective responses. Best studied is β-1,3-glucan, a glycan that activates complement and is recognized by dectin-1. Administration of antigens in association with β-1,3-glucan, either by direct conjugation or complexed in glucan particles, results in robust humoral and cellular immune responses. While the host has a host of mannose receptors, responses to fungal mannoproteins generally are amplified if cells are cooperatively stimulated with an additional danger signal such as a toll-like receptor agonist. Chitosan, a polycationic homopolymer of glucosamine manufactured by the deacetylation of chitin, is being studied as an adjuvant in DNA and protein-based vaccines. It appears particularly promising in mucosal vaccines. Finally, universal and organism-specific fungal vaccines have been formulated by conjugating fungal cell wall glycans to carrier proteins. A major challenge will be to advance these experimental findings so that at risk patients can be protected.

Exploiting fungal cell wall components in vaccines

PubMed Central

Levitz, Stuart M.; Huang, Haibin; Ostroff, Gary R.; Specht, Charles A.

2014-01-01

Innate recognition of fungi leads to strong adaptive immunity. Investigators are trying to exploit this observation in vaccine development by combining antigens with evolutionarily conserved fungal cell wall carbohydrates to induce protective responses. Best studied is β-1,3-glucan, a glycan that activates complement and is recognized by Dectin-1. Administration of antigens in association with β-1,3-glucan, either by direct conjugation or complexed in glucan particles, results in robust humoral and cellular immune responses. While the host has a host of mannose receptors, responses to fungal mannoproteins generally are amplified if cells are cooperatively stimulated with an additional danger signal such as a toll-like receptor agonist. Chitosan, a polycationic homopolymer of glucosamine manufactured by the deacetylation of chitin, is being studied as an adjuvant in DNA and protein-based vaccines. It appears particularly promising in mucosal vaccines. Finally, universal and organism-specific fungal vaccines have been formulated by conjugating fungal cell wall glycans to carrier proteins. A major challenge will be to advance these experimental findings so that at risk patients can be protected. PMID:25404118

Evolutionarily Conserved, Growth Plate Zone-Specific Regulation of the Matrilin-1 Promoter: L-Sox5/Sox6 and Nfi Factors Bound near TATA Finely Tune Activation by Sox9 ▿

PubMed Central

Nagy, Andrea; Kénesi, Erzsébet; Rentsendorj, Otgonchimeg; Molnár, Annamária; Szénási, Tibor; Sinkó, Ildikó; Zvara, Ágnes; Thottathil Oommen, Sajit; Barta, Endre; Puskás, László G.; Lefebvre, Veronique; Kiss, Ibolya

2011-01-01

To help uncover the mechanisms underlying the staggered expression of cartilage-specific genes in the growth plate, we dissected the transcriptional mechanisms driving expression of the matrilin-1 gene (Matn1). We show that a unique assembly of evolutionarily conserved cis-acting elements in the Matn1 proximal promoter restricts expression to the proliferative and prehypertrophic zones of the growth plate. These elements functionally interact with distal elements and likewise are capable of restricting the domain of activity of a pancartilaginous Col2a1 enhancer. The proximal elements include a Pe1 element binding the chondrogenic L-Sox5, Sox6, and Sox9 proteins, a SI element binding Nfi proteins, and an initiator Ine element binding the Sox trio and other factors. Sox9 binding to Pe1 is indispensable for functional interaction with the distal promoter. Binding of L-Sox5/Sox6 to Ine and Nfib to SI modulates Sox9 transactivation in a protein dose-dependent manner, possibly to enhance Sox9 activity in early stages of chondrogenesis and repress it at later stages. Hence, our data suggest a novel model whereby Sox and Nfi proteins bind to conserved Matn1 proximal elements and functionally interact with each other to finely tune gene expression in specific zones of the cartilage growth plate. PMID:21173167

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

PubMed Central

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-01-01

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

PubMed

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-07-16

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus.

PubMed

Bradel-Tretheway, Birgit G; Liu, Qian; Stone, Jacquelyn A; McInally, Samantha; Aguilar, Hector C

2015-07-01

Hendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within the Paramyxoviridae family. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses. Viral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2 to G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had roles that were very conserved between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between the two viruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses.

PubMed

Byrd-Leotis, Lauren; Liu, Renpeng; Bradley, Konrad C; Lasanajak, Yi; Cummings, Sandra F; Song, Xuezheng; Heimburg-Molinaro, Jamie; Galloway, Summer E; Culhane, Marie R; Smith, David F; Steinhauer, David A; Cummings, Richard D

2014-06-03

Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of "shotgun glycomics" to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

PubMed

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells.

PubMed

Srikrishna, G; Panneerselvam, K; Westphal, V; Abraham, V; Varki, A; Freeze, H H

2001-04-01

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

Galectins are human milk glycan receptors

PubMed Central

Noll, Alexander J; Gourdine, Jean-Philippe; Yu, Ying; Lasanajak, Yi; Smith, David F; Cummings, Richard D

2016-01-01

The biological recognition of human milk glycans (HMGs) is poorly understood. Because HMGs are rich in galactose we explored whether they might interact with human galectins, which bind galactose-containing glycans and are highly expressed in epithelial cells and other cell types. We screened a number of human galectins for their binding to HMGs on a shotgun glycan microarray consisting of 247 HMGs derived from human milk, as well as to a defined HMG microarray. Recombinant human galectins (hGal)-1, -3, -4, -7, -8 and -9 bound selectively to glycans, with each galectin recognizing a relatively unique binding motif; by contrast hGal-2 did not recognize HMGs, but did bind to the human blood group A Type 2 determinants on other microarrays. Unlike other galectins, hGal-7 preferentially bound to glycans expressing a terminal Type 1 (Galβ1-3GlcNAc) sequence, a motif that had eluded detection on non-HMG glycan microarrays. Interactions with HMGs were confirmed in a solution setting by isothermal titration microcalorimetry and hapten inhibition experiments. These results demonstrate that galectins selectively bind to HMGs and suggest the possibility that galectin–HMG interactions may play a role in infant immunity. PMID:26747425

The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

PubMed Central

Liu, Yan; Blaum, Bärbel S.; Reiter, Dirk M.; Feizi, Ten; Dermody, Terence S.; Stehle, Thilo

2012-01-01

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus. PMID:23236285

The GM2 glycan serves as a functional coreceptor for serotype 1 reovirus.

PubMed

Reiss, Kerstin; Stencel, Jennifer E; Liu, Yan; Blaum, Bärbel S; Reiter, Dirk M; Feizi, Ten; Dermody, Terence S; Stehle, Thilo

2012-01-01

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

Structural basis of glycan specificity of P[19] VP8*: Implications for rotavirus zoonosis and evolution.

PubMed

Liu, Yang; Xu, Shenyuan; Woodruff, Andrew L; Xia, Ming; Tan, Ming; Kennedy, Michael A; Jiang, Xi

2017-11-01

Recognition of specific cell surface glycans, mediated by the VP8* domain of the spike protein VP4, is the essential first step in rotavirus (RV) infection. Due to lack of direct structural information of virus-ligand interactions, the molecular basis of ligand-controlled host ranges of the major human RVs (P[8] and P[4]) in P[II] genogroup remains unknown. Here, through characterization of a minor P[II] RV (P[19]) that can infect both animals (pigs) and humans, we made an important advance to fill this knowledge gap by solving the crystal structures of the P[19] VP8* in complex with its ligands. Our data showed that P[19] RVs use a novel binding site that differs from the known ones of other genotypes/genogroups. This binding site is capable of interacting with two types of glycans, the mucin core and type 1 histo-blood group antigens (HBGAs) with a common GlcNAc as the central binding saccharide. The binding site is apparently shared by other P[II] RVs and possibly two genotypes (P[10] and P[12]) in P[I] as shown by their highly conserved GlcNAc-interacting residues. These data provide strong evidence of evolutionary connections among these human and animal RVs, pointing to a common ancestor in P[I] with a possible animal host origin. While the binding properties to GlcNAc-containing saccharides are maintained, changes in binding to additional residues, such as those in the polymorphic type 1 HBGAs may occur in the course of RV evolution, explaining the complex P[II] genogroup that mainly causes diseases in humans but also in some animals.

O2 sensing-associated glycosylation exposes the F-box-combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.

PubMed

Sheikh, M Osman; Thieker, David; Chalmers, Gordon; Schafer, Christopher M; Ishihara, Mayumi; Azadi, Parastoo; Woods, Robert J; Glushka, John N; Bendiak, Brad; Prestegard, James H; West, Christopher M

2017-11-17

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF ( S kp1/ C ullin-1/ F -box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box-binding domain. Prolyl hydroxylation of Skp1 contributes to O 2 -dependent Dictyostelium development, but full glycosylation at that position is required for optimal O 2 sensing. Previous studies have shown that the glycan promotes organization of the F-box-binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box-binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two α-helices of the F-box-combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding*

PubMed Central

Cabrera, Paula V.; Pang, Mabel; Marshall, Jamie L.; Kung, Raymond; Nelson, Stanley F.; Stalnaker, Stephanie H.; Wells, Lance; Crosbie-Watson, Rachelle H.; Baum, Linda G.

2012-01-01

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function. PMID:22570487

Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread

PubMed Central

Gulati, Shelly; Smith, David F.; Cummings, Richard D.; Couch, Robert B.; Griesemer, Sara B.; St. George, Kirsten; Webster, Robert G.; Air, Gillian M.

2013-01-01

It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population. PMID:23805213

Cell–cell adhesion in metazoans relies on evolutionarily conserved features of the α-catenin·β-catenin–binding interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Xiangqiang; Kang, Hyunook; Loveless, Timothy

Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and β-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/β-catenin is higher than that of α-catenin for β-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalianmore » α-catenin·β-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40–100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans. Our data provide novel insights into how cadherin-dependent cell–cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.« less

Cell–cell adhesion in metazoans relies on evolutionarily conserved features of the α-catenin·β-catenin–binding interface

DOE PAGES

Shao, Xiangqiang; Kang, Hyunook; Loveless, Timothy; ...

2017-08-25

Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and β-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/β-catenin is higher than that of α-catenin for β-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalianmore » α-catenin·β-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40–100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans. Our data provide novel insights into how cadherin-dependent cell–cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.« less

Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

DOE PAGES

Hu, Liya; Ramani, Sasirekha; Czako, Rita; ...

2015-09-30

We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type IImore » precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.« less

Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Liya; Ramani, Sasirekha; Czako, Rita

We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type IImore » precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.« less

Cloning, expression, purification and crystallographic studies of galectin-11 from domestic sheep (Ovis aries).

PubMed

Sakthivel, Dhanasekaran; Littler, Dene; Shahine, Adam; Troy, Sally; Johnson, Matthew; Rossjohn, Jamie; Piedrafita, David; Beddoe, Travis

2015-08-01

Galectins are an evolutionarily conserved family of proteins that translate glycan recognition into cellular effects. Galectin-11 is a unique member of the galectin family that is only expressed in ruminants such as sheep, goat and cattle and that plays a critical role in several important biological processes, such as reproduction and parasite-mediated innate immune responses. Currently, these two areas are of major importance for the sustainability of ruminant livestock production. Despite the emerging biological significance of galectin-11, no structural information is available. It is expected that structural studies will unravel the functional mechanisms of galectin-11 activity. Here, the expression, purification and crystallization of the ruminant-specific galectin-11 from domestic sheep and the collection of X-ray data to 2.0 Å resolution are reported.

Conformational Heterogeneity of the HIV Envelope Glycan Shield.

PubMed

Yang, Mingjun; Huang, Jing; Simon, Raphael; Wang, Lai-Xi; MacKerell, Alexander D

2017-06-30

To better understand the conformational properties of the glycan shield covering the surface of the HIV gp120/gp41 envelope (Env) trimer, and how the glycan shield impacts the accessibility of the underlying protein surface, we performed enhanced sampling molecular dynamics (MD) simulations of a model glycosylated HIV Env protein and related systems. Our simulation studies revealed a conformationally heterogeneous glycan shield with a network of glycan-glycan interactions more extensive than those observed to date. We found that partial preorganization of the glycans potentially favors binding by established broadly neutralizing antibodies; omission of several specific glycans could increase the accessibility of other glycans or regions of the protein surface to antibody or CD4 receptor binding; the number of glycans that can potentially interact with known antibodies is larger than that observed in experimental studies; and specific glycan conformations can maximize or minimize interactions with individual antibodies. More broadly, the enhanced sampling MD simulations described here provide a valuable tool to guide the engineering of specific Env glycoforms for HIV vaccine design.

Rapid assays for lectin toxicity and binding changes that reflect altered glycosylation in mammalian cells.

PubMed

Stanley, Pamela; Sundaram, Subha

2014-06-03

Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis. Copyright © 2014 John Wiley & Sons, Inc.

Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.

PubMed

Deng, Lingquan; Bensing, Barbara A; Thamadilok, Supaporn; Yu, Hai; Lau, Kam; Chen, Xi; Ruhl, Stefan; Sullam, Paul M; Varki, Ajit

2014-12-01

Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.

A beta-N-acetylglucosaminyl phosphate diester residue is attached to the glycosylphosphatidylinositol anchor of human placental alkaline phosphatase: a target of the channel-forming toxin aerolysin.

PubMed

Fukushima, Keiko; Ikehara, Yukio; Kanai, Michiko; Kochibe, Naohisa; Kuroki, Masahide; Yamashita, Katsuko

2003-09-19

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitous in eukaryotes. The minimum conserved GPI core structure of all GPI-anchored glycans has been determined as EtN-PO4-6Manalpha1-2Manalpha1-6Manalpha1-4GlcN-myo-inositol-PO3H. Human placental alkaline phosphatase (AP) has been reported to be a GPI-anchored membrane protein. AP carries one N-glycan, (NeuAcalpha2-->3)2Gal2GlcNAc2Man3GlcNAc(+/-Fuc)GlcNAc, and a GPI anchor, which contains an ethanolamine phosphate diester group, as a side chain. However, we found that both sialidase-treated soluble AP (sAP) and its GPI-anchored glycan bound to a Psathyrella velutina lectin (PVL)-Sepharose column, which binds beta-GlcNAc residues. PVL binding of asialo-sAP and its GPI-anchored glycan was diminished by digestion with diplococcal beta-N-acetylhexosaminidase or by mild acid treatment. After sequential digestion of asialo-sAP with beta-N-acetylhexosaminidase and acid phosphatase, the elution patterns on chromatofocusing gels were changed in accordance with the negative charges of phosphate residues. Trypsin-digested sAP was analyzed by liquid chromatography/electrospray ionization mass spectrometry, and the structures of two glycopeptides with GPI-anchored glycans were confirmed as peptide-EtN-PO4-6Manalpha1-->2(GlcNAcbeta1-PO4-->6)Manalpha1-6(+/-EtN-PO4-->)Manalpha1-->4GlcN, which may be produced by endo-alpha-glucosaminidase. In addition to AP, GPI-anchored carcinoembryonic antigen, cholinesterase, and Tamm-Horsfall glycoprotein also bound to a PVL-Sepharose column, suggesting that the beta-N-acetylglucosaminyl phosphate diester residue is widely distributed in human GPI-anchored glycans. Furthermore, we found that the beta-N-acetylglucosaminyl phosphate diester residue is important for GPI anchor recognition of aerolysin, a channel-forming toxin derived from Aeromonas hydrophila.

Recognition of microbial glycans by human intelectin-1

DOE PAGES

Wesener, Darryl A.; Wangkanont, Kittikhun; McBride, Ryan; ...

2015-07-06

The glycans displayed on mammalian cells can differ markedly from those on microbes. Such differences could, in principle, be 'read' by carbohydrate-binding proteins, or lectins. In this paper, we used glycan microarrays to show that human intelectin-1 (hIntL-1) does not bind known human glycan epitopes but does interact with multiple glycan epitopes found exclusively on microbes: β-linked D-galactofuranose (β-Galf), D-phosphoglycerol–modified glycans, heptoses, D-glycero-D-talo-oct-2-ulosonic acid (KO) and 3-deoxy-D-manno-oct-2-ulosonic acid (KDO). The 1.6-Å-resolution crystal structure of hIntL-1 complexed with β-Galf revealed that hIntL-1 uses a bound calcium ion to coordinate terminal exocyclic 1,2-diols. N-acetylneuraminic acid (Neu5Ac), a sialic acid widespread in humanmore » glycans, has an exocyclic 1,2-diol but does not bind hIntL-1, probably owing to unfavorable steric and electronic effects. hIntL-1 marks only Streptococcus pneumoniae serotypes that display surface glycans with terminal 1,2-diol groups. Finally, this ligand selectivity suggests that hIntL-1 functions in microbial surveillance.« less

Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

PubMed Central

Stencel-Baerenwald, Jennifer; Reiss, Kerstin; Blaum, Bärbel S.; Colvin, Daniel; Li, Xiao-Nan; Abel, Ty; Boyd, Kelli; Stehle, Thilo

2015-01-01

ABSTRACT Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease. PMID:25736887

Biogenesis of influenza a virus hemagglutinin cross-protective stem epitopes.

PubMed

Magadán, Javier G; Altman, Meghan O; Ince, William L; Hickman, Heather D; Stevens, James; Chevalier, Aaron; Baker, David; Wilson, Patrick C; Ahmed, Rafi; Bennink, Jack R; Yewdell, Jonathan W

2014-06-01

Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets.

PubMed

Singh, Anirudh K; Woodiga, Shireen A; Grau, Margaret A; King, Samantha J

2017-03-01

Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis , like Streptococcus gordonii and Streptococcus sanguinis , binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors. Copyright © 2017 American Society for Microbiology.

Antibody Evasion by a Gammaherpesvirus O-Glycan Shield

PubMed Central

Machiels, Bénédicte; Lété, Céline; Guillaume, Antoine; Mast, Jan; Stevenson, Philip G.; Vanderplasschen, Alain; Gillet, Laurent

2011-01-01

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog - gp180 - contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target. PMID:22114560

Mutation of Nogo-B receptor, a subunit of cis-prenyltransferase, causes a congenital disorder of glycosylation.

PubMed

Park, Eon Joo; Grabińska, Kariona A; Guan, Ziqiang; Stránecký, Viktor; Hartmannová, Hana; Hodaňová, Kateřina; Barešová, Veronika; Sovová, Jana; Jozsef, Levente; Ondrušková, Nina; Hansíková, Hana; Honzík, Tomáš; Zeman, Jiří; Hůlková, Helena; Wen, Rong; Kmoch, Stanislav; Sessa, William C

2014-09-02

Dolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice, and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provide a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation. Copyright © 2014 Elsevier Inc. All rights reserved.

Mutation of NgBR, a subunit of cis-prenyltransferase, causes a congenial disorder of glycosylation

PubMed Central

Park, Eon Joo; Grabińska, Kariona A.; Guan, Ziqiang; Stránecký, Viktor; Hartmannová, Hana; Hodaňová, Kateřina; Barešová, Veronika; Sovová, Jana; Jozsef, Levente; Ondrušková, Nina; Hansíková, Hana; Honzík, Tomáš; Zeman, Jiří; Hůlková, Helena; Wen, Rong; Kmoch, Stanislav; Sessa, William C.

2014-01-01

Summary Dolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. Cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provides a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation. PMID:25066056

A computational tool to predict the evolutionarily conserved protein-protein interaction hot-spot residues from the structure of the unbound protein.

PubMed

Agrawal, Neeraj J; Helk, Bernhard; Trout, Bernhardt L

2014-01-21

Identifying hot-spot residues - residues that are critical to protein-protein binding - can help to elucidate a protein's function and assist in designing therapeutic molecules to target those residues. We present a novel computational tool, termed spatial-interaction-map (SIM), to predict the hot-spot residues of an evolutionarily conserved protein-protein interaction from the structure of an unbound protein alone. SIM can predict the protein hot-spot residues with an accuracy of 36-57%. Thus, the SIM tool can be used to predict the yet unknown hot-spot residues for many proteins for which the structure of the protein-protein complexes are not available, thereby providing a clue to their functions and an opportunity to design therapeutic molecules to target these proteins. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

NASA Astrophysics Data System (ADS)

Shivatare, Sachin S.; Chang, Shih-Huang; Tsai, Tsung-I.; Tseng, Susan Yu; Shivatare, Vidya S.; Lin, Yih-Shyan; Cheng, Yang-Yu; Ren, Chien-Tai; Lee, Chang-Chun David; Pawar, Sujeet; Tsai, Charng-Sheng; Shih, Hao-Wei; Zeng, Yi-Fang; Liang, Chi-Hui; Kwong, Peter D.; Burton, Dennis R.; Wu, Chung-Yi; Wong, Chi-Huey

2016-04-01

A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120—a glycoprotein found on the surface of the virus envelope—thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

Conserved mRNA-binding proteomes in eukaryotic organisms.

PubMed

Matia-González, Ana M; Laing, Emma E; Gerber, André P

2015-12-01

RNA-binding proteins (RBPs) are essential for post-transcriptional regulation of gene expression. Recent high-throughput screens have dramatically increased the number of experimentally identified RBPs; however, comprehensive identification of RBPs within living organisms is elusive. Here we describe the repertoire of 765 and 594 proteins that reproducibly interact with polyadenylated mRNAs in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. Furthermore, we report the differential association of mRNA-binding proteins (mRPBs) upon induction of apoptosis in C. elegans L4-stage larvae. Strikingly, most proteins composing mRBPomes, including components of early metabolic pathways and the proteasome, are evolutionarily conserved between yeast and C. elegans. We speculate, on the basis of our evidence that glycolytic enzymes bind distinct glycolytic mRNAs, that enzyme-mRNA interactions relate to an ancient mechanism for post-transcriptional coordination of metabolic pathways that perhaps was established during the transition from the early 'RNA world' to the 'protein world'.

Cryptic glucocorticoid receptor-binding sites pervade genomic NF-κB response elements.

PubMed

Hudson, William H; Vera, Ian Mitchelle S de; Nwachukwu, Jerome C; Weikum, Emily R; Herbst, Austin G; Yang, Qin; Bain, David L; Nettles, Kendall W; Kojetin, Douglas J; Ortlund, Eric A

2018-04-06

Glucocorticoids (GCs) are potent repressors of NF-κB activity, making them a preferred choice for treatment of inflammation-driven conditions. Despite the widespread use of GCs in the clinic, current models are inadequate to explain the role of the glucocorticoid receptor (GR) within this critical signaling pathway. GR binding directly to NF-κB itself-tethering in a DNA binding-independent manner-represents the standing model of how GCs inhibit NF-κB-driven transcription. We demonstrate that direct binding of GR to genomic NF-κB response elements (κBREs) mediates GR-driven repression of inflammatory gene expression. We report five crystal structures and solution NMR data of GR DBD-κBRE complexes, which reveal that GR recognizes a cryptic response element between the binding footprints of NF-κB subunits within κBREs. These cryptic sequences exhibit high sequence and functional conservation, suggesting that GR binding to κBREs is an evolutionarily conserved mechanism of controlling the inflammatory response.

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCEPost-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

DOE PAGES

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.; ...

2017-11-28

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Lectin-mediated binding and sialoglycans of porcine surfactant protein D synergistically neutralize influenza A virus.

PubMed

van Eijk, Martin; Rynkiewicz, Michael J; Khatri, Kshitij; Leymarie, Nancy; Zaia, Joseph; White, Mitchell R; Hartshorn, Kevan L; Cafarella, Tanya R; Van Die, Irma; Hessing, Martin; Seaton, Barbara A; Haagsman, Henk P

2018-05-16

Innate immunity is critical in the early containment of influenza A virus (IAV) infection, and surfactant protein D (SP-D) plays a crucial role in the pulmonary defense against IAV. In pigs, which are important intermediate hosts during the generation of pandemic IAVs, SP-D uses its unique carbohydrate recognition domain (CRD) to interact with IAV. An N-linked CRD-glycosylation provides interactions with the sialic acid binding site of IAV, and a tripeptide loop at the lectin binding site facilitates enhanced interactions with IAV glycans. Here, to investigate both mechanisms of IAV neutralization in greater detail, we produced an N-glycosylated neckCRD fragment of porcine SP-D (RpNCRD) in HEK293 cells. X-ray crystallography disclosed that the N-glycan did not alter the CRD backbone structure including the lectin site conformation, but revealed a potential second non-lectin binding site for glycans. IAV hemagglutination inhibition, IAV aggregation and neutralization of IAV infection studies showed that RpNCRD, unlike the human analogue RhNCRD, exhibits potent neutralizing activity against pandemic A/Aichi/68 (H3N2), enabled by both porcine-specific structural features of its CRD. MS analysis revealed an N-glycan site-occupancy of >98% at Asn303 of RpNCRD with complex-type, heterogeneously branched and predominantly α(2,3) sialylated oligosaccharides. Glycan binding array data characterized both RpNCRD and RhNCRD as mannose-type lectins. RpNCRD also bound LewisY structures whereas RhNCRD bound polylactosamine-containing glycans. Presence of the N-glycan in the CRD increases the glycan binding specificity of RpNCRD. These insights increase our understanding of porcine-specific innate defense against pandemic IAV and may inform  the design of  recombinant SP-D-based antiviral drugs. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Solution NMR Analyses of the C-type Carbohydrate Recognition Domain of DC-SIGNR Protein Reveal Different Binding Modes for HIV-derived Oligosaccharides and Smaller Glycan Fragments

PubMed Central

Probert, Fay; Whittaker, Sara B.-M.; Crispin, Max; Mitchell, Daniel A.; Dixon, Ann M.

2013-01-01

The C-type lectin DC-SIGNR (dendritic cell-specific ICAM-3-grabbing non-integrin-related; also known as L-SIGN or CD299) is a promising drug target due to its ability to promote infection and/or within-host survival of several dangerous pathogens (e.g. HIV and severe acute respiratory syndrome coronavirus (SARS)) via interactions with their surface glycans. Crystallography has provided excellent insight into the mechanism by which DC-SIGNR interacts with small glycans, such as (GlcNAc)2Man3; however, direct observation of complexes with larger, physiological oligosaccharides, such as Man9GlcNAc2, remains elusive. We have utilized solution-state nuclear magnetic resonance spectroscopy to investigate DC-SIGNR binding and herein report the first backbone assignment of its active, calcium-bound carbohydrate recognition domain. Direct interactions with the small sugar fragments Man3, Man5, and (GlcNAc)2Man3 were investigated alongside Man9GlcNAc derived from recombinant gp120 (present on the HIV viral envelope), providing the first structural data for DC-SIGNR in complex with a virus-associated ligand, and unique binding modes were observed for each glycan. In particular, our data show that DC-SIGNR has a different binding mode for glycans on the HIV viral envelope compared with the smaller glycans previously observed in the crystalline state. This suggests that using the binding mode of Man9GlcNAc, instead of those of small glycans, may provide a platform for the design of DC-SIGNR inhibitors selective for high mannose glycans (like those on HIV). 15N relaxation measurements provided the first information on the dynamics of the carbohydrate recognition domain, demonstrating that it is a highly flexible domain that undergoes ligand-induced conformational and dynamic changes that may explain the ability of DC-SIGNR to accommodate a range of glycans on viral surfaces. PMID:23788638

Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts.

PubMed

Yoon, Seon-Joo; Utkina, Natalia; Sadilek, Martin; Yagi, Hirokazu; Kato, Koichi; Hakomori, Sen-itiroh

2013-07-01

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses*

PubMed Central

Yu, Ying; Lasanajak, Yi; Song, Xuezheng; Hu, Liya; Ramani, Sasirekha; Mickum, Megan L.; Ashline, David J.; Prasad, B. V. Venkataram; Estes, Mary K.; Reinhold, Vernon N.; Cummings, Richard D.; Smith, David F.

2014-01-01

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MSn analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MSn are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures. PMID:25048705

Notable Aspects of Glycan-Protein Interactions

PubMed Central

Cohen, Miriam

2015-01-01

This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

Unraveling transcriptional control and cis-regulatory codes using the software suite GeneACT

PubMed Central

Cheung, Tom Hiu; Kwan, Yin Lam; Hamady, Micah; Liu, Xuedong

2006-01-01

Deciphering gene regulatory networks requires the systematic identification of functional cis-acting regulatory elements. We present a suite of web-based bioinformatics tools, called GeneACT , that can rapidly detect evolutionarily conserved transcription factor binding sites or microRNA target sites that are either unique or over-represented in differentially expressed genes from DNA microarray data. GeneACT provides graphic visualization and extraction of common regulatory sequence elements in the promoters and 3'-untranslated regions that are conserved across multiple mammalian species. PMID:17064417

Evolution and Conservation of Plant NLR Functions

PubMed Central

Jacob, Florence; Vernaldi, Saskia; Maekawa, Takaki

2013-01-01

In plants and animals, nucleotide-binding domain and leucine-rich repeats (NLR)-containing proteins play pivotal roles in innate immunity. Despite their similar biological functions and protein architecture, comparative genome-wide analyses of NLRs and genes encoding NLR-like proteins suggest that plant and animal NLRs have independently arisen in evolution. Furthermore, the demonstration of interfamily transfer of plant NLR functions from their original species to phylogenetically distant species implies evolutionary conservation of the underlying immune principle across plant taxonomy. In this review we discuss plant NLR evolution and summarize recent insights into plant NLR-signaling mechanisms, which might constitute evolutionarily conserved NLR-mediated immune mechanisms. PMID:24093022

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

PubMed

Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

2017-07-05

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Comparative glycoproteomics of stem cells identifies new players in ricin toxicity.

PubMed

Stadlmann, Johannes; Taubenschmid, Jasmin; Wenzel, Daniel; Gattinger, Anna; Dürnberger, Gerhard; Dusberger, Frederico; Elling, Ulrich; Mach, Lukas; Mechtler, Karl; Penninger, Josef M

2017-09-28

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

PubMed

Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K; Lira-Navarrete, Erandi; Pedersen, Nis B; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G; Nielsen, Rikke; Christensen, Erik I; Hansen, Lars; Bennett, Eric P; Vakhrushev, Sergey Y; Schjoldager, Katrine T; Clausen, Henrik

2018-05-11

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O -glycan sites. Moreover, we found that O -glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O -glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O -glycosylation of LDLR-related proteins and identified conserved O -glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O -glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O -glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A novel core 1 O-linked glycan-specific binding lectin from the fruiting body of Hericium erinaceus.

PubMed

Kim, Seonghun

2018-02-01

Mucin-type O-glycans are involved in biological functions on the cell surface as well as the glycoproteins and can also be used as specific carbohydrate biomarkers of many diseases. In this study, I purified a novel core 1 O-linked glycan specific lectin, Hericium erinaceus lecin (HeL), from the fruiting body of the mushroom Hericium erinaceus, which is known as the natural source for a sialic acid-binding lectin. Upon optimization of the purification conditions, a sequence of ion exchange, affinity, ion exchange, and size-exclusion chromatography resulted in the highest yield and best quality of lectin without protease activity. The resulting purified HeL is an apparent hexameric protein with a subunit molecular weight of 15kDa, and a pI of 4.3. In hemagglutination inhibition assay, the purified lectin was only inhibited by glycoproteins containing mucin-type O-glycans and reacted weakly with Galβ(1,3)GalNAc. Glycan array analyses showed that HeL specifically interacts with core 1 O-linked glycans as well as extended O-glycan structures containing sialylation or fucosylation. The glycan binding specificity of HeL is comparable to that of peanut agglutinin for detection of a broader range of extended core 1 O-glycan structures. Taken together, these results provide an efficient and optimized procedure for the purification of HeL from the fruiting body of the mushroom Hericium erinaceus. Moreover, HeL represents a powerful tool for analyzing core 1 and extended core 1 O- glycan structures in diagnosis assays. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional Regulation of Sugar Assimilation by N-Glycan-specific Interaction of Pancreatic α-Amylase with Glycoproteins of Duodenal Brush Border Membrane*

PubMed Central

Asanuma-Date, Kimie; Hirano, Yuki; Le, Na; Sano, Kotone; Kawasaki, Nana; Hashii, Noritaka; Hiruta, Yoko; Nakayama, Ken-ichi; Umemura, Mariko; Ishikawa, Kazuhiko; Sakagami, Hiromi; Ogawa, Haruko

2012-01-01

Porcine pancreatic α-amylase (PPA) binds to N-linked glycans of glycoproteins (Matsushita, H., Takenaka, M., and Ogawa, H. (2002) J. Biol Chem., 277, 4680–4686). Immunostaining revealed that PPA is located at the brush-border membrane (BBM) of enterocytes in the duodenum and that the binding is inhibited by mannan but not galactan, indicating that PPA binds carbohydrate-specifically to BBM. The ligands for PPA in BBM were identified as glycoprotein N-glycans that are significantly involved in the assimilation of glucose, including sucrase-isomaltase (SI) and Na+/Glc cotransporter 1 (SGLT1). Binding of SI and SGLT1 in BBM to PPA was dose-dependent and inhibited by mannan. Using BBM vesicles, we found functional changes in PPA and its ligands in BBM due to the N-glycan-specific interaction. The starch-degrading activity of PPA and maltose-degrading activity of SI were enhanced to 240 and 175%, respectively, while Glc uptake by SGLT1 was markedly inhibited by PPA at high but physiologically possible concentrations, and the binding was attenuated by the addition of mannose-specific lectins, especially from Galanthus nivalis. Additionally, recombinant human pancreatic α-amylases expressed in yeast and purified by single-step affinity chromatography exhibited the same carbohydrate binding specificity as PPA in binding assays with sugar-biotinyl polymer probes. The results indicate that mammalian pancreatic α-amylases share a common carbohydrate binding activity and specifically bind to the intestinal BBM. Interaction with N-glycans in the BBM activated PPA and SI to produce much Glc on the one hand and to inhibit Glc absorption by enterocytes via SGLT1 in order to prevent a rapid increase in blood sugar on the other. PMID:22584580

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

PubMed Central

Shewell, Lucy K.; Harvey, Richard M.; Higgins, Melanie A.; Day, Christopher J.; Hartley-Tassell, Lauren E.; Chen, Austen Y.; Gillen, Christine M.; James, David B. A.; Alonzo, Francis; Torres, Victor J.; Walker, Mark J.; Paton, Adrienne W.; Paton, James C.; Jennings, Michael P.

2014-01-01

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a Kd of 1.88 × 10−5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism. PMID:25422425

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

PubMed

Shewell, Lucy K; Harvey, Richard M; Higgins, Melanie A; Day, Christopher J; Hartley-Tassell, Lauren E; Chen, Austen Y; Gillen, Christine M; James, David B A; Alonzo, Francis; Torres, Victor J; Walker, Mark J; Paton, Adrienne W; Paton, James C; Jennings, Michael P

2014-12-09

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

A nonself sugar mimic of the HIV glycan shield shows enhanced antigenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doores, Katie J.; Fulton, Zara; Hong, Vu

2011-08-24

Antibody 2G12 uniquely neutralizes a broad range of HIV-1 isolates by binding the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. Antigens that resemble these natural epitopes of 2G12 would be highly desirable components for an HIV-1 vaccine. However, host-produced (self)-carbohydrate motifs have been unsuccessful so far at eliciting 2G12-like antibodies that cross-react with gp120. Based on the surprising observation that 2G12 binds nonproteinaceous monosaccharide D-fructose with higher affinity than D-mannose, we show here that a designed set of nonself, synthetic monosaccharides are potent antigens. When introduced to the terminus of the D1 arm of protein glycans recognized by 2G12,more » their antigenicity is significantly enhanced. Logical variation of these unnatural sugars pinpointed key modifications, and the molecular basis of this increased antigenicity was elucidated using high-resolution crystallographic analyses. Virus-like particle protein conjugates containing such nonself glycans are bound more tightly by 2G12. As immunogens they elicit higher titers of antibodies than those immunogenic conjugates containing the self D1 glycan motif. These antibodies generated from nonself immunogens also cross-react with this self motif, which is found in the glycan shield, when it is presented in a range of different conjugates and glycans. However, these antibodies did not bind this glycan motif when present on gp120.« less

CORECLUST: identification of the conserved CRM grammar together with prediction of gene regulation.

PubMed

Nikulova, Anna A; Favorov, Alexander V; Sutormin, Roman A; Makeev, Vsevolod J; Mironov, Andrey A

2012-07-01

Identification of transcriptional regulatory regions and tracing their internal organization are important for understanding the eukaryotic cell machinery. Cis-regulatory modules (CRMs) of higher eukaryotes are believed to possess a regulatory 'grammar', or preferred arrangement of binding sites, that is crucial for proper regulation and thus tends to be evolutionarily conserved. Here, we present a method CORECLUST (COnservative REgulatory CLUster STructure) that predicts CRMs based on a set of positional weight matrices. Given regulatory regions of orthologous and/or co-regulated genes, CORECLUST constructs a CRM model by revealing the conserved rules that describe the relative location of binding sites. The constructed model may be consequently used for the genome-wide prediction of similar CRMs, and thus detection of co-regulated genes, and for the investigation of the regulatory grammar of the system. Compared with related methods, CORECLUST shows better performance at identification of CRMs conferring muscle-specific gene expression in vertebrates and early-developmental CRMs in Drosophila.

Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans

NASA Astrophysics Data System (ADS)

Zhao, Nan; Martin, Brigitte E.; Yang, Chun-Kai; Luo, Feng; Wan, Xiu-Feng

2015-10-01

Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2-6Galβ, human-origin viruses could bind glycans with Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galβ1-4GlcNAc substructures; Galβ and GlcNAcβ terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2-3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms.

Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears.

PubMed

Kautto, Liisa; Nguyen-Khuong, Terry; Everest-Dass, Arun; Leong, Andrea; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H; Peterson, Robyn

2016-04-01

The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin; 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins; 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells; 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with α2-3,6,8 neuraminidase (sialidase) and α1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Localization of an evolutionarily conserved protein proton pyrophosphatase in evolutionarily distant plants oryza sativa and physcomitrella patens

USDA-ARS?s Scientific Manuscript database

Proton Pyrophosphatase (H+-PPase) is a highly evolutionarily conserved protein that is prevalent in the plant kingdom. One of the salient features of H+-PPase expression pattern, at least in vascular plants like Arabidopsis, is its conspicuous localization in both actively dividing cells and the phl...

Host specific glycans are correlated with susceptibility to infection by lagoviruses, but not with their virulence.

PubMed

Lopes, Ana M; Breiman, Adrien; Lora, Mónica; Le Moullac-Vaidye, Béatrice; Galanina, Oxana; Nyström, Kristina; Marchandeau, Stephane; Le Gall-Reculé, Ghislaine; Strive, Tanja; Neimanis, Aleksija; Bovin, Nicolai V; Ruvoën-Clouet, Nathalie; Esteves, Pedro J; Abrantes, Joana; Le Pendu, Jacques

2017-11-29

The rabbit hemorrhagic disease virus (RHDV) and the European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus , respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged and many non-pathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, non-pathogenic lagoviruses and EBHSV potentially play a role in determining host range and virulence of lagoviruses. We observed binding to A, B or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits ( Oryctolagus cuniculus ), that have recently been classified as GI strains. Yet, we could not explain the emergence of virulence since similar glycan specificities were found between several pathogenic and non-pathogenic strains. By contrast, EBHSV, recently classified as GII.1, bound to terminal β-linked N-acetylglucosamine residues of O-glycans. Expression of these attachment factors in the upper respiratory and digestive tracts in three lagomorph species ( Oryctolagus cuniculus, Lepus europaeus and Sylvilagus floridanus ) showed species-specific patterns regarding the susceptibility to infection by these viruses, indicating that species-specific glycan expression is likely a major contributor to lagoviruses host specificity and range. IMPORTANCE Lagoviruses constitute a genus of the Caliciviridae family, comprising highly pathogenic viruses, RHDV and EBHSV, which infect rabbits and hares, respectively. Recently, non-pathogenic strains were discovered and new pathogenic strains have emerged. In addition, host jumps between lagomorphs are observed. The mechanisms responsible for the emergence of pathogenicity and host-species range are unknown. Previous studies showed that RHDV strains attach to glycans expressed in the upper respiratory and digestive tracts of rabbits, the likely doors of virus entry. Here we studied the glycan-binding properties of novel pathogenic and non-pathogenic strains looking for a link between glycan-binding and virulence or between glycan specificity and host range. We found that glycan binding did not correlate with virulence. However, expression of glycan motifs in the upper respiratory and digestive tracts of lagomorphs revealed species-specific patterns associated with the host range of the virus strains, suggesting that glycan diversity contributes to lagoviruses' host range. Copyright © 2017 American Society for Microbiology.

C-type lectins: their network and roles in pathogen recognition and immunity.

PubMed

Mayer, Sabine; Raulf, Marie-Kristin; Lepenies, Bernd

2017-02-01

C-type lectins (CTLs) represent the most complex family of animal/human lectins that comprises 17 different groups. During evolution, CTLs have developed by diversification to cover a broad range of glycan ligands. However, ligand binding by CTLs is not necessarily restricted to glycans as some CTLs also bind to proteins, lipids, inorganic molecules, or ice crystals. CTLs share a common fold that harbors a Ca 2+ for contact to the sugar and about 18 invariant residues in a phylogenetically conserved pattern. In vertebrates, CTLs have numerous functions, including serum glycoprotein homeostasis, pathogen sensing, and the initiation of immune responses. Myeloid CTLs in innate immunity are mainly expressed by antigen-presenting cells and play a prominent role in the recognition of a variety of pathogens such as fungi, bacteria, viruses, and parasites. However, myeloid CTLs such as the macrophage inducible CTL (Mincle) or Clec-9a may also bind to self-antigens and thus contribute to immune homeostasis. While some CTLs induce pro-inflammatory responses and thereby lead to activation of adaptive immune responses, other CTLs act as inhibitory receptors and dampen cellular functions. Since CTLs are key players in pathogen recognition and innate immunity, targeting CTLs may be a promising strategy for cell-specific delivery of drugs or vaccine antigens and to modulate immune responses.

A chicken influenza virus recognizes fucosylated α2,3 sialoglycan receptors on the epithelial cells lining upper respiratory tracts of chickens.

PubMed

Hiono, Takahiro; Okamatsu, Masatoshi; Nishihara, Shoko; Takase-Yoden, Sayaka; Sakoda, Yoshihiro; Kida, Hiroshi

2014-05-01

Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid α2,3 galactose (SAα2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR) bound to fucose-branched SAα2,3Gal glycans, whereas the binding towards linear SAα2,3Gal glycans was weak. On the epithelial cells of the upper respiratory tracts of chickens, fucose-branched SAα2,3Gal glycans were detected, but not linear SAα2,3Gal glycans. The growth of Ck/IBR in MDCK-FUT cells, which were genetically prepared to express fucose-branched SAα2,3Gal glycans, was significantly higher than that in the parental MDCK cells. The present results indicate that fucose-branched SAα2,3Gal glycans existing on the epithelial cells lining the upper respiratory tracts of chickens are critical for recognition by Ck/IBR. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs.

PubMed

Arata-Kawai, Hanayo; Singer, Mark S; Bistrup, Annette; Zante, Annemieke van; Wang, Yang-Qing; Ito, Yuki; Bao, Xingfeng; Hemmerich, Stefan; Fukuda, Minoru; Rosen, Steven D

2011-01-01

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

The glycan structure of albumin Redhill, a glycosylated variant of human serum albumin.

PubMed

Kragh-Hansen, U; Donaldson, D; Jensen, P H

2001-11-26

Although human serum albumin is synthesized without carbohydrate, glycosylated variants of the protein can be found. We have determined the structure of the glycan bound to the double-mutant albumin Redhill (-1 Arg, 320 Ala-->Thr). The oligosaccharide was released from the protein using anhydrous hydrazine, and its structure was investigated using neuraminidase and a reagent array analysis method, which is based on the use of specific exoglycosidases. The glycan was shown to be a disialylated biantennary complex type oligosaccharide N-linked to 318 Asn. However, a minor part (11 mol%) of the glycan was without sialic acid. The structure is principally the same as that of glycans bound to two other types of glycosylated albumin variants. Glycosylation can affect, for example, the fatty acid binding properties of albumin. Taking the present information into account, it is apparent that different effects on binding are caused not by different glycan structures but by different locations of attachment, with the possible addition of local conformational changes in the protein molecule.

Distinct roles of N- and O-glycans in cellulase activity and stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T.

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalyticmore » domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

Molecular Mechanism of Flocculation Self-Recognition in Yeast and Its Role in Mating and Survival

PubMed Central

Goossens, Katty V. Y.; Ielasi, Francesco S.; Nookaew, Intawat; Stals, Ingeborg; Alonso-Sarduy, Livan; Daenen, Luk; Van Mulders, Sebastiaan E.; Stassen, Catherine; van Eijsden, Rudy G. E.; Siewers, Verena; Delvaux, Freddy R.; Kasas, Sandor; Nielsen, Jens; Devreese, Bart

2015-01-01

ABSTRACT We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca2+ takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca2+. These results unify the generally accepted lectin hypothesis with the historically first-proposed “Ca2+-bridge” hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating. PMID:25873380

Distinct roles of N- and O-glycans in cellulase activity and stability

DOE PAGES

Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T.; ...

2017-12-11

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalyticmore » domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells

PubMed Central

Taverna, Elena; Mora-Bermúdez, Felipe; Strzyz, Paulina J.; Florio, Marta; Icha, Jaroslav; Haffner, Christiane; Norden, Caren; Wilsch-Bräuninger, Michaela; Huttner, Wieland B.

2016-01-01

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change. PMID:26879757

Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin.

PubMed

Viswanathan, Karthik; Koh, Xiaoying; Chandrasekaran, Aarthi; Pappas, Claudia; Raman, Rahul; Srinivasan, Aravind; Shriver, Zachary; Tumpey, Terrence M; Sasisekharan, Ram

2010-10-29

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.

Determinants of Glycan Receptor Specificity of H2N2 Influenza A Virus Hemagglutinin

PubMed Central

Chandrasekaran, Aarthi; Pappas, Claudia; Raman, Rahul; Srinivasan, Aravind; Shriver, Zachary; Tumpey, Terrence M.; Sasisekharan, Ram

2010-01-01

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA. PMID:21060797

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man*

PubMed Central

Macauley, Matthew S.; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M.; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C.

2015-01-01

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. PMID:26507663

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man.

PubMed

Macauley, Matthew S; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C

2015-12-11

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Hierarchical sampling for metastable conformers determines biomolecular recognition: the case of malectin and diglucosylated N-glycan interactions.

PubMed

Mamidi, Ashalatha Sreshty; Surolia, Avadhesha

2015-01-01

Structural information over the entire course of binding interactions based on the analyses of energy landscapes is described, which provides a framework to understand the events involved during biomolecular recognition. Conformational dynamics of malectin's exquisite selectivity for diglucosylated N-glycan (Dig-N-glycan), a highly flexible oligosaccharide comprising of numerous dihedral torsion angles, are described as an example. For this purpose, a novel approach based on hierarchical sampling for acquiring metastable molecular conformations constituting low-energy minima for understanding the structural features involved in a biologic recognition is proposed. For this purpose, four variants of principal component analysis were employed recursively in both Cartesian space and dihedral angles space that are characterized by free energy landscapes to select the most stable conformational substates. Subsequently, k-means clustering algorithm was implemented for geometric separation of the major native state to acquire a final ensemble of metastable conformers. A comparison of malectin complexes was then performed to characterize their conformational properties. Analyses of stereochemical metrics and other concerted binding events revealed surface complementarity, cooperative and bidentate hydrogen bonds, water-mediated hydrogen bonds, carbohydrate-aromatic interactions including CH-π and stacking interactions involved in this recognition. Additionally, a striking structural transition from loop to β-strands in malectin CRD upon specific binding to Dig-N-glycan is observed. The interplay of the above-mentioned binding events in malectin and Dig-N-glycan supports an extended conformational selection model as the underlying binding mechanism.

Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

PubMed Central

Chatterjee, Aparajita; Ratner, Daniel M.; Ryan, Christopher M.; Johnson, Patricia J.; O’Keefe, Barry R.; Secor, W. Evan; Anderson, Deborah J.; Robbins, Phillips W.; Samuelson, John

2015-01-01

Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas. PMID:26252012

Multiple N-glycans cooperate in the subcellular targeting and functioning of Arabidopsis KORRIGAN1.

PubMed

Rips, Stephan; Bentley, Nolan; Jeong, In Sil; Welch, Justin L; von Schaewen, Antje; Koiwa, Hisashi

2014-09-01

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-β1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Δall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells. © 2014 American Society of Plant Biologists. All rights reserved.

Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides

NASA Astrophysics Data System (ADS)

Chai, Wengang; Zhang, Yibing; Mauri, Laura; Ciampa, Maria G.; Mulloy, Barbara; Sonnino, Sandro; Feizi, Ten

2018-05-01

Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.

A recombinant fungal lectin for labeling truncated glycans on human cancer cells.

PubMed

Audfray, Aymeric; Beldjoudi, Mona; Breiman, Adrien; Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

2015-01-01

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.

A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

PubMed Central

Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

2015-01-01

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases. PMID:26042789

The α-galactomannan Davanat binds galectin-1 at a site different from the conventional galectin carbohydrate binding domain

PubMed Central

Miller, Michelle C; Klyosov, Anatole; Mayo, Kevin H

2009-01-01

Galectins are a sub-family of lectins, defined by their highly conserved β-sandwich structures and ability to bind to β-galactosides, like Gal β1-4 Glc (lactose). Here, we used 15N-1H HSQC and pulse field gradient (PFG) NMR spectroscopy to demonstrate that galectin-1 (gal-1) binds to the relatively large galactomannan Davanat, whose backbone is composed of β1-4-linked d-mannopyranosyl units to which single d-galactopyranosyl residues are periodically attached via α1-6 linkage (weight-average MW of 59 kDa). The Davanat binding domain covers a relatively large area on the surface of gal-1 that runs across the dimer interface primarily on that side of the protein opposite to the lactose binding site. Our data show that gal-1 binds Davanat with an apparent equilibrium dissociation constant (Kd) of 10 × 10−6 M, compared to 260 × 10−6 M for lactose, and a stiochiometry of about 3 to 6 gal-1 molecules per Davanat molecule. Mannan also interacts at the same galactomannan binding domain on gal-1, but with at least 10-fold lower avidity, supporting the role of galactose units in Davanat for relatively strong binding to gal-1. We also found that the β-galactoside binding domain remains accessible in the gal-1/Davanat complex, as lactose can still bind with no apparent loss in affinity. In addition, gal-1 binding to Davanat also modifies the supermolecular structure of the galactomannan and appears to reduce its hydrodynamic radius and disrupt inter-glycan interactions thereby reducing glycan-mediated solution viscosity. Overall, our findings contribute to understanding gal-1–carbohydrate interactions and provide insight into gal-1 function with potentially significant biological consequences. PMID:19541770

N-Glycans Modulate the Function of Human Corticosteroid-Binding Globulin*

PubMed Central

Sumer-Bayraktar, Zeynep; Kolarich, Daniel; Campbell, Matthew P.; Ali, Sinan; Packer, Nicolle H.; Thaysen-Andersen, Morten

2011-01-01

Human corticosteroid-binding globulin (CBG), a heavily glycosylated protein containing six N-linked glycosylation sites, transports cortisol and other corticosteroids in blood circulation. Here, we investigate the biological importance of the N-glycans of CBG derived from human serum by performing a structural and functional characterization of CBG N-glycosylation. Liquid chromatography-tandem MS-based glycoproteomics and glycomics combined with exoglycosidase treatment revealed 26 complex type N-glycoforms, all of which were terminated with α2,3-linked neuraminic acid (NeuAc) residues. The CBG N-glycans showed predominantly bi- and tri-antennary branching, but higher branching was also observed. N-glycans from all six N-glycosylation sites were identified with high site occupancies (70.5–99.5%) and glycoforms from all sites contained a relatively low degree of core-fucosylation (0–34.9%). CBG showed site-specific glycosylation and the site-to-site differences in core-fucosylation and branching could be in silico correlated with the accessibility to the individual glycosylation sites on the maturely folded protein. Deglycosylated and desialylated CBG analogs were generated to investigate the biological importance of CBG N-glycans. As a functional assay, MCF-7 cells were challenged with native and glycan-modified CBG and the amount of cAMP, which is produced as a quantitative response upon CBG binding to its cell surface receptor, was used to evaluate the CBG:receptor interaction. The removal of both CBG N-glycans and NeuAc residues increased the production of cAMP significantly. This confirms that N-glycans are involved in the CBG:receptor interaction and indicates that the modulation is performed by steric and/or electrostatic means through the terminal NeuAc residues. PMID:21558494

Phyloscan: locating transcription-regulating binding sites in mixed aligned and unaligned sequence data.

PubMed

Palumbo, Michael J; Newberg, Lee A

2010-07-01

The transcription of a gene from its DNA template into an mRNA molecule is the first, and most heavily regulated, step in gene expression. Especially in bacteria, regulation is typically achieved via the binding of a transcription factor (protein) or small RNA molecule to the chromosomal region upstream of a regulated gene. The protein or RNA molecule recognizes a short, approximately conserved sequence within a gene's promoter region and, by binding to it, either enhances or represses expression of the nearby gene. Since the sought-for motif (pattern) is short and accommodating to variation, computational approaches that scan for binding sites have trouble distinguishing functional sites from look-alikes. Many computational approaches are unable to find the majority of experimentally verified binding sites without also finding many false positives. Phyloscan overcomes this difficulty by exploiting two key features of functional binding sites: (i) these sites are typically more conserved evolutionarily than are non-functional DNA sequences; and (ii) these sites often occur two or more times in the promoter region of a regulated gene. The website is free and open to all users, and there is no login requirement. Address: (http://bayesweb.wadsworth.org/phyloscan/).

Host adaptation of a bacterial toxin from the human pathogen Salmonella Typhi

PubMed Central

Deng, Lingquan; Song, Jeongmin; Gao, Xiang; Wang, Jiawei; Yu, Hai; Chen, Xi; Varki, Nissi; Naito-Matsui, Yuko; Galán, Jorge E.; Varki, Ajit

2014-01-01

Salmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here we report that typhoid toxin binds to and is toxic towards cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid toxin. The atomic structure of typhoid toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi’s host specificity and may help the development of therapies for typhoid fever. PMID:25480294

A Steric-inhibition model for regulation of nucleotide exchange via the Dock180 family of GEFs.

PubMed

Lu, Mingjian; Kinchen, Jason M; Rossman, Kent L; Grimsley, Cynthia; Hall, Matthew; Sondek, John; Hengartner, Michael O; Yajnik, Vijay; Ravichandran, Kodi S

2005-02-22

CDM (CED-5, Dock180, Myoblast city) family members have been recently identified as novel, evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho-family GTPases . They regulate multiple processes, including embryonic development, cell migration, apoptotic-cell engulfment, tumor invasion, and HIV-1 infection, in diverse model systems . However, the mechanism(s) of regulation of CDM proteins has not been well understood. Here, our studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 family of GEFs. At basal state, the N-terminal SH3 domain of Dock180 binds to the distant catalytic Docker domain and negatively regulates the function of Dock180. Further studies revealed that the SH3:Docker interaction sterically blocks Rac access to the Docker domain. Interestingly, ELMO binding to the SH3 domain of Dock180 disrupted the SH3:Docker interaction, facilitated Rac access to the Docker domain, and contributed to the GEF activity of the Dock180/ELMO complex. Additional genetic rescue studies in C. elegans suggested that the regulation of the Docker-domain-mediated GEF activity by the SH3 domain and its adjoining region is evolutionarily conserved. This steric-inhibition model may be a general mechanism for regulating multiple SH3-domain-containing Dock180 family members and may have implications for a variety of biological processes.

The conserved role of Krox-20 in directing Hox gene expression during vertebrate hindbrain segmentation.

PubMed

Nonchev, S; Maconochie, M; Vesque, C; Aparicio, S; Ariza-McNaughton, L; Manzanares, M; Maruthainar, K; Kuroiwa, A; Brenner, S; Charnay, P; Krumlauf, R

1996-09-03

Transient segmentation in the hindbrain is a fundamental morphogenetic phenomenon in the vertebrate embryo, and the restricted expression of subsets of Hox genes in the developing rhombomeric units and their derivatives is linked with regional specification. Here we show that patterning of the vertebrate hindbrain involves the direct upregulation of the chicken and pufferfish group 2 paralogous genes, Hoxb-2 and Hoxa-2, in rhombomeres 3 and 5 (r3 and r5) by the zinc finger gene Krox-20. We identified evolutionarily conserved r3/r5 enhancers that contain high affinity Krox-20. binding sites capable of mediating transactivation by Krox-20. In addition to conservation of binding sites critical for Krox-20 activity in the chicken Hoxa-2 and pufferfish Hoxb-2 genes, the r3/r5 enhancers are also characterized by the presence of a number of identical motifs likely to be involved in cooperative interactions with Krox-20 during the process of hindbrain patterning in vertebrates.

An Evolutionarily Conserved DOF-CONSTANS Module Controls Plant Photoperiodic Signaling1[OPEN

PubMed Central

2015-01-01

The response to daylength is a crucial process that evolved very early in plant evolution, entitling the early green eukaryote to predict seasonal variability and attune its physiological responses to the environment. The photoperiod responses evolved into the complex signaling pathways that govern the angiosperm floral transition today. The Chlamydomonas reinhardtii DNA-Binding with One Finger (CrDOF) gene controls transcription in a photoperiod-dependent manner, and its misexpression influences algal growth and viability. In short days, CrDOF enhances CrCO expression, a homolog of plant CONSTANS (CO), by direct binding to its promoter, while it reduces the expression of cell division genes in long days independently of CrCO. In Arabidopsis (Arabidopsis thaliana), transgenic plants overexpressing CrDOF show floral delay and reduced expression of the photoperiodic genes CO and FLOWERING LOCUS T. The conservation of the DOF-CO module during plant evolution could be an important clue to understanding diversification by the inheritance of conserved gene toolkits in key developmental programs. PMID:25897001

Structure-function Analysis of Receptor-binding in Adeno-Associated Virus Serotype 6 (AAV-6)

PubMed Central

Xie, Qing; Lerch, Thomas F.; Meyer, Nancy L.; Chapman, Michael S.

2011-01-01

Crystal structures of the AAV-6 capsid at 3 Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests that receptor-attachment is mediated by four residues: Arg576, Lys493, Lys459 and Lys531, defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys531 and Lys459. Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2’s neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality. PMID:21917284

Inhibition of glycosylation on a camelid antibody uniquely affects its FcγRI binding activity.

PubMed

Krahn, Natalie; Spearman, Maureen; Meier, Markus; Dorion-Thibaudeau, July; McDougall, Matthew; Patel, Trushar R; De Crescenzo, Gregory; Durocher, Yves; Stetefeld, Jörg; Butler, Michael

2017-01-01

Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity are well known. However, little is understood about the effect that these modifications have on binding to the high affinity FcγRI receptor. This study analyzed the effect of variable N-glycosylation on a human-llama hybrid mAb (EG2-hFc, 80kDa) binding to FcγRI including a comparison to a full-sized IgG1 (DP-12, 150kDa). This was achieved by the addition of three glycosylation inhibitors (swainsonine, castanospermine, and kifunensine) independently to Chinese hamster ovary (CHO) cell cultures to generate hybrid and high mannose glycan structures. Biophysical analysis by circular dichroism, dynamic light scattering and analytical ultra-centrifugation confirmed that the solution-behaviour of the mAbs remained constant over multiple concentrations and glycan treatments. However, changes were observed when studying the interaction of FcγRI with variously glycosylated mAbs. Both mAbs were observed to have a decreased binding affinity upon treatment with swainsonine which produced hybrid glycans. Following de-glycosylation the binding affinity for EG2-hFc was only marginally reduced (6-fold) compared to a drastic (118-fold) decrease for DP-12. In summary, our data suggest that the relatively low molecular weight of chimeric EG2-hFc may contribute to its enhanced stability against glycan changes making it a highly suitable mAb candidate for therapeutic applications. Copyright © 2016 Elsevier B.V. All rights reserved.

PUF Proteins: Cellular Functions and Potential Applications.

PubMed

Kiani, Seyed Jalal; Taheri, Tahereh; Rafati, Sima; Samimi-Rad, Katayoun

2017-01-01

RNA-binding proteins play critical roles in the regulation of gene expression. Among several families of RNA-binding proteins, PUF (Pumilio and FBF) proteins have been the subject of extensive investigations, as they can bind RNA in a sequence-specific manner and they are evolutionarily conserved among a wide range of organisms. The outstanding feature of these proteins is a highly conserved RNA-binding domain, which is known as the Pumilio-homology domain (PUM-HD) that mostly consists of eight tandem repeats. Each repeat recognizes an RNA base with a simple three-letter code that can be programmed in order to change the sequence-specificity of the protein. Using this tailored architecture, researchers have been able to change the specificity of the PUM-HD and target desired transcripts in the cell, even in subcellular compartments. The potential applications of this versatile tool in molecular cell biology seem unbounded and the use of these factors in pharmaceutics might be an interesting field of study in near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J.

PubMed

Wacker, Stephan Armin; Alvarado, Cristobal; von Wichert, Götz; Knippschild, Uwe; Wiedenmann, Jörg; Clauss, Karen; Nienhaus, Gerd Ulrich; Hameister, Horst; Baumann, Bernd; Borggrefe, Tilman; Knöchel, Walter; Oswald, Franz

2011-01-05

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

Oligosaccharide Binding Proteins from Bifidobacterium longum subsp. infantis Reveal a Preference for Host Glycans

PubMed Central

Garrido, Daniel; Kim, Jae Han; German, J. Bruce; Raybould, Helen E.; Mills, David A.

2011-01-01

Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process. PMID:21423604

Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications

NASA Astrophysics Data System (ADS)

Geissner, Andreas; Seeberger, Peter H.

2016-06-01

A major branch of glycobiology and glycan-focused biomedicine studies the interaction between carbohydrates and other biopolymers, most importantly, glycan-binding proteins. Today, this research into glycan-biopolymer interaction is unthinkable without glycan arrays, tools that enable high-throughput analysis of carbohydrate interaction partners. Glycan arrays offer many applications in basic biochemical research, for example, defining the specificity of glycosyltransferases and lectins such as immune receptors. Biomedical applications include the characterization and surveillance of influenza strains, identification of biomarkers for cancer and infection, and profiling of immune responses to vaccines. Here, we review major applications of glycan arrays both in basic and applied research. Given the dynamic nature of this rapidly developing field, we focus on recent findings.

Structural evolution of glycan recognition by a family of potent HIV antibodies.

PubMed

Garces, Fernando; Sok, Devin; Kong, Leopold; McBride, Ryan; Kim, Helen J; Saye-Francisco, Karen F; Julien, Jean-Philippe; Hua, Yuanzi; Cupo, Albert; Moore, John P; Paulson, James C; Ward, Andrew B; Burton, Dennis R; Wilson, Ian A

2014-09-25

The HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here, we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g., Ab PGT124) or engage multiple glycans (e.g., Abs PGT121-123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans. Copyright © 2014 Elsevier Inc. All rights reserved.

High-Throughput Lectin Microarray-Based Analysis of Live Cell Surface Glycosylation

PubMed Central

Li, Yu; Tao, Sheng-ce; Zhu, Heng; Schneck, Jonathan P.

2011-01-01

Lectins, plant-derived glycan-binding proteins, have long been used to detect glycans on cell surfaces. However, the techniques used to characterize serum or cells have largely been limited to mass spectrometry, blots, flow cytometry, and immunohistochemistry. While these lectin-based approaches are well established and they can discriminate a limited number of sugar isomers by concurrently using a limited number of lectins, they are not amenable for adaptation to a high-throughput platform. Fortunately, given the commercial availability of lectins with a variety of glycan specificities, lectins can be printed on a glass substrate in a microarray format to profile accessible cell-surface glycans. This method is an inviting alternative for analysis of a broad range of glycans in a high-throughput fashion and has been demonstrated to be a feasible method of identifying binding-accessible cell surface glycosylation on living cells. The current unit presents a lectin-based microarray approach for analyzing cell surface glycosylation in a high-throughput fashion. PMID:21400689

A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

PubMed

Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah; Dallabernadina, Pietro; Boos, Irene; Andersen, Mathias C F; Kotake, Toshihisa; Knox, J Paul; Hahn, Michael G; Clausen, Mads H; Pfrengle, Fabian

2017-11-01

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision. © 2017 American Society of Plant Biologists. All Rights Reserved.

Violation of an Evolutionarily Conserved Immunoglobulin Diversity Gene Sequence Preference Promotes Production of dsDNA-Specific IgG Antibodies

PubMed Central

Silva-Sanchez, Aaron; Liu, Cun Ren; Vale, Andre M.; Khass, Mohamed; Kapoor, Pratibha; Elgavish, Ada; Ivanov, Ivaylo I.; Ippolito, Gregory C.; Schelonka, Robert L.; Schoeb, Trenton R.; Burrows, Peter D.; Schroeder, Harry W.

2015-01-01

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies. PMID:25706374

Structure and Function of Mammalian Carbohydrate-Lectin Interactions

NASA Astrophysics Data System (ADS)

Anderson, Kevin; Evers, David; Rice, Kevin G.

Over the past three decades the field of glycobiology has expanded beyond a basic understanding of the structure and biosynthesis of glycoprotein, proteoglycans, and glycolipids toward a more detailed picture of how these molecules afford communication through binding to mammalian lectins. Although the number of different mammalian lectin domains appears to be finite and even much smaller than early estimates predicated based on the diversity of glycan structures, nature appears capable of using these in numerous combinations to fine tune specificity. The following provides an overview of the major classes of mammalian lectins and discusses their glycan binding specificity. The review provides a snapshot of the field of glycobiology that continues to grow providing an increasing number of examples of biological processes that rely upon glycan-lectin binding.

Automated glycan assembly of galactosylated xyloglucan oligosaccharides and their recognition by plant cell wall glycan-directed antibodies.

PubMed

Dallabernardina, Pietro; Ruprecht, Colin; Smith, Peter J; Hahn, Michael G; Urbanowicz, Breeanna R; Pfrengle, Fabian

2017-12-06

We report the automated glycan assembly of oligosaccharides related to the plant cell wall hemicellulosic polysaccharide xyloglucan. The synthesis of galactosylated xyloglucan oligosaccharides was enabled by introducing p-methoxybenzyl (PMB) as a temporary protecting group for automated glycan assembly. The generated oligosaccharides were printed as microarrays, and the binding of a collection of xyloglucan-directed monoclonal antibodies (mAbs) to the oligosaccharides was assessed. We also demonstrated that the printed glycans can be further enzymatically modified while appended to the microarray surface by Arabidopsis thaliana xyloglucan xylosyltransferase 2 (AtXXT2).

The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku

PubMed Central

di Fagagna, Fabrizio d'Adda; Weller, Geoffrey R.; Doherty, Aidan J.; Jackson, Stephen P.

2003-01-01

Mu bacteriophage inserts its DNA into the genome of host bacteria and is used as a model for DNA transposition events in other systems. The eukaryotic Ku protein has key roles in DNA repair and in certain transposition events. Here we show that the Gam protein of phage Mu is conserved in bacteria, has sequence homology with both subunits of Ku, and has the potential to adopt a similar architecture to the core DNA-binding region of Ku. Through biochemical studies, we demonstrate that Gam and the related protein of Haemophilus influenzae display DNA binding characteristics remarkably similar to those of human Ku. In addition, we show that Gam can interfere with Ty1 retrotransposition in Saccharomyces cerevisiae. These data reveal structural and functional parallels between bacteriophage Gam and eukaryotic Ku and suggest that their functions have been evolutionarily conserved. PMID:12524520

Solution Binding and Structural Analyses Reveal Potential Multidrug Resistance Functions for SAV2435 and CTR107 and Other GyrI-like Proteins.

PubMed

Moreno, Andrew; Froehlig, John R; Bachas, Sharrol; Gunio, Drew; Alexander, Teressa; Vanya, Aaron; Wade, Herschel

2016-08-30

Multidrug resistance (MDR) refers to the acquired ability of cells to tolerate a broad range of toxic compounds. One mechanism cells employ is to increase the level of expression of efflux pumps for the expulsion of xenobiotics. A key feature uniting efflux-related mechanisms is multidrug (MD) recognition, either by efflux pumps themselves or by their transcriptional regulators. However, models describing MD binding by MDR effectors are incomplete, underscoring the importance of studies focused on the recognition elements and key motifs that dictate polyspecific binding. One such motif is the GyrI-like domain, which is found in several MDR proteins and is postulated to have been adapted for small-molecule binding and signaling. Here we report the solution binding properties and crystal structures of two proteins containing GyrI-like domains, SAV2435 and CTR107, bound to various ligands. Furthermore, we provide a comparison with deposited crystal structures of GyrI-like proteins, revealing key features of GyrI-like domains that not only support polyspecific binding but also are conserved among GyrI-like domains. Together, our studies suggest that GyrI-like domains perform evolutionarily conserved functions connected to multidrug binding and highlight the utility of these types of studies for elucidating mechanisms of MDR.

Complement Factor H and Simian Virus 40 bind the GM1 ganglioside in distinct conformations.

PubMed

Blaum, Bärbel S; Frank, Martin; Walker, Ross C; Neu, Ursula; Stehle, Thilo

2016-05-01

Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 μs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

PubMed

Lahr, Roni M; Mack, Seshat M; Héroux, Annie; Blagden, Sarah P; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc; Berman, Andrea J

2015-09-18

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes

PubMed Central

Bielska, Ewa; Schuster, Martin; Roger, Yvonne; Berepiki, Adokiye; Soanes, Darren M.; Talbot, Nicholas J.

2014-01-01

Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood. In this paper, we report that a Hook protein, Hok1, is essential for kinesin-3– and dynein-dependent early endosome (EE) motility in the fungus Ustilago maydis. Hok1 binds to EEs via its C-terminal region, where it forms a complex with homologues of human fused toes (FTS) and its interactor FTS- and Hook-interacting protein. A highly conserved N-terminal region is required to bind dynein and kinesin-3 to EEs. To change the direction of EE transport, kinesin-3 is released from organelles, and dynein binds subsequently. A chimaera of human Hook3 and Hok1 rescues the hok1 mutant phenotype, suggesting functional conservation between humans and fungi. We conclude that Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional EE trafficking by controlling attachment of both kinesin-3 and dynein. PMID:24637326

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

DOE PAGES

Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

2015-07-22

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

Role of siglecs and related glycan-binding proteins in immune responses and immunoregulation.

PubMed

Bochner, Bruce S; Zimmermann, Nives

2015-03-01

Virtually all cells and extracellular material are heavily decorated by various glycans, yet our understanding of the structure and function of these moieties lags behind the understanding of nucleic acids, lipids, and proteins. Recent years have seen a tremendous acceleration of knowledge in the field of glycobiology, revealing many intricacies and functional contributions that were previously poorly appreciated or even unrecognized. This review highlights several topics relevant to glycoimmunology in which mammalian and pathogen-derived glycans displayed on glycoproteins and other scaffolds are recognized by specific glycan-binding proteins (GBPs), leading to a variety of proinflammatory and anti-inflammatory cellular responses. The focus for this review is mainly on 2 families of GBPs, sialic acid-binding immunoglobulin-like lectins (siglecs) and selectins, that are involved in multiple steps of the immune response, including distinguishing pathogens from self, cell trafficking to sites of inflammation, fine-tuning of immune responses leading to activation or tolerance, and regulation of cell survival. Importantly for the clinician, accelerated rates of discovery in the field of glycoimmunology are being translated into innovative medical approaches that harness the interaction of glycans and GBPs to the benefit of the host and might soon lead to novel diagnostics and therapeutics. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

PubMed Central

Halder, Sujata; Cotmore, Susan; Heimburg-Molinaro, Jamie; Smith, David F.; Cummings, Richard D.; Chen, Xi; Trollope, Alana J.; North, Simon J.; Haslam, Stuart M.; Dell, Anne; Tattersall, Peter; McKenna, Robert; Agbandje-McKenna, Mavis

2014-01-01

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen. PMID:24475195

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy.

PubMed

Poiroux, Guillaume; Barre, Annick; van Damme, Els J M; Benoist, Hervé; Rougé, Pierre

2017-06-09

Aberrant O -glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O -glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola , and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O -glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors.

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy

PubMed Central

Poiroux, Guillaume; Barre, Annick; van Damme, Els J. M.; Benoist, Hervé; Rougé, Pierre

2017-01-01

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors. PMID:28598369

The Double Face of Mucin-Type O-Glycans in Lectin-Mediated Infection and Immunity.

PubMed

Morozov, Vasily; Borkowski, Julia; Hanisch, Franz-Georg

2018-05-11

Epithelial human blood group antigens (HBGAs) on O-glycans play roles in pathogen binding and the initiation of infection, while similar structures on secretory mucins exert protective functions. These double-faced features of O-glycans in infection and innate immunity are reviewed based on two instructive examples of bacterial and viral pathogens. Helicobacter pylori represents a class 1 carcinogen in the human stomach. By expressing blood group antigen-binding adhesin ( BabA ) and LabA adhesins that bind to Lewis-b and LacdiNAc, respectively, H. pylori colocalizes with the mucin MUC5AC in gastric surface epithelia, but not with MUC6, which is cosecreted with trefoil factor family 2 ( TFF2 ) by deep gastric glands. Both components of the glandular secretome are concertedly up-regulated upon infection. While MUC6 expresses GlcNAc-capped glycans as natural antibiotics for H. pylori growth control, TFF2 may function as a probiotic lectin. In viral infection human noroviruses of the GII genogroup interact with HBGAs via their major capsid protein, VP1. HBGAs on human milk oligosaccharides (HMOs) may exert protective functions by binding to the P2 domain pocket on the capsid. We discuss structural details of the P2 carbohydrate-binding pocket in interaction with blood group H/Lewis-b HMOs and fucoidan-derived oligofucoses as effective interactors for the most prevalent norovirus strains, GII.4 and GII.17.

Anti-GM1 antibodies as a model of the immune response to self-glycans.

PubMed

Nores, Gustavo A; Lardone, Ricardo D; Comín, Romina; Alaniz, María E; Moyano, Ana L; Irazoqui, Fernando J

2008-03-01

Glycans are a class of molecules with high structural variability, frequently found in the plasma membrane facing the extracellular space. Because of these characteristics, glycans are often considered as recognition molecules involved in cell social functions, and as targets of pathogenic factors. Induction of anti-glycan antibodies is one of the early events in immunological defense against bacteria that colonize the body. Because of this natural infection, antibodies recognizing a variety of bacterial glycans are found in sera of adult humans and animals. The immune response to glycans is restricted by self-tolerance, and no antibodies to self-glycans should exist in normal subjects. However, antibodies recognizing structures closely related to self-glycans do exist, and can lead to production of harmful anti-self antibodies. Normal human sera contain low-affinity anti-GM1 IgM-antibodies. Similar antibodies with higher affinity or different isotype are found in some neuropathy patients. Two hypotheses have been developed to explain the origin of disease-associated anti-GM1 antibodies. According to the "molecular mimicry" hypothesis, similarity between GM1 and Campylobacter jejuni lipopolysaccharide carrying a GM1-like glycan is the cause of Guillain-Barré syndrome associated with anti-GM1 IgG-antibodies. According to the "binding site drift" hypothesis, IgM-antibodies associated with disease originate through changes in the binding site of normally occurring anti-GM1 antibodies. We now present an "integrated" hypothesis, combining the "mimicry" and "drift" concepts, which satisfactorily explains most of the published data on anti-GM1 antibodies.

Chemoenzymatic assembly of mammalian O-mannose glycans.

PubMed

Cao, Hongzhi; Meng, Caicai; Sasmal, Aniruddha; Zhang, Yan; Gao, Tian; Liu, Chang-Cheng; Khan, Naazneen; Varki, Ajit; Wang, Fengshan

2018-05-26

O-Mannose glycans account up to 30% of total O-glycans in brain. Previous synthesis and functional studies only focused on the Core M3 O-mannose glycans of α-dystroglycan which are a causative factor for various muscular diseases. In this study, a highly efficient chemoenzymatic strategy was developed that enabled the first collective synthesis of 63 Core M1 and Core M2 O-mannose glycans. This chemoenzymatic strategy features the gram-scale chemical synthesis of 5 judiciously designed core structures, and the diversity-oriented modification of the core structures with 3 enzyme modules to provide 58 complex O-mannose glycans in a linear sequence that does not exceed 4 steps. The binding profiles of synthetic O-mannose glycans with a panel of lectins, antibodies and brain proteins were also explored using the printed O-mannose glycan array. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Galectins as Cancer Biomarkers

PubMed Central

Balan, Vitaly; Nangia-Makker, Pratima; Raz, Avraham

2010-01-01

Galectins are a group of proteins that bind β-galactosides through evolutionarily conserved sequence elements of the carbohydrate recognition domain (CRD). Proteins similar to galectins can be found in very primitive animals such as sponges. Each galectin has an individual carbohydrate binding preference and can be found in cytoplasm as well as in the nucleus. They also can be secreted through non-classical pathways and function extra-cellularly. Experimental and clinical data demonstrate a correlation between galectin expression and tumor progression and metastasis, and therefore, galectins have the potential to serve as reliable tumor markers. In this review, we describe the expression and role of galectins in different cancers and their clinical applications for diagnostic use. PMID:23658855

Removal of either N-glycan site from the envelope receptor binding domain of Moloney and Friend but not AKV mouse ecotropic gammaretroviruses alters receptor usage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knoper, Ryan C.; Ferrarone, John; Yan Yuhe

2009-09-01

Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIHmore » 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.« less

Defining the interaction of human soluble lectin ZG16p and mycobacterial phosphatidylinositol mannosides

PubMed Central

Ikeda, Akemi; Kojima-Aikawa, Kyoko; Taniguchi, Naoyuki; Varón Silva, Daniel; Feizi, Ten; Seeberger, Peter H.; Yamaguchi, Yoshiki

2018-01-01

ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analyses of the interactions of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs), using glycan microarray and NMR. Pathogen-related glycan microarray analysis identified phosphatidylinositol mono- and di-mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation Transfer Difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interacts with ZG16p using the mannose residues. Binding site of PIMs is identified by chemical shift perturbation experiments using uniformly 15N-labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan, which would help to consider the physiological role of ZG16p. PMID:25919894

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yue; Sheng, Ju; Baggen, Jim

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon’ on the virus surface. The sialic acid receptor induces a cascade of conformational changes inmore » the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Furthermore, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry.« less

Myosin MyTH4-FERM structures highlight important principles of convergent evolution.

PubMed

Planelles-Herrero, Vicente José; Blanc, Florian; Sirigu, Serena; Sirkia, Helena; Clause, Jeffrey; Sourigues, Yannick; Johnsrud, Daniel O; Amigues, Beatrice; Cecchini, Marco; Gilbert, Susan P; Houdusse, Anne; Titus, Margaret A

2016-05-24

Myosins containing MyTH4-FERM (myosin tail homology 4-band 4.1, ezrin, radixin, moesin, or MF) domains in their tails are found in a wide range of phylogenetically divergent organisms, such as humans and the social amoeba Dictyostelium (Dd). Interestingly, evolutionarily distant MF myosins have similar roles in the extension of actin-filled membrane protrusions such as filopodia and bind to microtubules (MT), suggesting that the core functions of these MF myosins have been highly conserved over evolution. The structures of two DdMyo7 signature MF domains have been determined and comparison with mammalian MF structures reveals that characteristic features of MF domains are conserved. However, across millions of years of evolution conserved class-specific insertions are seen to alter the surfaces and the orientation of subdomains with respect to each other, likely resulting in new sites for binding partners. The MyTH4 domains of Myo10 and DdMyo7 bind to MT with micromolar affinity but, surprisingly, their MT binding sites are on opposite surfaces of the MyTH4 domain. The structural analysis in combination with comparison of diverse MF myosin sequences provides evidence that myosin tail domain features can be maintained without strict conservation of motifs. The results illustrate how tuning of existing features can give rise to new structures while preserving the general properties necessary for myosin tails. Thus, tinkering with the MF domain enables it to serve as a multifunctional platform for cooperative recruitment of various partners, allowing common properties such as autoinhibition of the motor and microtubule binding to arise through convergent evolution.

The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2G12 Recognizes a Cluster of α1→2 Mannose Residues on the Outer Face of gp120

PubMed Central

Scanlan, Christopher N.; Pantophlet, Ralph; Wormald, Mark R.; Ollmann Saphire, Erica; Stanfield, Robyn; Wilson, Ian A.; Katinger, Hermann; Dwek, Raymond A.; Rudd, Pauline M.; Burton, Dennis R.

2002-01-01

2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N→A substitution produced significant decreases in 2G12 binding affinity to gp120JR-CSF. Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120JR-CSF. Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with Aspergillus saitoi mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Manα1→2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or N-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue N-butyl-deoxynojirimycin similarly implicated Manα1→2Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Manα1→2Man-linked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation. PMID:12072529

Diversity in the protein N-glycosylation pathways within the Campylobacter genus.

PubMed

Nothaft, Harald; Scott, Nichollas E; Vinogradov, Evgeny; Liu, Xin; Hu, Rui; Beadle, Bernadette; Fodor, Christopher; Miller, William G; Li, Jianjun; Cordwell, Stuart J; Szymanski, Christine M

2012-11-01

The foodborne bacterial pathogen, Campylobacter jejuni, possesses an N-linked protein glycosylation (pgl) pathway involved in adding conserved heptasaccharides to asparagine-containing motifs of >60 proteins, and releasing the same glycan into its periplasm as free oligosaccharides. In this study, comparative genomics of all 30 fully sequenced Campylobacter taxa revealed conserved pgl gene clusters in all but one species. Structural, phylogenetic and immunological studies showed that the N-glycosylation systems can be divided into two major groups. Group I includes all thermotolerant taxa, capable of growth at the higher body temperatures of birds, and produce the C. jejuni-like glycans. Within group I, the niche-adapted C. lari subgroup contain the smallest genomes among the epsilonproteobacteria, and are unable to glucosylate their pgl pathway glycans potentially reminiscent of the glucosyltransferase regression observed in the O-glycosylation system of Neisseria species. The nonthermotolerant Campylobacters, which inhabit a variety of hosts and niches, comprise group II and produce an unexpected diversity of N-glycan structures varying in length and composition. This includes the human gut commensal, C. hominis, which produces at least four different N-glycan structures, akin to the surface carbohydrate diversity observed in the well-studied commensal, Bacteroides. Both group I and II glycans are immunogenic and cell surface exposed, making these structures attractive targets for vaccine design and diagnostics.

Restricted N-glycan conformational space in the PDB and its implication in glycan structure modeling.

PubMed

Jo, Sunhwan; Lee, Hui Sun; Skolnick, Jeffrey; Im, Wonpil

2013-01-01

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

PubMed Central

Jo, Sunhwan; Lee, Hui Sun; Skolnick, Jeffrey; Im, Wonpil

2013-01-01

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures. PMID:23516343

An Evolutionarily Conserved DOF-CONSTANS Module Controls Plant Photoperiodic Signaling.

PubMed

Lucas-Reina, Eva; Romero-Campero, Francisco J; Romero, José M; Valverde, Federico

2015-06-01

The response to daylength is a crucial process that evolved very early in plant evolution, entitling the early green eukaryote to predict seasonal variability and attune its physiological responses to the environment. The photoperiod responses evolved into the complex signaling pathways that govern the angiosperm floral transition today. The Chlamydomonas reinhardtii DNA-Binding with One Finger (CrDOF) gene controls transcription in a photoperiod-dependent manner, and its misexpression influences algal growth and viability. In short days, CrDOF enhances CrCO expression, a homolog of plant CONSTANS (CO), by direct binding to its promoter, while it reduces the expression of cell division genes in long days independently of CrCO. In Arabidopsis (Arabidopsis thaliana), transgenic plants overexpressing CrDOF show floral delay and reduced expression of the photoperiodic genes CO and FLOWERING LOCUS T. The conservation of the DOF-CO module during plant evolution could be an important clue to understanding diversification by the inheritance of conserved gene toolkits in key developmental programs. © 2015 American Society of Plant Biologists. All Rights Reserved.

The PRC2-binding long non-coding RNAs in human and mouse genomes are associated with predictive sequence features

NASA Astrophysics Data System (ADS)

Tu, Shiqi; Yuan, Guo-Cheng; Shao, Zhen

2017-01-01

Recently, long non-coding RNAs (lncRNAs) have emerged as an important class of molecules involved in many cellular processes. One of their primary functions is to shape epigenetic landscape through interactions with chromatin modifying proteins. However, mechanisms contributing to the specificity of such interactions remain poorly understood. Here we took the human and mouse lncRNAs that were experimentally determined to have physical interactions with Polycomb repressive complex 2 (PRC2), and systematically investigated the sequence features of these lncRNAs by developing a new computational pipeline for sequences composition analysis, in which each sequence is considered as a series of transitions between adjacent nucleotides. Through that, PRC2-binding lncRNAs were found to be associated with a set of distinctive and evolutionarily conserved sequence features, which can be utilized to distinguish them from the others with considerable accuracy. We further identified fragments of PRC2-binding lncRNAs that are enriched with these sequence features, and found they show strong PRC2-binding signals and are more highly conserved across species than the other parts, implying their functional importance.

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

Functions of galectins as 'self/non-self'-recognition and effector factors.

PubMed

Vasta, Gerardo R; Feng, Chiguang; González-Montalbán, Nuria; Mancini, Justin; Yang, Lishi; Abernathy, Kelsey; Frost, Graeme; Palm, Cheyenne

2017-07-31

Carbohydrate structures on the cell surface encode complex information that through specific recognition by carbohydrate-binding proteins (lectins) modulates interactions between cells, cells and the extracellular matrix, or mediates recognition of potential microbial pathogens. Galectins are a family of ß-galactoside-binding lectins, which are evolutionary conserved and have been identified in most organisms, from fungi to invertebrates and vertebrates, including mammals. Since their discovery in the 1970s, their biological roles, initially understood as limited to recognition of endogenous carbohydrate ligands in embryogenesis and development, have expanded in recent years by the discovery of their roles in tissue repair and regulation of immune homeostasis. More recently, evidence has accumulated to support the notion that galectins can also bind glycans on the surface of potentially pathogenic microbes, and function as recognition and effector factors in innate immunity, thus establishing a new paradigm. Furthermore, some parasites 'subvert' the recognition roles of the vector/host galectins for successful attachment or invasion. These recent findings have revealed a striking functional diversification in this structurally conserved lectin family. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Solid-phase glycan isolation for glycomics analysis

PubMed Central

Yang, Shuang; Zhang, Hui

2013-01-01

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis. PMID:23090885

Solid-phase glycan isolation for glycomics analysis.

PubMed

Yang, Shuang; Zhang, Hui

2012-12-01

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

PubMed Central

Rydzak, Joanna; Kaczmarek, Radoslaw; Czerwinski, Marcin; Lukasiewicz, Jolanta; Tyborowska, Jolanta; Szewczyk, Boguslaw; Jaskiewicz, Ewa

2015-01-01

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands. PMID:25588042

Top-Down Chemoenzymatic Approach to Synthesizing Diverse High-Mannose N-Glycans and Related Neoglycoproteins for Carbohydrate Microarray Analysis.

PubMed

Toonstra, Christian; Wu, Lisa; Li, Chao; Wang, Denong; Wang, Lai-Xi

2018-05-22

High-mannose-type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose-type N-glycans, and glycan array technology has provided an avenue to probe these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesize a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the Man 9 GlcNAc 2 Asn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins containing the oligomannose moieties. The glycoepitopes displayed were characterized using an array of glycan-binding proteins, including the broadly virus-neutralizing agents, glycan-specific antibody 2G12, Galanthus nivalis lectin (GNA), and Narcissus pseudonarcissus lectin (NPA).

Development of a Schistosoma mansoni shotgun O-glycan microarray and application to the discovery of new antigenic schistosome glycan motifs.

PubMed

van Diepen, Angela; van der Plas, Arend-Jan; Kozak, Radoslaw P; Royle, Louise; Dunne, David W; Hokke, Cornelis H

2015-06-01

Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

PubMed Central

Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

2006-01-01

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

Computational approaches to define a human milk metaglycome

PubMed Central

Agravat, Sanjay B.; Song, Xuezheng; Rojsajjakul, Teerapat; Cummings, Richard D.; Smith, David F.

2016-01-01

Motivation: The goal of deciphering the human glycome has been hindered by the lack of high-throughput sequencing methods for glycans. Although mass spectrometry (MS) is a key technology in glycan sequencing, MS alone provides limited information about the identification of monosaccharide constituents, their anomericity and their linkages. These features of individual, purified glycans can be partly identified using well-defined glycan-binding proteins, such as lectins and antibodies that recognize specific determinants within glycan structures. Results: We present a novel computational approach to automate the sequencing of glycans using metadata-assisted glycan sequencing, which combines MS analyses with glycan structural information from glycan microarray technology. Success in this approach was aided by the generation of a ‘virtual glycome’ to represent all potential glycan structures that might exist within a metaglycomes based on a set of biosynthetic assumptions using known structural information. We exploited this approach to deduce the structures of soluble glycans within the human milk glycome by matching predicted structures based on experimental data against the virtual glycome. This represents the first meta-glycome to be defined using this method and we provide a publically available web-based application to aid in sequencing milk glycans. Availability and implementation: http://glycomeseq.emory.edu Contact: sagravat@bidmc.harvard.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26803164

A Lectin from Platypodium elegans with Unusual Specificity and Affinity for Asymmetric Complex N-Glycans*

PubMed Central

Benevides, Raquel Guimarães; Ganne, Géraldine; Simões, Rafael da Conceição; Schubert, Volker; Niemietz, Mathäus; Unverzagt, Carlo; Chazalet, Valérie; Breton, Christelle; Varrot, Annabelle; Cavada, Benildo Sousa; Imberty, Anne

2012-01-01

Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume plant from the Dalbergieae tribe. The gene of Platypodium elegans lectin A has been cloned, and the resulting 261-amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin from the same tribe. The recombinant lectin has been expressed in Escherichia coli and refolded from inclusion bodies. Analysis of specificity by glycan array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6-arm of the N-glycan, whereas extensions by GlcNAc, Gal, and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn-linked heptasaccharides prepared by the semi-synthetic method. Strong affinity with Kd of 4.5 μm was obtained for both ligands. Crystal structures of Platypodium elegans lectin A complexed with branched trimannose and symmetrical complex-type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution, respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighboring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site with extensive additional contacts on both arms. The GlcNAc on the 6-arm is bound in a constrained conformation that may rationalize the higher affinity observed on the glycan array for N-glycans with only a mannose on the 6-arm. PMID:22692206

The Interaction of N-Glycans in Fcγ Receptor I α-Chain with Escherichia coli K1 Outer Membrane Protein A for Entry into Macrophages

PubMed Central

Krishnan, Subramanian; Liu, Fan; Abrol, Ravinder; Hodges, Jacqueline; Goddard, William A.; Prasadarao, Nemani V.

2014-01-01

Neonatal meningitis, caused by Escherichia coli K1, is a serious central nervous system disease. We have established that macrophages serve as permissive niches for E. coli K1 to multiply in the host and for attaining a threshold level of bacterial load, which is a prerequisite for the onset of the disease. Here, we demonstrate experimentally that three N-glycans in FcγRIa interact with OmpA of E. coli K1 for binding to and entering the macrophages. Adoptive transfer of FcγRIa−/− bone marrow-derived macrophages transfected with FcγRIa into FcγRIa−/− newborn mice renders them susceptible to E. coli K1-induced meningitis. In contrast, mice that received bone marrow-derived macrophages transfected with FcγRIa in which N-glycosylation sites 1, 4, and 5 are mutated to alanines exhibit resistance to E. coli K1 infection. Our molecular dynamics and simulation studies predict that N-glycan 5 exhibits strong binding at the barrel site of OmpA formed by loops 3 and 4, whereas N-glycans 1 and 4 interact with loops 1, 3, and 4 of OmpA at tip regions. Molecular modeling data also suggest no role for the IgG binding site in the invasion process. In agreement, experimental mutations in IgG binding site had no effect on the E. coli K1 entry into macrophages in vitro or on the onset of meningitis in newborn mice. Together, this integration of experimental and computational studies reveals how the N-glycans in FcγRIa interact with the OmpA of E. coli K1 for inducing the disease pathogenesis. PMID:25231998

Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module*

PubMed Central

Taylor, Courtney B.; Talib, M. Faiz; McCabe, Clare; Bu, Lintao; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3–6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function. PMID:22147693

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

PubMed

Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

2013-05-01

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

Structures of Human Pumilio with Noncognate RNAs Reveal Molecular Mechanisms for Binding Promiscuity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta,Y.; Nair, D.; Wharton, R.

2008-01-01

Pumilio is a founder member of the evolutionarily conserved Puf family of RNA-binding proteins that control a number of physiological processes in eukaryotes. A structure of human Pumilio (hPum) Puf domain bound to a Drosophila regulatory sequence showed that each Puf repeat recognizes a single nucleotide. Puf domains in general bind promiscuously to a large set of degenerate sequences, but the structural basis for this promiscuity has been unclear. Here, we describe the structures of hPum Puf domain complexed to two noncognate RNAs, CycBreverse and Puf5. In each complex, one of the nucleotides is ejected from the binding surface, inmore » effect, acting as a 'spacer.' The complexes also reveal the plasticity of several Puf repeats, which recognize noncanonical nucleotides. Together, these complexes provide a molecular basis for recognition of degenerate binding sites, which significantly increases the number of mRNAs targeted for regulation by Puf proteins in vivo.« less

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins.

PubMed

Norman, Michael; Rivers, Caroline; Lee, Youn-Bok; Idris, Jalilah; Uney, James

2016-12-01

RNA-binding proteins play a central role in cellular metabolism by orchestrating the complex interactions of coding, structural and regulatory RNA species. The SAFB (scaffold attachment factor B) proteins (SAFB1, SAFB2 and SAFB-like transcriptional modulator, SLTM), which are highly conserved evolutionarily, were first identified on the basis of their ability to bind scaffold attachment region DNA elements, but attention has subsequently shifted to their RNA-binding and protein-protein interactions. Initial studies identified the involvement of these proteins in the cellular stress response and other aspects of gene regulation. More recently, the multifunctional capabilities of SAFB proteins have shown that they play crucial roles in DNA repair, processing of mRNA and regulatory RNA, as well as in interaction with chromatin-modifying complexes. With the advent of new techniques for identifying RNA-binding sites, enumeration of individual RNA targets has now begun. This review aims to summarise what is currently known about the functions of SAFB proteins. © 2016 The Author(s).

Preparation and biological activities of anti-HER2 monoclonal antibodies with fully core-fucosylated homogeneous bi-antennary complex-type glycans.

PubMed

Tsukimura, Wataru; Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Matsuda, Akio; Takegawa, Kaoru; Furukawa, Kiyoshi; Shirai, Takashi

2017-12-01

Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs.

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray.

PubMed

Mickum, Megan L; Prasanphanich, Nina Salinger; Song, Xuezheng; Dorabawila, Nelum; Mandalasi, Msano; Lasanajak, Yi; Luyai, Anthony; Secor, W Evan; Wilkins, Patricia P; Van Die, Irma; Smith, David F; Nyame, A Kwame; Cummings, Richard D; Rivera-Marrero, Carlos A

2016-05-01

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

PubMed Central

Mickum, Megan L.; Prasanphanich, Nina Salinger; Song, Xuezheng; Dorabawila, Nelum; Mandalasi, Msano; Lasanajak, Yi; Luyai, Anthony; Secor, W. Evan; Wilkins, Patricia P.; Van Die, Irma; Smith, David F.; Nyame, A. Kwame

2016-01-01

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. PMID:26883596

Crystal Structure of the Heterotrimeric Integrin-Binding Region of Laminin-111.

PubMed

Pulido, David; Hussain, Sadaf-Ahmahni; Hohenester, Erhard

2017-03-07

Laminins are cell-adhesive glycoproteins that are essential for basement membrane assembly and function. Integrins are important laminin receptors, but their binding site on the heterotrimeric laminins is poorly defined structurally. We report the crystal structure at 2.13 Å resolution of a minimal integrin-binding fragment of mouse laminin-111, consisting of ∼50 residues of α1β1γ1 coiled coil and the first three laminin G-like (LG) domains of the α1 chain. The LG domains adopt a triangular arrangement, with the C terminus of the coiled coil situated between LG1 and LG2. The critical integrin-binding glutamic acid residue in the γ1 chain tail is surface exposed and predicted to bind to the metal ion-dependent adhesion site in the integrin β1 subunit. Additional contacts to the integrin are likely to be made by the LG1 and LG2 surfaces adjacent to the γ1 chain tail, which are notably conserved and free of obstructing glycans. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Modulation of IgG1 immunoeffector function by glycoengineering of the GDP-fucose biosynthesis pathway.

PubMed

Kelly, Ronan M; Kowle, Ronald L; Lian, Zhirui; Strifler, Beth A; Witcher, Derrick R; Parekh, Bhavin S; Wang, Tongtong; Frye, Christopher C

2018-03-01

Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG C H 2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function. © 2017 Wiley Periodicals, Inc.

Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics.

PubMed

Price, Joshua L; Shental-Bechor, Dalit; Dhar, Apratim; Turner, Maurice J; Powers, Evan T; Gruebele, Martin; Levy, Yaakov; Kelly, Jeffery W

2010-11-03

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible loops. Our data are consistent with the hypothesis that specific, evolved protein-glycan contacts must also play an important role in mediating the beneficial energetic effects on protein folding that glycosylation can confer.

Sialoglycoproteins and N-glycans from secreted exosomes of ovarian carcinoma cells.

PubMed

Escrevente, Cristina; Grammel, Nicolas; Kandzia, Sebastian; Zeiser, Johannes; Tranfield, Erin M; Conradt, Harald S; Costa, Júlia

2013-01-01

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.

Glycan-independent binding and internalization of human IgM to FCMR, its cognate cellular receptor

NASA Astrophysics Data System (ADS)

Lloyd, Katy A.; Wang, Jiabin; Urban, Britta C.; Czajkowsky, Daniel M.; Pleass, Richard J.

2017-02-01

IgM is the first antibody to be produced in immune responses and plays an important role in the neutralization of bacteria and viruses. Human IgM is heavily glycosylated, featuring five N-linked glycan sites on the μ chain and one on the J-chain. Glycosylation of IgG is known to modulate the effector functions of Fcγ receptors. In contrast, little is known about the effect of glycosylation on IgM binding to the human Fcμ receptor (hFCMR). In this study, we identify the Cμ4 domain of IgM as the target of hFCMR, and show that binding and internalization of IgM by hFCMR is glycan-independent. We generated a homology-based structure for hFCMR and used molecular dynamic simulations to show how this interaction with IgM may occur. Finally, we reveal an inhibitory function for IgM in the proliferation of T cells.

CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells.

PubMed

Peng, Wenjie; Paulson, James C

2017-09-13

CD22 is a sialic acid-binding immunoglobulin-like lectin (Siglec) that is highly expressed on B-cells and B cell lymphomas, and is a validated target for antibody and nanoparticle based therapeutics. However, cell targeted therapeutics are limited by their complexity, heterogeneity, and difficulties in production. We describe here a chemically defined natural N-linked glycan scaffold that displays high affinity CD22 glycan ligands and outcompetes the natural ligand for the receptor, resulting in single molecule binding to CD22 and endocytosis into cells. Binding affinity is increased by up to 1500-fold compared to the monovalent ligand, while maintaining the selectivity for hCD22 over other Siglecs. Conjugates of these multivalent ligands with auristatin and saporin toxins are efficiently internalized via hCD22 resulting in killing of B-cell lymphoma cells. This single molecule ligand targeting strategy represents an alternative to antibody- and nanoparticle-mediated approaches for delivery of agents to cells expressing CD22 and other Siglecs.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

Modifications of Glycans: Biological Significance and Therapeutic Opportunities

PubMed Central

Muthana, Saddam M.; Campbell, Christopher; Gildersleeve, Jeffrey C.

2012-01-01

Carbohydrates play a central role in a wide range of biological processes. As with nucleic acids and proteins, modifications of specific sites within the glycan chain can modulate a carbohydrate’s overall biological function. For example, acylation, methylation, sulfation, epimerization, and phosphorylation can occur at various positions within a carbohydrate to modulate bioactivity. Therefore, there is significant interest in identifying discrete carbohydrate modifications and understanding their biological effects. Additionally, enzymes that catalyze those modifications and proteins that bind modified glycans provide numerous targets for therapeutic intervention. This review will focus on modifications of glycans that occur after the oligomer/polymer has been assembled, generally referred to as postglycosylational modifications. PMID:22195988

Glycobiology of the ocular surface: Mucins and lectins

PubMed Central

Argüeso, Pablo

2013-01-01

Glycosylation is an important and common form of posttranscriptional modification of proteins in cells. A vast array of biological functions has been ascribed to glycans during the last decade thanks to a rapid evolution in glycomic technologies. Glycogenes highly expressed at the human ocular surface include families of glycosyltransferases, proteoglycans, glycan degradation proteins, as well as mucins and carbohydrate-binding proteins such as the galectins. On the apical glycocalyx, mucin O-glycans promote boundary lubrication, prevent bacterial adhesion and endocytic activity, and maintain epithelial barrier function through interactions with galectins. The emerging roles attributed to glycans are contributing to the appreciation of their biological capabilities at the ocular surface. PMID:23325272

The glycan-mediated mechanism on the interactions of gp120 with CD4 and antibody: Insights from molecular dynamics simulation.

PubMed

Zhang, Yan; Niu, Yuzhen; Tian, Jiaqi; Liu, Xuewei; Yao, Xiaojun; Liu, Huanxiang

2017-12-01

N-linked glycans such as 234 and 276 gp 120 glycans are vital components of HIV evasion from humoral immunity and important for HIV-1 neutralization of many broadly neutralizing antibodies (bNAbs). However, it is unknown the action mechanism of two glycans. To investigate the roles of the glycans on the interactions of gp120 with CD4 and antibody, molecular dynamic simulations based on gp120-CD4-8ANC195 complex with 234 and 276 gp 120 glycans, 234 gp 120 glycan, 276 gp 120 glycan, and without glycan were performed. Our results reveal that 276 gp 120 glycan can enhance gp120-CD4 and gp120-antibody interactions through the formation of hydrogen bonds of the glycan with CD4 and antibody and make the binding interface of gp120, CD4 and antibody stable; 234 gp 120 glycan primarily reinforces gp120-antibody interactions and weakly affects gp120-CD4 interactions as it mainly lies between gp120 and antibody. The co-operating of two glycans can enhance gp120-CD4 and gp120-antibody associations. Through the structural analysis, it can be seen that 234 gp 120 glycan leads to moving upward of two glycans and the variable region of heavy chain, which is favorable for the interactions of gp120 with CD4 and antibody. The information obtained in this study can provide the guidance for design vaccines and small molecule inhibitors. © 2017 John Wiley & Sons A/S.

Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue

USDA-ARS?s Scientific Manuscript database

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The...

Trichuris suis-induced modulation of human dendritic cell function is glycan-mediated.

PubMed

Klaver, Elsenoor J; Kuijk, Loes M; Laan, Lisa C; Kringel, Helene; van Vliet, Sandra J; Bouma, Gerd; Cummings, Richard D; Kraal, Georg; van Die, Irma

2013-03-01

Human monocyte-derived dendritic cells (DCs) show remarkable phenotypic changes upon direct contact with soluble products (SPs) of Trichuris suis, a pig whipworm that is experimentally used in therapies to ameliorate inflammation in patients with Crohn's disease and multiple sclerosis. These changes may contribute to the observed induction of a T helper 2 (Th2) response and the suppression of Toll-like receptor (TLR)-induced Th1 and Th17 responses by human DCs primed with T. suis SPs. Here it is demonstrated that glycans of T. suis SPs contribute significantly to the suppression of the lipopolysaccharide (LPS)-induced expression in DCs of a broad variety of cytokines and chemokines, including important pro-inflammatory mediators such as TNF-α, IL-6, IL-12, lymphotoxin α (LTA), C-C Motif Ligand (CCL)2, C-X-C Motif Ligands (CXCL)9 and CXCL10. In addition, the data show that human DCs strongly bind T. suis SP-glycans via the C-type lectin receptors (CLRs) mannose receptor (MR) and DC-specific ICAM-3-grabbing non-integrin (DC-SIGN). The interaction of DCs with T. suis glycans likely involves mannose-type glycans, rather than fucosylated glycans, which differs from DC binding to soluble egg antigens of the human worm parasite, Schistosoma mansoni. In addition, macrophage galactose-type lectin (MGL) recognises T. suis SPs, which may contribute to the interaction with immature DCs or other MGL-expressing immune cells such as macrophages. The interaction of T. suis glycans with CLRs of human DCs may be essential for the ability of T. suis to suppress a pro-inflammatory phenotype of human DCs. The finding that the T. suis-induced modulation of human DC function is glycan-mediated is novel and indicates that helminth glycans contribute to the dampening of inflammation in a wide range of human inflammatory diseases. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Massive Gene Transfer and Extensive RNA Editing of a Symbiotic Dinoflagellate Plastid Genome

PubMed Central

Mungpakdee, Sutada; Shinzato, Chuya; Takeuchi, Takeshi; Kawashima, Takeshi; Koyanagi, Ryo; Hisata, Kanako; Tanaka, Makiko; Goto, Hiroki; Fujie, Manabu; Lin, Senjie; Satoh, Nori; Shoguchi, Eiichi

2014-01-01

Genome sequencing of Symbiodinium minutum revealed that 95 of 109 plastid-associated genes have been transferred to the nuclear genome and subsequently expanded by gene duplication. Only 14 genes remain in plastids and occur as DNA minicircles. Each minicircle (1.8–3.3 kb) contains one gene and a conserved noncoding region containing putative promoters and RNA-binding sites. Nine types of RNA editing, including a novel G/U type, were discovered in minicircle transcripts but not in genes transferred to the nucleus. In contrast to DNA editing sites in dinoflagellate mitochondria, which tend to be highly conserved across all taxa, editing sites employed in DNA minicircles are highly variable from species to species. Editing is crucial for core photosystem protein function. It restores evolutionarily conserved amino acids and increases peptidyl hydropathy. It also increases protein plasticity necessary to initiate photosystem complex assembly. PMID:24881086

Rational and Computational Design of Stabilized Variants of Cyanovirin-N which Retain Affinity and Specificity for Glycan Ligands

PubMed Central

Patsalo, Vadim; Raleigh, Daniel P.; Green, David F.

2011-01-01

Cyanovirin-N (CVN) is an 11-kDa pseudo-symmetric cyanobacterial lectin that has been shown to inhibit infection by the Human Immunodeficiency Virus (HIV) by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work we describe rationally-designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson–Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 m of GuaHCl against chemical denaturation, relative to a previously-characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations, and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding. PMID:22032696

Databases and Associated Tools for Glycomics and Glycoproteomics.

PubMed

Lisacek, Frederique; Mariethoz, Julien; Alocci, Davide; Rudd, Pauline M; Abrahams, Jodie L; Campbell, Matthew P; Packer, Nicolle H; Ståhle, Jonas; Widmalm, Göran; Mullen, Elaine; Adamczyk, Barbara; Rojas-Macias, Miguel A; Jin, Chunsheng; Karlsson, Niclas G

2017-01-01

The access to biodatabases for glycomics and glycoproteomics has proven to be essential for current glycobiological research. This chapter presents available databases that are devoted to different aspects of glycobioinformatics. This includes oligosaccharide sequence databases, experimental databases, 3D structure databases (of both glycans and glycorelated proteins) and association of glycans with tissue, disease, and proteins. Specific search protocols are also provided using tools associated with experimental databases for converting primary glycoanalytical data to glycan structural information. In particular, researchers using glycoanalysis methods by U/HPLC (GlycoBase), MS (GlycoWorkbench, UniCarb-DB, GlycoDigest), and NMR (CASPER) will benefit from this chapter. In addition we also include information on how to utilize glycan structural information to query databases that associate glycans with proteins (UniCarbKB) and with interactions with pathogens (SugarBind).

Development of Rare Bacterial Monosaccharide Analogs for Metabolic Glycan Labeling in Pathogenic Bacteria.

PubMed

Clark, Emily L; Emmadi, Madhu; Krupp, Katharine L; Podilapu, Ananda R; Helble, Jennifer D; Kulkarni, Suvarn S; Dube, Danielle H

2016-12-16

Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here, we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino d-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.

A Conserved Non-Canonical Docking Mechanism Regulates the Binding of Dual Specificity Phosphatases to Cell Integrity Mitogen-Activated Protein Kinases (MAPKs) in Budding and Fission Yeasts

PubMed Central

Sacristán-Reviriego, Almudena; Madrid, Marisa; Cansado, José; Martín, Humberto; Molina, María

2014-01-01

Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi. PMID:24465549

Sialic acid-dependent cell entry of human enterovirus D68

DOE PAGES

Liu, Yue; Sheng, Ju; Baggen, Jim; ...

2015-11-13

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon’ on the virus surface. The sialic acid receptor induces a cascade of conformational changes inmore » the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Furthermore, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry.« less

Sialic acid-dependent cell entry of human enterovirus D68

PubMed Central

Liu, Yue; Sheng, Ju; Baggen, Jim; Meng, Geng; Xiao, Chuan; Thibaut, Hendrik J.; van Kuppeveld, Frank J. M.; Rossmann, Michael G.

2015-01-01

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon' on the virus surface. The sialic acid receptor induces a cascade of conformational changes in the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Thus, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry. PMID:26563423

A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP.

PubMed

Vigil, Dominico; Lin, Jung-Hsin; Sotriffer, Christoph A; Pennypacker, Juniper K; McCammon, J Andrew; Taylor, Susan S

2006-01-01

Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RIalpha R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.

The Role of Conserved N-Linked Glycans on Ebola Virus Glycoprotein 2.

PubMed

Lennemann, Nicholas J; Walkner, Madeline; Berkebile, Abigail R; Patel, Neil; Maury, Wendy

2015-10-01

N-linked glycosylation is a common posttranslational modification found on viral glycoproteins (GPs) and involved in promoting expression, cellular attachment, protection from proteases, and antibody evasion. The GP subunit GP2 of filoviruses contains 2 completely conserved N-linked glycosylation sites (NGSs) at N563 and N618, suggesting that they have been maintained through selective pressures. We assessed mutants lacking these glycans for expression and function to understand the role of these sites during Ebola virus entry. Elimination of either GP2 glycan individually had a modest effect on GP expression and no impact on antibody neutralization of vesicular stomatitis virus pseudotyped with Ebola virus GP. However, loss of the N563 glycan enhanced entry by 2-fold and eliminated GP detection by a well-characterized monoclonal antibody KZ52. Loss of both sites dramatically decreased GP expression and abolished entry. Surprisingly, a GP that retained a single NGS at N563, eliminating the remaining 16 NGSs from GP1 and GP2, had detectable expression, a modest increase in entry, and pronounced sensitivity to antibody neutralization. Our findings support the importance of the GP2 glycans in GP expression/structure, transduction efficiency, and antibody neutralization, particularly when N-linked glycans are also removed from GP1. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Proton movement and coupling in the POT family of peptide transporters

PubMed Central

Parker, Joanne L.; Li, Chenghan; Brinth, Allete; Wang, Zhi; Vogeley, Lutz; Solcan, Nicolae; Ledderboge-Vucinic, Gregory; Swanson, Jessica M. J.; Caffrey, Martin; Voth, Gregory A.

2017-01-01

POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate. PMID:29180426

Proton movement and coupling in the POT family of peptide transporters.

PubMed

Parker, Joanne L; Li, Chenghan; Brinth, Allete; Wang, Zhi; Vogeley, Lutz; Solcan, Nicolae; Ledderboge-Vucinic, Gregory; Swanson, Jessica M J; Caffrey, Martin; Voth, Gregory A; Newstead, Simon

2017-12-12

POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate. Copyright © 2017 the Author(s). Published by PNAS.

The N14 anti-afamin antibody Fab: a rare VL1 CDR glycosylation, crystallographic re-sequencing, molecular plasticity and conservative versus enthusiastic modelling.

PubMed

Naschberger, Andreas; Fürnrohr, Barbara G; Lenac Rovis, Tihana; Malic, Suzana; Scheffzek, Klaus; Dieplinger, Hans; Rupp, Bernhard

2016-12-01

The monoclonal antibody N14 is used as a detection antibody in ELISA kits for the human glycoprotein afamin, a member of the albumin family, which has recently gained interest in the capture and stabilization of Wnt signalling proteins, and for its role in metabolic syndrome and papillary thyroid carcinoma. As a rare occurrence, the N14 Fab is N-glycosylated at Asn26L at the onset of the V L 1 antigen-binding loop, with the α-1-6 core fucosylated complex glycan facing out of the L1 complementarity-determining region. The crystal structures of two non-apparent (pseudo) isomorphous crystals of the N14 Fab were analyzed, which differ significantly in the elbow angles, thereby cautioning against the overinterpretation of domain movements upon antigen binding. In addition, the map quality at 1.9 Å resolution was sufficient to crystallographically re-sequence the variable V L and V H domains and to detect discrepancies in the hybridoma-derived sequence. Finally, a conservatively refined parsimonious model is presented and its statistics are compared with those from a less conservatively built model that has been modelled more enthusiastically. Improvements to the PDB validation reports affecting ligands, clashscore and buried surface calculations are suggested.

NKG2D and CD94 bind to multimeric alpha2,3-linked N-acetylneuraminic acid.

PubMed

Imaizumi, Yuzo; Higai, Koji; Suzuki, Chiho; Azuma, Yutaro; Matsumoto, Kojiro

2009-05-08

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to alpha2,3-sialylated human alpha(1)-acid glycoprotein (AGP) but not to alpha2,6-sialylated AGP. Mutagenesis revealed that (152)Y of NKG2D and (144)F and (160)N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to alpha2,3-sialylated but not to alpha2,6-sialylated multi-antennary N-glycans.

Glycomics-based analysis of chicken red blood cells provides insight into the selectivity of the viral agglutination assay.

PubMed

Aich, Udayanath; Beckley, Nia; Shriver, Zachary; Raman, Rahul; Viswanathan, Karthik; Hobbie, Sven; Sasisekharan, Ram

2011-05-01

Agglutination of red blood cells (RBCs), including chicken RBCs (cRBCs), has been used extensively to estimate viral titer, to screen glycan-receptor binding preference, and to assess the protective response of vaccines. Although this assay enjoys widespread use, some virus strains do not agglutinate RBCs. To address these underlying issues and to increase the usefulness of cRBCs as tools for studying viruses, such as influenza, we analyzed the cell surface N-glycans of cRBCs. On the basis of the results obtained from complementary analytical strategies, including MS, 1D and 2D-NMR spectroscopy, exoglycosidase digestions, and HPLC profiling, we report the major glycan structures present on cRBCs. By comparing the glycan structures of cBRCs with those of representative human upper respiratory cells, we offer a possible explanation for the fact that certain influenza strains do not agglutinate cRBCs, using specific human-adapted influenza hemagglutinins as examples. Finally, recent understanding of the role of various glycan structures in high affinity binding to influenza hemagglutinins provides context to our findings. These results illustrate that the field of glycomics can provide important information with respect to the experimental systems used to characterize, detect and study viruses. © 2011 The Authors Journal compilation © 2011 FEBS.

Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates

PubMed Central

Yoshino, Timothy P.; Wu, Xiao-Jun; Gonzalez, Laura A.; Hokke, Cornelis H.

2013-01-01

Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval S. mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins, as well as the ability or hemocytes to acquire shared glycans by the selective binding of parasite-released LTP. Unraveling the functional significance of these naturally expressed and acquired shared glycans on specific hemocyte populations represents an important challenge for future investigations. PMID:23085445

Analysis of sDMA modifications of PIWI proteins

PubMed Central

Honda, Shozo; Kirino, Yoriko; Kirino, Yohei

2015-01-01

Summary Arginine methylation is an important post-translational protein modification that modulates protein function for a wide range of biological processes. PIWI proteins, a subclade of the Argonaute family proteins, contain evolutionarily conserved symmetrical dimethylarginines (sDMAs). It has become increasingly apparent that the sDMAs of PIWI proteins serve as binding elements for TUDOR-domain containing proteins and that sDMA-dependent protein interactions play crucial roles in the biogenesis and function of PIWI-interacting RNAs (piRNAs). We describe a method for detecting PIWI sDMAs and purifying PIWI/piRNA complexes using anti-sDMA antibodies. PMID:24178562

Tribbles in normal and malignant haematopoiesis.

PubMed

Stein, Sarah J; Mack, Ethan A; Rome, Kelly S; Pear, Warren S

2015-10-01

The tribbles protein family, an evolutionarily conserved group of pseudokinases, have been shown to regulate multiple cellular events including those involved in normal and malignant haematopoiesis. The three mammalian Tribbles homologues, Trib1, Trib2 and Trib3 are characterized by conserved motifs, including a pseudokinase domain and a C-terminal E3 ligase-binding domain. In this review, we focus on the role of Trib (mammalian Tribbles homologues) proteins in mammalian haematopoiesis and leukaemia. The Trib proteins show divergent expression in haematopoietic cells, probably indicating cell-specific functions. The roles of the Trib proteins in oncogenesis are also varied and appear to be tissue-specific. Finally, we discuss the potential mechanisms by which the Trib proteins preferentially regulate these processes in multiple cell types. © 2015 Authors; published by Portland Press Limited.

Mutations in FLNB cause boomerang dysplasia

PubMed Central

Bicknell, L; Morgan, T; Bonafe, L; Wessels, M; Bialer, M; Willems, P; Cohn, D; Krakow, D; Robertson, S

2005-01-01

Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans. PMID:15994868

Mutations in FLNB cause boomerang dysplasia.

PubMed

Bicknell, L S; Morgan, T; Bonafé, L; Wessels, M W; Bialer, M G; Willems, P J; Cohn, D H; Krakow, D; Robertson, S P

2005-07-01

Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans.

Corticotropin-releasing hormone-binding protein and stress: from invertebrates to humans.

PubMed

Ketchesin, Kyle D; Stinnett, Gwen S; Seasholtz, Audrey F

2017-09-01

Corticotropin-releasing hormone (CRH) is a key regulator of the stress response. This peptide controls the hypothalamic-pituitary-adrenal (HPA) axis as well as a variety of behavioral and autonomic stress responses via the two CRH receptors, CRH-R1 and CRH-R2. The CRH system also includes an evolutionarily conserved CRH-binding protein (CRH-BP), a secreted glycoprotein that binds CRH with subnanomolar affinity to modulate CRH receptor activity. In this review, we discuss the current literature on CRH-BP and stress across multiple species, from insects to humans. We describe the regulation of CRH-BP in response to stress, as well as genetic mouse models that have been utilized to elucidate the in vivo role(s) of CRH-BP in modulating the stress response. Finally, the role of CRH-BP in the human stress response is examined, including single nucleotide polymorphisms in the human CRHBP gene that are associated with stress-related affective disorders and addiction. Lay summary The stress response is controlled by corticotropin-releasing hormone (CRH), acting via CRH receptors. However, the CRH system also includes a unique CRH-binding protein (CRH-BP) that binds CRH with an affinity greater than the CRH receptors. In this review, we discuss the role of this highly conserved CRH-BP in regulation of the CRH-mediated stress response from invertebrates to humans.

Exposure of Trypanosoma brucei to an N-acetylglucosamine-Binding Lectin Induces VSG Switching and Glycosylation Defects Resulting in Reduced Infectivity

PubMed Central

Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Van Damme, Els J. M.; Balzarini, Jan; González-Pacanowska, Dolores

2015-01-01

Trypanosoma brucei variant surface glycoproteins (VSG) are glycosylated by both paucimannose and oligomannose structures which are involved in the formation of a protective barrier against the immune system. Here, we report that the stinging nettle lectin (UDA), with predominant N-acetylglucosamine-binding specificity, interacts with glycosylated VSGs and kills parasites by provoking defects in endocytosis together with impaired cytokinesis. Prolonged exposure to UDA induced parasite resistance based on a diminished capacity to bind the lectin due to an enrichment of biantennary paucimannose and a reduction of triantennary oligomannose structures. Two molecular mechanisms involved in resistance were identified: VSG switching and modifications in N-glycan composition. Glycosylation defects were correlated with the down-regulation of the TbSTT3A and/or TbSTT3B genes (coding for oligosaccharyltransferases A and B, respectively) responsible for glycan specificity. Furthermore, UDA-resistant trypanosomes exhibited severely impaired infectivity indicating that the resistant phenotype entails a substantial fitness cost. The results obtained further support the modification of surface glycan composition resulting from down-regulation of the genes coding for oligosaccharyltransferases as a general resistance mechanism in response to prolonged exposure to carbohydrate-binding agents. PMID:25746926

Arabidopsis F-box protein containing a Nictaba-related lectin domain interacts with N-acetyllactosamine structures.

PubMed

Stefanowicz, Karolina; Lannoo, Nausicaä; Proost, Paul; Van Damme, Els J M

2012-01-01

The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N- and O-glycans containing N-acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly-N-acetyllactosamine ([Galβ1-4GlcNAc]n) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides.

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers*

PubMed Central

Yu, Ying; Mishra, Shreya; Song, Xuezheng; Lasanajak, Yi; Bradley, Konrad C.; Tappert, Mary M.; Air, Gillian M.; Steinhauer, David A.; Halder, Sujata; Cotmore, Susan; Tattersall, Peter; Agbandje-McKenna, Mavis; Cummings, Richard D.; Smith, David F.

2012-01-01

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens. PMID:23115247

Vesicular Stomatitis Virus Pseudotyped with Ebola Virus Glycoprotein Serves as a Protective, Noninfectious Vaccine against Ebola Virus Challenge in Mice

PubMed Central

Lennemann, Nicholas J.; Herbert, Andrew S.; Brouillette, Rachel; Rhein, Bethany; Bakken, Russell A.; Perschbacher, Katherine J.; Cooney, Ashley L.; Miller-Hunt, Catherine L.; Ten Eyck, Patrick; Biggins, Julia; Olinger, Gene; Dye, John M.

2017-01-01

ABSTRACT The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVΔG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSVΔG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVΔG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSVΔG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSVΔG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success. IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSVΔG pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSVΔG pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy. PMID:28615211

Mechanism of pathogen recognition by human dectin-2.

PubMed

Feinberg, Hadar; Jégouzo, Sabine A F; Rex, Maximus J; Drickamer, Kurt; Weis, William I; Taylor, Maureen E

2017-08-11

Dectin-2, a C-type lectin on macrophages and other cells of the innate immune system, functions in response to pathogens, particularly fungi. The carbohydrate-recognition domain (CRD) in dectin-2 is linked to a transmembrane sequence that interacts with the common Fc receptor γ subunit to initiate immune signaling. The molecular mechanism by which dectin-2 selectively binds to pathogens has been investigated by characterizing the CRD expressed in a bacterial system. Competition binding studies indicated that the CRD binds to monosaccharides with modest affinity and that affinity was greatly enhanced for mannose-linked α1-2 or α1-4 to a second mannose residue. Glycan array analysis confirmed selective binding of the CRD to glycans that contain Manα1-2Man epitopes. Crystals of the CRD in complex with a mammalian-type high-mannose Man 9 GlcNAc 2 oligosaccharide exhibited interaction with Manα1-2Man on two different termini of the glycan, with the reducing-end mannose residue ligated to Ca 2+ in a primary binding site and the nonreducing terminal mannose residue occupying an adjacent secondary site. Comparison of the binding sites in DC-SIGN and langerin, two other pathogen-binding receptors of the innate immune system, revealed why these two binding sites accommodate only terminal Manα1-2Man structures, whereas dectin-2 can bind Manα1-2Man in internal positions in mannans and other polysaccharides. The specificity and geometry of the dectin-2-binding site provide the molecular mechanism for binding of dectin-2 to fungal mannans and also to bacterial lipopolysaccharides, capsular polysaccharides, and lipoarabinomannans that contain the Manα1-2Man disaccharide unit. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

PubMed Central

Castilho, Alexandra; Gruber, Clemens; Thader, Andreas; Oostenbrink, Chris; Pechlaner, Maria; Steinkellner, Herta; Altmann, Friedrich

2015-01-01

We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (α1,3- and α1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF3, and GnGnF6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core α1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies. PMID:26067753

GlycReSoft: A Software Package for Automated Recognition of Glycans from LC/MS Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maxwell, Evan; Tan, Yan; Tan, Yuxiang

2012-09-26

Glycosylation modifies the physicochemical properties and protein binding functions of glycoconjugates. These modifications are biosynthesized in the endoplasmic reticulum and Golgi apparatus by a series of enzymatic transformations that are under complex control. As a result, mature glycans on a given site are heterogeneous mixtures of glycoforms. This gives rise to a spectrum of adhesive properties that strongly influences interactions with binding partners and resultant biological effects. In order to understand the roles glycosylation plays in normal and disease processes, efficient structural analysis tools are necessary. In the field of glycomics, liquid chromatography/mass spectrometry (LC/MS) is used to profile themore » glycans present in a given sample. This technology enables comparison of glycan compositions and abundances among different biological samples, i.e. normal versus disease, normal versus mutant, etc. Manual analysis of the glycan profiling LC/MS data is extremely time-consuming and efficient software tools are needed to eliminate this bottleneck. In this work, we have developed a tool to computationally model LC/MS data to enable efficient profiling of glycans. Using LC/MS data deconvoluted by Decon2LS/DeconTools, we built a list of unique neutral masses corresponding to candidate glycan compositions summarized over their various charge states, adducts and range of elution times. Our work aims to provide confident identification of true compounds in complex data sets that are not amenable to manual interpretation. This capability is an essential part of glycomics work flows. We demonstrate this tool, GlycReSoft, using an LC/MS dataset on tissue derived heparan sulfate oligosaccharides. The software, code and a test data set are publically archived under an open source license.« less

Context-specific target definition in influenza a virus hemagglutinin-glycan receptor interactions.

PubMed

Shriver, Zachary; Raman, Rahul; Viswanathan, Karthik; Sasisekharan, Ram

2009-08-28

Protein-glycan interactions are important regulators of a variety of biological processes, ranging from immune recognition to anticoagulation. An important area of active research is directed toward understanding the role of host cell surface glycans as recognition sites for pathogen protein receptors. Recognition of cell surface glycans is a widely employed strategy for a variety of pathogens, including bacteria, parasites, and viruses. We present here a representative example of such an interaction: the binding of influenza A hemagglutinin (HA) to specific sialylated glycans on the cell surface of human upper airway epithelial cells, which initiates the infection cycle. We detail a generalizable strategy to understand the nature of protein-glycan interactions both structurally and biochemically, using HA as a model system. This strategy combines a top-down approach using available structural information to define important contacts between glycans and HA, with a bottom-up approach using data-mining and informatics approaches to identify the common motifs that distinguish glycan binders from nonbinders. By probing protein-glycan interactions simultaneously through top-down and bottom-up approaches, we can scientifically validate a series of observations. This in turn provides additional confidence and surmounts known challenges in the study of protein-glycan interactions, such as accounting for multivalency, and thus truly defines concepts such as specificity, affinity, and avidity. With the advent of new technologies for glycomics-including glycan arrays, data-mining solutions, and robust algorithms to model protein-glycan interactions-we anticipate that such combination approaches will become tractable for a wide variety of protein-glycan interactions.

The interaction of N-glycans in Fcγ receptor I α-chain with Escherichia coli K1 outer membrane protein A for entry into macrophages: experimental and computational analysis.

PubMed

Krishnan, Subramanian; Liu, Fan; Abrol, Ravinder; Hodges, Jacqueline; Goddard, William A; Prasadarao, Nemani V

2014-11-07

Neonatal meningitis, caused by Escherichia coli K1, is a serious central nervous system disease. We have established that macrophages serve as permissive niches for E. coli K1 to multiply in the host and for attaining a threshold level of bacterial load, which is a prerequisite for the onset of the disease. Here, we demonstrate experimentally that three N-glycans in FcγRIa interact with OmpA of E. coli K1 for binding to and entering the macrophages. Adoptive transfer of FcγRIa(-/-) bone marrow-derived macrophages transfected with FcγRIa into FcγRIa(-/-) newborn mice renders them susceptible to E. coli K1-induced meningitis. In contrast, mice that received bone marrow-derived macrophages transfected with FcγRIa in which N-glycosylation sites 1, 4, and 5 are mutated to alanines exhibit resistance to E. coli K1 infection. Our molecular dynamics and simulation studies predict that N-glycan 5 exhibits strong binding at the barrel site of OmpA formed by loops 3 and 4, whereas N-glycans 1 and 4 interact with loops 1, 3, and 4 of OmpA at tip regions. Molecular modeling data also suggest no role for the IgG binding site in the invasion process. In agreement, experimental mutations in IgG binding site had no effect on the E. coli K1 entry into macrophages in vitro or on the onset of meningitis in newborn mice. Together, this integration of experimental and computational studies reveals how the N-glycans in FcγRIa interact with the OmpA of E. coli K1 for inducing the disease pathogenesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert

Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

DOE PAGES

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; ...

2015-06-02

Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

Identification of DNA-Binding Proteins Using Structural, Electrostatic and Evolutionary Features

PubMed Central

Nimrod, Guy; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir

2009-01-01

Summary DNA binding proteins (DBPs) often take part in various crucial processes of the cell's life cycle. Therefore, the identification and characterization of these proteins are of great importance. We present here a random forests classifier for identifying DBPs among proteins with known three-dimensional structures. First, clusters of evolutionarily conserved regions (patches) on the protein's surface are detected using the PatchFinder algorithm; previous studies showed that these regions are typically the proteins' functionally important regions. Next, we train a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein including its dipole moment. Using 10-fold cross validation on a dataset of 138 DNA-binding proteins and 110 proteins which do not bind DNA, the classifier achieved a sensitivity and a specificity of 0.90, which is overall better than the performance of previously published methods. Furthermore, when we tested 5 different methods on 11 new DBPs which did not appear in the original dataset, only our method annotated all correctly. The resulting classifier was applied to a collection of 757 proteins of known structure and unknown function. Of these proteins, 218 were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA. PMID:19233205

Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action.

PubMed

Upton, Rosie; Bell, Leonard; Guy, Colin; Caldwell, Paul; Estdale, Sian; Barran, Perdita E; Firth, David

2016-10-18

In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.

Glycosylation of random IgG distinguishes seropositive and seronegative rheumatoid arthritis.

PubMed

Magorivska, I; Döncző, B; Dumych, T; Karmash, A; Boichuk, M; Hychka, K; Mihalj, M; Szabó, M; Csánky, E; Rech, J; Guttman, A; Vari, S G; Bilyy, R

2018-05-01

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

Biological roles of glycans

PubMed Central

Varki, Ajit

2017-01-01

Abstract Simple and complex carbohydrates (glycans) have long been known to play major metabolic, structural and physical roles in biological systems. Targeted microbial binding to host glycans has also been studied for decades. But such biological roles can only explain some of the remarkable complexity and organismal diversity of glycans in nature. Reviewing the subject about two decades ago, one could find very few clear-cut instances of glycan-recognition-specific biological roles of glycans that were of intrinsic value to the organism expressing them. In striking contrast there is now a profusion of examples, such that this updated review cannot be comprehensive. Instead, a historical overview is presented, broad principles outlined and a few examples cited, representing diverse types of roles, mediated by various glycan classes, in different evolutionary lineages. What remains unchanged is the fact that while all theories regarding biological roles of glycans are supported by compelling evidence, exceptions to each can be found. In retrospect, this is not surprising. Complex and diverse glycans appear to be ubiquitous to all cells in nature, and essential to all life forms. Thus, >3 billion years of evolution consistently generated organisms that use these molecules for many key biological roles, even while sometimes coopting them for minor functions. In this respect, glycans are no different from other major macromolecular building blocks of life (nucleic acids, proteins and lipids), simply more rapidly evolving and complex. It is time for the diverse functional roles of glycans to be fully incorporated into the mainstream of biological sciences. PMID:27558841

Property Graph vs RDF Triple Store: A Comparison on Glycan Substructure Search

PubMed Central

Alocci, Davide; Mariethoz, Julien; Horlacher, Oliver; Bolleman, Jerven T.; Campbell, Matthew P.; Lisacek, Frederique

2015-01-01

Resource description framework (RDF) and Property Graph databases are emerging technologies that are used for storing graph-structured data. We compare these technologies through a molecular biology use case: glycan substructure search. Glycans are branched tree-like molecules composed of building blocks linked together by chemical bonds. The molecular structure of a glycan can be encoded into a direct acyclic graph where each node represents a building block and each edge serves as a chemical linkage between two building blocks. In this context, Graph databases are possible software solutions for storing glycan structures and Graph query languages, such as SPARQL and Cypher, can be used to perform a substructure search. Glycan substructure searching is an important feature for querying structure and experimental glycan databases and retrieving biologically meaningful data. This applies for example to identifying a region of the glycan recognised by a glycan binding protein (GBP). In this study, 19,404 glycan structures were selected from GlycomeDB (www.glycome-db.org) and modelled for being stored into a RDF triple store and a Property Graph. We then performed two different sets of searches and compared the query response times and the results from both technologies to assess performance and accuracy. The two implementations produced the same results, but interestingly we noted a difference in the query response times. Qualitative measures such as portability were also used to define further criteria for choosing the technology adapted to solving glycan substructure search and other comparable issues. PMID:26656740

Fixation of Oligosaccharides to a Surface May Increase the Susceptibility to Human Parainfluenza Virus 1, 2, or 3 Hemagglutinin-Neuraminidase▿†

PubMed Central

Tappert, Mary M.; Smith, David F.; Air, Gillian M.

2011-01-01

The hemagglutinin-neuraminidase (HN) protein of human parainfluenza viruses (hPIVs) both binds (H) and cleaves (N) oligosaccharides that contain N-acetylneuraminic acid (Neu5Ac). H is thought to correspond to receptor binding and N to receptor-destroying activity. At present, N′s role in infection remains unclear: does it destroy only receptors, or are there other targets? We previously demonstrated that hPIV1 and 3 HNs bind to oligosaccharides containing the motif Neu5Acα2-3Galβ1-4GlcNAc (M. Amonsen, D. F. Smith, R. D. Cummings, and G. M. Air, J. Virol. 81:8341–8345, 2007). In the present study, we tested the binding specificity of hPIV2 on the Consortium for Functional Glycomics' glycan array and found that hPIV2 binds to oligosaccharides containing the same motif. We determined the specificities of N on red blood cells, soluble small-molecule and glycoprotein substrates, and the glycan array and compared them to the specificities of H. hPIV2 and -3, but not hPIV1, cleaved their ligands on red blood cells. hPIV1, -2, and -3 cleaved their NeuAcα2-3 ligands on the glycan array; hPIV2 and -3 also cleaved NeuAcα2-6 ligands bound by influenza A virus. While all three HNs exhibited similar affinities for all cleavable soluble substrates, their activities were 5- to 10-fold higher on small molecules than on glycoproteins. In addition, some soluble glycoproteins were not cleaved, despite containing oligosaccharides that were cleaved on the glycan array. We conclude that the susceptibility of an oligosaccharide substrate to N increases when the substrate is fixed to a surface. These findings suggest that HN may undergo a conformational change that activates N upon receptor binding at a cell surface. PMID:21917945

An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

PubMed Central

Chen, Minyong; Shi, Xiaofeng; Duke, Rebecca M.; Ruse, Cristian I.; Dai, Nan; Taron, Christopher H.; Samuelson, James C.

2017-01-01

A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity. PMID:28534482

Endothelial Galectin-1 Binds to Specific Glycans on Nipah Virus Fusion Protein and Inhibits Maturation, Mobility, and Function to Block Syncytia Formation

PubMed Central

Garner, Omai B.; Aguilar, Hector C.; Fulcher, Jennifer A.; Levroney, Ernest L.; Harrison, Rebecca; Wright, Lacey; Robinson, Lindsey R.; Aspericueta, Vanessa; Panico, Maria; Haslam, Stuart M.; Morris, Howard R.; Dell, Anne

2010-01-01

Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell. PMID:20657665

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

PubMed

Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

2014-03-14

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Therapeutic Targeting of Siglecs using Antibody- and Glycan-based Approaches

PubMed Central

Angata, Takashi; Nycholat, Corwin M.; Macauley, Matthew S.

2015-01-01

The sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of immunomodulatory receptors whose functions are regulated by their glycan ligands. Siglecs are attractive therapeutic targets because of their cell-type specific expression pattern, endocytic properties, high expression on certain lymphomas/leukemias, and ability to modulate receptor signaling. Siglec-targeting approaches with therapeutic potential encompass antibody- and glycan-based strategies. Several antibody-based therapies are in clinical trials and continue to be developed for the treatment of lymphoma/leukemia and autoimmune disease, while the therapeutic potential of glycan-based strategies for cargo-delivery and immunomodulation is a promising new approach. Here, we review these strategies with special emphasis on emerging approaches and disease areas that may benefit from targeting the Siglec family. PMID:26435210

Glycan gimmickry by parasitic helminths: a strategy for modulating the host immune response?

PubMed

van Die, Irma; Cummings, Richard D

2010-01-01

Parasitic helminths (worms) co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Among their survival strategies, worms use their glycans within glycoproteins and glycolipids, which are abundant on helminth surfaces and in their excretory/ secretory products, to regulate and suppress host immune responses. Many helminths express unusual and antigenic (nonhost-like) glycans, including those containing polyfucose, tyvelose, terminal GalNAc, phosphorylcholine, methyl groups, and sugars in unusual linkages. In addition, some glycan antigens are expressed that share structural features with those in their intermediate and vertebrate hosts (host-like glycans), including Le(X) (Galbeta1-4[Fucalpha1-3]GlcNAc-), LDNF (GalNAcbeta1-4[Fucalpha1-3]GlcNAc-), LDN (GalNAcbeta1-4GlcNAc-), and Tn (GalNAcalpha1-O-Thr/Ser) antigens. The expression of host-like glycan determinants is remarkable and suggests that helminths may gain advantages by synthesizing such glycans. The expression of host-like glycans by parasites previously led to the concept of "molecular mimicry," in which molecules are either derived from the pathogen or acquired from the host to evade recognition by the host immune system. However, recent discoveries into the potential of host glycan-binding proteins (GBPs), such as C-type lectin receptors and galectins, to functionally interact with various host-like helminth glycans provide new insights. Host GBPs through their interactions with worm-derived glycans participate in shaping innate and adaptive immune responses upon infection. We thus propose an alternative concept termed "glycan gimmickry," which is defined as an active strategy of parasites to use their glycans to target GBPs within the host to promote their survival.

Identification of regulatory targets for the bacterial Nus factor complex.

PubMed

Baniulyte, Gabriele; Singh, Navjot; Benoit, Courtney; Johnson, Richard; Ferguson, Robert; Paramo, Mauricio; Stringer, Anne M; Scott, Ashley; Lapierre, Pascal; Wade, Joseph T

2017-12-11

Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. Here, we identify an additional cellular function for the Nus factor complex in Escherichia coli: repression of the Nus factor-encoding gene, suhB. This repression occurs primarily by translation inhibition, followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Conservation of putative NusB/E binding sites upstream of Nus factor genes suggests that Nus factor autoregulation occurs in many bacterial species. Additionally, many putative NusB/E binding sites are also found upstream of other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory.

Zebrafish (Danio rerio) androgen receptor: sequence homology and up-regulation by the fungicide vinclozolin.

PubMed

Smolinsky, Amanda N; Doughman, Jennifer M; Kratzke, Liên-Thành C; Lassiter, Christopher S

2010-03-01

Steroid hormones regulate gene expression in organisms by binding to receptor proteins. These hormones include the androgens, which signal through androgen receptors (ARs). Endocrine disrupters (EDCs) are chemicals in the environment that adversely affect organisms by binding to nuclear receptors, including ARs. Vinclozolin, a fungicide used on fruit and vegetable crops, is a known anti-androgen, a type of EDC that blocks signals from testosterone and its derivatives. In order to better understand the effects of EDCs, further research on androgen receptors and other hormone signaling pathways is necessary. In this study, we demonstrate the evolutionary conservation between the genomic structure of the human and zebrafish ar genes and find that ar mRNA expression increases in zebrafish embryos exposed to vinclozolin, which may be evolutionarily conserved as well. At 48 and 72 h post-fertilization, vinclozolin-treated embryos express ar mRNA 8-fold higher than the control level. These findings suggest that zebrafish embryos attempt to compensate for the presence of an anti-androgen by increasing the number of androgen receptors available.

Characterizing carbohydrate-protein interactions by NMR

PubMed Central

Bewley, Carole A.; Shahzad-ul-Hussan, Syed

2013-01-01

Interactions between proteins and soluble carbohydrates and/or surface displayed glycans are central to countless recognition, attachment and signaling events in biology. The physical chemical features associated with these binding events vary considerably, depending on the biological system of interest. For example, carbohydrate-protein interactions can be stoichiometric or multivalent, the protein receptors can be monomeric or oligomeric, and the specificity of recognition can be highly stringent or rather promiscuous. Equilibrium dissociation constants for carbohydrate binding are known to vary from micromolar to millimolar, with weak interactions being far more prevalent; and individual carbohydrate binding sites can be truly symmetrical or merely homologous, and hence, the affinities of individual sites within a single protein can vary, as can the order of binding. Several factors, including the weak affinities with which glycans bind their protein receptors, the dynamic nature of the glycans themselves, and the non-equivalent interactions among oligomeric carbohydrate receptors, have made NMR an especially powerful tool for studying and defining carbohydrate-protein interactions. Here we describe those NMR approaches that have proven to be the most robust in characterizing these systems, and explain what type of information can (or cannot) be obtained from each. Our goal is to provide to the reader the information necessary for selecting the correct experiment or sets of experiments to characterize their carbohydrate-protein interaction of interest. PMID:23784792

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

PubMed

Zhang, Meiling; Chekan, Jonathan R; Dodd, Dylan; Hong, Pei-Ying; Radlinski, Lauren; Revindran, Vanessa; Nair, Satish K; Mackie, Roderick I; Cann, Isaac

2014-09-02

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods.

PubMed

Sato, Shigeru; Ikeda, Keiko; Shioi, Go; Nakao, Kazuki; Yajima, Hiroshi; Kawakami, Kiyoshi

2012-08-01

The Six1 homeobox gene plays critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in human SIX1 cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing loss and branchial defects. The present study was designed to identify enhancers responsible for the dynamic expression pattern of Six1 during mouse embryogenesis. The results showed distinct enhancer activities of seven conserved non-coding sequences (CNSs) retained in tetrapod Six1 loci. The activities were detected in all cranial placodes (excluding the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-expression domains during development were covered by the sum of activities of these enhancers, together with the previously identified enhancer for the pre-placodal region and foregut endoderm. Thus, the eight CNSs identified in a series of our study represent major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. The results also confirmed that chick electroporation is a robust means to decipher regulatory information stored in vertebrate genomes. Mutational analysis of the most conserved placode-specific enhancer, Six1-21, indicated that the enhancer integrates a variety of inputs from Sox, Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. Positive autoregulation of Six1 is achieved through the regulation of Six protein-binding sites. The identified Six1 enhancers provide valuable tools to understand the mechanism of Six1 regulation and to manipulate gene expression in the developing embryo, particularly in the sensory organs. Copyright © 2012 Elsevier Inc. All rights reserved.

1H, 15N and 13C backbone and side-chain resonance assignments of a family 32 carbohydrate-binding module from the Clostridium perfringens NagH.

PubMed

Grondin, Julie M; Chitayat, Seth; Ficko-Blean, Elizabeth; Boraston, Alisdair B; Smith, Steven P

2012-10-01

The Gram-positive anaerobe Clostridium perfringens is an opportunistic bacterial pathogen that secretes a battery of enzymes involved in glycan degradation. These glycoside hydrolases are thought to be involved in turnover of mucosal layer glycans, and in the spread of major toxins commonly associated with the development of gastrointestinal diseases and gas gangrene in humans. These enzymes employ multi-modularity and carbohydrate-binding function to degrade extracellular eukaryotic host sugars. Here, we report the full (1)H, (15)N and (13)C chemical shift resonance assignments of the first family 32 carbohydrate-binding module from NagH, a secreted family 84 glycoside hydrolase.

Evaluation of galectin binding by frontal affinity chromatography (FAC).

PubMed

Iwaki, Jun; Hirabayashi, Jun

2015-01-01

Frontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail. As a result, their minimal requirements for recognition of disaccharides, i.e., β-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K d's for interactions between a series of standard glycans and various galectins.

How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans.

PubMed

Cartmell, Alan; Lowe, Elisabeth C; Baslé, Arnaud; Firbank, Susan J; Ndeh, Didier A; Murray, Heath; Terrapon, Nicolas; Lombard, Vincent; Henrissat, Bernard; Turnbull, Jeremy E; Czjzek, Mirjam; Gilbert, Harry J; Bolam, David N

2017-07-03

The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron , a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides , indicating that the model developed is of generic relevance to this important microbial community.

Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs

PubMed Central

García-Chapa, Meritxell; López-Moya, Juan José; Burgyán, József

2010-01-01

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC. PMID:20657820

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family.

PubMed

Stone, Jacquelyn A; Nicola, Anthony V; Baum, Linda G; Aguilar, Hector C

2016-02-01

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.

Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning

PubMed Central

Rondard, Philippe; Huang, Siluo; Monnier, Carine; Tu, Haijun; Blanchard, Bertrand; Oueslati, Nadia; Malhaire, Fanny; Li, Ying; Trinquet, Eric; Labesse, Gilles; Pin, Jean-Philippe; Liu, Jianfeng

2008-01-01

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABAB1 and GABAB2. GABAB1 binds agonists, whereas GABAB2 is required for trafficking GABAB1 to the cell surface, increasing agonist affinity to GABAB1, and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABAB1 VFT leads to GABAB2 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABAB VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABAB2, including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation. PMID:18388862

Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of multispecificity through domain duplication and divergent evolution

PubMed Central

Van Damme, Els J. M.; Nakamura-Tsuruta, Sachiko; Smith, David F.; Ongenaert, Maté; Winter, Harry C.; Rougé, Pierre; Goldstein, Irwin J.; Mo, Hanqing; Kominami, Junko; Culerrier, Raphaël; Barre, Annick; Hirabayashi, Jun; Peumans, Willy J.

2007-01-01

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes. PMID:17288538

The Recognition of N-Glycans by the Lectin ArtinM Mediates Cell Death of a Human Myeloid Leukemia Cell Line

PubMed Central

Carvalho, Fernanda Caroline; Soares, Sandro Gomes; Tamarozzi, Mirela Barros; Rego, Eduardo Magalhães; Roque-Barreira, Maria-Cristina

2011-01-01

ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment. PMID:22132163

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

Structural and Functional Analysis of the Interaction Between the Nucleoporin Nup98 and the mRNA Export Facto Rae1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Ren; H Seo; G Blobel

The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 {angstrom} resolution. Rae1 forms a seven-bladed {beta}-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an {approx}50-{angstrom}-long hairpinmore » that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 {beta}-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1 {center_dot} Nup98{sup GLEBS} surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1 {center_dot} Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.« less

Identification of DNA-binding proteins using structural, electrostatic and evolutionary features.

PubMed

Nimrod, Guy; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir

2009-04-10

DNA-binding proteins (DBPs) participate in various crucial processes in the life-cycle of the cells, and the identification and characterization of these proteins is of great importance. We present here a random forests classifier for identifying DBPs among proteins with known 3D structures. First, clusters of evolutionarily conserved regions (patches) on the surface of proteins were detected using the PatchFinder algorithm; earlier studies showed that these regions are typically the functionally important regions of proteins. Next, we trained a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein, including its dipole moment. Using 10-fold cross-validation on a dataset of 138 DBPs and 110 proteins that do not bind DNA, the classifier achieved a sensitivity and a specificity of 0.90, which is overall better than the performance of published methods. Furthermore, when we tested five different methods on 11 new DBPs that did not appear in the original dataset, only our method annotated all correctly. The resulting classifier was applied to a collection of 757 proteins of known structure and unknown function. Of these proteins, 218 were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA.

Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV.

PubMed

Inagaki, M; Muranishi, H; Yamada, K; Kakehi, K; Uchida, K; Suzuki, T; Yabe, T; Nakagomi, T; Nakagomi, O; Kanamaru, Y

2014-05-01

Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. κ-Casein (κ-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine κ-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, κ-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in κ-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95°C for 30 min) in a viral titer-dependent manner. Although the heated κ-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine κ-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in κ-CN may play an important role in maintenance of κ-CN binding to HRV. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

N-glycosylation at the SynCAM (synaptic cell adhesion molecule) immunoglobulin interface modulates synaptic adhesion.

PubMed

Fogel, Adam I; Li, Yue; Giza, Joanna; Wang, Qing; Lam, Tukiet T; Modis, Yorgo; Biederer, Thomas

2010-11-05

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn(60). Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn(70)/Asn(104) flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn(60) reduces adhesion, N-glycans at Asn(70)/Asn(104) of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion*

PubMed Central

Fogel, Adam I.; Li, Yue; Giza, Joanna; Wang, Qing; Lam, TuKiet T.; Modis, Yorgo; Biederer, Thomas

2010-01-01

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn60. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn70/Asn104 flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn60 reduces adhesion, N-glycans at Asn70/Asn104 of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion. PMID:20739279

Inhibition of homologous phosphorolytic ribonucleases by citrate may represent an evolutionarily conserved communicative link between RNA degradation and central metabolism

PubMed Central

Stone, Carlanne M.; Butt, Louise E.; Bufton, Joshua C.; Lourenco, Daniel C.; Gowers, Darren M.; Pickford, Andrew R.; Cox, Paul A.

2017-01-01

Abstract Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution. PMID:28334892

Massive gene transfer and extensive RNA editing of a symbiotic dinoflagellate plastid genome.

PubMed

Mungpakdee, Sutada; Shinzato, Chuya; Takeuchi, Takeshi; Kawashima, Takeshi; Koyanagi, Ryo; Hisata, Kanako; Tanaka, Makiko; Goto, Hiroki; Fujie, Manabu; Lin, Senjie; Satoh, Nori; Shoguchi, Eiichi

2014-05-31

Genome sequencing of Symbiodinium minutum revealed that 95 of 109 plastid-associated genes have been transferred to the nuclear genome and subsequently expanded by gene duplication. Only 14 genes remain in plastids and occur as DNA minicircles. Each minicircle (1.8-3.3 kb) contains one gene and a conserved noncoding region containing putative promoters and RNA-binding sites. Nine types of RNA editing, including a novel G/U type, were discovered in minicircle transcripts but not in genes transferred to the nucleus. In contrast to DNA editing sites in dinoflagellate mitochondria, which tend to be highly conserved across all taxa, editing sites employed in DNA minicircles are highly variable from species to species. Editing is crucial for core photosystem protein function. It restores evolutionarily conserved amino acids and increases peptidyl hydropathy. It also increases protein plasticity necessary to initiate photosystem complex assembly. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Structural Insights into the Allosteric Operation of the Lon AAA+ Protease.

PubMed

Lin, Chien-Chu; Su, Shih-Chieh; Su, Ming-Yuan; Liang, Pi-Hui; Feng, Chia-Cheng; Wu, Shih-Hsiung; Chang, Chung-I

2016-05-03

The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine. Copyright © 2016 Elsevier Ltd. All rights reserved.

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

PubMed

Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

2016-02-01

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

PubMed

Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

2016-04-01

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and cDNA cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per a litter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48-1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

An optics-based variable-temperature assay system for characterizing thermodynamics of biomolecular reactions on solid support

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fei, Yiyan; Landry, James P.; Zhu, X. D., E-mail: xdzhu@physics.ucdavis.edu

A biological state is equilibrium of multiple concurrent biomolecular reactions. The relative importance of these reactions depends on physiological temperature typically between 10 °C and 50 °C. Experimentally the temperature dependence of binding reaction constants reveals thermodynamics and thus details of these biomolecular processes. We developed a variable-temperature opto-fluidic system for real-time measurement of multiple (400–10 000) biomolecular binding reactions on solid supports from 10 °C to 60 °C within ±0.1 °C. We illustrate the performance of this system with investigation of binding reactions of plant lectins (carbohydrate-binding proteins) with 24 synthetic glycans (i.e., carbohydrates). We found that the lectin-glycan reactions in general can be enthalpy-driven,more » entropy-driven, or both, and water molecules play critical roles in the thermodynamics of these reactions.« less

An optics-based variable-temperature assay system for characterizing thermodynamics of biomolecular reactions on solid support

NASA Astrophysics Data System (ADS)

Fei, Yiyan; Landry, James P.; Li, Yanhong; Yu, Hai; Lau, Kam; Huang, Shengshu; Chokhawala, Harshal A.; Chen, Xi; Zhu, X. D.

2013-11-01

A biological state is equilibrium of multiple concurrent biomolecular reactions. The relative importance of these reactions depends on physiological temperature typically between 10 °C and 50 °C. Experimentally the temperature dependence of binding reaction constants reveals thermodynamics and thus details of these biomolecular processes. We developed a variable-temperature opto-fluidic system for real-time measurement of multiple (400-10 000) biomolecular binding reactions on solid supports from 10 °C to 60 °C within ±0.1 °C. We illustrate the performance of this system with investigation of binding reactions of plant lectins (carbohydrate-binding proteins) with 24 synthetic glycans (i.e., carbohydrates). We found that the lectin-glycan reactions in general can be enthalpy-driven, entropy-driven, or both, and water molecules play critical roles in the thermodynamics of these reactions.

Integrated analyses of proteins and their glycans in a magnetic bead-based multiplex assay format.

PubMed

Li, Danni; Chiu, Hanching; Chen, Jing; Zhang, Hui; Chan, Daniel W

2013-01-01

Well-annotated clinical samples are valuable resources for biomarker discovery and validation. Multiplex and integrated methods that simultaneously measure multiple analytes and generate integrated information about these analytes from a single measurement are desirable because these methods help conserve precious samples. We developed a magnetic bead-based system for multiplex and integrated glycoprotein quantification by immunoassays and glycan detection by lectin immunosorbent assays (LISAs). Magnetic beads coupled with antibodies were used for capturing proteins of interest. Biotinylated antibodies in combination with streptavidin-labeled phycoerythrin were used for protein quantification. In the LISAs, biotinylated detection antibodies were replaced by biotinylated lectins for glycan detection. Using tissue inhibitor of metallopeptidase 1 (TIMP-1), tissue plasminogen activator, membrane metallo-endopeptidase, and dipeptidyl peptidase-IV (DPP-4) as models, we found that the multiplex integrated system was comparable to single immunoassays in protein quantification and LISAs in glycan detection. The merits of this system were demonstrated when applied to well-annotated prostate cancer tissues for validation of biomarkers in aggressive prostate cancer. Because of the system's multiplex ability, we used only 300 ng of tissue protein for the integrated detection of glycans in these proteins. Fucosylated TIMP-1 and DPP-4 offered improved performance over the proteins in distinguishing aggressive and nonaggressive prostate cancer. The multiplex and integrated system conserves samples and is a useful tool for validation of glycoproteins and their glycoforms as biomarkers. © 2012 American Association for Clinical Chemistry

Organic Electrochemical Transistors for the Detection of Cell Surface Glycans.

PubMed

Chen, Lizhen; Fu, Ying; Wang, Naixiang; Yang, Anneng; Li, Yuanzhe; Wu, Jie; Ju, Huangxian; Yan, Feng

2018-05-23

Cell surface glycans play critical roles in diverse biological processes, such as cell-cell communication, immunity, infection, development, and differentiation. Their expressions are closely related to cancer growth and metastasis. This work demonstrates an organic electrochemical transistor (OECT)-based biosensor for the detection of glycan expression on living cancer cells. Herein, mannose on human breast cancer cells (MCF-7) as the target glycan model, poly dimethyl diallyl ammonium chloride-multiwall carbon nanotubes (PDDA-MWCNTs) as the loading interface, concanavalin A (Con A) with active mannose binding sites, aptamer and horseradish peroxidase co-immobilized gold nanoparticles (HRP-aptamer-Au NPs) as specific nanoprobes are used to fabricate the OECT biosensor. In this strategy, PDDA-MWCNT interfaces can enhance the loading of Con A, and the target cells can be captured through Con A via active mannose binding sites. Thus, the expression of cell surface can be reflected by the amount of cells captured on the gate. Specific nanoprobes are introduced to the captured cells to produce an OECT signal because of the reduction of hydrogen peroxide catalyzed by HRP conjugated on Au nanoparticles, while the aptamer on nanoprobes can selectively recognize the MCF-7 cells. It is reasonable that more target cells are captured on the gate electrode, more HRP-nanoprobes are loaded thus a larger signal response. The device shows an obvious response to MCF-7 cells down to 10 cells/μL and can be used to selectively monitor the change of mannose expression on cell surfaces upon a treatment with the N-glycan inhibitor. The OECT-based biosensor is promising for the analysis of glycan expressions on the surfaces of different types of cells.

Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

PubMed Central

Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

2015-01-01

The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120–gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120–gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

Lectin-Array Blotting.

PubMed

Pazos, Raquel; Echevarria, Juan; Hernandez, Alvaro; Reichardt, Niels-Christian

2017-09-01

Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands.

PubMed

Sun, Xiaoman; Wang, Lihong; Qi, Jianxun; Li, Dandi; Wang, Mengxuan; Cong, Xin; Peng, Ruchao; Chai, Wengang; Zhang, Qing; Wang, Hong; Wen, Hongling; Gao, George F; Tan, Ming; Duan, Zhaojun

2018-06-01

Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines. IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs. Copyright © 2018 American Society for Microbiology.

Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kronewitter, Scott R.; Marginean, Ioan; Cox, Jonathan T.

The N-glycan diversity of human serum glycoproteins, i.e. the human blood serum N-glycome, is complex due to the range of glycan structures potentially synthesizable by human glycosylation enzymes. The reported glycome, however, is limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report, several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to provide an improved view of glycan diversity. Sample preparation improvements include acidified, microwave-accelerated, PNGase F N-glycan release, and sodium borohydride reduction were optimized to improvemore » quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased the sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. On-line separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient which provides additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) has been utilized. When method improvements are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described1 these technologies demonstrate the ability to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrate application of these advances in the context of the human serum glycome, and for which our initial observations include detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.« less

Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

PubMed

Borges, Chad R; Rehder, Douglas S

2016-09-15

Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

PubMed

Pochechueva, Tatiana; Jacob, Francis; Goldstein, Darlene R; Huflejt, Margaret E; Chinarev, Alexander; Caduff, Rosemarie; Fink, Daniel; Hacker, Neville; Bovin, Nicolai V; Heinzelmann-Schwarz, Viola

2011-12-01

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection. © The Author(s) 2011. This article is published with open access at Springerlink.com

Direct Enzymatic Branch-End Extension of Glycocluster-Presented Glycans: An Effective Strategy for Programming Glycan Bioactivity.

PubMed

Bayón, Carlos; He, Ning; Deir-Kaspar, Mario; Blasco, Pilar; André, Sabine; Gabius, Hans-Joachim; Rumbero, Ángel; Jiménez-Barbero, Jesús; Fessner, Wolf-Dieter; Hernáiz, María J

2017-01-31

The sequence of a glycan and its topology of presentation team up to determine the specificity and selectivity of recognition by saccharide receptors (lectins). Structure-activity analysis would be furthered if the glycan part of a glycocluster could be efficiently elaborated in situ while keeping all other parameters constant. By using a bacterial α2,6-sialyltransferase and a small library of bi- to tetravalent glycoclusters, we illustrate the complete conversion of scaffold-presented lactoside units into two different sialylated ligands based on N-acetyl/glycolyl-neuraminic acid incorporation. We assess the ensuing effect on their bioactivity for a plant toxin, and present an analysis of the noncovalent substrate binding contacts that the added sialic acid moiety makes to the lectin. Enzymatic diversification of a scaffold-presented glycan can thus be brought to completion in situ, offering a versatile perspective for rational glycocluster engineering. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120

PubMed Central

Kong, Leopold; Lee, Jeong Hyun; Doores, Katie J.; Murin, Charles D.; Julien, Jean-Philippe; McBride, Ryan; Liu, Yan; Marozsan, Andre; Cupo, Albert; Klasse, Per-Johan; Hoffenberg, Simon; Caulfield, Michael; King, C. Richter; Hua, Yuanzi; Le, Khoa M.; Khayat, Reza; Deller, Marc C.; Clayton, Thomas; Tien, Henry; Feizi, Ten; Sanders, Rogier W.; Paulson, James C.; Moore, John P.; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.

2013-01-01

A substantial fraction of broadly neutralizing antibodies (bnAbs) in certain HIV-infected donors recognizes glycan-dependent epitopes on HIV-1 gp120. Here, we elucidate how bnAb PGT 135 recognizes its Asn332 glycan-dependent epitope from its crystal structure with gp120, CD4 and Fab 17b at 3.1 Å resolution. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield to access the gp120 protein surface. Electron microscopy reveals PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. The combined structural studies of PGT 135, PGT 128 and 2G12 show this Asn332-dependent epitope is highly accessible and much more extensive than initially appreciated, allowing for multiple binding modes and varied angles of approach, thereby representing a supersite of vulnerability for antibody neutralization. PMID:23708606

Mechanistic Study on Electron Capture Dissociation of the Oligosaccharide-Mg2+ Complex

NASA Astrophysics Data System (ADS)

Huang, Yiqun; Pu, Yi; Yu, Xiang; Costello, Catherine E.; Lin, Cheng

2014-08-01

Electron capture dissociation (ECD) has shown great potential in structural characterization of glycans. However, our current understanding of the glycan ECD process is inadequate for accurate interpretation of the complex glycan ECD spectra. Here, we present the first comprehensive theoretical investigation on the ECD fragmentation behavior of metal-adducted glycans, using the cellobiose-Mg2+ complex as the model system. Molecular dynamics simulation was carried out to determine the typical glycan-Mg2+ binding patterns and the lowest-energy conformer identified was used as the initial geometry for density functional theory-based theoretical modeling. It was found that the electron is preferentially captured by Mg2+ and the resultant Mg+• can abstract a hydroxyl group from the glycan moiety to form a carbon radical. Subsequent radical migration and α-cleavage(s) result in the formation of a variety of product ions. The proposed hydroxyl abstraction mechanism correlates well with the major features in the ECD spectrum of the Mg2+-adducted cellohexaose. The mechanism presented here also predicts the presence of secondary, radical-induced fragmentation pathways. These secondary fragment ions could be misinterpreted, leading to erroneous structural determination. The present study highlights an urgent need for continuing investigation of the glycan ECD mechanism, which is imperative for successful development of bioinformatics tools that can take advantage of the rich structural information provided by ECD of metal-adducted glycans.

Investigations on therapeutic glucocerebrosidases through paired detection with fluorescent activity-based probes

PubMed Central

Kallemeijn, Wouter W.; Scheij, Saskia; Hoogendoorn, Sascha; Witte, Martin D.; Herrera Moro Chao, Daniela; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Overkleeft, Herman S.; Boot, Rolf G.; Aerts, Johannes M. F. G.

2017-01-01

Deficiency of glucocerebrosidase (GBA) causes Gaucher disease (GD). In the common non-neuronopathic GD type I variant, glucosylceramide accumulates primarily in the lysosomes of visceral macrophages. Supplementing storage cells with lacking enzyme is accomplished via chronic intravenous administration of recombinant GBA containing mannose-terminated N-linked glycans, mediating the selective uptake by macrophages expressing mannose-binding lectin(s). Two recombinant GBA preparations with distinct N-linked glycans are registered in Europe for treatment of type I GD: imiglucerase (Genzyme), contains predominantly Man(3) glycans, and velaglucerase (Shire PLC) Man(9) glycans. Activity-based probes (ABPs) enable fluorescent labeling of recombinant GBA preparations through their covalent attachment to the catalytic nucleophile E340 of GBA. We comparatively studied binding and uptake of ABP-labeled imiglucerase and velaglucerase in isolated dendritic cells, cultured human macrophages and living mice, through simultaneous detection of different GBAs by paired measurements. Uptake of ABP-labeled rGBAs by dendritic cells was comparable, as well as the bio-distribution following equimolar intravenous administration to mice. ABP-labeled rGBAs were recovered largely in liver, white-blood cells, bone marrow and spleen. Lungs, brain and skin, affected tissues in severe GD types II and III, were only poorly supplemented. Small, but significant differences were noted in binding and uptake of rGBAs in cultured human macrophages, in the absence and presence of mannan. Mannan-competed binding and uptake were largest for velaglucerase, when determined with single enzymes or as equimolar mixtures of both enzymes. Vice versa, imiglucerase showed more prominent binding and uptake not competed by mannan. Uptake of recombinant GBAs by cultured macrophages seems to involve multiple receptors, including several mannose-binding lectins. Differences among cells from different donors (n = 12) were noted, but the same trends were always observed. Our study suggests that further insight in targeting and efficacy of enzyme therapy of individual Gaucher patients could be obtained by the use of recombinant GBA, trace-labeled with an ABP, preferably equipped with an infrared fluorophore or other reporter tag suitable for in vivo imaging. PMID:28207759

A conserved function for pericentromeric satellite DNA

PubMed Central

Jagannathan, Madhav; Cummings, Ryan

2018-01-01

A universal and unquestioned characteristic of eukaryotic cells is that the genome is divided into multiple chromosomes and encapsulated in a single nucleus. However, the underlying mechanism to ensure such a configuration is unknown. Here, we provide evidence that pericentromeric satellite DNA, which is often regarded as junk, is a critical constituent of the chromosome, allowing the packaging of all chromosomes into a single nucleus. We show that the multi-AT-hook satellite DNA-binding proteins, Drosophila melanogaster D1 and mouse HMGA1, play an evolutionarily conserved role in bundling pericentromeric satellite DNA from heterologous chromosomes into ‘chromocenters’, a cytological association of pericentromeric heterochromatin. Defective chromocenter formation leads to micronuclei formation due to budding from the interphase nucleus, DNA damage and cell death. We propose that chromocenter and satellite DNA serve a fundamental role in encapsulating the full complement of the genome within a single nucleus, the universal characteristic of eukaryotic cells. PMID:29578410

Direct uptake and degradation of DNA by lysosomes

PubMed Central

Fujiwara, Yuuki; Kikuchi, Hisae; Aizawa, Shu; Furuta, Akiko; Hatanaka, Yusuke; Konya, Chiho; Uchida, Kenko; Wada, Keiji; Kabuta, Tomohiro

2013-01-01

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered “RNautophagy,” an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term “DNautophagy,” is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa. PMID:23839276

Organization, chromosomal localization and promoter analysis of the gene encoding human acidic fibroblast growth factor intracellular binding protein.

PubMed Central

Kolpakova, E; Frengen, E; Stokke, T; Olsnes, S

2000-01-01

Acidic fibroblast growth factor (aFGF) intracellular binding protein (FIBP) is a protein found mainly in the nucleus that might be involved in the intracellular function of aFGF. Here we present a comparative analysis of the deduced amino acid sequences of human, murine and Drosophila FIBP analogues and demonstrate that FIBP is an evolutionarily conserved protein. The human gene spans more than 5 kb, comprising ten exons and nine introns, and maps to chromosome 11q13.1. Two slightly different splice variants found in different tissues were isolated and characterized. Sequence analysis of the region surrounding the translation start revealed a CpG island, a classical feature of widely expressed genes. Functional studies of the promoter region with a luciferase reporter system suggested a strong transcriptional activity residing within 600 bp of the 5' flanking region. PMID:11104667

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

PubMed

Galloway, Alison; Saveliev, Alexander; Łukasiak, Sebastian; Hodson, Daniel J; Bolland, Daniel; Balmanno, Kathryn; Ahlfors, Helena; Monzón-Casanova, Elisa; Mannurita, Sara Ciullini; Bell, Lewis S; Andrews, Simon; Díaz-Muñoz, Manuel D; Cook, Simon J; Corcoran, Anne; Turner, Martin

2016-04-22

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint. Copyright © 2016, American Association for the Advancement of Science.

Structural Basis for the Interaction of Mutasome Assembly Factor REV1 with Ubiquitin.

PubMed

Cui, Gaofeng; Botuyan, Maria Victoria; Mer, Georges

2018-05-18

REV1 is an evolutionarily conserved translesion synthesis (TLS) DNA polymerase and an assembly factor key for the recruitment of other TLS polymerases to DNA damage sites. REV1-mediated recognition of ubiquitin in the proliferative cell nuclear antigen is thought to be the trigger for TLS activation. Here we report the solution NMR structure of a 108-residue fragment of human REV1 encompassing the two putative ubiquitin-binding motifs UBM1 and UBM2 in complex with ubiquitin. While in mammals UBM1 and UBM2 are both required for optimal association of REV1 with replication factories after DNA damage, we show that only REV1 UBM2 binds ubiquitin. Structure-guided mutagenesis in Saccharomyces cerevisiae further highlights the importance of UBM2 for REV1-mediated mutagenesis and DNA damage tolerance. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

2003-12-31

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involvedmore » in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.« less

Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Rui; McBride, Ryan; Paulson, James C.

2010-03-04

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which representmore » the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.« less

Natively glycosylated HIV-1 Env structure reveals new mode for antibody recognition of the CD4-binding site

PubMed Central

West, Anthony P; Schamber, Michael; Gazumyan, Anna; Golijanin, Jovana; Seaman, Michael S; Fätkenheuer, Gerd; Klein, Florian; Nussenzweig, Michel C; Bjorkman, Pamela J

2016-01-01

HIV-1 vaccine design is informed by structural studies elucidating mechanisms by which broadly neutralizing antibodies (bNAbs) recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env). Variability in high-mannose and complex-type Env glycoforms leads to heterogeneity that usually precludes visualization of the native glycan shield. We present 3.5-Å- and 3.9-Å-resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation, revealing a glycan shield of high-mannose and complex-type N-glycans, which we used to define complete epitopes of two bNAbs. Env trimer was complexed with 10-1074 (against the V3-loop) and IOMA, a new CD4-binding site (CD4bs) antibody. Although IOMA derives from VH1-2*02, the germline gene of CD4bs-targeting VRC01-class bNAbs, its light chain lacks the short CDRL3 that defines VRC01-class bNAbs. Thus IOMA resembles 8ANC131-class/VH1-46–derived CD4bs bNAbs, which have normal-length CDRL3s. The existence of bNAbs that combine features of VRC01-class and 8ANC131-class antibodies has implications for immunization strategies targeting VRC01-like bNAbs. PMID:27617431

Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding, Endocytosis, and Degradation*

PubMed Central

Date, Kimie; Satoh, Ayano; Iida, Kaoruko; Ogawa, Haruko

2015-01-01

α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na+/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104–23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption stage in the intestine. PMID:26023238

An evolutionarily conserved gene, FUWA, plays a role in determining panicle architecture, grain shape and grain weight in rice.

PubMed

Chen, Jun; Gao, He; Zheng, Xiao-Ming; Jin, Mingna; Weng, Jian-Feng; Ma, Jin; Ren, Yulong; Zhou, Kunneng; Wang, Qi; Wang, Jie; Wang, Jiu-Lin; Zhang, Xin; Cheng, Zhijun; Wu, Chuanyin; Wang, Haiyang; Wan, Jian-Min

2015-08-01

Plant breeding relies on creation of novel allelic combinations for desired traits. Identification and utilization of beneficial alleles, rare alleles and evolutionarily conserved genes in the germplasm (referred to as 'hidden' genes) provide an effective approach to achieve this goal. Here we show that a chemically induced null mutation in an evolutionarily conserved gene, FUWA, alters multiple important agronomic traits in rice, including panicle architecture, grain shape and grain weight. FUWA encodes an NHL domain-containing protein, with preferential expression in the root meristem, shoot apical meristem and inflorescences, where it restricts excessive cell division. Sequence analysis revealed that FUWA has undergone a bottleneck effect, and become fixed in landraces and modern cultivars during domestication and breeding. We further confirm a highly conserved role of FUWA homologs in determining panicle architecture and grain development in rice, maize and sorghum through genetic transformation. Strikingly, knockdown of the FUWA transcription level by RNA interference results in an erect panicle and increased grain size in both indica and japonica genetic backgrounds. This study illustrates an approach to create new germplasm with improved agronomic traits for crop breeding by tapping into evolutionary conserved genes. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere Sequestration.

PubMed

Hafner, Lukas; Lezaja, Aleksandra; Zhang, Xu; Lemmens, Laure; Shyian, Maksym; Albert, Benjamin; Follonier, Cindy; Nunes, Jose Manuel; Lopes, Massimo; Shore, David; Mattarocci, Stefano

2018-04-24

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Insights into the regioselectivity and RNA-binding affinity of HIV-1 nucleocapsid protein from linear-scaling quantum methods.

PubMed

Khandogin, Jana; Musier-Forsyth, Karin; York, Darrin M

2003-07-25

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) plays several important roles in the viral life-cycle and presents an attractive target for rational drug design. Here, the macromolecular reactivity of NC and its binding to RNA is characterized through determination of electrostatic and chemical descriptors derived from linear-scaling quantum calculations in solution. The computational results offer a rationale for the experimentally observed susceptibility of the Cys49 thiolate toward small-molecule electrophilic agents, and support the recently proposed stepwise protonation mechanism of the C-terminal Zn-coordination complex. The distinctive binding mode of NC to SL2 and SL3 stem-loops of the HIV-1 genomic RNA packaging signal is studied on the basis of protein side-chain contributions to the electrostatic binding energies. These results indicate the importance of several basic residues in the 3(10) helical region and the N-terminal zinc finger, and rationalize the presence of several evolutionarily conserved residues in NC. The combined reactivity and RNA-binding study provides new insights that may contribute toward the structure-based design of anti-HIV therapies.

An evolutionarily conserved motif in the TAB1 C-terminal region is necessary for interaction with and activation of TAK1 MAPKKK.

PubMed

Ono, K; Ohtomo, T; Sato, S; Sugamata, Y; Suzuki, M; Hisamoto, N; Ninomiya-Tsuji, J; Tsuchiya, M; Matsumoto, K

2001-06-29

TAK1, a member of the MAPKKK family, is involved in the intracellular signaling pathways mediated by transforming growth factor beta, interleukin 1, and Wnt. TAK1 kinase activity is specifically activated by the TAK1-binding protein TAB1. The C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation. Analysis of various truncated versions of TAB1-C68 defined a C-terminal 30-amino acid sequence (TAB1-C30) necessary for TAK1 binding and activation. NMR studies revealed that the TAB1-C30 region has a unique alpha-helical structure. We identified a conserved sequence motif, PYVDXA/TXF, in the C-terminal domain of mammalian TAB1, Xenopus TAB1, and its Caenorhabditis elegans homolog TAP-1, suggesting that this motif constitutes a specific TAK1 docking site. Alanine substitution mutagenesis showed that TAB1 Phe-484, located in the conserved motif, is crucial for TAK1 binding and activation. The C. elegans homolog of TAB1, TAP-1, was able to interact with and activate the C. elegans homolog of TAK1, MOM-4. However, the site in TAP-1 corresponding to Phe-484 of TAB1 is an alanine residue (Ala-364), and changing this residue to Phe abrogates the ability of TAP-1 to interact with and activate MOM-4. These results suggest that the Phe or Ala residue within the conserved motif of the TAB1-related proteins is important for interaction with and activation of specific TAK1 MAPKKK family members in vivo.

Chlorophyll-Derivative Modulation of Rhodopsin Signaling Properties through Evolutionarily Conserved Interaction Pathways

PubMed Central

Woods, Kristina N.; Pfeffer, Jürgen; Klein-Seetharaman, Judith

2017-01-01

Retinal is the light-absorbing chromophore that is responsible for the activation of visual pigments and light-driven ion pumps. Evolutionary changes in the intermolecular interactions of the retinal with specific amino acids allow for adaptation of the spectral characteristics, referred to as spectral tuning. However, it has been proposed that a specific species of dragon fish has bypassed the adaptive evolutionary process of spectral tuning and replaced it with a single evolutionary event: photosensitization of rhodopsin by chlorophyll derivatives. Here, by using a combination of experimental measurements and computational modeling to probe retinal-receptor interactions in rhodopsin, we show how the binding of the chlorophyll derivative, chlorin-e6 (Ce6) in the intracellular domain (ICD) of the receptor allosterically excites G-protein coupled receptor class A (GPCR-A) conserved long-range correlated fluctuations that connect distant parts of the receptor. These long-range correlated motions are associated with regulating the dynamics and intermolecular interactions of specific amino acids in the retinal ligand-binding pocket that have been associated with shifts in the absorbance peak maximum (λmax) and hence, spectral sensitivity of the visual system. Moreover, the binding of Ce6 affects the overall global properties of the receptor. Specifically, we find that Ce6-induced dynamics alter the thermal stability of rhodopsin by adjusting hydrogen-bonding interactions near the receptor active-site that consequently also influences the intrinsic conformational equilibrium of the receptor. Due to the conservation of the ICD residues amongst different receptors in this class and the fact that all GPCR-A receptors share a common mechanism of activation, it is possible that the allosteric associations excited in rhodopsin with Ce6 binding are a common feature in all class A GPCRs. PMID:29312953

Chicken galectin-1B inhibits Newcastle disease virus adsorption and replication through binding to hemagglutinin-neuraminidase (HN) glycoprotein.

PubMed

Sun, Junfeng; Han, Zongxi; Qi, Tianming; Zhao, Ran; Liu, Shengwang

2017-12-08

Galectin-1 is an important immunoregulatory factor and can mediate the host-pathogen interaction via binding glycans on the surface of various viruses. We previously reported that avian respiratory viruses, including lentogenic Newcastle disease virus (NDV), can induce up-regulation of chicken galectin (CG)-1B in the primary target organ. In this study, we investigated whether CG-1B participated in the infectious process of NDV in chickens. We demonstrated that velogenic NDV induced up-regulation of CG-1B in target organs. We also found that CG-1B directly bound to NDV virions and inhibited their hemagglutination activity in vitro We confirmed that CG-1B interacted with NDV hemagglutinin-neuraminidase (HN) glycoprotein, in which the specific G4 N -glycans significantly contributed to the interaction between CG-1B and HN glycoprotein. The presence of extracellular CG-1B, rather than the internalization process, inhibited adsorption of NDV. The interaction between intracellular CG-1B and NDV HN glycoproteins inhibited cell-surface expression of HN glycoprotein and reduced the titer of progeny virus in NDV-infected DF-1 cells. Significantly, the replication of parental and HN glycosylation mutant viruses in CG-1B knockdown and overexpression cells demonstrated that the replication of NDV was correlated with the expression of CG-1B in a specific glycan-dependent manner. Collectively, our results indicate that CG-1B has anti-NDV activity by binding to N -glycans on HN glycoprotein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Antigenic potential of a highly conserved Neisseria meningitidis lipopolysaccharide inner core structure defined by chemical synthesis.

PubMed

Reinhardt, Anika; Yang, You; Claus, Heike; Pereira, Claney L; Cox, Andrew D; Vogel, Ulrich; Anish, Chakkumkal; Seeberger, Peter H

2015-01-22

Neisseria meningitidis is a leading cause of bacterial meningitis worldwide. We studied the potential of synthetic lipopolysaccharide (LPS) inner core structures as broadly protective antigens against N. meningitidis. Based on the specific reactivity of human serum antibodies to synthetic LPS cores, we selected a highly conserved LPS core tetrasaccharide as a promising antigen. This LPS inner core tetrasaccharide induced a robust IgG response in mice when formulated as an immunogenic glycoconjugate. Binding of raised mouse serum to a broad collection of N. meningitidis strains demonstrated the accessibility of the LPS core on viable bacteria. The distal trisaccharide was identified as the crucial epitope, whereas the proximal Kdo moiety was immunodominant and induced mainly nonprotective antibodies that are responsible for lack of functional protection in polyclonal serum. Our results identified key antigenic determinants of LPS core glycan and, hence, may aid the design of a broadly protective immunization against N. meningitidis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Galectins: Double-edged Swords in the Cross-roads of Pregnancy Complications and Female Reproductive Tract Inflammation and Neoplasia.

PubMed

Than, Nandor Gabor; Romero, Roberto; Balogh, Andrea; Karpati, Eva; Mastrolia, Salvatore Andrea; Staretz-Chacham, Orna; Hahn, Sinuhe; Erez, Offer; Papp, Zoltan; Kim, Chong Jai

2015-05-01

Galectins are an evolutionarily ancient and widely expressed family of lectins that have unique glycan-binding characteristics. They are pleiotropic regulators of key biological processes, such as cell growth, proliferation, differentiation, apoptosis, signal transduction, and pre-mRNA splicing, as well as homo- and heterotypic cell-cell and cell-extracellular matrix interactions. Galectins are also pivotal in immune responses since they regulate host-pathogen interactions, innate and adaptive immune responses, acute and chronic inflammation, and immune tolerance. Some galectins are also central to the regulation of angiogenesis, cell migration and invasion. Expression and functional data provide convincing evidence that, due to these functions, galectins play key roles in shared and unique pathways of normal embryonic and placental development as well as oncodevelopmental processes in tumorigenesis. Therefore, galectins may sometimes act as double-edged swords since they have beneficial but also harmful effects for the organism. Recent advances facilitate the use of galectins as biomarkers in obstetrical syndromes and in various malignancies, and their therapeutic applications are also under investigation. This review provides a general overview of galectins and a focused review of this lectin subfamily in the context of inflammation, infection and tumors of the female reproductive tract as well as in normal pregnancies and those complicated by the great obstetrical syndromes.

Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-μ chains in the early secretory pathway.

PubMed

Giannone, Chiara; Fagioli, Claudio; Valetti, Caterina; Sitia, Roberto; Anelli, Tiziana

2017-02-03

The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4-5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers.

Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection

PubMed Central

Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A.; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D.

2015-01-01

Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein’s globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-μ chains in the early secretory pathway

PubMed Central

Giannone, Chiara; Fagioli, Claudio; Valetti, Caterina; Sitia, Roberto; Anelli, Tiziana

2017-01-01

The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4–5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers. PMID:28157181

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus*

PubMed Central

Hendricks, Gabriel L.; Weirich, Kim L.; Viswanathan, Karthik; Li, Jing; Shriver, Zachary H.; Ashour, Joseph; Ploegh, Hidde L.; Kurt-Jones, Evelyn A.; Fygenson, Deborah K.; Finberg, Robert W.; Comolli, James C.; Wang, Jennifer P.

2013-01-01

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains. PMID:23362274

Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly▿ †

PubMed Central

Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Jones, E. Yvonne; Stuart, David I.

2010-01-01

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses. PMID:20375167

Mucin-type O-glycans in Tears of Normal Subjects and Patients with Non-Sjögren’s Dry Eye

PubMed Central

Guzman-Aranguez, Ana; Mantelli, Flavio; Argüeso, Pablo

2009-01-01

Purpose O-linked carbohydrates (O-glycans) contribute to the hydrophilic character of mucins in mucosal tissues. This study aimed to identify the repertoire of O-glycans in the tear film, and the glycosyltransferases associated with their biosynthesis, in normal subjects and patients with non-Sjögren’s dry eye. Methods Human tear fluid was collected from the inferior conjunctival fornix. O-glycans were released by hydrazinolysis, labeled with 2-aminobenzamide, and analyzed by fluorometric, high-performance liquid chromatography (HPLC) coupled with exoglycosidase digestions. O-glycan structures identified in tears were related to potential biosynthetic pathways in human conjunctival epithelium using a glycogene microarray database. Lectin-binding analyses were performed using agglutinins from Arachis hypogaea, Maackia amurensis, and Sambucus nigra. Results The O-glycan profile of human tears consisted primarily of core 1 (Galβ1-3GalNAcα1-Ser/Thr)-based structures. Mono-sialyl O-glycans represented approximately 66% of the glycan pool, being α2-6-sialyl core 1 the predominant O-glycan structure in human tears (48%). Four families of glycosyltranferases potentially related to the biosynthesis of these structures were identified in human conjunctiva. These included thirteen polypeptide-GalNAc-transferases (GALNT), the core 1 β-3-galactosyltransferase (T-synthase), three α2-6-sialyltransferases (ST6GalNAc), and two α2-3-sialyltransferases (ST3Gal). No significant differences in total amount of O-glycans were detected between tears of normal subjects and dry eye patients, by HPLC and lectin blot. Likewise, no differences in glycosyltransferase expression were found by glycogene microarray. Conclusions This study identifies the most common mucin-type O-glycans in human tears and their expected biosynthetic pathways in ocular surface epithelia. Patients with non-Sjögren’s dry eye show no alterations in composition and amount of O-glycans in the tear fluid. PMID:19407012

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients

PubMed Central

Pochechueva, Tatiana; Chinarev, Alexander; Schoetzau, Andreas; Fedier, André; Bovin, Nicolai V.; Hacker, Neville F.; Jacob, Francis; Heinzelmann-Schwarz, Viola

2016-01-01

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2–6 vs. α2–3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers. PMID:27764122

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients.

PubMed

Pochechueva, Tatiana; Chinarev, Alexander; Schoetzau, Andreas; Fedier, André; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2016-01-01

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2-6 vs. α2-3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers.

LC-MS/MS Analysis of Permethylated Free Oligosaccharides and N-glycans Derived from Human, Bovine, and Goat Milk Samples

PubMed Central

Dong, Xue; Zhou, Shiyue; Mechref, Yehia

2016-01-01

Oligosaccharides in milk not only provide nutrition to the infants, but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat and human milk using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat and human milk samples (without isomeric consideration) were 11, 8 and 11 respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by PGC LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using PGC column. Permethylation of the glycan structures facilitated the interpretation of tandem MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers. PMID:26959529

LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples.

PubMed

Dong, Xue; Zhou, Shiyue; Mechref, Yehia

2016-06-01

Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans.

PubMed

Schmidt, Deborah; Schuhmacher, Frank; Geissner, Andreas; Seeberger, Peter H; Pfrengle, Fabian

2015-04-07

Monoclonal antibodies that recognize plant cell wall glycans are used for high-resolution imaging, providing important information about the structure and function of cell wall polysaccharides. To characterize the binding epitopes of these powerful molecular probes a library of eleven plant arabinoxylan oligosaccharides was produced by automated solid-phase synthesis. Modular assembly of oligoarabinoxylans from few building blocks was enabled by adding (2-naphthyl)methyl (Nap) to the toolbox of orthogonal protecting groups for solid-phase synthesis. Conjugation-ready oligosaccharides were obtained and the binding specificities of xylan-directed antibodies were determined on microarrays. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission.

PubMed

Mathys, Leen; Balzarini, Jan

2015-01-01

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

PubMed Central

Mathys, Leen; Balzarini, Jan

2015-01-01

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission. PMID:26121645

Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.

PubMed

Fujisawa, Takao; Filippakopoulos, Panagis

2017-04-01

Bromodomains (BRDs) are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions and are present in most tissues. BRDs selectively recognize and bind to acetylated Lys residues - particularly in histones - and thereby have important roles in the regulation of gene expression. BRD-containing proteins are frequently dysregulated in cancer, they participate in gene fusions that generate diverse, frequently oncogenic proteins, and many cancer-causing mutations have been mapped to the BRDs themselves. Importantly, BRDs can be targeted by small-molecule inhibitors, which has stimulated many translational research projects that seek to attenuate the aberrant functions of BRD-containing proteins in disease.

Origins and activity of the Mediator complex.

PubMed

Conaway, Ronald C; Conaway, Joan Weliky

2011-09-01

The Mediator is a large, multisubunit RNA polymerase II transcriptional regulator that was first identified in Saccharomyces cerevisiae as a factor required for responsiveness of Pol II and the general initiation factors to DNA binding transactivators. Since its discovery in yeast, Mediator has been shown to be an integral and highly evolutionarily conserved component of the Pol II transcriptional machinery with critical roles in multiple stages of transcription, from regulation of assembly of the Pol II initiation complex to regulation of Pol II elongation. Here we provide a brief overview of the evolutionary origins of Mediator, its subunit composition, and its remarkably diverse collection of activities in Pol II transcription. Copyright © 2011 Elsevier Ltd. All rights reserved.

Recent Advances in Immobilization Strategies for Glycosidases

PubMed Central

Karav, Sercan; Cohen, Joshua L.; Barile, Daniela; de Moura Bell, Juliana Maria Leite Nobrega

2017-01-01

Glycans play important biological roles in cell-to-cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large-scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo-β-N-acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI-1) that cleaves N-N′-diacetyl chitobiose moieties found in the N-linked glycan (N-glycan) core of high mannose, hybrid, and complex N-glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N-glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state-of-the-art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices. PMID:27718339

Subtle Differences in Symbiont Cell Surface Glycan Profiles Do Not Explain Species-Specific Colonization Rates in a Model Cnidarian-Algal Symbiosis

PubMed Central

Parkinson, John E.; Tivey, Trevor R.; Mandelare, Paige E.; Adpressa, Donovon A.; Loesgen, Sandra; Weis, Virginia M.

2018-01-01

Mutualisms between cnidarian hosts and dinoflagellate endosymbionts are foundational to coral reef ecosystems. These symbioses are often re-established every generation with high specificity, but gaps remain in our understanding of the cellular mechanisms that control symbiont recognition and uptake dynamics. Here, we tested whether differences in glycan profiles among different symbiont species account for the different rates at which they initially colonize aposymbiotic polyps of the model sea anemone Aiptasia (Exaiptasia pallida). First, we used a lectin array to characterize the glycan profiles of colonizing Symbiodinium minutum (ITS2 type B1) and noncolonizing Symbiodinium pilosum (ITS2 type A2), finding subtle differences in the binding of lectins Euonymus europaeus lectin (EEL) and Urtica dioica agglutinin lectin (UDA) that distinguish between high-mannoside and hybrid-type protein linked glycans. Next, we enzymatically cleaved glycans from the surfaces of S. minutum cultures and followed their recovery using flow cytometry, establishing a 48–72 h glycan turnover rate for this species. Finally, we exposed aposymbiotic host polyps to cultured S. minutum cells masked by EEL or UDA lectins for 48 h, then measured cell densities the following day. We found no effect of glycan masking on symbiont density, providing further support to the hypothesis that glycan-lectin interactions are more important for post-phagocytic persistence of specific symbionts than they are for initial uptake. We also identified several methodological and biological factors that may limit the utility of studying glycan masking in the Aiptasia system. PMID:29765363

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system

PubMed Central

Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D.; Baba, Hiroko; Ikenaka, Kazuhiro

2017-01-01

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy. PMID:28186137

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system.

PubMed

Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D; Baba, Hiroko; Ikenaka, Kazuhiro

2017-02-10

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P 0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy.

Crystal structure of Urtica dioica agglutinin, a superantigen presented by MHC molecules of class I and class II.

PubMed

Saul, F A; Rovira, P; Boulot, G; Damme, E J; Peumans, W J; Truffa-Bachi, P; Bentley, G A

2000-06-15

Urtica dioica agglutinin (UDA), a monomeric lectin extracted from stinging nettle rhizomes, is specific for saccharides containing N-acetylglucosamine (GlcNAc). The lectin behaves as a superantigen for murine T cells, inducing the exclusive proliferation of Vbeta8.3(+) lymphocytes. UDA is unique among known T cell superantigens because it can be presented by major histocompatibility complex (MHC) molecules of both class I and II. The crystal structure of UDA has been determined in the ligand-free state, and in complex with tri-acetylchitotriose and tetra-acetylchitotetraose at 1.66 A, 1.90 A and 1.40 A resolution, respectively. UDA comprises two hevein-like domains, each with a saccharide-binding site. A serine and three aromatic residues at each site form the principal contacts with the ligand. The N-terminal domain binding site can centre on any residue of a chito-oligosaccharide, whereas that of the C-terminal domain is specific for residues at the nonreducing terminus of the ligand. We have shown previously that oligomers of GlcNAc inhibit the superantigenic activity of UDA and that the lectin binds to glycans on the MHC molecule. We show that UDA also binds to glycans on the T cell receptor (TCR). The presence of two saccharide-binding sites observed in the structure of UDA suggests that its superantigenic properties arise from the simultaneous fixation of glycans on the TCR and MHC molecules of the T cell and antigen-presenting cell, respectively. The well defined spacing between the two binding sites of UDA is probably a key factor in determining the specificity for Vbeta8.3(+) lymphocytes.

A Novel High-Mannose Specific Lectin from the Green Alga Halimeda renschii Exhibits a Potent Anti-Influenza Virus Activity through High-Affinity Binding to the Viral Hemagglutinin

PubMed Central

Mu, Jinmin; Hirayama, Makoto; Sato, Yuichiro; Morimoto, Kinjiro; Hori, Kanji

2017-01-01

We have isolated a novel lectin, named HRL40 from the green alga Halimeda renschii. In hemagglutination-inhibition test and oligosaccharide-binding experiment with 29 pyridylaminated oligosaccharides, HRL40 exhibited a strict binding specificity for high-mannose N-glycans having an exposed (α1-3) mannose residue in the D2 arm of branched mannosides, and did not have an affinity for monosaccharides and other oligosaccharides examined, including complex N-glycans, an N-glycan core pentasaccharide, and oligosaccharides from glycolipids. The carbohydrate binding profile of HRL40 resembled those of Type I high-mannose specific antiviral algal lectins, or the Oscillatoria agardhii agglutinin (OAA) family, which were previously isolated from red algae and a blue-green alga (cyanobacterium). HRL40 potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells with half-maximal effective dose (ED50) of 2.45 nM through high-affinity binding to a viral envelope hemagglutinin (KD, 3.69 × 10−11 M). HRL40 consisted of two isolectins (HRL40-1 and HRL40-2), which could be separated by reverse-phase HPLC. Both isolectins had the same molecular weight of 46,564 Da and were a disulfide -linked tetrameric protein of a 11,641 Da polypeptide containing at least 13 half-cystines. Thus, HRL40, which is the first Type I high-mannose specific antiviral lectin from the green alga, had the same carbohydrate binding specificity as the OAA family, but a molecular structure distinct from the family. PMID:28813016

NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

PubMed

Higai, Koji; Imaizumi, Yuzo; Suzuki, Chiho; Azuma, Yutaro; Matsumoto, Kojiro

2009-09-04

Killer lectin-like receptors NKG2D and CD94 on natural killer cells trigger cytotoxicity through binding of glycans on target cells including sialyl Lewis X antigen. We previously reported that NKG2D and CD94 recognize alpha2,3-linked NeuAc on multi-antennary N-glycans. Here we further investigated polysaccharide binding by these receptors, using glutathione-S-transferase-fused extracellular domains of NKG2D AA 73-216 (rNKG2Dlec) and CD94 AA 68-179 (rCD94lec). We found that rNKG2Dlec and rCD94lec bind in a dose-dependent manner to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA). Binding to heparin-BSA was suppressed by soluble sulfate-containing polysaccharides, but minimally impacted by 2-O-, 6-O-, and 2-N-desulfated heparin. Mutagenesis revealed that (152)Y and (199)Y of NKG2D and (144)F, (160)N, and (166)C of CD94 were critical for binding to heparin-BSA. The present manuscript provides the first evidence that NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

Glycosylation status of bone sialoprotein and its role in mineralization.

PubMed

Xu, Lan; Zhang, Zhenqing; Sun, Xue; Wang, Jingjing; Xu, Wei; Shi, Lv; Lu, Jiaojiao; Tang, Juan; Liu, Jingjing; Su, Xiong

2017-11-15

The highly glycosylated bone sialoprotein (BSP) is an abundant non-collagenous phosphoprotein in bone which enhances osteoblast differentiation and new bone deposition in vitro and in vivo. However, the structural details of its different glycosylation linkages have not been well studied and their functions in bone homeostasis are not clear. Previous studies suggested that the O-glycans, but not the N-glycans on BSP, are highly sialylated. Herein, we employed tandem mass spectrometry (MS/MS) to demonstrate that the N-glycanson the recombinant human integrin binding sialoprotein (rhiBSP) are also enriched in sialic acids (SAs) at their termini. We also identified multiple novel sites of N-glycan modification. Treatment of rhiBSP enhances osteoblast differentiation and mineralization of MC3T3-E1 cells and this effect could be partially reversed by efficient enzymatic removal of its N-glycans. Removal of all terminal SAs has a greater effect in reversing the effect of rhiBSP on osteogenesis, especially on mineralization, suggesting that sialylation at the termini of both N-glycans and O-glycans plays an important role in this regulation. Moreover, BSP-conjugated SAs may affect mineralization via ERK activation of VDR expression. Collectively, our results identified novel N-glycans enriched in SAs on the rhiBSP and demonstrated that SAs at both N- and O-glycans are important for BSP regulation of osteoblast differentiation and mineralization in vitro. Copyright © 2017 Elsevier Inc. All rights reserved.

Structures of glycans bound to receptors from saturation transfer difference (STD) NMR spectroscopy: quantitative analysis by using CORCEMA-ST.

PubMed

Enríquez-Navas, Pedro M; Guzzi, Cinzia; Muñoz-García, Juan C; Nieto, Pedro M; Angulo, Jesús

2015-01-01

Glycan-receptor interactions are of fundamental relevance for a large number of biological processes, and their kinetics properties (medium/weak binding affinities) make them appropriated to be studied by ligand observed NMR techniques, among which saturation transfer difference (STD) NMR spectroscopy has been shown to be a very robust and powerful approach. The quantitative analysis of the results from a STD NMR study of a glycan-receptor interaction is essential to be able to translate the resulting spectral intensities into a 3D molecular model of the complex. This chapter describes how to carry out such a quantitative analysis by means of the Complete Relaxation and Conformational Exchange Matrix Approach for STD NMR (CORCEMA-ST), in general terms, and an example of a previous work on an antibody-glycan interaction is also shown.

An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins.

PubMed

Xiao, Haopeng; Chen, Weixuan; Smeekens, Johanna M; Wu, Ronghu

2018-04-27

Protein glycosylation is ubiquitous in biological systems and essential for cell survival. However, the heterogeneity of glycans and the low abundance of many glycoproteins complicate their global analysis. Chemical methods based on reversible covalent interactions between boronic acid and glycans have great potential to enrich glycopeptides, but the binding affinity is typically not strong enough to capture low-abundance species. Here, we develop a strategy using dendrimer-conjugated benzoboroxole to enhance the glycopeptide enrichment. We test the performance of several boronic acid derivatives, showing that benzoboroxole markedly increases glycopeptide coverage from human cell lysates. The enrichment is further improved by conjugating benzoboroxole to a dendrimer, which enables synergistic benzoboroxole-glycan interactions. This robust and simple method is highly effective for sensitive glycoproteomics analysis, especially capturing low-abundance glycopeptides. Importantly, the enriched glycopeptides remain intact, making the current method compatible with mass-spectrometry-based approaches to identify glycosylation sites and glycan structures.

Binding affinities of NKG2D and CD94 to sialyl Lewis X-expressing N-glycans and heparin.

PubMed

Higai, Koji; Suzuki, Chiho; Imaizumi, Yuzo; Xin, Xin; Azuma, Yutaro; Matsumoto, Kojiro

2011-01-01

Lectin-like receptors natural killer group 2D (NKG2D) and CD94 on natural killer (NK) cells bind to α2,3-NeuAc-containing N-glycans and heparin/heparan sulfate (HS). Using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rGST-NKG2Dlec) and CD94 (rGST-CD94lec), we evaluated their binding affinities (K(d)) to high sialyl Lewis X (sLeX)-expressing transferrin secreted by HepG2 cells (HepTf) and heparin-conjugated bovine serum albumin (Heparin-BSA), using quartz crystal microbalance (QCM) and enzyme immunoassay (EIA) microplate methods. K(d) values obtained by linear reciprocal plots revealed good coincidence between the two methods. K(d) values of rGST-NKG2Dlec obtained by QCM and EIA, respectively, were 1.19 and 1.11 µM for heparin-BSA >0.30 and 0.20 µM for HepTf, while those of rGST-CD94lec were 1.31 and 1.45 µM for HepTf >0.37 and 0.36 µM for heparin-BSA. These results suggested that these glycans can interact with NKG2D and CD94 to modulate NK cell-dependent cytotoxicity.

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

PubMed

Håvik, B; Ragnhildstveit, E; Lorens, J B; Saelemyr, K; Fauske, O; Knudsen, L K; Fjose, A

1999-12-20

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences. Copyright 1999 Academic Press.

Mechanisms of stable lipid loss in a social insect

PubMed Central

Ament, Seth A.; Chan, Queenie W.; Wheeler, Marsha M.; Nixon, Scott E.; Johnson, S. Peir; Rodriguez-Zas, Sandra L.; Foster, Leonard J.; Robinson, Gene E.

2011-01-01

SUMMARY Worker honey bees undergo a socially regulated, highly stable lipid loss as part of their behavioral maturation. We used large-scale transcriptomic and proteomic experiments, physiological experiments and RNA interference to explore the mechanistic basis for this lipid loss. Lipid loss was associated with thousands of gene expression changes in abdominal fat bodies. Many of these genes were also regulated in young bees by nutrition during an initial period of lipid gain. Surprisingly, in older bees, which is when maximum lipid loss occurs, diet played less of a role in regulating fat body gene expression for components of evolutionarily conserved nutrition-related endocrine systems involving insulin and juvenile hormone signaling. By contrast, fat body gene expression in older bees was regulated more strongly by evolutionarily novel regulatory factors, queen mandibular pheromone (a honey bee-specific social signal) and vitellogenin (a conserved yolk protein that has evolved novel, maturation-related functions in the bee), independent of nutrition. These results demonstrate that conserved molecular pathways can be manipulated to achieve stable lipid loss through evolutionarily novel regulatory processes. PMID:22031746

Mechanisms of stable lipid loss in a social insect.

PubMed

Ament, Seth A; Chan, Queenie W; Wheeler, Marsha M; Nixon, Scott E; Johnson, S Peir; Rodriguez-Zas, Sandra L; Foster, Leonard J; Robinson, Gene E

2011-11-15

Worker honey bees undergo a socially regulated, highly stable lipid loss as part of their behavioral maturation. We used large-scale transcriptomic and proteomic experiments, physiological experiments and RNA interference to explore the mechanistic basis for this lipid loss. Lipid loss was associated with thousands of gene expression changes in abdominal fat bodies. Many of these genes were also regulated in young bees by nutrition during an initial period of lipid gain. Surprisingly, in older bees, which is when maximum lipid loss occurs, diet played less of a role in regulating fat body gene expression for components of evolutionarily conserved nutrition-related endocrine systems involving insulin and juvenile hormone signaling. By contrast, fat body gene expression in older bees was regulated more strongly by evolutionarily novel regulatory factors, queen mandibular pheromone (a honey bee-specific social signal) and vitellogenin (a conserved yolk protein that has evolved novel, maturation-related functions in the bee), independent of nutrition. These results demonstrate that conserved molecular pathways can be manipulated to achieve stable lipid loss through evolutionarily novel regulatory processes.

Repertoire of BALB/c Mice Natural Anti-Carbohydrate Antibodies: Mice vs. Humans Difference, and Otherness of Individual Animals

PubMed Central

Bello-Gil, Daniel; Khasbiullina, Nailya; Shilova, Nadezhda; Bovin, Nicolai; Mañez, Rafael

2017-01-01

One of the most common genetic backgrounds for mice used as a model to investigate human diseases is the inbred BALB/c strain. This work is aimed to characterize the pattern of natural anti-carbohydrate antibodies present in the serum of 20 BALB/c mice by printed glycan array technology and to compare their binding specificities with that of human natural anti-carbohydrate antibodies. Natural antibodies (NAbs) from the serum of BALB/c mice interacted with 71 glycans from a library of 419 different carbohydrate structures. However, only seven of these glycans were recognized by the serum of all the animals studied, and other five glycans by at least 80% of mice. The pattern of the 12 glycans mostly recognized by the circulating antibodies of BALB/c mice differed significantly from that observed with natural anti-carbohydrate antibodies in humans. This lack of identical repertoires of natural anti-carbohydrate antibodies between individual inbred mice, and between mice and humans, should be taken into consideration when mouse models are intended to be used for investigation of NAbs in biomedical research. PMID:29163519

Computationally Discovered Potentiating Role of Glycans on NMDA Receptors

NASA Astrophysics Data System (ADS)

Sinitskiy, Anton V.; Stanley, Nathaniel H.; Hackos, David H.; Hanson, Jesse E.; Sellers, Benjamin D.; Pande, Vijay S.

2017-04-01

N-methyl-D-aspartate receptors (NMDARs) are glycoproteins in the brain central to learning and memory. The effects of glycosylation on the structure and dynamics of NMDARs are largely unknown. In this work, we use extensive molecular dynamics simulations of GluN1 and GluN2B ligand binding domains (LBDs) of NMDARs to investigate these effects. Our simulations predict that intra-domain interactions involving the glycan attached to residue GluN1-N440 stabilize closed-clamshell conformations of the GluN1 LBD. The glycan on GluN2B-N688 shows a similar, though weaker, effect. Based on these results, and assuming the transferability of the results of LBD simulations to the full receptor, we predict that glycans at GluN1-N440 might play a potentiator role in NMDARs. To validate this prediction, we perform electrophysiological analysis of full-length NMDARs with a glycosylation-preventing GluN1-N440Q mutation, and demonstrate an increase in the glycine EC50 value. Overall, our results suggest an intramolecular potentiating role of glycans on NMDA receptors.

Glycan microarray analysis of the carbohydrate-recognition specificity of native and recombinant forms of the lectin ArtinM.

PubMed

Liu, Y; Cecílio, N T; Carvalho, F C; Roque-Barreira, M C; Feizi, T

2015-12-01

This article contains data related to the researc.h article entitled "Yeast-derived ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM" by Cecílio et al. (2015) [1]. ArtinM, a D-mannose-binding lectin isolated from the seeds of Artocarpus heterophyllus, exerts immunomodulatory and regenerative activities through its Carbohydrate Recognition Domain (CRD) (Souza et al., 2013; Mariano et al., 2014 [2], [3]). The limited availability of the native lectin (n-ArtinM) led us to characterize a recombinant form of the protein, obtained by expression in Saccharomyces cerevisiae (y-ArtinM). We compared the carbohydrate-binding specificities of y-ArtinM and n-ArtinM by analyzing the binding of biotinylated preparations of the two lectin forms using a neoglycolipid (NGL)-based glycan microarray. Data showed that y-ArtinM mirrored the specificity exhibited by n-ArtinM.

A lectin HPLC method to enrich selectively-glycosylated peptides from complex biological samples.

PubMed

Johansen, Eric; Schilling, Birgit; Lerch, Michael; Niles, Richard K; Liu, Haichuan; Li, Bensheng; Allen, Simon; Hall, Steven C; Witkowska, H Ewa; Regnier, Fred E; Gibson, Bradford W; Fisher, Susan J; Drake, Penelope M

2009-10-01

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls-fucosylated and sialylated human lactoferrin glycopeptides-and negative controls-high mannose glycopeptides from Saccharomyces cerevisiae-that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Core 1-derived O-glycans are essential E-selectin ligands on neutrophils.

PubMed

Yago, Tadayuki; Fu, Jianxin; McDaniel, J Michael; Miner, Jonathan J; McEver, Rodger P; Xia, Lijun

2010-05-18

Neutrophils roll on E-selectin in inflamed venules through interactions with cell-surface glycoconjugates. The identification of physiologic E-selectin ligands on neutrophils has been elusive. Current evidence suggests that P-selectin glycoprotein ligand-1 (PSGL-1), E-selectin ligand-1 (ESL-1), and CD44 encompass all glycoprotein ligands for E-selectin; that ESL-1 and CD44 use N-glycans to bind to E-selectin; and that neutrophils lacking core 2 O-glycans have partially defective interactions with E-selectin. These data imply that N-glycans on ESL-1 and CD44 and O-glycans on PSGL-1 constitute all E-selectin ligands, with neither glycan subset having a dominant role. The enzyme T-synthase transfers Gal to GalNAcalpha1-Ser/Thr to form the core 1 structure Galbeta1-3GalNAcalpha1-Ser/Thr, a precursor for core 2 and extended core 1 O-glycans that might serve as selectin ligands. Here, using mice lacking T-synthase in endothelial and hematopoietic cells, we found that E-selectin bound to CD44 and ESL-1 in lysates of T-synthase-deficient neutrophils. However, the cells exhibited markedly impaired rolling on E-selectin in vitro and in vivo, failed to activate beta2 integrins while rolling, and did not emigrate into inflamed tissues. These defects were more severe than those of neutrophils lacking PSGL-1, CD44, and the mucin CD43. Our results demonstrate that core 1-derived O-glycans are essential E-selectin ligands; that some of these O-glycans are on protein(s) other than PSGL-1, CD44, and CD43; and that PSGL-1, CD44, and ESL-1 do not constitute all glycoprotein ligands for E-selectin.

TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability

PubMed Central

Germann, Susanne M.; Schramke, Vera; Pedersen, Rune Troelsgaard; Gallina, Irene; Eckert-Boulet, Nadine; Oestergaard, Vibe H.

2014-01-01

DNA anaphase bridges are a potential source of genome instability that may lead to chromosome breakage or nondisjunction during mitosis. Two classes of anaphase bridges can be distinguished: DAPI-positive chromatin bridges and DAPI-negative ultrafine DNA bridges (UFBs). Here, we establish budding yeast Saccharomyces cerevisiae and the avian DT40 cell line as model systems for studying DNA anaphase bridges and show that TopBP1/Dpb11 plays an evolutionarily conserved role in their metabolism. Together with the single-stranded DNA binding protein RPA, TopBP1/Dpb11 binds to UFBs, and depletion of TopBP1/Dpb11 led to an accumulation of chromatin bridges. Importantly, the NoCut checkpoint that delays progression from anaphase to abscission in yeast was activated by both UFBs and chromatin bridges independently of Dpb11, and disruption of the NoCut checkpoint in Dpb11-depleted cells led to genome instability. In conclusion, we propose that TopBP1/Dpb11 prevents accumulation of anaphase bridges via stimulation of the Mec1/ATR kinase and suppression of homologous recombination. PMID:24379413

The Many Roles of Galectin-3, a Multifaceted Molecule, in Innate Immune Responses against Pathogens

PubMed Central

Díaz-Alvarez, Laura

2017-01-01

Galectins are a group of evolutionarily conserved proteins with the ability to bind β-galactosides through characteristic carbohydrate-recognition domains (CRD). Galectin-3 is structurally unique among all galectins as it contains a C-terminal CRD linked to an N-terminal protein-binding domain, being the only chimeric galectin. Galectin-3 participates in many functions, both intra- and extracellularly. Among them, a prominent role for Galectin-3 in inflammation has been recognized. Galectin-3 has also been shown to directly bind to pathogens and to have various effects on the functions of the cells of the innate immune system. Thanks to these two properties, Galectin-3 participates in several ways in the innate immune response against invading pathogens. Galectin-3 has been proposed to function not only as a pattern-recognition receptor (PRR) but also as a danger-associated molecular pattern (DAMP). In this review, we analyze the various roles that have been assigned to Galectin-3, both as a PRR and as a DAMP, in the context of immune responses against pathogenic microorganisms. PMID:28607536

TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability.

PubMed

Germann, Susanne M; Schramke, Vera; Pedersen, Rune Troelsgaard; Gallina, Irene; Eckert-Boulet, Nadine; Oestergaard, Vibe H; Lisby, Michael

2014-01-06

DNA anaphase bridges are a potential source of genome instability that may lead to chromosome breakage or nondisjunction during mitosis. Two classes of anaphase bridges can be distinguished: DAPI-positive chromatin bridges and DAPI-negative ultrafine DNA bridges (UFBs). Here, we establish budding yeast Saccharomyces cerevisiae and the avian DT40 cell line as model systems for studying DNA anaphase bridges and show that TopBP1/Dpb11 plays an evolutionarily conserved role in their metabolism. Together with the single-stranded DNA binding protein RPA, TopBP1/Dpb11 binds to UFBs, and depletion of TopBP1/Dpb11 led to an accumulation of chromatin bridges. Importantly, the NoCut checkpoint that delays progression from anaphase to abscission in yeast was activated by both UFBs and chromatin bridges independently of Dpb11, and disruption of the NoCut checkpoint in Dpb11-depleted cells led to genome instability. In conclusion, we propose that TopBP1/Dpb11 prevents accumulation of anaphase bridges via stimulation of the Mec1/ATR kinase and suppression of homologous recombination.

Beta-propeller crystal structure of Psathyrella velutina lectin: an integrin-like fungal protein interacting with monosaccharides and calcium.

PubMed

Cioci, Gianluca; Mitchell, Edward P; Chazalet, Valerie; Debray, Henri; Oscarson, Stefan; Lahmann, Martina; Gautier, Catherine; Breton, Christelle; Perez, Serge; Imberty, Anne

2006-04-14

The lectin from the mushroom Psathyrella velutina recognises specifically N-acetylglucosamine and N-acetylneuraminic acid containing glycans. The crystal structure of the 401 amino acid residue lectin shows that it adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. In the complex with N-acetylglucosamine, six monosaccharides are bound in pockets located between two consecutive propeller blades. Due to the repeats shown by the sequence the binding sites are very similar. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups stabilize the complex, together with the hydrophobic interactions with a conserved tyrosine and histidine. The complex with N-acetylneuraminic acid shows molecular mimicry with the same hydrogen bond network, but with different orientations of the carbohydrate ring in the binding site. The beta-hairpin loops connecting the two inner beta-strands of each blade are metal binding sites and two to three calcium ions were located in the structure. The multispecificity and high multivalency of this mushroom lectin, combined with its similarity to the extracellular domain of an important class of cell adhesion molecules, integrins, are another example of the outstanding success of beta-propeller structures as molecular binding machines in nature.

A rho-binding protein kinase C-like activity is required for the function of protein kinase N in Drosophila development.

PubMed

Betson, Martha; Settleman, Jeffrey

2007-08-01

The Rho GTPases interact with multiple downstream effectors to exert their biological functions, which include important roles in tissue morphogenesis during the development of multicellular organisms. Among the Rho effectors are the protein kinase N (PKN) proteins, which are protein kinase C (PKC)-like kinases that bind activated Rho GTPases. The PKN proteins are well conserved evolutionarily, but their biological role in any organism is poorly understood. We previously determined that the single Drosophila ortholog of mammalian PKN proteins, Pkn, is a Rho/Rac-binding kinase essential for Drosophila development. By performing "rescue" studies with various Pkn mutant constructs, we have defined the domains of Pkn required for its role during Drosophila development. These studies suggested that Rho, but not Rac binding is important for Pkn function in development. In addition, we determined that the kinase domain of PKC53E, a PKC family kinase, can functionally substitute for the kinase domain of Pkn during development, thereby exemplifying the evolutionary strategy of "combining" functional domains to produce proteins with distinct biological activities. Interestingly, we also identified a requirement for Pkn in wing morphogenesis, thereby revealing the first postembryonic function for Pkn.

PAPERCLIP identifies microRNA targets and a role of CstF64/64tau in promoting non-canonical poly(A) site usage

PubMed Central

Hwang, Hun-Way; Park, Christopher Y.; Goodarzi, Hani; Fak, John J.; Mele, Aldo; Moore, Michael J.; Saito, Yuhki; Darnell, Robert B.

2016-01-01

Accurate and precise annotation of the 3′ untranslated regions (3′ UTRs) is critical in understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here we describe a method, PAPERCLIP (Poly(A) binding Protein-mediated mRNA 3′ End Retrieval by CrossLinking ImmunoPrecipitation), which shows high specificity for the mRNA 3′ ends and compares favorably to existing 3′ end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of them containing a downstream GUKKU motif. Furthermore, in mouse brain, PAPERCLIP discovers extended 3′ UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts including one in Atp2b2 that is evolutionarily conserved in human and results in a gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo. PMID:27050522

Identifying mRNA sequence elements for target recognition by human Argonaute proteins

PubMed Central

Li, Jingjing; Kim, TaeHyung; Nutiu, Razvan; Ray, Debashish; Hughes, Timothy R.; Zhang, Zhaolei

2014-01-01

It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (∼10 nucleotides in length) that alone can accurately predict AGO–mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity. PMID:24663241

Identification and characterization of Hoxa9 binding sites in hematopoietic cells

PubMed Central

Huang, Yongsheng; Sitwala, Kajal; Bronstein, Joel; Sanders, Daniel; Dandekar, Monisha; Collins, Cailin; Robertson, Gordon; MacDonald, James; Cezard, Timothee; Bilenky, Misha; Thiessen, Nina; Zhao, Yongjun; Zeng, Thomas; Hirst, Martin; Hero, Alfred; Jones, Steven

2012-01-01

The clustered homeobox proteins play crucial roles in development, hematopoiesis, and leukemia, yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 cobind at hundreds of highly evolutionarily conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation, and transcriptional activation of a network of proto-oncogenes, including Erg, Flt3, Lmo2, Myb, and Sox4. Collectively, this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia. PMID:22072553

Metabolic Glyco-Engineering in Eukaryotic Cells and Selected Applications.

PubMed

Piller, Friedrich; Mongis, Aline; Piller, Véronique

2015-01-01

By metabolic glyco-engineering cellular glycoconjugates are modified through the incorporation of synthetic monosaccharides which are usually analogues of naturally present sugars. In order to get incorporated, the monosaccharides need to enter the cytoplasm and to be substrates for the enzymes necessary for their transformation into activated sugars, most often nucleotide sugars. These have to be substrates for glycosyltransferases which finally catalyze their incorporation into glycans. Such pathways are difficult to reconstitute in vitro and therefore new monosaccharide analogues have to be tested in tissue culture for their suitability in metabolic glyco-engineering. For this, glycosylation mutants are the most appropriate since they are unable to synthesize specific glycans but through the introduction of the monosaccharide analogues they may express some glycans at the cell surface with the unnatural sugar incorporated. The presence of those glycans can be easily and quantitatively detected by lectin binding or by chemical methods identifying specific sugars. Monosaccharide analogues can also block the pathways leading to sugar incorporation, thus inhibiting the synthesis of glycan structures which is also easily detectable at the cell surface by lectin labeling. The most useful and most frequently employed application of metabolic glyco-engineering is the introduction of reactive groups which can undergo bio-orthogonal click reactions for the efficient labeling of glycans at the surface of live cells.

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3)

PubMed Central

Koymans, Kirsten J.; Feitsma, Louris J.; Brondijk, T. Harma C.; Aerts, Piet C.; Lukkien, Eddie; Lössl, Philip; van Kessel, Kok P. M.; de Haas, Carla J. C.; van Strijp, Jos A. G.; Huizinga, Eric G.

2015-01-01

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2–glycans with the conserved LewisX binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2–Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2–lipopeptide complex, it prevents TLR heterodimerization and downstream signaling. PMID:26283364

The Long Non-Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed-Batch Conditions.

PubMed

Amann, Thomas; Hansen, Anders Holmgaard; Kol, Stefan; Lee, Gyun Min; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup

2018-06-03

In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, we aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2 and-T3 with and without a disrupted B4Gal-T4 sequence resulted in only ∼1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. We revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ∼6% and ∼3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, we present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, we provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins. This article is protected by copyright. All rights reserved.

Recognition of Mannosylated Ligands and Influenza A Virus by Human Surfactant Protein D: Contributions of an Extended Site and Residue 343

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crouch, E.; Hartshorn, K; Horlacher, T

2009-01-01

Surfactant protein D (SP-D) plays important roles in antiviral host defense. Although SP-D shows a preference for glucose/maltose, the protein also recognizes d-mannose and a variety of mannose-rich microbial ligands. This latter preference prompted an examination of the mechanisms of mannose recognition, particularly as they relate to high-mannose viral glycans. Trimeric neck plus carbohydrate recognition domains from human SP-D (hNCRD) preferred ?1-2-linked dimannose (DM) over the branched trimannose (TM) core, ?1-3 or ?1-6 DM, or d-mannose. Previous studies have shown residues flanking the carbohydrate binding site can fine-tune ligand recognition. A mutant with valine at 343 (R343V) showed enhanced bindingmore » to mannan relative to wild type and R343A. No alteration in affinity was observed for d-mannose or for ?1-3- or ?1-6-linked DM; however, substantially increased affinity was observed for ?1-2 DM. Both proteins showed efficient recognition of linear and branched subdomains of high-mannose glycans on carbohydrate microarrays, and R343V showed increased binding to a subset of the oligosaccharides. Crystallographic analysis of an R343V complex with 1,2-DM showed a novel mode of binding. The disaccharide is bound to calcium by the reducing sugar ring, and a stabilizing H-bond is formed between the 2-OH of the nonreducing sugar ring and Arg349. Although hNCRDs show negligible binding to influenza A virus (IAV), R343V showed markedly enhanced viral neutralizing activity. Hydrophobic substitutions for Arg343 selectively blocked binding of a monoclonal antibody (Hyb 246-05) that inhibits IAV binding activity. Our findings demonstrate an extended ligand binding site for mannosylated ligands and the significant contribution of the 343 side chain to specific recognition of multivalent microbial ligands, including high-mannose viral glycans.« less

Poly-LacNAc as an Age-Specific Ligand for Rotavirus P[11] in Neonates and Infants

PubMed Central

Liu, Yang; Huang, Pengwei; Jiang, Baoming; Tan, Ming; Morrow, Ardythe L.; Jiang, Xi

2013-01-01

Rotavirus (RV) P[11] is an unique genotype that infects neonates. The mechanism of such age-specific host restriction remains unknown. In this study, we explored host mucosal glycans as a potential age-specific factor for attachment of P[11] RVs. Using in vitro binding assays, we demonstrated that VP8* of a P[11] RV (N155) could bind saliva of infants (60.3%, N = 151) but not of adults (0%, N = 48), with a significantly negative correlation between binding of VP8* and ages of infants (P<0.01). Recognition to the infant saliva did not correlate with the ABO, secretor and Lewis histo-blood group antigens (HBGAs) but with the binding of the lectin Lycopersicon esculentum (LEA) that is known to recognize the oligomers of N-acetyllactosamine (LacNAc), a precursor of human HBGAs. Direct evidence of LacNAc involvement in P[11] binding was obtained from specific binding of VP8* with homopolymers of LacNAc in variable lengths through a glycan array analysis of 611 glycans. These results were confirmed by strong binding of VP8* to the Lec2 cell line that expresses LacNAc oligomers but not to the Lec8 cell line lacking the LacNAc. In addition, N155 VP8* and authentic P[11] RVs (human 116E and bovine B223) hemagglutinated human red blood cells that are known to express poly-LacNAc. The potential role of poly-LacNAc in host attachment and infection of RVs has been obtained by abrogation of 116E replication by the PAA-conjugated poly-LacNAc, human milk, and LEA positive infant saliva. Overall, our results suggested that the poly-LacNAc could serve as an age-specific receptor for P[11] RVs and well explained the epidemiology that P[11] RVs mainly infect neonates and young children. PMID:24244290

Glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy.

PubMed

Nguyen-Khuong, Terry; Everest-Dass, Arun V; Kautto, Liisa; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H

2015-03-01

As a secreted fluid, the state of tear glycosylation is particularly important in the role of immunity of the ocular surface. Tears are a valuable source of non-invasive biomarkers for disease and there are continued efforts to characterize their components thoroughly. In this study, a small volume of basal tears (5 μL) was collected from healthy controls, patients with diabetes without retinopathy and patients with diabetes and retinopathy. The detailed N- and O-linked tear protein glycome was characterized and the relative abundance of each structure determined. Of the 50 N-linked glycans found, 89% were complex with 50% containing a bisecting N-acetylglucosamine, 65% containing a core fucose whilst 33% were sialylated. Of the 8 O-linked glycans detected, 3 were of cores 1 and 5 of core 2 type, with a majority of them being sialylated (90%). Additionally, these glycan structures were profiled across the three diabetic disease groups. Whilst the higher abundant structures did not alter across the three groups, only five low abundance N-linked glycans and 1 O-linked glycan did alter with the onset of diabetes mellitus and diabetic retinopathy (DR). These results suggest the conservation of glycan types on basal tear proteins between individuals and point to only small changes in glycan expression on the proteins in tears with the development of diabetes and DR. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Inducible SUMO modification of TANK alleviates its repression of TLR7 signalling.

PubMed

Renner, Florian; Saul, Vera V; Pagenstecher, Axel; Wittwer, Tobias; Schmitz, Michael Lienhard

2011-02-01

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK-binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin-related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ɛ (IKKɛ) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKɛ and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7-dependent target genes, providing a molecular mechanism that allows the control of TANK function.

Genome Organization Drives Chromosome Fragility.

PubMed

Canela, Andres; Maman, Yaakov; Jung, Seolkyoung; Wong, Nancy; Callen, Elsa; Day, Amanda; Kieffer-Kwon, Kyong-Rim; Pekowska, Aleksandra; Zhang, Hongliang; Rao, Suhas S P; Huang, Su-Chen; Mckinnon, Peter J; Aplan, Peter D; Pommier, Yves; Aiden, Erez Lieberman; Casellas, Rafael; Nussenzweig, André

2017-07-27

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT. Published by Elsevier Inc.

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

PubMed Central

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-01-01

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

Global priorities for conserving the evolutionary history of sharks, rays and chimaeras.

PubMed

Stein, R William; Mull, Christopher G; Kuhn, Tyler S; Aschliman, Neil C; Davidson, Lindsay N K; Joy, Jeffrey B; Smith, Gordon J; Dulvy, Nicholas K; Mooers, Arne O

2018-02-01

In an era of accelerated biodiversity loss and limited conservation resources, systematic prioritization of species and places is essential. In terrestrial vertebrates, evolutionary distinctness has been used to identify species and locations that embody the greatest share of evolutionary history. We estimate evolutionary distinctness for a large marine vertebrate radiation on a dated taxon-complete tree for all 1,192 chondrichthyan fishes (sharks, rays and chimaeras) by augmenting a new 610-species molecular phylogeny using taxonomic constraints. Chondrichthyans are by far the most evolutionarily distinct of all major radiations of jawed vertebrates-the average species embodies 26 million years of unique evolutionary history. With this metric, we identify 21 countries with the highest richness, endemism and evolutionary distinctness of threatened species as targets for conservation prioritization. On average, threatened chondrichthyans are more evolutionarily distinct-further motivating improved conservation, fisheries management and trade regulation to avoid significant pruning of the chondrichthyan tree of life.

Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III.

PubMed

Matsutani, Sachiko

2004-08-09

In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

Structures of the Streptococcus sanguinis SrpA Binding Region with Human Sialoglycans Suggest Features of the Physiological Ligand.

PubMed

Loukachevitch, Lioudmila V; Bensing, Barbara A; Yu, Hai; Zeng, Jie; Chen, Xi; Sullam, Paul M; Iverson, T M

2016-10-11

Streptococcus sanguinis is a leading cause of bacterial infective endocarditis, a life-threatening infection of heart valves. S. sanguinis binds to human platelets with high avidity, and this adherence is likely to enhance virulence. Previous studies suggest that a serine-rich repeat adhesin termed SrpA mediates the binding of S. sanguinis to human platelets via its interaction with sialoglycans on the receptor GPIbα. However, in vitro binding assays with SrpA and defined sialoglycans failed to identify specific high-affinity ligands. To improve our understanding of the interaction between SrpA and human platelets, we determined cocrystal structures of the SrpA sialoglycan binding region (SrpA BR ) with five low-affinity ligands: three sialylated trisaccharides (sialyl-T antigen, 3'-sialyllactose, and 3'-sialyl-N-acetyllactosamine), a sialylated tetrasaccharide (sialyl-Lewis X ), and a sialyl galactose disaccharide component common to these sialoglyans. We then combined structural analysis with mutagenesis to further determine whether our observed interactions between SrpA BR and glycans are important for binding to platelets and to better map the binding site for the physiological receptor. We found that the sialoglycan binding site of SrpA BR is significantly larger than the sialoglycans cocrystallized in this study, which suggests that binding of SrpA to platelets either is multivalent or occurs via a larger, disialylated glycan.

Structures of the Streptococcus sanguinis SrpA Binding Region with Human Sialoglycans Suggest Features of the Physiological Ligand

PubMed Central

Loukachevitch, Lioudmila V.; Bensing, Barbara A.; Yu, Hai; Zeng, Jie; Chen, Xi; Sullam, Paul M.; Iverson, T M

2016-01-01

Streptococcus sanguinis is a leading cause of bacterial infective endocarditis, a life threatening infection of heart valves. S. sanguinis binds to human platelets with high avidity, and this adherence is likely to enhance virulence. Previous studies suggest that a serine-rich repeat adhesin termed SrpA mediates the binding of S. sanguinis to human platelets via its interaction with sialoglycans on the receptor GPIbα. However, in vitro binding assays between SrpA and defined sialoglycans failed to identify specific high-affinity ligands. To better understand the interaction between SrpA and human platelets, we determined cocrystal structures of the SrpA sialoglycan binding region (SrpABR) with five low-affinity ligands: three sialylated trisaccharides (sialyl-T antigen, 3'-sialyllactose, and 3'-sialyl-N-acetyllactosamine), a sialylated tetrasaccharide (sialyl-LewisX) and a sialyl galactose disaccharide component common to these sialoglyans. We then combined structural analysis with mutagenesis to further determine whether our observed interactions between SrpABR and glycans are important for binding to platelets and to better map the binding site for the physiological receptor. We found that the sialoglycan binding site of SrpABR is significantly larger than the sialoglycans cocrystallized in this study, which suggests that SrpA binding to platelets is either multivalent or occurs via a larger, disialylated glycan. PMID:27685666

Directing stem cell trafficking via GPS.

PubMed

Sackstein, Robert

2010-01-01

The success of stem-cell-based regenerative therapeutics critically hinges on delivering relevant stem/progenitor cells to sites of tissue injury. To achieve adequate parenchymal infiltration following intravascular administration, it is first necessary that circulating cells bind to target tissue endothelium with sufficient strength to overcome the prevailing forces of hemodynamic shear. The principal mediators of these shear-resistant binding interactions consist of a family of C-type lectins known as "selectins" that bind discrete sialofucosylated glycans on their respective ligands. One member of this family, E-selectin, is an endothelial molecule that is inducibly expressed on postcapillary venules at all sites of tissue injury, but is also constitutively expressed on the luminal surface of bone marrow and dermal microvascular endothelium. Most stem/progenitor cells express high levels of CD44, and, in particular, human hematopoietic stem cells express a specialized sialofucosylated glycoform of CD44 known as "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a potent E-selectin ligand. This chapter describes a method called "glycosyltransferase-programmed stereosubstitution" (GPS) for custom-modifying CD44 glycans to create HCELL on the surface of living cells that natively lack HCELL. Ex vivo glycan engineering of HCELL via GPS licenses trafficking of infused cells to endothelial beds that express E-selectin, thereby enabling efficient vascular delivery of stem/progenitor cells to sites where they are needed. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.

PubMed

Chokhawala, Harshal A; Huang, Shengshu; Lau, Kam; Yu, Hai; Cheng, Jiansong; Thon, Vireak; Hurtado-Ziola, Nancy; Guerrero, Juan A; Varki, Ajit; Chen, Xi

2008-09-19

Although the vital roles of structures containing sialic acid in biomolecular recognition are well documented, limited information is available on how sialic acid structural modifications, sialyl linkages, and the underlying glycan structures affect the binding or the activity of sialic acid-recognizing proteins and related downstream biological processes. A novel combinatorial chemoenzymatic method has been developed for the highly efficient synthesis of biotinylated sialosides containing different sialic acid structures and different underlying glycans in 96-well plates from biotinylated sialyltransferase acceptors and sialic acid precursors. By transferring the reaction mixtures to NeutrAvidin-coated plates and assaying for the yields of enzymatic reactions using lectins recognizing sialyltransferase acceptors but not the sialylated products, the biotinylated sialoside products can be directly used, without purification, for high-throughput screening to quickly identify the ligand specificity of sialic acid-binding proteins. For a proof-of-principle experiment, 72 biotinylated alpha2,6-linked sialosides were synthesized in 96-well plates from 4 biotinylated sialyltransferase acceptors and 18 sialic acid precursors using a one-pot three-enzyme system. High-throughput screening assays performed in NeutrAvidin-coated microtiter plates show that whereas Sambucus nigra Lectin binds to alpha2,6-linked sialosides with high promiscuity, human Siglec-2 (CD22) is highly selective for a number of sialic acid structures and the underlying glycans in its sialoside ligands.

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

PubMed Central

Jones, Meredith B.; Rosenberg, Julian N.; Betenbaugh, Michael J.; Krag, Sharon S.

2009-01-01

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide. PMID:19348869

Mucin-Type O-Glycosylation in Invertebrates.

PubMed

Staudacher, Erika

2015-06-09

O-Glycosylation is one of the most important posttranslational modifications of proteins. It takes part in protein conformation, protein sorting, developmental processes and the modulation of enzymatic activities. In vertebrates, the basics of the biosynthetic pathway of O-glycans are already well understood. However, the regulation of the processes and the molecular aspects of defects, especially in correlation with cancer or developmental abnormalities, are still under investigation. The knowledge of the correlating invertebrate systems and evolutionary aspects of these highly conserved biosynthetic events may help improve the understanding of the regulatory factors of this pathway. Invertebrates display a broad spectrum of glycosylation varieties, providing an enormous potential for glycan modifications which may be used for the design of new pharmaceutically active substances. Here, overviews of the present knowledge of invertebrate mucin-type O-glycan structures and the currently identified enzymes responsible for the biosynthesis of these oligosaccharides are presented, and the few data dealing with functional aspects of O-glycans are summarised.

Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

NASA Astrophysics Data System (ADS)

Both, P.; Green, A. P.; Gray, C. J.; Šardzík, R.; Voglmeir, J.; Fontana, C.; Austeri, M.; Rejzek, M.; Richardson, D.; Field, R. A.; Widmalm, G.; Flitsch, S. L.; Eyers, C. E.

2014-01-01

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

Suppression of grp78 core promoter element-mediated stress induction by the dbpA and dbpB (YB-1) cold shock domain proteins.

PubMed Central

Li, W W; Hsiung, Y; Wong, V; Galvin, K; Zhou, Y; Shi, Y; Lee, A S

1997-01-01

The highly conserved grp78 core promoter element plays an important role in the induction of grp78 under diverse stress signals. Previous studies have established a functional region in the 3' half of the core (stress-inducible change region [SICR]) which exhibits stress-inducible changes in stressed nuclei. The human transcription factor YY1 is shown to bind the SICR and transactivate the core element under stress conditions. Here we report that expression library screening with the core element has identified two new core binding proteins, YB-1 and dbpA. Both proteins belong to the Y-box family of proteins characterized by an evolutionarily conserved DNA binding motif, the cold shock domain (CSD). In contrast to YY1, which binds only double-stranded SICR, the Y-box/CSD proteins much prefer the lower strand of the SICR. The Y-box proteins can repress the inducibility of the grp78 core element mediated by treatment of cells with A23187, thapsigargin, and tunicamycin. In gel shift assays, YY1 binding to the core element is inhibited by either YB-1 or dbpA. A yeast interaction trap screen using LexA-YY1 as a bait and a HeLa cell cDNA-acid patch fusion library identified YB-1 as a YY1-interacting protein. In cotransfection experiments, the Y-box proteins antagonize the YY1-mediated enhancement of transcription directed by the grp78 core in stressed cells. Thus, the CSD proteins may be part of the stress signal transduction mechanism in the mammalian system. PMID:8972186

Evolutionarily Conserved Epitopes on Human Immunodeficiency Virus Type 1 (HIV-1) and Feline Immunodeficiency Virus Reverse Transcriptases Detected by HIV-1-Infected Subjects

PubMed Central

Sanou, Missa P.; Roff, Shannon R.; Mennella, Antony; Sleasman, John W.; Rathore, Mobeen H.; Levy, Jay A.

2013-01-01

Anti-human immunodeficiency virus (HIV) cytotoxic T lymphocyte (CTL)-associated epitopes, evolutionarily conserved on both HIV type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptases (RT), were identified using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and carboxyfluorescein diacetate succinimide ester (CFSE) proliferation assays followed by CTL-associated cytotoxin analysis. The peripheral blood mononuclear cells (PBMC) or T cells from HIV-1-seropositive (HIV+) subjects were stimulated with overlapping RT peptide pools. The PBMC from the HIV+ subjects had more robust IFN-γ responses to the HIV-1 peptide pools than to the FIV peptide pools, except for peptide-pool F3. In contrast, much higher and more frequent CD8+ T-cell proliferation responses were observed with the FIV peptide pools than with the HIV peptide pools. HIV-1-seronegative subjects had no proliferation or IFN-γ responses to the HIV and FIV peptide pools. A total of 24% (40 of 166) of the IFN-γ responses to HIV pools and 43% (23 of 53) of the CD8+ T-cell proliferation responses also correlated to responses to their counterpart FIV pools. Thus, more evolutionarily conserved functional epitopes were identified by T-cell proliferation than by IFN-γ responses. In the HIV+ subjects, peptide-pool F3, but not the HIV H3 counterpart, induced the most IFN-γ and proliferation responses. These reactions to peptide-pool F3 were highly reproducible and persisted over the 1 to 2 years of testing. All five individual peptides and epitopes of peptide-pool F3 induced IFN-γ and/or proliferation responses in addition to inducing CTL-associated cytotoxin responses (perforin, granzyme A, granzyme B). The epitopes inducing polyfunctional T-cell activities were highly conserved among human, simian, feline, and ungulate lentiviruses, which indicated that these epitopes are evolutionarily conserved. These results suggest that FIV peptides could be used in an HIV-1 vaccine. PMID:23824804

SoxNeuro orchestrates central nervous system specification and differentiation in Drosophila and is only partially redundant with Dichaete

PubMed Central

2014-01-01

Background Sox proteins encompass an evolutionarily conserved family of transcription factors with critical roles in animal development and stem cell biology. In common with vertebrates, the Drosophila group B proteins SoxNeuro and Dichaete are involved in central nervous system development, where they play both similar and unique roles in gene regulation. Sox genes show extensive functional redundancy across metazoans, but the molecular basis underpinning functional compensation mechanisms at the genomic level are currently unknown. Results Using a combination of genome-wide binding analysis and gene expression profiling, we show that SoxNeuro directs embryonic neural development from the early specification of neuroblasts through to the terminal differentiation of neurons and glia. To address the issue of functional redundancy and compensation at a genomic level, we compare SoxNeuro and Dichaete binding, identifying common and independent binding events in wild-type conditions, as well as instances of compensation and loss of binding in mutant backgrounds. Conclusions We find that early aspects of group B Sox functions in the central nervous system, such as stem cell maintenance and dorsoventral patterning, are highly conserved. However, in contrast to vertebrates, we find that Drosophila group B1 proteins also play prominent roles during later aspects of neural morphogenesis. Our analysis of the functional relationship between SoxNeuro and Dichaete uncovers evidence for redundant and independent functions for each protein, along with unexpected examples of compensation and interdependency, thus providing new insights into the general issue of transcription factor functional redundancy. PMID:24886562

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures

DOE PAGES

Hawkes, Emily J.; Hennelly, Scott P.; Novikova, Irina V.; ...

2016-09-20

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. Here, we investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probingmore » and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.« less

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hawkes, Emily J.; Hennelly, Scott P.; Novikova, Irina V.

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. Here, we investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probingmore » and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.« less

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins.

PubMed

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Joshi, Hiren Jitendra; Siukstaite, Lina; Harrison, Oliver J; Brasch, Julia; Goodman, Kerry M; Hansen, Lars; Shapiro, Lawrence; Honig, Barry; Vakhrushev, Sergey Y; Clausen, Henrik; Halim, Adnan

2017-10-17

The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1-4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific β-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3.

Playing Hide and Seek: How Glycosylation of the Influenza Virus Hemagglutinin Can Modulate the Immune Response to Infection

PubMed Central

Tate, Michelle D.; Job, Emma R.; Deng, Yi-Mo; Gunalan, Vithiagaran; Maurer-Stroh, Sebastian; Reading, Patrick C.

2014-01-01

Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the hemagglutinin (HA) glycoprotein. The viral HA is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection. However, addition of glycans can also interfere with the receptor binding properties of HA and this must be compensated for by additional mutations, creating a fitness barrier to accumulation of glycosylation sites. In addition, glycans on HA are also recognized by phylogenetically ancient lectins of the innate immune system and the benefit provided by evasion of humoral immunity is balanced by attenuation of infection. Therefore, a fine balance must exist regarding the optimal pattern of HA glycosylation to offset competing pressures associated with recognition by innate defenses, evasion of humoral immunity and maintenance of virus fitness. In this review, we examine HA glycosylation patterns of IAV associated with pandemic and seasonal influenza and discuss recent advancements in our understanding of interactions between IAV glycans and components of innate and adaptive immunity. PMID:24638204

Comparative promoter analysis allows de novo identification of specialized cell junction-associated proteins.

PubMed

Cohen, Clemens D; Klingenhoff, Andreas; Boucherot, Anissa; Nitsche, Almut; Henger, Anna; Brunner, Bodo; Schmid, Holger; Merkle, Monika; Saleem, Moin A; Koller, Klaus-Peter; Werner, Thomas; Gröne, Hermann-Josef; Nelson, Peter J; Kretzler, Matthias

2006-04-11

Shared transcription factor binding sites that are conserved in distance and orientation help control the expression of gene products that act together in the same biological context. New bioinformatics approaches allow the rapid characterization of shared promoter structures and can be used to find novel interacting molecules. Here, these principles are demonstrated by using molecules linked to the unique functional unit of the glomerular slit diaphragm. An evolutionarily conserved promoter model was generated by comparative genomics in the proximal promoter regions of the slit diaphragm-associated molecule nephrin. Phylogenetic promoter fingerprints of known elements of the slit diaphragm complex identified the nephrin model in the promoter region of zonula occludens-1 (ZO-1). Genome-wide scans using this promoter model effectively predicted a previously unrecognized slit diaphragm molecule, cadherin-5. Nephrin, ZO-1, and cadherin-5 mRNA showed stringent coexpression across a diverse set of human glomerular diseases. Comparative promoter analysis can identify regulatory pathways at work in tissue homeostasis and disease processes.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

DOE PAGES

Zhang, Chunhua; Brown, Michelle Q.; van de Ven, Wilhelmina; ...

2015-11-25

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells andmore » enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. Ultimately, this study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N.

Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure ofmore » VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N.

Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered thatVibrio parahaemolyticusVtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC withmore » bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.« less

The human sirtuin family: Evolutionary divergences and functions

PubMed Central

2011-01-01

The sirtuin family of proteins is categorised as class III histone deacetylases that play complex and important roles in ageing-related pathological conditions such as cancer and the deregulation of metabolism. There are seven members in humans, divided into four classes, and evolutionarily conserved orthologues can be found in most forms of life, including both eukaryotes and prokaryotes. The highly conserved catalytic core domain composed of a large oxidised nicotinamide adenine dinucleotide (NAD+)-binding Rossmann fold subunit suggests that these proteins belong to a family of nutrient-sensing regulators. Along with their function in regulating cellular metabolism in response to stressful conditions, they are implicated in modifying a wide variety of substrates; this increases the complexity of unravelling the interplay of sirtuins and their partners. Over the past few years, all of these new findings have attracted the interest of researchers exploring potential therapeutic implications related to the function of sirtuins. It remains to be elucidated whether, indeed, sirtuins can serve as molecular targets for the treatment of human illnesses. PMID:21807603

ROCC, a conserved region in cohesin's Mcd1 subunit, is essential for the proper regulation of the maintenance of cohesion and establishment of condensation

PubMed Central

Eng, Thomas; Guacci, Vincent; Koshland, Doug

2014-01-01

Cohesin helps orchestrate higher-order chromosome structure, thereby promoting sister chromatid cohesion, chromosome condensation, DNA repair, and transcriptional regulation. To elucidate how cohesin facilitates these diverse processes, we mutagenized Mcd1p, the kleisin regulatory subunit of budding yeast cohesin. In the linker region of Mcd1p, we identified a novel evolutionarily conserved 10–amino acid cluster, termed the regulation of cohesion and condensation (ROCC) box. We show that ROCC promotes cohesion maintenance by protecting a second activity of cohesin that is distinct from its stable binding to chromosomes. The existence of this second activity is incompatible with the simple embrace mechanism of cohesion. In addition, we show that the ROCC box is required for the establishment of condensation. We provide evidence that ROCC controls cohesion maintenance and condensation establishment through differential functional interactions with Pds5p and Wpl1p. PMID:24966169

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chunhua; Brown, Michelle Q.; van de Ven, Wilhelmina

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells andmore » enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. Ultimately, this study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.« less

Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function

PubMed Central

Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

2014-01-01

Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations

PubMed Central

2013-01-01

Background Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC. Methods Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. Results A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) <5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR <5%, p-value ≤0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p <0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes’ as the main enriched category. A module map analysis of the protein-coding genes significantly (p <0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p <0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p <0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. Conclusion Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation. PMID:24238219

A genome-wide association study identifies multiple loci for variation in human ear morphology.

PubMed

Adhikari, Kaustubh; Reales, Guillermo; Smith, Andrew J P; Konka, Esra; Palmen, Jutta; Quinto-Sanchez, Mirsha; Acuña-Alonzo, Victor; Jaramillo, Claudia; Arias, William; Fuentes, Macarena; Pizarro, María; Barquera Lozano, Rodrigo; Macín Pérez, Gastón; Gómez-Valdés, Jorge; Villamil-Ramírez, Hugo; Hunemeier, Tábita; Ramallo, Virginia; Silva de Cerqueira, Caio C; Hurtado, Malena; Villegas, Valeria; Granja, Vanessa; Gallo, Carla; Poletti, Giovanni; Schuler-Faccini, Lavinia; Salzano, Francisco M; Bortolini, Maria-Cátira; Canizales-Quinteros, Samuel; Rothhammer, Francisco; Bedoya, Gabriel; Calderón, Rosario; Rosique, Javier; Cheeseman, Michael; Bhutta, Mahmood F; Humphries, Steve E; Gonzalez-José, Rolando; Headon, Denis; Balding, David; Ruiz-Linares, Andrés

2015-06-24

Here we report a genome-wide association study for non-pathological pinna morphology in over 5,000 Latin Americans. We find genome-wide significant association at seven genomic regions affecting: lobe size and attachment, folding of antihelix, helix rolling, ear protrusion and antitragus size (linear regression P values 2 × 10(-8) to 3 × 10(-14)). Four traits are associated with a functional variant in the Ectodysplasin A receptor (EDAR) gene, a key regulator of embryonic skin appendage development. We confirm expression of Edar in the developing mouse ear and that Edar-deficient mice have an abnormally shaped pinna. Two traits are associated with SNPs in a region overlapping the T-Box Protein 15 (TBX15) gene, a major determinant of mouse skeletal development. Strongest association in this region is observed for SNP rs17023457 located in an evolutionarily conserved binding site for the transcription factor Cartilage paired-class homeoprotein 1 (CART1), and we confirm that rs17023457 alters in vitro binding of CART1.

Alternative Polyadenylation Regulates CELF1/CUGBP1 Target Transcripts Following T Cell Activation

PubMed Central

Beisang, Daniel; Reilly, Cavan; Bohjanen, Paul R.

2014-01-01

Alternative polyadenylation (APA) is an evolutionarily conserved mechanism for regulating gene expression. Transcript 3′ end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3′ untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3′ end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation PMID:25123787

Multiple transcription factor codes activate epidermal wound–response genes in Drosophila

PubMed Central

Pearson, Joseph C.; Juarez, Michelle T.; Kim, Myungjin; Drivenes, Øyvind; McGinnis, William

2009-01-01

Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes—Ddc, ple, msn, and kkv—that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound–response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view. PMID:19168633

Symbiont-induced odorant binding proteins mediate insect host hematopoiesis

PubMed Central

Benoit, Joshua B; Vigneron, Aurélien; Broderick, Nichole A; Wu, Yineng; Sun, Jennifer S; Carlson, John R; Aksoy, Serap; Weiss, Brian L

2017-01-01

Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies (Glossina spp.) house maternally transmitted symbionts that regulate the development and function of their host’s immune system. Herein we demonstrate that the obligate mutualist, Wigglesworthia, up-regulates expression of odorant binding protein six in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor lozenge and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval Drosophila’s indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems. DOI: http://dx.doi.org/10.7554/eLife.19535.001 PMID:28079523

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2*

PubMed Central

Hosokawa, Hiroyuki; Dip, Phat Vinh; Merkulova, Maria; Bakulina, Anastasia; Zhuang, Zhenjie; Khatri, Ashok; Jian, Xiaoying; Keating, Shawn M.; Bueler, Stephanie A.; Rubinstein, John L.; Randazzo, Paul A.; Ausiello, Dennis A.; Grüber, Gerhard; Marshansky, Vladimir

2013-01-01

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H+-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe5, Met10, and Gln14 involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor. PMID:23288846

Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Xueyong; Viswanathan, Karthik; Raman, Rahul

Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA) mutants from ferret-transmissible H5N1 viruses of A/Viet Nam/1203/04 and A/Indonesia/5/05 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6 linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3 linked sialosides.more » Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogues reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.« less

Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

DOE PAGES

Zhu, Xueyong; Viswanathan, Karthik; Raman, Rahul; ...

2015-11-01

Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA) mutants from ferret-transmissible H5N1 viruses of A/Viet Nam/1203/04 and A/Indonesia/5/05 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6 linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3 linked sialosides.more » Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogues reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.« less

N-glycans of Human Protein C Inhibitor: Tissue-Specific Expression and Function

PubMed Central

Engström, Åke; Sooriyaarachchi, Sanjeewani; Ubhayasekera, Wimal; Hreinsson, Julius; Wånggren, Kjell; Clark, Gary F.; Dell, Anne; Schedin-Weiss, Sophia

2011-01-01

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewisx. A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M−1s−1 for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M−1s−1 for the corresponding PNGase F-treated forms. The 7–8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA. PMID:22205989

Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation

PubMed Central

Taladriz, Soraya; Hanke, Tobias; Ramiro, María J.; García-Díaz, Miguel; Lacoba, Mario García de; Blanco, Luis; Larraga, Vicente

2001-01-01

We have identified a novel polymerase beta (Pol β)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol β, shows a nuclear localization that contrasts with the mitochondrial localization of Pol β from Crithidia fasciculata, a closely related parasite, the only polymerase β described so far in Trypanosomatidae. Li Pol β, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol β-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol β, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol β mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol β at the infective phase of the parasite. The data suggest a role of Li Pol β in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells. PMID:11557814

Spliced X-box Binding Protein 1 Couples the Unfolded Protein Response to Hexosamine Biosynthetic Pathway

PubMed Central

Wang, Zhao V.; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L.; Morales, Cyndi R.; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A.; Rothermel, Beverly A.; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P.A.; Ferdous, Anwarul; Gillette, Thomas G.; Scherer, Philipp E.; Hill, Joseph A.

2014-01-01

SUMMARY The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis, by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. PMID:24630721

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

PubMed

Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

2014-03-13

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Galectins: Double-edged Swords in the Cross-roads of Pregnancy Complications and Female Reproductive Tract Inflammation and Neoplasia

PubMed Central

Than, Nandor Gabor; Romero, Roberto; Balogh, Andrea; Karpati, Eva; Mastrolia, Salvatore Andrea; Staretz-Chacham, Orna; Hahn, Sinuhe; Erez, Offer; Papp, Zoltan; Kim, Chong Jai

2015-01-01

Galectins are an evolutionarily ancient and widely expressed family of lectins that have unique glycan-binding characteristics. They are pleiotropic regulators of key biological processes, such as cell growth, proliferation, differentiation, apoptosis, signal transduction, and pre-mRNA splicing, as well as homo- and heterotypic cell-cell and cell-extracellular matrix interactions. Galectins are also pivotal in immune responses since they regulate host-pathogen interactions, innate and adaptive immune responses, acute and chronic inflammation, and immune tolerance. Some galectins are also central to the regulation of angiogenesis, cell migration and invasion. Expression and functional data provide convincing evidence that, due to these functions, galectins play key roles in shared and unique pathways of normal embryonic and placental development as well as oncodevelopmental processes in tumorigenesis. Therefore, galectins may sometimes act as double-edged swords since they have beneficial but also harmful effects for the organism. Recent advances facilitate the use of galectins as biomarkers in obstetrical syndromes and in various malignancies, and their therapeutic applications are also under investigation. This review provides a general overview of galectins and a focused review of this lectin subfamily in the context of inflammation, infection and tumors of the female reproductive tract as well as in normal pregnancies and those complicated by the great obstetrical syndromes. PMID:26018511

Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function.

PubMed

Scherer, Paul C; Ding, Yan; Liu, Zhiqing; Xu, Jing; Mao, Haibin; Barrow, James C; Wei, Ning; Zheng, Ning; Snyder, Solomon H; Rao, Feng

2016-03-29

The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.

Transcriptional regulation of human eosinophil RNases by an evolutionary- conserved sequence motif in primate genome

PubMed Central

Wang, Hsiu-Yu; Chang, Hao-Teng; Pai, Tun-Wen; Wu, Chung-I; Lee, Yuan-Hung; Chang, Yen-Hsin; Tai, Hsiu-Ling; Tang, Chuan-Yi; Chou, Wei-Yao; Chang, Margaret Dah-Tsyr

2007-01-01

Background Human eosinophil-derived neurotoxin (edn) and eosinophil cationic protein (ecp) are members of a subfamily of primate ribonuclease (rnase) genes. Although they are generated by gene duplication event, distinct edn and ecp expression profile in various tissues have been reported. Results In this study, we obtained the upstream promoter sequences of several representative primate eosinophil rnases. Bioinformatic analysis revealed the presence of a shared 34-nucleotide (nt) sequence stretch located at -81 to -48 in all edn promoters and macaque ecp promoter. Such a unique sequence motif constituted a region essential for transactivation of human edn in hepatocellular carcinoma cells. Gel electrophoretic mobility shift assay, transient transfection and scanning mutagenesis experiments allowed us to identify binding sites for two transcription factors, Myc-associated zinc finger protein (MAZ) and SV-40 protein-1 (Sp1), within the 34-nt segment. Subsequent in vitro and in vivo binding assays demonstrated a direct molecular interaction between this 34-nt region and MAZ and Sp1. Interestingly, overexpression of MAZ and Sp1 respectively repressed and enhanced edn promoter activity. The regulatory transactivation motif was mapped to the evolutionarily conserved -74/-65 region of the edn promoter, which was guanidine-rich and critical for recognition by both transcription factors. Conclusion Our results provide the first direct evidence that MAZ and Sp1 play important roles on the transcriptional activation of the human edn promoter through specific binding to a 34-nt segment present in representative primate eosinophil rnase promoters. PMID:17927842

Targeting B lymphoma with nanoparticles bearing glycan ligands of CD22.

PubMed

Chen, Weihsu C; Sigal, Darren S; Saven, Alan; Paulson, James C

2012-02-01

CD22 is a member of the siglec (sialic acid-binding immunoglobulin-like lectin) family expressed on B cells that recognizes glycans of glycoproteins as ligands. Because siglecs exhibit restricted expression on one or a few leukocyte cell types, they have gained attention as attractive targets for cell-directed therapies. Several antibody-based therapies targeting CD22 (Siglec-2) are currently in clinical trials for the treatment of hairy cell leukemia and other B cell lymphomas. As an alternative to antibodies we have developed liposomal nanoparticles decorated with glycan ligands of CD22 that selectively target B cells. Because CD22 is an endocytic receptor, ligand-decorated liposomes are bound by CD22 and rapidly internalized by the cell. When loaded with a toxic cargo such as doxorubicin, they are efficacious in prolonging life in a Daudi B cell lymphoma model. These B cell targeted nanoparticles have been demonstrated to bind and kill malignant B cells from patients with hairy cell leukemia, marginal zone lymphoma and chronic lymphocytic leukemia. The results demonstrate the potential of using CD22 ligand-targeted liposomal nanoparticles as an alternative approach for the treatment of B cell malignancies.

Combining Crystallography and Hydrogen-Deuterium Exchange to Study Galectin-Ligand Complexes.

PubMed

Ruiz, Federico M; Gilles, Ulrich; Lindner, Ingo; André, Sabine; Romero, Antonio; Reusch, Dietmar; Gabius, Hans-Joachim

2015-09-21

The physiological significance arising from translating information stored in glycans into cellular effects explains the interest in structurally defining lectin-carbohydrate recognition. The relatively small set of adhesion/growth-regulatory galectins in chicken makes this system attractive to study the origins of specificity and divergence. Cell binding by using glycosylation mutants reveals binding of the N-terminal domain of chicken galectin-8 (CG-8N) to α-2,3-sialylated and galactose-terminated glycan chains. Cocrystals with lactose and its 3'-sialylated derivative disclose Arg58 as a key contact for the carboxylic acid and differences in loop lengths to the three homodimeric chicken galectins. Monitoring hydrogen-deuterium exchange by mass spectrometry revealed an effective reduction of deuteration after ligand binding within the contact area. In addition, evidence for changes in solvent accessibility of amide protons beyond this site was obtained. Their detection, which highlights the sensor capacity of this technique, encourages systematic studies on galectins and beyond. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Starch Catabolism by a Prominent Human Gut Symbiont Is Directed by the Recognition of Amylose Helices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koropatkin, Nicole M.; Martens, Eric C.; Gordon, Jeffrey I.

2009-01-12

The human gut microbiota performs functions that are not encoded in our Homo sapiens genome, including the processing of otherwise undigestible dietary polysaccharides. Defining the structures of proteins involved in the import and degradation of specific glycans by saccharolytic bacteria complements genomic analysis of the nutrient-processing capabilities of gut communities. Here, we describe the atomic structure of one such protein, SusD, required for starch binding and utilization by Bacteroides thetaiotaomicron, a prominent adaptive forager of glycans in the distal human gut microbiota. The binding pocket of this unique {alpha}-helical protein contains an arc of aromatic residues that complements the naturalmore » helical structure of starch and imposes this conformation on bound maltoheptaose. Furthermore, SusD binds cyclic oligosaccharides with higher affinity than linear forms. The structures of several SusD/oligosaccharide complexes reveal an inherent ligand recognition plasticity dominated by the three-dimensional conformation of the oligosaccharides rather than specific interactions with the composite sugars.« less

Microrheology study of human mucins varying in Helicobacter pylori binding affinity

NASA Astrophysics Data System (ADS)

Su, Clover; Sharba, Sinan; Linden, Sara; Bansil, Rama

Helicobacter pylori is the pathogen that colonizes the human stomach and causes gastric ulcers and cancer. One of the key mechanisms by which H. pylori establishes an infection on the gastric mucosa is by expressing adhesins that facilitate the binding of the bacterium to the host epithelial cell. We present the motility and microrheology study of a clinical isolate strain of H. pylori, J99, and its mutant with and without particular adhesins that bind to mucins with specific alterations in their glycans coat. Our microrheology experiments show that mucin viscosity depends on the glycans coat and decreases in the presence of bacteria. We found no significant changes in bacterial motility between J99 wild type and mutant in culture broth. Unlike previous observations made with other H. pylori strains, we did not see reversals in J99 strains. Bacteria tracking measurements are underway to examine the motility in these altered mucin solutions. Supported by NSF PHY 1410798.

Self-recognition and Ca2+-dependent carbohydrate-carbohydrate cell adhesion provide clues to the cambrian explosion.

PubMed

Fernàndez-Busquets, Xavier; Körnig, André; Bucior, Iwona; Burger, Max M; Anselmetti, Dario

2009-11-01

The Cambrian explosion of life was a relatively short period approximately 540 Ma that marked a generalized acceleration in the evolution of most animal phyla, but the trigger of this key biological event remains elusive. Sponges are the oldest extant Precambrian metazoan phylum and thus a valid model to study factors that could have unleashed the rise of multicellular animals. One such factor is the advent of self-/non-self-recognition systems, which would be evolutionarily beneficial to organisms to prevent germ-cell parasitism or the introduction of deleterious mutations resulting from fusion with genetically different individuals. However, the molecules responsible for allorecognition probably evolved gradually before the Cambrian period, and some other (external) factor remains to be identified as the missing triggering event. Sponge cells associate through calcium-dependent, multivalent carbohydrate-carbohydrate interactions of the g200 glycan found on extracellular proteoglycans. Single molecule force spectroscopy analysis of g200-g200 binding indicates that calcium affects the lifetime (+Ca/-Ca: 680 s/3 s) and bond reaction length (+Ca/-Ca: 3.47 A/2.27 A). Calculation of mean g200 dissociation times in low and high calcium within the theoretical framework of a cooperative binding model indicates the nonlinear and divergent characteristics leading to either disaggregated cells or stable multicellular assemblies, respectively. This fundamental phenomenon can explain a switch from weak to strong adhesion between primitive metazoan cells caused by the well-documented rise in ocean calcium levels at the end of Precambrian time. We propose that stronger cell adhesion allowed the integrity of genetically uniform animals composed only of "self" cells, facilitating genetic constitutions to remain within the metazoan individual and be passed down inheritance lines. The Cambrian explosion might have been triggered by the coincidence in time of primitive animals endowed with self-/non-self-recognition and of a surge in seawater calcium that increased the binding forces between their calcium-dependent cell adhesion molecules.

Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.

PubMed

Mathew, Mohit P; Donaldson, Julie G

2018-05-11

Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 molecule (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin-glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin-glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes.

Bivalent Carbohydrate Binding Is Required for Biological Activity of Clitocybe nebularis Lectin (CNL), the N,N′-Diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc)-specific Lectin from Basidiomycete C. nebularis*

PubMed Central

Pohleven, Jure; Renko, Miha; Magister, Špela; Smith, David F.; Künzler, Markus; Štrukelj, Borut; Turk, Dušan; Kos, Janko; Sabotič, Jerica

2012-01-01

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N′-diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency. PMID:22298779

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

2013-01-01

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

PubMed

Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

2018-05-15

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk. Copyright © 2018 American Society for Microbiology.

Identification of evolutionarily conserved Momordica charantia microRNAs using computational approach and its utility in phylogeny analysis.

PubMed

Thirugnanasambantham, Krishnaraj; Saravanan, Subramanian; Karikalan, Kulandaivelu; Bharanidharan, Rajaraman; Lalitha, Perumal; Ilango, S; HairulIslam, Villianur Ibrahim

2015-10-01

Momordica charantia (bitter gourd, bitter melon) is a monoecious Cucurbitaceae with anti-oxidant, anti-microbial, anti-viral and anti-diabetic potential. Molecular studies on this economically valuable plant are very essential to understand its phylogeny and evolution. MicroRNAs (miRNAs) are conserved, small, non-coding RNA with ability to regulate gene expression by bind the 3' UTR region of target mRNA and are evolved at different rates in different plant species. In this study we have utilized homology based computational approach and identified 27 mature miRNAs for the first time from this bio-medically important plant. The phylogenetic tree developed from binary data derived from the data on presence/absence of the identified miRNAs were noticed to be uncertain and biased. Most of the identified miRNAs were highly conserved among the plant species and sequence based phylogeny analysis of miRNAs resolved the above difficulties in phylogeny approach using miRNA. Predicted gene targets of the identified miRNAs revealed their importance in regulation of plant developmental process. Reported miRNAs held sequence conservation in mature miRNAs and the detailed phylogeny analysis of pre-miRNA sequences revealed genus specific segregation of clusters. Copyright © 2015 Elsevier Ltd. All rights reserved.

Restricted HIV-1 Env glycan engagement by lectin-reengineered DAVEI protein chimera is sufficient for lytic inactivation of the virus

PubMed Central

Parajuli, Bibek; Acharya, Kriti; Bach, Harry C.; Parajuli, Bijay; Zhang, Shiyu; Smith, Amos B.; Abrams, Cameron F.; Chaiken, Irwin

2018-01-01

We previously reported a first-generation recombinant DAVEI construct, a dual action virus entry inhibitor composed of cyanovirin-N (CVN) fused to a membrane proximal external region or its derivative peptide Trp3. DAVEI exhibits potent and irreversible inactivation of HIV-1 (human immunodeficiency virus) viruses by dual engagement of gp120 and gp41. However, the promiscuity of CVN to associate with multiple glycosylation sites in gp120 and its multivalency limit current understanding of the molecular arrangement of the DAVEI molecules on trimeric spike. Here, we constructed and investigated the virolytic function of second-generation DAVEI molecules using a simpler lectin, microvirin (MVN). MVN is a monovalent lectin with a single glycan-binding site in gp120, is structurally similar to CVN and exhibits no toxicity or mitogenicity, both of which are liabilities with CVN. We found that, like CVN-DAVEI-L2-3Trp (peptide sequence DKWASLWNW), MVN-DAVEI2-3Trp exploits a similar mechanism of action for inducing HIV-1 lytic inactivation, but by more selective gp120 glycan engagement. By sequence redesign, we significantly increased the potency of MVN-DAVEI2-3Trp protein. Unlike CVN-DAVEI2-3Trp, re-engineered MVN-DAVEI2-3Trp(Q81K/M83R) virolytic activity and its interaction with gp120 were both competed by 2G12 antibody. That the lectin domain in DAVEIs can utilize MVN without loss of virolytic function argues that restricted HIV-1 Env (envelope glycoprotein) glycan engagement is sufficient for virolysis. It also shows that DAVEI lectin multivalent binding with gp120 is not required for virolysis. MVN-DAVEI2-3Trp(Q81K/M83R) provides an improved tool to elucidate productive molecular arrangements of Env-DAVEI enabling virolysis and also opens the way to form DAVEI fusions made up of gp120-binding small molecules linked to Trp3 peptide. PMID:29343613

O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

PubMed

Tzeng, Sheue-Fen; Tsai, Chin-Hsien; Chao, Tai-Kuang; Chou, Yu-Ching; Yang, Yu-Chih; Tsai, Mong-Hsun; Cha, Tai-Lung; Hsiao, Pei-Wen

2018-06-15

Disseminated castration-resistant prostate cancer (CRPC) is a common disease in men that is characterized by limited survival and resistance to androgen-deprivation therapy. The increase in human epidermal growth factor receptor 2 (HER2) signaling contributes to androgen receptor activity in a subset of patients with CRPC; however, enigmatically, HER2-targeted therapies have demonstrated a lack of efficacy in patients with CRPC. Aberrant glycosylation is a hallmark of cancer and involves key processes that support cancer progression. Using transcriptomic analysis of prostate cancer data sets, histopathologic examination of clinical specimens, and in vivo experiments of xenograft models, we reveal in this study a coordinated increase in glycan-binding protein, galectin-4, specific glycosyltransferases of core 1 synthase, glycoprotein- N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) and ST3 beta-galactoside α-2,3-sialyltransferase 1 (ST3GAL1), and resulting mucin-type O-glycans during the progression of CRPC. Furthermore, galectin-4 engaged with C1GALT1-dependent O-glycans to promote castration resistance and metastasis by activating receptor tyrosine kinase signaling and cancer cell stemness properties mediated by SRY-box 9 (SOX9). This galectin-glycan interaction up-regulated the MYC-dependent expression of C1GALT1 and ST3GAL1, which altered cellular mucin-type O-glycosylation to allow for galectin-4 binding. In clinical prostate cancer, high-level expression of C1GALT1 and galectin-4 together predict poor overall survival compared with low-level expression of C1GALT1 and galectin-4. In summary, MYC regulates abnormal O-glycosylation, thus priming cells for binding to galectin-4 and downstream signaling, which promotes castration resistance and metastasis.-Tzeng, S.-F., Tsai, C.-H., Chao, T.-K., Chou, Y.-C., Yang, Y.-C., Tsai, M.-H., Cha, T.-L., Hsiao, P.-W. O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

Molecular-Scale Features that Govern the Effects of O-Glycosylation on a Carbohydrate-Binding Module

DOE PAGES

Guan, Xiaoyang; Chaffey, Patrick K.; Zeng, Chen; ...

2015-09-21

The protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. This study suggests the possibility ofmore » designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.« less

Carbohydrate moieties of myelin-associated glycoprotein, major glycoprotein of the peripheral nervous system myelin and other myelin glycoproteins potentially involved in cell adhesion.

PubMed

Badache, A; Burger, D; Villarroya, H; Robert, Y; Kuchler, S; Steck, A J; Zanetta, J P

1992-01-01

The myelin-associated glycoprotein (MAG) and the major glycoprotein of the peripheral nervous system myelin (P0) are two members of the family of cell adhesion molecules (CAMs). A role in cell adhesion of the carbohydrate moiety of these molecules has been attributed to the presence of N-glycans bearing the HNK-1 carbohydrate epitope. On the other hand, it has been suggested that these glycoproteins could be ligands of an endogenous mannose-binding lectin present in myelin, the cerebellar soluble lectin (CSL). In order to further document the heterogeneity of the glycans of these two CAMs, we have used several probes: an anti-carbohydrate antibody of the HNK-1 type, called Elec-39, the plant lectin concanavalin A (ConA), and the endogenous lectin CSL involved in myelin compaction. This study shows that CSL binds to a small proportion of the polypeptide chains of MAG found in adult CNS of rats and man and the polypeptide chains of P0 molecules from adult human and rat sciatic nerve. For MAG from adult rat brain, the binding of CSL is restricted to glycans of polypeptide chains which could be separated from the others according to their solubility properties. These MAG molecular entities react also with the Elec-39 antibody and with ConA. These results confirm that P0 and MAG are heterogeneous in their carbohydrate moieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Scaffolding protein RanBPM and its interactions in diverse signaling pathways in health and disease.

PubMed

Das, Soumyadip; Haq, Saba; Ramakrishna, Suresh

2018-04-01

Ran-binding protein in the microtubule-organizing center (RanBPM) is an evolutionarily conserved, nucleocytoplasmic scaffolding protein involved in various cellular processes and several signal transduction pathways. RanBPM has a crucial role in mediating disease pathology by interacting with diverse proteins to regulate their functions. Previously, we compiled diverse cellular functions of RanBPM. Since then the functions of RanBPM have increased exponentially. In this article, we have updated the functions of RanBPM through its manifold interactions that have been investigated to date, according to their roles in protein stability, transcriptional activity, cellular development, neurobiology, and the cell cycle. Our review provides a complete guide on RanBPM interactors, the physiological role of RanBPM in cellular functions, and potential applications in disease therapeutics.

Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies

PubMed Central

Olson, Linda J.; Jensen, Davin R.; Volkman, Brian F.; Kim, Jung-Ja P.; Peterson, Francis C.; Gundry, Rebekah L.; Dahms, Nancy M.

2015-01-01

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that interacts with diverse ligands and plays central roles in autophagy, development, and tumor suppression. By delivering newly synthesized phosphomannosyl-containing acid hydrolases from the Golgi to endosomal compartments, CI-MPR is an essential component in the generation of lysosomes that are critical for the maintenance of cellular homeostasis. The ability of CI-MPR to interact with ~60 different acid hydrolases is facilitated by its large extracellular region, with four out of its 15 domains binding phosphomannosyl residues. Although the glycan specificity of CI-MPR has been elucidated, the molecular basis of carbohydrate binding has not been determined for two out of these four carbohydrate recognition domains (CRD). Here we report expression of CI-MPR’s CRD located in domain 5 that preferentially binds phosphodiester-containing glycans. Domain 5 of CI-MPR was expressed in Escherichia coli BL21 (DE3) cells as a fusion protein containing an N-terminal histidine tag and the small ubiquitin-like modifier (SUMO) protein. The His6-SUMO-CRD construct was recovered from inclusion bodies, refolded in buffer to facilitate disulfide bond formation, and subjected to Ni-NTA affinity chromatography and size exclusion chromatography. Surface plasmon resonance analyses demonstrated that the purified protein was active and bound phosphorylated glycans. Characterization by NMR spectroscopy revealed high quality 1H–15N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8 Å resolution. Together, these studies demonstrate the feasibility of producing CI-MPR’s CRD suitable for three-dimensional structure determination by NMR spectroscopic and X-ray crystallographic approaches. PMID:25863146

CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pujari, Radha; Eligar, Sachin M.; Kumar, Natesh

2012-03-23

Highlights: Black-Right-Pointing-Pointer RBL, a potent mitogenic and complex N-glycan specific lectin binds to CD45 on PBMC. Black-Right-Pointing-Pointer RBL triggers CD45-mediated signaling involved in activation of p38MAPK and STAT-5. Black-Right-Pointing-Pointer Inhibition of CD45 PTPase signaling blocks RBL-induced ZAP70 phosphorylation. Black-Right-Pointing-Pointer RBL-CD45 mediated signaling is crucial for RBL-induced immunodulatory activities. -- Abstract: We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate themore » involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.« less

Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

PubMed Central

Benatar, Alejandro F.; García, Gabriela A.; Bua, Jacqeline; Cerliani, Juan P.; Postan, Miriam; Tasso, Laura M.; Scaglione, Jorge; Stupirski, Juan C.; Toscano, Marta A.

2015-01-01

Background Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection. Methodology and Principal Findings Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1 -/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain. Conclusion/Significance Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions. PMID:26451839

Krüppel-like factors are effectors of nuclear receptor signaling

PubMed Central

Knoedler, Joseph R.; Denver, Robert J.

2015-01-01

Binding of steroid and thyroid hormones to their cognate nuclear receptors (NRs) impacts virtually every aspect of postembryonic development, physiology and behavior, and inappropriate signaling by NRs may contribute to disease. While NRs regulate genes by direct binding to hormone response elements in the genome, their actions may depend on the activity of other transcription factors (TFs) that may or may not bind DNA. The Krüppel-like family of transcription factors (KLF) is an evolutionarily conserved class of DNA-binding proteins that influence many aspects of development and physiology. Several members of this family have been shown to play diverse roles in NR signaling. For example, KLFs 1) act as accessory transcription factors for NR actions, 2) regulate expression of NR genes, and 3) as gene products of primary NR response genes function as key players in NR-dependent transcriptional networks. In mouse models, deletion of different KLFs leads to aberrant transcriptional and physiological responses to hormones, underscoring the importance of these proteins in the regulation of hormonal signaling. Understanding the functional relationships between NRs and KLFs will yield important insights into mechanisms of NR signaling. In this review we present a conceptual framework for understanding how KLFs participate in NR signaling, and we provide examples of how these proteins function to effect hormone action. PMID:24642391

Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.

PubMed

Newburg, D S

2009-04-01

This review discusses the role of human milk glycans in protecting infants, but the conclusion that the human milk glycans constitute an innate immune system whereby the mother protects her offspring may have general applicability in all mammals, including species of commercial importance. Infants that are not breastfed have a greater incidence of severe diarrhea and respiratory diseases than those who are breastfed. In the past, this had been attributed primarily to human milk secretory antibodies. However, the oligosaccharides are major components of human milk, and milk is also rich in other glycans, including glycoproteins, mucins, glycosaminoglycans, and glycolipids. These milk glycans, especially the oligosaccharides, are composed of thousands of components. The milk factor that promotes gut colonization by Bifidobacterium bifidum was found to be a glycan, and such prebiotic characteristics may contribute to protection against infectious agents. However, the ability of human milk glycans to protect the neonate seems primarily to be due to their inhibition of pathogen binding to their host cell target ligands. Many such examples include specific fucosylated oligosaccharides and glycans that inhibit specific pathogens. Most human milk oligosaccharides are fucosylated, and their production depends on fucosyltransferase enzymes; mutations in these fucosyltransferase genes are common and underlie the various Lewis blood types in humans. Variable expression of specific fucosylated oligosaccharides in milk, also a function of these genes (and maternal Lewis blood type), is significantly associated with the risk of infectious disease in breastfed infants. Human milk also contains major quantities and large numbers of sialylated oligosaccharides, many of which are also present in bovine colostrum. These could similarly inhibit several common viral pathogens. Moreover, human milk oligosaccharides strongly attenuate inflammatory processes in the intestinal mucosa. These results support the hypothesis that oligosaccharides and other glycans are the major constituents of an innate immune system of human milk whereby the mother protects her infant from enteric and other pathogens through breastfeeding. These protective glycans may prove useful as a basis for the development of novel prophylactic and therapeutic agents that inhibit disease by mucosal pathogens in many species.

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins

PubMed Central

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Siukstaite, Lina; Harrison, Oliver J.; Brasch, Julia; Goodman, Kerry M.; Hansen, Lars; Shapiro, Lawrence; Honig, Barry; Vakhrushev, Sergey Y.; Clausen, Henrik

2017-01-01

The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1–4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific β-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3. PMID:28973932

How to Crack the Sugar Code.

PubMed

Gabius, H-J

2017-01-01

The known ubiquitous presence of glycans fulfils an essential prerequisite for fundamental roles in cell sociology. Since carbohydrates are chemically predestined to form biochemical messages of a maximum of structural diversity in a minimum of space, coding of biological information by sugars is the reason for the broad occurrence of cellular glycoconjugates. Their glycans originate from sophisticated enzymatic assembly and dynamically adaptable remodelling. These signals are read and translated into effects by receptors (lectins). The functional pairing between lectins and their counterreceptor(s) is highly specific, often orchestrated by intimate co-regulation of the receptor, the cognate glycan and the bioactive scaffold (e.g., an integrin). Bottom-up approaches, teaming up synthetic and supramolecular chemistry to prepare fully programmable nanoparticles as binding partners with systematic network analysis of lectins and rational design of variants, enable us to delineate the rules of the sugar code.

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development.

PubMed

Suzuki, Atsushi; Niimi, Yuki; Shinmyozu, Kaori; Zhou, Zhi; Kiso, Makoto; Saga, Yumiko

2016-01-01

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development. © 2015 The Authors.

A novel role for Gtb1p in glucose trimming of N-linked glycans

PubMed Central

Quinn, Robert P; Mahoney, Sarah J; Wilkinson, Barrie M; Thornton, David J; Stirling, Colin J

2009-01-01

Glucosidase II (GluII) is a glycan-trimming enzyme active on nascent glycoproteins in the endoplasmic reticulum (ER). It trims the middle and innermost glucose residues (Glc2 and Glc1) from N-linked glycans. The monoglucosylated glycan produced by the first GluII trimming reaction is recognized by calnexin/calreticulin and serves as the signal for entry into this folding pathway. GluII is a heterodimer of α and β subunits corresponding to yeast Gls2p and Gtb1p, respectively. While Gls2p contains the glucosyl hydrolase active site, the Gtb1p subunit has previously been shown to be essential for the Glc1 trimming event. Here we demonstrate that Gtb1p also determines the rate of Glc2 trimming. In order to further dissect these activities we mutagenized a number of conserved residues across the protein. Our data demonstrate that both the MRH and G2B domains of Gtb1p contribute to the Glc2 trimming event but that the MRH domain is essential for Glc1 trimming. PMID:19542522

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

PubMed

Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2017-02-10

Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

OsMOGS is required for N-glycan formation and auxin-mediated root development in rice (Oryza sativa L.)

PubMed Central

Zhang, SaiNa; Lim, Jae-Min; Lee, Kyun Oh; Li, ChuanYou; Qian, Qian; Jiang, De An; Qi, YanHua

2014-01-01

SUMMARY N-glycosylation is a major modification of glycoproteins in eukaryotic cells. In Arabidopsis, great progress has been made in functional analysis of N-glycan production; however, there are few studies in monocotyledons. Here, we characterized a rice (Oryza sativa L.) osmogs mutant with shortened roots and isolated a gene coding a putative mannosyl-oligosaccharide glucosidase (OsMOGS), an ortholog of α-glucosidase I in Arabidopsis, which trims the terminal glucosyl residue of the oligosaccharide chain of nascent peptides in the endoplasmic reticulum (ER). OsMOGS is strongly expressed in rapidly cell-dividing tissues and OsMOGS protein is localized in the ER. Mutation of OsMOGS entirely blocked N-glycan maturation and inhibited high-mannose N-glycan formation. The osmogs mutant exhibited severe defects in root cell division and elongation, resulting in a short-root phenotype. In addition, osmogs plants had impaired root hair formation and elongation, and reduced root epidemic cell wall thickness due to decreased cellulose synthesis. Further analysis showed that auxin content and polar transport in osmogs roots were reduced due to incomplete N-glycosylation of the B subfamily of ATP-binding cassette transporter proteins (ABCBs). Our results demonstrate that involvement of OsMOGS in N-glycan formation is required for auxin-mediated root development in rice. PMID:24597623

[Amphioxus ortholog of ECSIT, an evolutionarily conserved adaptor in the Toll and BMP signaling pathways].

PubMed

Lin, Y H; Zhang, W; Li, J W; Zhang, H W; Chen, D Y

2017-01-01

In vertebrates, evolutionarily conserved signaling intermediate in the Toll pathway (ECSIT) interacts with the TNF-receptor associated factor 6 (TRAF6) to regulate the processing of MEKK1, activate NF-κB, and also control BMP target genes. However, the role of ECSIT in invertebrates remains largely unexplored. We performed comparative investigations of the expression, gene structure, and phylogeny of ECSIT, Toll-like receptor (TLR), and Smad4 in the cephalochordate Branchiostoma belcheri. Phylogenetic analysis indicated that, in amphioxus, ECSIT, TLR, and Smad4 form independent clusters at the base of Chordate   clusters. Interestingly, overall gene structures were comparable to those in vertebrate orthologs. Transcripts of AmphiECSIT were detectable at the mid-neural stage, and continued to be expressed in the epithelium of the pharyngeal region at later stages. In adult animals, strong expression was observed in the nerve cord, endostyle, epithelial cells of the gut and wheel organ, genital membrane of the testis, and coelom and lymphoid cavities, what is highly similar to AmphiTLR and AmphiSmad4 expression patterns during development and in adult organisms. Our data suggests that ECSIT is evolutionarily conserved. Its amphioxus ortholog functions during embryonic development and as part of the innate immune system and may be involved in TLR/BMP signaling.

Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin

PubMed Central

Byres, Emma; Paton, Adrienne W.; Paton, James C.; Löfling, Jonas C.; Smith, David F.; Wilce, Matthew C.J.; Talbot, Ursula M.; Chong, Damien C.; Yu, Hai; Huang, Shengshu; Chen, Xi; Varki, Nissi M.; Varki, Ajit; Rossjohn, Jamie; Beddoe, Travis

2009-01-01

AB5 toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB5 toxin secreted by Shiga toxigenic Escherichia coli (STEC)1, which causes serious gastrointestinal disease in humans2. SubAB causes haemolytic uraemic syndrome-like pathology in mice3 via SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone4. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesised in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite human lack of Neu5Gc biosynthesis, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, together with the human lack of Neu5Gc-containing body fluid competitors, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin’s receptor is generated by metabolic incorporation of an exogenous factor derived from food. PMID:18971931

Interaction of MYC with host cell factor-1 is mediated by the evolutionarily conserved Myc box IV motif.

PubMed

Thomas, L R; Foshage, A M; Weissmiller, A M; Popay, T M; Grieb, B C; Qualls, S J; Ng, V; Carboneau, B; Lorey, S; Eischen, C M; Tansey, W P

2016-07-07

The MYC family of oncogenes encodes a set of three related transcription factors that are overexpressed in many human tumors and contribute to the cancer-related deaths of more than 70,000 Americans every year. MYC proteins drive tumorigenesis by interacting with co-factors that enable them to regulate the expression of thousands of genes linked to cell growth, proliferation, metabolism and genome stability. One effective way to identify critical co-factors required for MYC function has been to focus on sequence motifs within MYC that are conserved throughout evolution, on the assumption that their conservation is driven by protein-protein interactions that are vital for MYC activity. In addition to their DNA-binding domains, MYC proteins carry five regions of high sequence conservation known as Myc boxes (Mb). To date, four of the Mb motifs (MbI, MbII, MbIIIa and MbIIIb) have had a molecular function assigned to them, but the precise role of the remaining Mb, MbIV, and the reason for its preservation in vertebrate Myc proteins, is unknown. Here, we show that MbIV is required for the association of MYC with the abundant transcriptional coregulator host cell factor-1 (HCF-1). We show that the invariant core of MbIV resembles the tetrapeptide HCF-binding motif (HBM) found in many HCF-interaction partners, and demonstrate that MYC interacts with HCF-1 in a manner indistinguishable from the prototypical HBM-containing protein VP16. Finally, we show that rationalized point mutations in MYC that disrupt interaction with HCF-1 attenuate the ability of MYC to drive tumorigenesis in mice. Together, these data expose a molecular function for MbIV and indicate that HCF-1 is an important co-factor for MYC.

Glycans: bioactive signals decoded by lectins.

PubMed

Gabius, Hans-Joachim

2008-12-01

The glycan part of cellular glycoconjugates affords a versatile means to build biochemical signals. These oligosaccharides have an exceptional talent in this respect. They surpass any other class of biomolecule in coding capacity within an oligomer (code word). Four structural factors account for this property: the potential for variability of linkage points, anomeric position and ring size as well as the aptitude for branching (first and second dimensions of the sugar code). Specific intermolecular recognition is favoured by abundant potential for hydrogen/co-ordination bonds and for C-H/pi-interactions. Fittingly, an array of protein folds has developed in evolution with the ability to select certain glycans from the natural diversity. The thermodynamics of this reaction profits from the occurrence of these ligands in only a few energetically favoured conformers, comparing favourably with highly flexible peptides (third dimension of the sugar code). Sequence, shape and local aspects of glycan presentation (e.g. multivalency) are key factors to regulate the avidity of lectin binding. At the level of cells, distinct glycan determinants, a result of enzymatic synthesis and dynamic remodelling, are being defined as biomarkers. Their presence gains a functional perspective by co-regulation of the cognate lectin as effector, for example in growth regulation. The way to tie sugar signal and lectin together is illustrated herein for two tumour model systems. In this sense, orchestration of glycan and lectin expression is an efficient means, with far-reaching relevance, to exploit the coding potential of oligosaccharides physiologically and medically.

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

PubMed

Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

2009-03-23

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

Crossroads between Bacterial and Mammalian Glycosyltransferases

PubMed Central

Brockhausen, Inka

2014-01-01

Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen–Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures. PMID:25368613

Glycosylation potential of human prostate cancer cell lines

PubMed Central

Gao, Yin; Chachadi, Vishwanath B.; Cheng, Pi-Wan

2014-01-01

Altered glycosylation is a universal feature of cancer cells and altered glycans can help cancer cells escape immune surveillance, facilitate tumor invasion, and increase malignancy. The goal of this study was to identify specific glycoenzymes, which could distinguish prostate cancer cells from normal prostatic cells. We investigated enzymatic activities and gene expression levels of key glycosyl- and sulfotransferases responsible for the assembly of O- and N-glycans in several prostatic cells. These cells included immortalized RWPE-1 cells derived from normal prostatic tissues, and prostate cancer cells derived from metastasis in bone (PC-3), brain (DU145), lymph node (LNCaP), and vertebra (VCaP). We found that all cells were capable of synthesizing complex N-glycans and O-glycans with the core 1 structure, and each cell line had characteristic bio-synthetic pathways to modify these structures. The in vitro measured activities corresponded well to the mRNA levels of glycosyltransferases and sulfotransferases. Lectin and antibody binding to whole cells supported these results, which form the basis for the development of tumor cell-specific targeting strategies. PMID:22843320

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

PubMed

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-04-15

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

N-linked (N-) glycoproteomics of urinary exosomes. [Corrected].

PubMed

Saraswat, Mayank; Joenväära, Sakari; Musante, Luca; Peltoniemi, Hannu; Holthofer, Harry; Renkonen, Risto

2015-02-01

Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Fucose-binding Lectin from Photorhabdus luminescens (PLL) with an Unusual Heptabladed β-Propeller Tetrameric Structure*

PubMed Central

Kumar, Atul; Sýkorová, Petra; Demo, Gabriel; Dobeš, Pavel; Hyršl, Pavel

2016-01-01

Photorhabdus luminescens is known for its symbiosis with the entomopathogenic nematode Heterorhabditis bacteriophora and its pathogenicity toward insect larvae. A hypothetical protein from P. luminescens was identified, purified from the native source, and characterized as an l-fucose-binding lectin, named P. luminescens lectin (PLL). Glycan array and biochemical characterization data revealed PLL to be specific toward l-fucose and the disaccharide glycan 3,6-O-Me2-Glcβ1–4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2. PLL was discovered to be a homotetramer with an intersubunit disulfide bridge. The crystal structures of native and recombinant PLL revealed a seven-bladed β-propeller fold creating seven putative fucose-binding sites per monomer. The crystal structure of the recombinant PLL·l-fucose complex confirmed that at least three sites were fucose-binding. Moreover, the crystal structures indicated that some of the other sites are masked either by the tetrameric nature of the lectin or by incorporation of the C terminus of the lectin into one of these sites. PLL exhibited an ability to bind to insect hemocytes and the cuticular surface of a nematode, H. bacteriophora. PMID:27758853

The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.

PubMed

Saito, Motoki; Ishikawa, Fuyuki

2002-09-20

Although mammalian MBD3 contains the mCpG-binding domain (MBD) and is highly homologous with the authentic mCpG-binding protein MBD2, it was reported that the protein does not bind to mCpG specifically. Using recombinant human wild type and mutant MBD3 proteins, we demonstrated that atypical amino acids found in MBD3 MBD, namely, His-30 and Phe-34, are responsible for the inability of MBD3 to bind to mCpG. Interestingly, although H30K/F34Y MBD3 mutant protein binds to mCpG efficiently in vitro, it was not localized at the mCpG-rich pericentromeric regions in mouse cells. We also showed that Y34F MBD2b MBD, which possesses not the mCpG-specific DNA-binding activity but the nonspecific DNA-binding activity, was localized at the pericentromeric regions. These results suggested that the mCpG-specific DNA-binding activity is largely dispensable, and another factor(s) is required for the localization of MBD proteins in vivo. MBD3 was identified as a component of the NuRD/Mi2 complex that shows chromatin remodeling and histone deacetylase activities. We demonstrated that MBD3 MBD is necessary and sufficient for binding to HDAC1 and MTA2, two components of the NuRD/Mi2 complex. It was therefore suggested that mCpG-binding-defective MBD3 has evolutionarily conserved its MBD because of the secondary role played by the MBD in protein-protein interactions.

Galectin-9 binds to O-glycans on protein disulfide isomerase.

PubMed

Schaefer, Katrin; Webb, Nicholas E; Pang, Mabel; Hernandez-Davies, Jenny E; Lee, Katharine P; Gonzalez, Pascual; Douglass, Martin V; Lee, Benhur; Baum, Linda G

2017-09-01

Changes in the T cell surface redox environment regulate critical cell functions, such as cell migration, viral entry and cytokine production. Cell surface protein disulfide isomerase (PDI) contributes to the regulation of T cell surface redox status. Cell surface PDI can be released into the extracellular milieu or can be internalized by T cells. We have found that galectin-9, a soluble lectin expressed by T cells, endothelial cells and dendritic cells, binds to and retains PDI on the cell surface. While endogenous galectin-9 is not required for basal cell surface PDI expression, exogenous galectin-9 mediated retention of cell surface PDI shifted the disulfide/thiol equilibrium on the T cell surface. O-glycans on PDI are required for galectin-9 binding, and PDI recognition appears to be specific for galectin-9, as galectin-1 and galectin-3 do not bind PDI. Galectin-9 is widely expressed by immune and endothelial cells in inflamed tissues, suggesting that T cells would be exposed to abundant galectin-9, in cis and in trans, in infectious or autoimmune conditions. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

H2B ubiquitination: Conserved molecular mechanism, diverse physiologic functions of the E3 ligase during meiosis.

PubMed

Wang, Liying; Cao, Chunwei; Wang, Fang; Zhao, Jianguo; Li, Wei

2017-09-03

RNF20/Bre1 mediated H2B ubiquitination (H2Bub) has various physiologic functions. Recently, we found that H2Bub participates in meiotic recombination by promoting chromatin relaxation during meiosis. We then analyzed the phylogenetic relationships among the E3 ligase for H2Bub, its E2 Rad6 and their partner WW domain-containing adaptor with a coiled-coil (WAC) or Lge1, and found that the molecular mechanism underlying H2Bub is evolutionarily conserved from yeast to mammals. However, RNF20 has diverse physiologic functions in different organisms, which might be caused by the evolutionary divergency of their domain/motif architectures. In the current extra view, we not only elucidate the evolutionarily conserved molecular mechanism underlying H2Bub, but also discuss the diverse physiologic functions of RNF20 during meiosis.

Epigenetic Pattern on the Human Y Chromosome Is Evolutionarily Conserved

PubMed Central

Meng, Hao; Agbagwa, Ikechukwu O.; Wang, Ling-Xiang; Wang, Yingzhi; Yan, Shi; Ren, Shancheng; Sun, Yinghao; Pei, Gang; Liu, Xin; Liu, Jiang; Jin, Li; Li, Hui; Sun, Yingli

2016-01-01

DNA methylation plays an important role for mammalian development. However, it is unclear whether the DNA methylation pattern is evolutionarily conserved. The Y chromosome serves as a powerful tool for the study of human evolution because it is transferred between males. In this study, based on deep-rooted pedigrees and the latest Y chromosome phylogenetic tree, we performed epigenetic pattern analysis of the Y chromosome from 72 donors. By comparing their respective DNA methylation level, we found that the DNA methylation pattern on the Y chromosome was stable among family members and haplogroups. Interestingly, two haplogroup-specific methylation sites were found, which were both genotype-dependent. Moreover, the African and Asian samples also had similar DNA methylation pattern with a remote divergence time. Our findings indicated that the DNA methylation pattern on the Y chromosome was conservative during human male history. PMID:26760298

Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

PubMed Central

Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

2013-01-01

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions.

PubMed

Khatri, Kshitij; Klein, Joshua A; White, Mitchell R; Grant, Oliver C; Leymarie, Nancy; Woods, Robert J; Hartshorn, Kevan L; Zaia, Joseph

2016-06-01

Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the primary determinant of antigenicity, virulence and zoonotic potential. Mutations leading to changes in the number of HA glycosylation sites are often reported. Such genetic sequencing studies predict at best the disruption or creation of sequons for N-linked glycosylation; they do not reflect actual phenotypic changes in HA structure. Therefore, combined analysis of glycan micro and macro-heterogeneity and bioassays will better define the relationships among glycosylation, viral bioactivity and evolution. We present a study that integrates proteomics, glycomics and glycoproteomics of HA before and after adaptation to innate immune system pressure. We combined this information with glycan array and immune lectin binding data to correlate the phenotypic changes with biological activity. Underprocessed glycoforms predominated at the glycosylation sites found to be involved in viral evolution in response to selection pressures and interactions with innate immune-lectins. To understand the structural basis for site-specific glycan microheterogeneity at these sites, we performed structural modeling and molecular dynamics simulations. We observed that the presence of immature, high-mannose type glycans at a particular site correlated with reduced accessibility to glycan remodeling enzymes. Further, the high mannose glycans at sites implicated in immune lectin recognition were predicted to be capable of forming trimeric interactions with the immune-lectin surfactant protein-D. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

N-glycosylation profile of undifferentiated and adipogenically differentiated human bone marrow mesenchymal stem cells: towards a next generation of stem cell markers.

PubMed

Hamouda, Houda; Ullah, Mujib; Berger, Markus; Sittinger, Michael; Tauber, Rudolf; Ringe, Jochen; Blanchard, Véronique

2013-12-01

Mesenchymal stem cells (MSCs) are multipotent cells that are easy to isolate and expand, develop into several tissues, including fat, migrate to diseased organs, have immunosuppressive properties and secrete regenerative factors. This makes MSCs ideal for regenerative medicine. For application and regulatory purposes, knowledge of (bio)markers characterizing MSCs and their development stages is of paramount importance. The cell surface is coated with glycans that possess lineage-specific nature, which makes glycans to be promising candidate markers. In the context of soft tissue generation, we aimed to identify glycans that could be markers for MSCs and their adipogenically differentiated progeny. MSCs were isolated from human bone marrow, adipogenically stimulated for 15 days and adipogenesis was verified by staining the lipid droplets and quantitative real time polymerase chain reaction of the marker genes peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid binding protein-4 (FABP4). Using matrix-assisted laser desorption-ionization-time of flight mass spectrometry combined with exoglycosidase digestions, we report for the first time the N-glycome of MSCs during adipogenic differentiation. We were able to detect more than 100 different N-glycans, including high-mannose, hybrid, and complex N-glycans, as well as poly-N-acetyllactosamine chains. Adipogenesis was accompanied by an increased amount of biantennary fucosylated structures, decreased amount of fucosylated, afucosylated tri- and tetraantennary structures and increased sialylation. N-glycans H6N5F1 and H7N6F1 were significantly overexpressed in undifferentiated MSCs while H3N4F1 and H5N4F3 were upregulated in adipogenically differentiated MSCs. These glycan structures are promising candidate markers to detect and distinguish MSCs and their adipogenic progeny.

An evolutionary analysis identifies a conserved pentapeptide stretch containing the two essential lysine residues for rice L-myo-inositol 1-phosphate synthase catalytic activity

PubMed Central

Basak, Papri; Maitra-Majee, Susmita; Das, Jayanta Kumar; Mukherjee, Abhishek; Ghosh Dastidar, Shubhra; Pal Choudhury, Pabitra

2017-01-01

A molecular evolutionary analysis of a well conserved protein helps to determine the essential amino acids in the core catalytic region. Based on the chemical properties of amino acid residues, phylogenetic analysis of a total of 172 homologous sequences of a highly conserved enzyme, L-myo-inositol 1-phosphate synthase or MIPS from evolutionarily diverse organisms was performed. This study revealed the presence of six phylogenetically conserved blocks, out of which four embrace the catalytic core of the functional protein. Further, specific amino acid modifications targeting the lysine residues, known to be important for MIPS catalysis, were performed at the catalytic site of a MIPS from monocotyledonous model plant, Oryza sativa (OsMIPS1). Following this study, OsMIPS mutants with deletion or replacement of lysine residues in the conserved blocks were made. Based on the enzyme kinetics performed on the deletion/replacement mutants, phylogenetic and structural comparison with the already established crystal structures from non-plant sources, an evolutionarily conserved peptide stretch was identified at the active pocket which contains the two most important lysine residues essential for catalytic activity. PMID:28950028

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

PubMed Central

Mahdavi, Jafar; Pirinccioglu, Necmettin; Oldfield, Neil J.; Carlsohn, Elisabet; Stoof, Jeroen; Aslam, Akhmed; Self, Tim; Cawthraw, Shaun A.; Petrovska, Liljana; Colborne, Natalie; Sihlbom, Carina; Borén, Thomas; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

2014-01-01

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis. PMID:24451549

Conservation and diversification of Msx protein in metazoan evolution.

PubMed

Takahashi, Hirokazu; Kamiya, Akiko; Ishiguro, Akira; Suzuki, Atsushi C; Saitou, Naruya; Toyoda, Atsushi; Aruga, Jun

2008-01-01

Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family proteins contributed to the diversification of animal body organization.

Identification of Oligosaccharides in Feces of Breast-fed Infants and Their Correlation with the Gut Microbial Community *

PubMed Central

Davis, Jasmine C. C.; Totten, Sarah M.; Huang, Julie O.; Nagshbandi, Sadaf; Kirmiz, Nina; Garrido, Daniel A.; Lewis, Zachery T.; Wu, Lauren D.; Smilowitz, Jennifer T.; German, J. Bruce; Mills, David A.; Lebrilla, Carlito B.

2016-01-01

Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population. In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan. Marker gene sequencing of the fecal samples revealed bifidobacteria as the dominant inhabitants of the infant gastrointestinal tracts. A glycosidase from Bifidobacterium longum subsp. longum was then expressed to digest HMOs in vitro, which showed that the digested oligosaccharides in feces corresponded to the action of glycosidases on HMOs. Similar expression of endoglycosidases also showed that N-glycans were released by bacterial enzymes. Although bifidobacteria may dominate the gut, it is possible that specific minority species are also responsible for the major products observed in feces. Nonetheless, the enzymatic activity correlated well with the known glycosidases in the respective bacteria, suggesting a direct relationship between microbial abundances and catabolic activity. PMID:27435585

Identification of Oligosaccharides in Feces of Breast-fed Infants and Their Correlation with the Gut Microbial Community.

PubMed

Davis, Jasmine C C; Totten, Sarah M; Huang, Julie O; Nagshbandi, Sadaf; Kirmiz, Nina; Garrido, Daniel A; Lewis, Zachery T; Wu, Lauren D; Smilowitz, Jennifer T; German, J Bruce; Mills, David A; Lebrilla, Carlito B

2016-09-01

Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population. In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan. Marker gene sequencing of the fecal samples revealed bifidobacteria as the dominant inhabitants of the infant gastrointestinal tracts. A glycosidase from Bifidobacterium longum subsp. longum was then expressed to digest HMOs in vitro, which showed that the digested oligosaccharides in feces corresponded to the action of glycosidases on HMOs. Similar expression of endoglycosidases also showed that N-glycans were released by bacterial enzymes. Although bifidobacteria may dominate the gut, it is possible that specific minority species are also responsible for the major products observed in feces. Nonetheless, the enzymatic activity correlated well with the known glycosidases in the respective bacteria, suggesting a direct relationship between microbial abundances and catabolic activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Conservation and variability of West Nile virus proteins.

PubMed

Koo, Qi Ying; Khan, Asif M; Jung, Keun-Ok; Ramdas, Shweta; Miotto, Olivo; Tan, Tin Wee; Brusic, Vladimir; Salmon, Jerome; August, J Thomas

2009-01-01

West Nile virus (WNV) has emerged globally as an increasingly important pathogen for humans and domestic animals. Studies of the evolutionary diversity of the virus over its known history will help to elucidate conserved sites, and characterize their correspondence to other pathogens and their relevance to the immune system. We describe a large-scale analysis of the entire WNV proteome, aimed at identifying and characterizing evolutionarily conserved amino acid sequences. This study, which used 2,746 WNV protein sequences collected from the NCBI GenPept database, focused on analysis of peptides of length 9 amino acids or more, which are immunologically relevant as potential T-cell epitopes. Entropy-based analysis of the diversity of WNV sequences, revealed the presence of numerous evolutionarily stable nonamer positions across the proteome (entropy value of < or = 1). The representation (frequency) of nonamers variant to the predominant peptide at these stable positions was, generally, low (< or = 10% of the WNV sequences analyzed). Eighty-eight fragments of length 9-29 amino acids, representing approximately 34% of the WNV polyprotein length, were identified to be identical and evolutionarily stable in all analyzed WNV sequences. Of the 88 completely conserved sequences, 67 are also present in other flaviviruses, and several have been associated with the functional and structural properties of viral proteins. Immunoinformatic analysis revealed that the majority (78/88) of conserved sequences are potentially immunogenic, while 44 contained experimentally confirmed human T-cell epitopes. This study identified a comprehensive catalogue of completely conserved WNV sequences, many of which are shared by other flaviviruses, and majority are potential epitopes. The complete conservation of these immunologically relevant sequences through the entire recorded WNV history suggests they will be valuable as components of peptide-specific vaccines or other therapeutic applications, for sequence-specific diagnosis of a wide-range of Flavivirus infections, and for studies of homologous sequences among other flaviviruses.

N-glycan based ER molecular chaperone and protein quality control system: the calnexin binding cycle

PubMed Central

Lamriben, Lydia; Graham, Jill B.; Adams, Benjamin M.; Hebert, Daniel N.

2015-01-01

Helenius and colleagues proposed over twenty-years ago a paradigm-shifting model for how chaperone binding in the endoplasmic reticulum was mediated and controlled for a new type of molecular chaperone- the carbohydrate binding chaperones, calnexin and calreticulin. While the originally established basics for this lectin chaperone binding cycle holds true today, there has been a number of important advances that have expanded our understanding of its mechanisms of action, role in protein homeostasis, and its connection to disease states that are highlighted in this review. PMID:26676362

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

PubMed

Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

2015-01-01

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation

PubMed Central

Sammond, Deanne W.; Payne, Christina M.; Brunecky, Roman; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions. PMID:23139804

Preferential recognition of avian-like receptors in human influenza A H7N9 viruses.

PubMed

Xu, Rui; de Vries, Robert P; Zhu, Xueyong; Nycholat, Corwin M; McBride, Ryan; Yu, Wenli; Paulson, James C; Wilson, Ian A

2013-12-06

The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu(226), a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the H7 hemagglutinin reveals negligible binding to humanlike α2-6-linked receptors and strong preference for a subset of avian-like α2-3-linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission.

Bacterial Surface Glycans: Microarray and QCM Strategies for Glycophenotyping and Exploration of Recognition by Host Receptors.

PubMed

Kalograiaki, Ioanna; Campanero-Rhodes, María A; Proverbio, Davide; Euba, Begoña; Garmendia, Junkal; Aastrup, Teodor; Solís, Dolores

2018-01-01

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria-host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment. © 2018 Elsevier Inc. All rights reserved.

An rbcL mRNA-binding protein is associated with C3 to C4 evolution and light-induced production of Rubisco in Flaveria.

PubMed

Yerramsetty, Pradeep; Agar, Erin M; Yim, Won C; Cushman, John C; Berry, James O

2017-07-20

Nuclear-encoded RLSB protein binds chloroplastic rbcL mRNA encoding the Rubisco large subunit. RLSB is highly conserved across all groups of land plants and is associated with positive post-transcriptional regulation of rbcL expression. In C3 leaves, RLSB and Rubisco occur in all chlorenchyma cell chloroplasts, while in C4 leaves these accumulate only within bundle sheath (BS) chloroplasts. RLSB's role in rbcL expression makes modification of its localization a likely prerequisite for the evolutionary restriction of Rubisco to BS cells. Taking advantage of evolutionarily conserved RLSB orthologs in several C3, C3-C4, C4-like, and C4 photosynthetic types within the genus Flaveria, we show that low level RLSB sequence divergence and modification to BS specificity coincided with ontogeny of Rubisco specificity and Kranz anatomy during C3 to C4 evolution. In both C3 and C4 species, Rubisco production reflected RLSB production in all cell types, tissues, and conditions examined. Co-localization occurred only in photosynthetic tissues, and both proteins were co-ordinately induced by light at post-transcriptional levels. RLSB is currently the only mRNA-binding protein to be associated with rbcL gene regulation in any plant, with variations in sequence and acquisition of cell type specificity reflecting the progression of C4 evolution within the genus Flaveria. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Warts phosphorylates Mud to promote Pins-mediated mitotic spindle orientation in Drosophila independent of Yorkie

PubMed Central

Dewey, Evan B.; Sanchez, Desiree; Johnston, Christopher A.

2015-01-01

SUMMARY Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily-conserved cell proliferation pathway. PMID:26592339

Warts phosphorylates mud to promote pins-mediated mitotic spindle orientation in Drosophila, independent of Yorkie.

PubMed

Dewey, Evan B; Sanchez, Desiree; Johnston, Christopher A

2015-11-02

Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here, we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily conserved cell proliferation pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes.

PubMed

Nandy, Suman Kumar; Seal, Alpana

2016-01-01

Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases.

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

NASA Astrophysics Data System (ADS)

Kochańczyk, Tomasz; Nowakowski, Michał; Wojewska, Dominika; Kocyła, Anna; Ejchart, Andrzej; Koźmiński, Wiktor; Krężel, Artur

2016-11-01

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

PubMed Central

Kochańczyk, Tomasz; Nowakowski, Michał; Wojewska, Dominika; Kocyła, Anna; Ejchart, Andrzej; Koźmiński, Wiktor; Krężel, Artur

2016-01-01

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits. PMID:27808280

The epitope of monoclonal antibodies blocking erythrocyte invasion by Plasmodium falciparum map to the dimerization and receptor glycan binding sites of EBA-175.

PubMed

Ambroggio, Xavier; Jiang, Lubin; Aebig, Joan; Obiakor, Harold; Lukszo, Jan; Narum, David L

2013-01-01

The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175- glycophorin A complex.

Bile salt receptor complex activates a pathogenic type III secretion system

DOE PAGES

Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N.; ...

2016-07-05

Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure ofmore » VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.« less

INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1

PubMed Central

Ainsztein, Alexandra M.; Kandels-Lewis, Stefanie E.; Mackay, Alastair M.; Earnshaw, William C.

1998-01-01

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1Hsα. Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle. PMID:9864353

Cardiovascular Small Heat Shock Protein HSPB7 Is a Kinetically Privileged Reactive Electrophilic Species (RES) Sensor.

PubMed

Surya, Sanjna L; Long, Marcus J C; Urul, Daniel A; Zhao, Yi; Mercer, Emily J; EIsaid, Islam M; Evans, Todd; Aye, Yimon

2018-02-08

Small heat shock protein (sHSP)-B7 (HSPB7) is a muscle-specific member of the non-ATP-dependent sHSPs. The precise role of HSPB7 is enigmatic. Here, we disclose that zebrafish Hspb7 is a kinetically privileged sensor that is able to react rapidly with native reactive electrophilic species (RES), when only substoichiometric amounts of RES are available in proximity to Hspb7 expressed in living cells. Among the two Hspb7-cysteines, this RES sensing is fulfilled by a single cysteine (C117). Purification and characterizations in vitro reveal that the rate for RES adduction is among the most efficient reported for protein-cysteines with native carbonyl-based RES. Covalent-ligand binding is accompanied by structural changes (increase in β-sheet-content), based on circular dichroism analysis. Among the two cysteines, only C117 is conserved across vertebrates; we show that the human ortholog is also capable of RES sensing in cells. Furthermore, a cancer-relevant missense mutation reduces this RES-sensing property. This evolutionarily conserved cysteine-biosensor may play a redox-regulatory role in cardioprotection.

Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence.

PubMed

Benko, Sabina; Fantes, Judy A; Amiel, Jeanne; Kleinjan, Dirk-Jan; Thomas, Sophie; Ramsay, Jacqueline; Jamshidi, Negar; Essafi, Abdelkader; Heaney, Simon; Gordon, Christopher T; McBride, David; Golzio, Christelle; Fisher, Malcolm; Perry, Paul; Abadie, Véronique; Ayuso, Carmen; Holder-Espinasse, Muriel; Kilpatrick, Nicky; Lees, Melissa M; Picard, Arnaud; Temple, I Karen; Thomas, Paul; Vazquez, Marie-Paule; Vekemans, Michel; Roest Crollius, Hugues; Hastie, Nicholas D; Munnich, Arnold; Etchevers, Heather C; Pelet, Anna; Farlie, Peter G; Fitzpatrick, David R; Lyonnet, Stanislas

2009-03-01

Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06-1.23 Mb upstream of SOX9, and microdeletions both approximately 1.5 Mb centromeric and approximately 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

Conserved Lipid and Small-Molecule Modulation of COQ8 Reveals Regulation of the Ancient Kinase-like UbiB Family.

PubMed

Reidenbach, Andrew G; Kemmerer, Zachary A; Aydin, Deniz; Jochem, Adam; McDevitt, Molly T; Hutchins, Paul D; Stark, Jaime L; Stefely, Jonathan A; Reddy, Thiru; Hebert, Alex S; Wilkerson, Emily M; Johnson, Isabel E; Bingman, Craig A; Markley, John L; Coon, Joshua J; Dal Peraro, Matteo; Pagliarini, David J

2018-02-15

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

NASA Astrophysics Data System (ADS)

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

PubMed Central

Hawryluk-Gara, Lisa A.; Platani, Melpomeni; Santarella, Rachel

2008-01-01

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53–Nup155 complex plays a critical role in the processes of NPC and NE assembly. PMID:18256286

Non-canonical autophagy: an exception or an underestimated form of autophagy?

PubMed

Scarlatti, Francesca; Maffei, Roberta; Beau, Isabelle; Ghidoni, Riccardo; Codogno, Patrice

2008-11-01

Macroautophagy (hereafter called autophagy) is a dynamic and evolutionarily conserved process used to sequester and degrade cytoplasm and entire organelles in a sequestering vesicle with a double membrane, known as the autophagosome, which ultimately fuses with a lysosome to degrade its autophagic cargo. Recently, we have unraveled two distinct forms of autophagy in cancer cells, which we term canonical and non-canonical autophagy. In contrast to classical or canonical autophagy, non-canonical autophagy is a process that does not require the entire set of autophagy-related (Atg) proteins in particular Beclin 1, to form the autophagosome. Non-canonical autophagy is therefore not blocked by the knockdown of Beclin 1 or of its binding partner hVps34. Moreover overexpression of Bcl-2, which is known to block canonical starvation-induced autophagy by binding to Beclin 1, is unable to reverse the non-canonical autophagy triggered by the polyphenol resveratrol in the breast cancer MCF-7 cell line. In MCF-7 cells, at least, non-canonical autophagy is involved in the caspase-independent cell death induced by resveratrol.

Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure

PubMed Central

Nicklisch, Sascha C. T.; Rees, Steven D.; McGrath, Aaron P.; Gökirmak, Tufan; Bonito, Lindsay T.; Vermeer, Lydia M.; Cregger, Cristina; Loewen, Greg; Sandin, Stuart; Chang, Geoffrey; Hamdoun, Amro

2016-01-01

The world’s oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)–100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals. PMID:27152359

Control of mitotic chromosome condensation by the fission yeast transcription factor Zas1.

PubMed

Schiklenk, Christoph; Petrova, Boryana; Kschonsak, Marc; Hassler, Markus; Klein, Carlo; Gibson, Toby J; Haering, Christian H

2018-05-07

Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C 2 H 2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation. © 2018 Schiklenk et al.

Identification of a transient Sox5 expressing progenitor population in the neonatal ventral forebrain by a novel cis-regulatory element

PubMed Central

Hao, Hailing; Li, Ying; Tzatzalos, Evangeline; Gilbert, Jordana; Zala, Dhara; Bhaumik, Mantu; Cai, Li

2014-01-01

Precise control of lineage-specific gene expression in the neural stem/progenitor cells is crucial for generation of the diversity of neuronal and glial cell types in the central nervous system (CNS). The mechanism underlying such gene regulation, however, is not fully elucidated. Here, we report that a 377 bp evolutionarily conserved DNA fragment (CR5), located approximately 32 kbp upstream of Olig2 transcription start site, acts as a cis-regulator for gene expression in the development of the neonatal forebrain. CR5 is active in a time-specific and brain region-restricted manner. CR5 activity is not detected in the embryonic stage, but it is exclusively in a subset of Sox5+ cells in the neonatal ventral forebrain. Furthermore, we show that Sox5 binding motif in CR5 is important for this cell-specific gene regulatory activity; mutation of Sox5 binding motif in CR5 alters reporter gene expression with different cellular composition. Together, our study provides new insights into the regulation of cell-specific gene expression during CNS development. PMID:24954155

Structural and functional analysis of mRNA export regulation by the nuclear pore complex.

PubMed

Lin, Daniel H; Correia, Ana R; Cai, Sarah W; Huber, Ferdinand M; Jette, Claudia A; Hoelz, André

2018-06-13

The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1•Nxt1 from mRNAs. Here, we report crystal structures of Gle1•Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP 6 , unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1•Nup42•DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans.

Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation

PubMed Central

Zhang, Zhengjian; Boskovic, Zarko; Hussain, Mahmud M; Hu, Wenxin; Inouye, Carla; Kim, Han-Je; Abole, A Katherine; Doud, Mary K; Lewis, Timothy A; Koehler, Angela N; Schreiber, Stuart L; Tjian, Robert

2015-01-01

Intrinsically disordered proteins/regions (IDPs/IDRs) are proteins or peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. In this study, we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions. DOI: http://dx.doi.org/10.7554/eLife.07777.001 PMID:26314865

Suppression of nemo-like kinase by miR-71 in Echinococcus multilocularis.

PubMed

Guo, Xiaola; Zhang, Xueyong; Yang, Jing; Jin, Xiaoliang; Ding, Juntao; Xiang, Haitao; Ayaz, Mazhar; Luo, Xuenong; Zheng, Yadong

2017-12-01

Echinococcus multilocularis metacestodes are a causative pathogen for alveolar echinococcosis in human beings, and have been found to express miRNAs including emu-miR-71. miR-71 is evolutionarily conserved and highly expressed across platyhelminths, but little is known about its role. Here it was shown that emu-miR-71 was differentially expressed in protoscoleces and was unlikely to be expressed in neoblasts. The results of the luciferase assay indicated that emu-miR-71 was able to bind in vitro to the 3'-UTR of emu-nlk, encoding a key regulator of cell division, causing significant downregulation of luciferase activity (p < 0.01) compared to the negative control and the construct with mutations in the binding site. Consistent with the decreased luciferase activity, transfection of emu-miR-71 mimics into protoscoleces notably repressed emu-NLK (p < 0.05). These results demonstrate the suppression of emu-nlk by emu-miR-71, potentially involved in the protoscolex development. Copyright © 2017 Elsevier Inc. All rights reserved.

Secreted and Transmembrane Wnt Inhibitors and Activators

PubMed Central

Cruciat, Cristina-Maria; Niehrs, Christof

2013-01-01

Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand–receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-β and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease. PMID:23085770

Structural basis of mammalian glycan targeting by Vibrio cholerae cytolysin and biofilm proteins

PubMed Central

De, Swastik; Kaus, Katherine; Sinclair, Shada

2018-01-01

Vibrio cholerae is an aquatic gram-negative microbe responsible for cholera, a pandemic disease causing life-threatening diarrheal outbreaks in populations with limited access to health care. Like most pathogenic bacteria, V. cholerae secretes virulence factors to assist colonization of human hosts, several of which bind carbohydrate receptors found on cell-surfaces. Understanding how pathogenic virulence proteins specifically target host cells is important for the development of treatment strategies to fight bacterial infections. Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin with a carboxy-terminal β-prism domain that targets complex N-glycans found on mammalian cell-surface proteins. To investigate glycan selectivity, we studied the VCC β-prism domain and two additional β-prism domains found within the V. cholerae biofilm matrix protein RbmC. We show that the two RbmC β-prism domains target a similar repertoire of complex N-glycan receptors as VCC and find through binding and modeling studies that a branched pentasaccharide core (GlcNAc2-Man3) represents the likely footprint interacting with these domains. To understand the structural basis of V. cholerae β-prism selectivity, we solved high-resolution crystal structures of fragments of the pentasaccharide core bound to one RbmC β-prism domain and conducted mutagenesis experiments on the VCC toxin. Our results highlight a common strategy for cell-targeting utilized by both toxin and biofilm matrix proteins in Vibrio cholerae and provide a structural framework for understanding the specificity for individual receptors. Our results suggest that a common strategy for disrupting carbohydrate interactions could affect multiple virulence factors produced by V. cholerae, as well as similar β-prism domains found in other vibrio pathogens. PMID:29432487

Inhibin binding protein in rats: alternative transcripts and regulation in the pituitary across the estrous cycle.

PubMed

Bernard, D J; Woodruff, T K

2001-04-01

Inhibin binding protein (InhBP) and the transforming growth factor-beta (TGF beta) type III receptor, beta glycan, have been identified as putative inhibin coreceptors. Here we cloned the InhBP cDNA in rats and predict that it encodes a large membrane-spanning protein that is part of the Ig superfamily, as has been described for humans. Two abundant InhBP transcripts (4.4 and 1.8 kb) were detected in the adult rat pituitary. The larger transcript encodes the full-length protein while the 1.8-kb transcript (InhBP-short or InhBP-S) corresponds to a splice variant of the receptor. This truncated isoform contains only the N-terminal signal peptide and first two (of 12) Ig-like domains observed in the full-length InhBP (InhBP-long or InhBP-L). InhBP-S does not contain a transmembrane domain and is predicted to be a soluble protein. Beta glycan was also detected in the pituitary; however, it was most abundant within the intermediate lobe. Although we also observed beta glycan immunopositive cells in the anterior pituitary, they rarely colocalized with FSH beta-producing cells. We next examined physiological regulation of the coreceptors across the rat estrous cycle. Like circulating inhibin A and inhibin B levels, pituitary InhBP-L and InhBP-S mRNA levels were dynamically regulated across the cycle and were negatively correlated with serum FSH levels. Expression of both forms of InhBP was also positively correlated with serum inhibin B, but not inhibin A, levels. These data are particularly interesting in light of our in vitro observations that InhBP may function as an inhibin B-specific coreceptor. Pituitary beta glycan mRNA levels did not fluctuate across the cycle nor did they correlate with serum FSH. These observations, coupled with its pattern of expression within the pituitary, indicate that beta glycan likely functions as more than merely an inhibin coreceptor within the pituitary. A direct role for InhBP or beta glycan in regulation of pituitary FSH by inhibin in vivo has yet to be determined, but the demonstration of dynamic regulation of pituitary InhBP and its negative relation to serum FSH across the estrous cycle is an important step in this direction.

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells

PubMed Central

Camp, J. Gray; Weiser, Matthew; Cocchiaro, Jordan L.; Kingsley, David M.; Furey, Terrence S.; Sheikh, Shehzad Z.; Rawls, John F.

2017-01-01

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology. PMID:28850571

Evolutionarily conserved structural and functional roles of the FYVE domain.

PubMed

Hayakawa, Akira; Hayes, Susan; Leonard, Deborah; Lambright, David; Corvera, Silvia

2007-01-01

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4'. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface

PubMed Central

Koo, Bon-Kyung; Park, Chin-Ju; Fernandez, Cesar F.; Chim, Nicholas; Ding, Yi; Chanfreau, Guillaume; Feigon, Juli

2011-01-01

H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that specifically binds to H/ACA RNAs. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent in eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of S. cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10- and RNA-binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA. PMID:21708174

Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans.

PubMed

David, Cynthia L; Smith, Harold E; Raynes, Deborah A; Pulcini, Elizabeth J; Whitesell, Luke

2003-01-01

In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol. Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide-binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases. To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake. Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span. However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90. Consistent with this observation, we found that solid phase-immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system. Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA. Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background. It heterodimerized with its vertebrate counterpart and showed no evidence of compromising its essential cellular functions. Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant. These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that target-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.

A Human Lectin Microarray for Sperm Surface Glycosylation Analysis *

PubMed Central

Sun, Yangyang; Cheng, Li; Gu, Yihua; Xin, Aijie; Wu, Bin; Zhou, Shumin; Guo, Shujuan; Liu, Yin; Diao, Hua; Shi, Huijuan; Wang, Guangyu; Tao, Sheng-ce

2016-01-01

Glycosylation is one of the most abundant and functionally important protein post-translational modifications. As such, technology for efficient glycosylation analysis is in high demand. Lectin microarrays are a powerful tool for such investigations and have been successfully applied for a variety of glycobiological studies. However, most of the current lectin microarrays are primarily constructed from plant lectins, which are not well suited for studies of human glycosylation because of the extreme complexity of human glycans. Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane-associated proteins including heat shock protein 90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays. PMID:27364157

Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

PubMed

Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

2018-04-01

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

Nuclear autophagy: An evolutionarily conserved mechanism of nuclear degradation in the cytoplasm.

PubMed

Luo, Majing; Zhao, Xueya; Song, Ying; Cheng, Hanhua; Zhou, Rongjia

2016-11-01

Macroautophagy/autophagy is a catabolic process that is essential for cellular homeostasis. Studies on autophagic degradation of cytoplasmic components have generated interest in nuclear autophagy. Although its mechanisms and roles have remained elusive, tremendous progress has been made toward understanding nuclear autophagy. Nuclear autophagy is evolutionarily conserved in eukaryotes that may target various nuclear components through a series of processes, including nuclear sensing, nuclear export, autophagic substrate encapsulation and autophagic degradation in the cytoplasm. However, the molecular processes and regulatory mechanisms involved in nuclear autophagy remain largely unknown. Numerous studies have highlighted the importance of nuclear autophagy in physiological and pathological processes such as cancer. This review focuses on current advances in nuclear autophagy and provides a summary of its research history and landmark discoveries to offer new perspectives.

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

PubMed Central

Cristóvão, Michele; Sisamakis, Evangelos; Hingorani, Manju M.; Marx, Andreas D.; Jung, Caroline P.; Rothwell, Paul J.; Seidel, Claus A. M.; Friedhoff, Peter

2012-01-01

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS–mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand. PMID:22367846

Absence of Capsule Reveals Glycan-Mediated Binding and Recognition of Salivary Mucin MUC7 by Streptococcus pneumoniae

PubMed Central

Thamadilok, Supaporn; Roche-Håkansson, Hazeline; Håkansson, Anders P.; Ruhl, Stefan

2015-01-01

SUMMARY Salivary proteins modulate bacterial colonization in the oral cavity and interact with systemic pathogens that pass through the oropharynx. An interesting example is the opportunistic respiratory pathogen Streptococcus pneumoniae that normally resides in the nasopharynx, but belongs to the greater Mitis group of streptococci, most of which colonize the oral cavity. S. pneumoniae also expresses a serine-rich repeat (SRR) adhesin, PsrP, that is a homologue to oral Mitis group SRR adhesins, such as Hsa of S. gordonii and SrpA of S. sanguinis. Since the latter bind to salivary glycoproteins through recognition of terminal sialic acids, we wanted to determine whether S. pneumoniae also binds to salivary proteins through possibly the same mechanism. We found that only a capsule-free mutant of S. pneumoniae TIGR4 binds to salivary proteins, most prominently to mucin MUC7, but that this binding was not mediated through PsrP or recognition of sialic acid. We also found, however, that PsrP is involved in agglutination of human red blood cells (RBCs). After removal of PsrP, an additional previously masked lectin-like adhesin activity mediating agglutination of sialidase-treated RBCs becomes revealed. Using a custom-spotted glycoprotein and neoglycoprotein dot blot array, we identify candidate glycan motifs recognized by PsrP and by the putative S. pneumoniae adhesin that could perhaps be responsible for pneumococcal binding to salivary MUC7 and glycoproteins on RBCs. PMID:26172471

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

Diversity in the protein N-glycosylation pathways among campylobacter species

USDA-ARS?s Scientific Manuscript database

The foodborne bacterial pathogen, Campylobacter jejuni, possesses an N-linked protein glycosylation (pgl) pathway involved in adding conserved heptasaccharides to asparaginecontaining motifs of >60 proteins, and releasing the same glycan into its periplasm as free oligosaccharides. In this study, co...

Fragments of the V1/V2 domain of HIV-1 glycoprotein 120 engineered for improved binding to the broadly neutralizing PG9 antibody.

PubMed

Morales, Javier F; Yu, Bin; Perez, Gerardo; Mesa, Kathryn A; Alexander, David L; Berman, Phillip W

2016-09-01

The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine development: (1) improving antibody responses to poorly immunogenic epitopes in the V1/V2 domain; (2) eliminating antibody responses to highly immunogenic (decoy) epitopes outside the V1/V2 domain; and (3) enabling the production of V1/V2 scaffolds in a cell line suitable for biopharmaceutical production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Deciphering the combinatorial architecture of a Drosophila homeotic gene enhancer

PubMed Central

Drewell, Robert A.; Nevarez, Michael J.; Kurata, Jessica S.; Winkler, Lauren N.; Li, Lily; Dresch, Jacqueline M.

2013-01-01

Summary In Drosophila, the 330 kb bithorax complex regulates cellular differentiation along the anterio-posterior axis during development in the thorax and abdomen and is comprised of three homeotic genes: Ultrabithorax, abdominal-A, and Abdominal-B. The expression of each of these genes is in turn controlled through interactions between transcription factors and a number of cis-regulatory modules in the neighboring intergenic regions. In this study, we examine how the sequence architecture of transcription factor binding sites mediates the functional activity of one of these cis-regulatory modules. Using computational, mathematical modeling and experimental molecular genetic approaches we investigate the IAB7b enhancer, which regulates Abdominal-B expression specifically in the presumptive seventh and ninth abdominal segments of the early embryo. A cross-species comparison of the IAB7b enhancer reveals an evolutionarily conserved signature motif containing two FUSHI-TARAZU activator transcription factor binding sites. We find that the transcriptional repressors KNIRPS, KRUPPEL and GIANT are able to restrict reporter gene expression to the posterior abdominal segments, using different molecular mechanisms including short-range repression and competitive binding. Additionally, we show the functional importance of the spacing between the two FUSHI-TARAZU binding sites and discuss the potential importance of cooperativity for transcriptional activation. Our results demonstrate that the transcriptional output of the IAB7b cis-regulatory module relies on a complex set of combinatorial inputs mediated by specific transcription factor binding and that the sequence architecture at this enhancer is critical to maintain robust regulatory function. PMID:24514265

The Sus operon: a model system for starch uptake by the human gut Bacteroidetes

PubMed Central

Foley, Matthew H.; Cockburn, Darrell W.; Koropatkin, Nicole M.

2016-01-01

Resident bacteria in the densely populated human intestinal tract must efficiently compete for carbohydrate nutrition. The Bacteroidetes, a dominant bacterial phylum in the mammalian gut, encode a plethora of discrete polysaccharide utilization loci (PULs) that are selectively activated to facilitate glycan capture at the cell surface. The most well-studied PUL-encoded glycan-up-take system is the starch utilization system (Sus) of Bacteroides thetaiotaomicron. The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. In this review, we discuss what is known about the eight Sus proteins of B. thetaiotaomicron that define the Sus-like paradigm of nutrient acquisition that is exclusive to the Gram-negative Bacteroidetes. We emphasize the well-characterized outer membrane proteins SusDEF and the α-amylase SusG, each of which have unique structural features that allow them to interact with starch on the cell surface. Despite the apparent redundancy in starch-binding sites among these proteins, each has a distinct role during starch catabolism. Additionally, we consider what is known about how these proteins dynamically interact and cooperate in the membrane and propose a model for the formation of the Sus outer membrane complex. PMID:27137179

Lectin-Based Assay for Glycoform-Specific Detection of α2,6-sialylated Transferrin and Carcinoembryonic Antigen in Tissue and Body Fluid.

PubMed

Ito, Hiromi; Hoshi, Kyoka; Honda, Takashi; Hashimoto, Yasuhiro

2018-05-30

Antibodies are useful for detecting glycoprotein antigens, but a conventional antibody recognizes only a protein epitope rather than a glycan. Thus, glycan isoform detection generally requires time- and labor-consuming processes such as lectin affinity column chromatography followed by sandwich ELISA. We recently found antigen-antibody reactions that were inhibited by lectin binding to glycans on the glycoprotein antigen, leading to a convenient glycoform-specific assay. Indeed, Sambucus sieboldiana agglutinin (SSA) lectin, a binder to sialylα2,6galactose residue, inhibited antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform-specific. SSA inhibition was also applicable for visualizing localization of α2,6-sialylated-Tf in a liver section. This is the first immunohistochemical demonstration of glycoform localization in a tissue section. SSA inhibition was utilized for establishing ELISA to quantify α2,6-sialylated carcinoembryonic antigen (CEA), a marker for various cancers. In addition, α2,6-sialylated-CEA was visualized in a colonic adenocarcinoma section by SSA inhibition. The method would further be applicable to a simple and rapid estimation of other α2,6-sialylated glycoproteins and have a potential aid to histopathological diagnosis.

Structure and Specificity of a Binary Tandem Domain F-Lectin from Striped Bass (Morone saxatilis)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bianchet, M.; Odom, E; Vasta, J

2010-01-01

The plasma of the striped bass Morone saxatilis contains a fucose-specific lectin (MsaFBP32) that consists of two F-type carbohydrate recognition domains (CRDs) in tandem. The crystal structure of the complex of MsaFBP32 with l-fucose reported here shows a cylindrical 81-A-long and 60-A-wide trimer divided into two globular halves: one containing N-terminal CRDs (N-CRDs) and the other containing C-terminal CRDs (C-CRDs). The resulting binding surfaces at the opposite ends of the cylindrical trimer have the potential to cross-link cell surface or humoral carbohydrate ligands. The N-CRDs and C-CRDs of MsaFBP32 exhibit significant structural differences, suggesting that they recognize different glycans. Analysismore » of the carbohydrate binding sites provides the structural basis for the observed specificity of MsaFBP32 for simple carbohydrates and suggests that the N-CRD recognizes more complex fucosylated oligosaccharides and with a relatively higher avidity than the C-CRD. Modeling of MsaFBP32 complexed with fucosylated glycans that are widely distributed in prokaryotes and eukaryotes rationalizes the observation that binary tandem CRD F-type lectins function as opsonins by cross-linking 'non-self' carbohydrate ligands and 'self' carbohydrate ligands, such as sugar structures displayed by microbial pathogens and glycans on the surface of phagocytic cells from the host.« less

Role of Dlx6 in regulation of an endothelin-1-dependent, dHAND branchial arch enhancer

PubMed Central

Charité, Jeroen; McFadden, David G.; Merlo, Giorgio; Levi, Giovanni; Clouthier, David E.; Yanagisawa, Masashi; Richardson, James A.; Olson, Eric N.

2001-01-01

Neural crest cells play a key role in craniofacial development. The endothelin family of secreted polypeptides regulates development of several neural crest sublineages, including the branchial arch neural crest. The basic helix–loop–helix transcription factor dHAND is also required for craniofacial development, and in endothelin-1 (ET-1) mutant embryos, dHAND expression in the branchial arches is down-regulated, implicating it as a transcriptional effector of ET-1 action. To determine the mechanism that links ET-1 signaling to dHAND transcription, we analyzed the dHAND gene for cis-regulatory elements that control transcription in the branchial arches. We describe an evolutionarily conserved dHAND enhancer that requires ET-1 signaling for activity. This enhancer contains four homeodomain binding sites that are required for branchial arch expression. By comparing protein binding to these sites in branchial arch extracts from endothelin receptor A (EdnrA) mutant and wild-type mouse embryos, we identified Dlx6, a member of the Distal-less family of homeodomain proteins, as an ET-1-dependent binding factor. Consistent with this conclusion, Dlx6 was down-regulated in branchial arches from EdnrA mutant mice. These results suggest that Dlx6 acts as an intermediary between ET-1 signaling and dHAND transcription during craniofacial morphogenesis. PMID:11711438

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Yanqi; Levy, Dan; Horton, John R.

SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-{kappa}B). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-l-methionine, reveals a V-like protein structure and suggests a model for NF-{kappa}B binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insightmore » into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation-phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.« less

Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP.

PubMed

Canosa, Andrea V; Faggiano, Serena; Marchetti, Marialaura; Armao, Stefano; Bettati, Stefano; Bruno, Stefano; Percudani, Riccardo; Campanini, Barbara; Mozzarelli, Andrea

2018-06-13

Serine racemase (SR) catalyses two reactions: the reversible racemisation of L-serine and the irreversible dehydration of L- and D-serine to pyruvate and ammonia. SRs are evolutionarily related to serine dehydratases (SDH) and degradative threonine deaminases (TdcB). Most SRs and TdcBs - but not SDHs - are regulated by nucleotides. SR binds ATP cooperatively and the nucleotide allosterically stimulates the serine dehydratase activity of the enzyme. A H-bond network comprising five residues (T52, N86, Q89, E283 and N316) and water molecules connects the active site with the ATP-binding site. Conservation analysis points to Q89 as a key residue for the allosteric communication, since its mutation to either Met or Ala is linked to the loss of control of activity by nucleotides. We verified this hypothesis by introducing the Q89M and Q89A point mutations in the human SR sequence. The allosteric communication between the active site and the allosteric site in both mutants is almost completely abolished. Indeed, the stimulation of the dehydratase activity by ATP is severely diminished and the binding of the nucleotide is no more cooperative. Ancestral state reconstruction suggests that the allosteric control by nucleotides established early in SR evolution and has been maintained in most eukaryotic lineages.

Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

PubMed Central

Choi, Sang Ki; Olsen, DeAnne S.; Roll-Mecak, Antonina; Martung, Agnes; Remo, Keith L.; Burley, Stephen K.; Hinnebusch, Alan G.; Dever, Thomas E.

2000-01-01

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site. PMID:10982835

Inflammasome - activated gasdermin D causes pyroptosis by forming membrane pores

PubMed Central

Liu, Xing; Zhang, Zhibin; Ruan, Jianbin; Pan, Youdong; Magupalli, Venkat Giri; Wu, Hao; Lieberman, Judy

2017-01-01

Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β1,2. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined. PMID:27383986

Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.

PubMed

Liu, Xing; Zhang, Zhibin; Ruan, Jianbin; Pan, Youdong; Magupalli, Venkat Giri; Wu, Hao; Lieberman, Judy

2016-07-07

Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.

Common structural features of cholesterol binding sites in crystallized soluble proteins

PubMed Central

Bukiya, Anna N.; Dopico, Alejandro M.

2017-01-01

Cholesterol-protein interactions are essential for the architectural organization of cell membranes and for lipid metabolism. While cholesterol-sensing motifs in transmembrane proteins have been identified, little is known about cholesterol recognition by soluble proteins. We reviewed the structural characteristics of binding sites for cholesterol and cholesterol sulfate from crystallographic structures available in the Protein Data Bank. This analysis unveiled key features of cholesterol-binding sites that are present in either all or the majority of sites: i) the cholesterol molecule is generally positioned between protein domains that have an organized secondary structure; ii) the cholesterol hydroxyl/sulfo group is often partnered by Asn, Gln, and/or Tyr, while the hydrophobic part of cholesterol interacts with Leu, Ile, Val, and/or Phe; iii) cholesterol hydrogen-bonding partners are often found on α-helices, while amino acids that interact with cholesterol’s hydrophobic core have a slight preference for β-strands and secondary structure-lacking protein areas; iv) the steroid’s C21 and C26 constitute the “hot spots” most often seen for steroid-protein hydrophobic interactions; v) common “cold spots” are C8–C10, C13, and C17, at which contacts with the proteins were not detected. Several common features we identified for soluble protein-steroid interaction appear evolutionarily conserved. PMID:28420706

Recombinant hepatitis B surface antigen and anionic phospholipids share a binding region in the fifth domain of β2-glycoprotein I (apolipoprotein H)

PubMed Central

Mehdi, Haider; Naqvi, Asma; Kamboh, M. lIyas

2008-01-01

Human β2-glycoprotein I (β2GPI) binds to recombinant hepatitis B surface antigen (rHBsAg), but the location of the binding domain on β2GPI is unknown. It has been suggested that the lipid rather than the protein moiety of rHBsAg binds to β2GPI. Since β2 GPI binds to anionic phospholipids (PL) through its lipid binding region in the fifth domain of β2GPI, we predicted that this lipid binding region may also be involved in binding rHBsAg. In this study, we examined rHBsAg binding to two naturally occurring mutants of β2GPI, Cys306Gly and Trp316Ser, or evolutionarily conserved hydrophobic amino acid sequence, Leu313-Ala314-Phe315 in the fifth domain of β2GPI. The two naturally occurring mutations and two mutagenized amino acids, Leu313Gly or Phe315Ser, disrupted the binding of recombinant β2GPI (rβ2GPI) to both rHBsAg and cardiolipin (CL), an anionic PL. These results suggest that rHBsAg and CL share the same region in the fifth domain of β2GPI. Credence to this conclusion was further provided by competitive ELISA, where CL-bound rβ2GPI was incubated with increasing amounts of rHBsAg. As expected, pre-incubation of rβ2GPI with CL precluded binding to rHBsAg, indicating that CL and rHBsAg bind to the same region on β2GPI. Our data provide evidence that the lipid (PL) rather than the protein moiety of rHBsAg binds to β2GPI and that this binding region is located in the fifth domain of β2GPI, which also binds to anionic PL. PMID:18230366

Glycomic Characterization of Respiratory Tract Tissues of Ferrets

PubMed Central

Jia, Nan; Barclay, Wendy S.; Roberts, Kim; Yen, Hui-Ling; Chan, Renee W. Y.; Lam, Alfred K. Y.; Air, Gillian; Peiris, J. S. Malik; Dell, Anne; Nicholls, John M.; Haslam, Stuart M.

2014-01-01

The initial recognition between influenza virus and the host cell is mediated by interactions between the viral surface protein hemagglutinin and sialic acid-terminated glycoconjugates on the host cell surface. The sialic acid residues can be linked to the adjacent monosaccharide by α2–3- or α2–6-type glycosidic bonds. It is this linkage difference that primarily defines the species barrier of the influenza virus infection with α2–3 binding being associated with avian influenza viruses and α2–6 binding being associated with human strains. The ferret has been extensively used as an animal model to study the transmission of influenza. To better understand the validity of this model system, we undertook glycomic characterization of respiratory tissues of ferret, which allows a comparison of potential viral receptors to be made between humans and ferrets. To complement the structural analysis, lectin staining experiments were performed to characterize the regional distributions of glycans along the respiratory tract of ferrets. Finally, the binding between the glycans identified and the hemagglutinins of different strains of influenza viruses was assessed by glycan array experiments. Our data indicated that the respiratory tissues of ferret heterogeneously express both α2–3- and α2–6-linked sialic acids. However, the respiratory tissues of ferret also expressed the Sda epitope (NeuAcα2-3(GalNAcβ1–4)Galβ1–4GlcNAc) and sialylated N,N′-diacetyllactosamine (NeuAcα2–6GalNAcβ1–4GlcNAc), which have not been observed in the human respiratory tract surface epithelium. The presence of the Sda epitope reduces potential binding sites for avian viruses and thus may have implications for the usefulness of the ferret in the study of influenza virus infection. PMID:25135641

Glycomic characterization of respiratory tract tissues of ferrets: implications for its use in influenza virus infection studies.

PubMed

Jia, Nan; Barclay, Wendy S; Roberts, Kim; Yen, Hui-Ling; Chan, Renee W Y; Lam, Alfred K Y; Air, Gillian; Peiris, J S Malik; Dell, Anne; Nicholls, John M; Haslam, Stuart M

2014-10-10

The initial recognition between influenza virus and the host cell is mediated by interactions between the viral surface protein hemagglutinin and sialic acid-terminated glycoconjugates on the host cell surface. The sialic acid residues can be linked to the adjacent monosaccharide by α2-3- or α2-6-type glycosidic bonds. It is this linkage difference that primarily defines the species barrier of the influenza virus infection with α2-3 binding being associated with avian influenza viruses and α2-6 binding being associated with human strains. The ferret has been extensively used as an animal model to study the transmission of influenza. To better understand the validity of this model system, we undertook glycomic characterization of respiratory tissues of ferret, which allows a comparison of potential viral receptors to be made between humans and ferrets. To complement the structural analysis, lectin staining experiments were performed to characterize the regional distributions of glycans along the respiratory tract of ferrets. Finally, the binding between the glycans identified and the hemagglutinins of different strains of influenza viruses was assessed by glycan array experiments. Our data indicated that the respiratory tissues of ferret heterogeneously express both α2-3- and α2-6-linked sialic acids. However, the respiratory tissues of ferret also expressed the Sda epitope (NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAc) and sialylated N,N'-diacetyllactosamine (NeuAcα2-6GalNAcβ1-4GlcNAc), which have not been observed in the human respiratory tract surface epithelium. The presence of the Sda epitope reduces potential binding sites for avian viruses and thus may have implications for the usefulness of the ferret in the study of influenza virus infection. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

CLEC4F Is an Inducible C-Type Lectin in F4/80-Positive Cells and Is Involved in Alpha-Galactosylceramide Presentation in Liver

PubMed Central

Yang, Chih-Ya; Chen, Jiun-Bo; Tsai, Ting-Fen; Tsai, Yi-Chen; Tsai, Ching-Yen; Liang, Pi-Hui; Hsu, Tsui-Ling; Wu, Chung-Yi; Netea, Mihai G.; Wong, Chi-Huey; Hsieh, Shie-Liang

2013-01-01

CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f−/−) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells. PMID:23762286

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus*

PubMed Central

Bagdonaite, Ieva; Nordén, Rickard; Joshi, Hiren J.; King, Sarah L.; Vakhrushev, Sergey Y.; Olofsson, Sigvard; Wandall, Hans H.

2016-01-01

Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans; thus we have recently developed a “bottom up” mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1. We found wide distribution of O-glycans on herpes simplex virus type 1 glycoproteins and demonstrated that elongated O-glycans were essential for the propagation of the virus. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family: varicella zoster virus, human cytomegalovirus, and Epstein-Barr virus. We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Because glycosylation is highly dependent on the host cell, we tested varicella zoster virus-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host immune system. This knowledge enables dissection of specific functional roles of individual glycosites and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation. PMID:27129252

Evolutionarily conserved regions and hydrophobic contacts at the superfamily level: The case of the fold-type I, pyridoxal-5′-phosphate-dependent enzymes

PubMed Central

Paiardini, Alessandro; Bossa, Francesco; Pascarella, Stefano

2004-01-01

The wealth of biological information provided by structural and genomic projects opens new prospects of understanding life and evolution at the molecular level. In this work, it is shown how computational approaches can be exploited to pinpoint protein structural features that remain invariant upon long evolutionary periods in the fold-type I, PLP-dependent enzymes. A nonredundant set of 23 superposed crystallographic structures belonging to this superfamily was built. Members of this family typically display high-structural conservation despite low-sequence identity. For each structure, a multiple-sequence alignment of orthologous sequences was obtained, and the 23 alignments were merged using the structural information to obtain a comprehensive multiple alignment of 921 sequences of fold-type I enzymes. The structurally conserved regions (SCRs), the evolutionarily conserved residues, and the conserved hydrophobic contacts (CHCs) were extracted from this data set, using both sequence and structural information. The results of this study identified a structural pattern of hydrophobic contacts shared by all of the superfamily members of fold-type I enzymes and involved in native interactions. This profile highlights the presence of a nucleus for this fold, in which residues participating in the most conserved native interactions exhibit preferential evolutionary conservation, that correlates significantly (r = 0.70) with the extent of mean hydrophobic contact value of their apolar fraction. PMID:15498941

Nuclear Pore Complexes: Global Conservation and Local Variation.

PubMed

Holzer, Guillaume; Antonin, Wolfram

2018-06-04

Nuclear pore complexes are the transport gates to the nucleus. Most proteins forming these huge complexes are evolutionarily conserved, as is the eightfold symmetry of these complexes. A new study reporting the structure of the yeast nuclear pore complex now shows striking differences from its human counterpart. Copyright © 2018 Elsevier Ltd. All rights reserved.

Individual N-Glycans Added at Intervals along the Stalk of the Nipah Virus G Protein Prevent Fusion but Do Not Block the Interaction with the Homologous F Protein

PubMed Central

Zhu, Qiyun; Biering, Scott B.; Mirza, Anne M.; Grasseschi, Brittany A.; Mahon, Paul J.; Lee, Benhur; Aguilar, Hector C.

2013-01-01

The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demonstrate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) and measles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDV HN and MV H that likely mediate the F interaction have been identified. However, despite extensive efforts, the F-interactive site(s) on the Nipah virus (NiV) G attachment glycoprotein has not been identified. In this study, we have introduced individual N-linked glycosylation sites at several positions spaced at intervals along the stalk of the NiV G protein. Five of the seven introduced sites are utilized as established by a retardation of electrophoretic mobility. Despite surface expression, ephrinB2 binding, and oligomerization comparable to those of the wild-type protein, four of the five added N-glycans completely eliminate the ability of the G protein to complement the homologous F protein in the promotion of fusion. The most membrane-proximal added N-glycan reduces fusion by 80%. However, unlike similar NDV HN and MV H mutants, the NiV G glycosylation stalk mutants retain the ability to bind F, indicating that the fusion deficiency of these mutants is not due to prevention of the G-F interaction. These findings suggest that the G-F interaction is not mediated entirely by the stalk domain of G and may be more complex than that of HN/H-F. PMID:23283956

Identification of endoplasmic reticulum proteins involved in glycan assembly: synthesis and characterization of P3-(4-azidoanilido)uridine 5'-triphosphate, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins.

PubMed Central

Rancour, D M; Menon, A K

1998-01-01

Much of the enzymic machinery required for the assembly of cell surface carbohydrates is located in the endoplasmic reticulum (ER) of eukaryotic cells. Structural information on these proteins is limited and the identity of the active polypeptide(s) is generally unknown. This paper describes the synthesis and characteristics of a photoaffinity reagent that can be used to identify and analyse members of the ER glycan assembly apparatus, specifically those glycosyltransferases, nucleotide phosphatases and nucleotide-sugar transporters that recognize uridine nucleotides or UDP-sugars. The photoaffinity reagent, P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP), was synthesized easily from commercially available precursors. AAUTP inhibited the activity of ER glycosyltransferases that utilize UDP-GlcNAc and UDP-Glc, indicating that it is recognized by UDP-sugar-binding proteins. In preliminary tests AAUTP[alpha-32P] labelled bovine milk galactosyltransferase, a model UDP-sugar-utilizing enzyme, in a UV-light-dependent, competitive and saturable manner. When incubated with rat liver ER vesicles, AAUTP[alpha-32P] labelled a discrete subset of ER proteins; labelling was light-dependent and metal ion-specific. Photolabelling of intact ER vesicles with AAUTP[alpha-32P] caused selective incorporation of radioactivity into proteins with cytoplasmically disposed binding sites; UDP-Glc:glycoprotein glucosyltransferase, a lumenal protein, was labelled only when the vesicle membrane was disrupted. These data indicate that AAUTP is a membrane topological probe of catalytic sites in target proteins. Strategies for using AAUTP to identify and study novel ER proteins involved in glycan assembly are discussed. PMID:9677326

Mechanisms of escape from the PGT128 family of anti-HIV broadly neutralizing antibodies.

PubMed

Krumm, Stefanie A; Mohammed, Hajer; Le, Khoa M; Crispin, Max; Wrin, Terri; Poignard, Pascal; Burton, Dennis R; Doores, Katie J

2016-02-02

Broadly neutralizing antibodies (bnAbs) directed against the mannose-patch on the HIV envelope glycoprotein gp120 have several features that make them desirable targets for vaccine design. The PGT125-131 bnAb family is of particular interest due to its superior breadth and potency. The overlapping epitopes recognized by this family are intricate and neutralization requires interaction with at least two N-linked glycans (N332/N334, N295 or N301) in addition to backbone-mediated contact with the (323)IGDIR(327) motif of the V3 loop. We have recently shown that this bnAb family consists of two distinct antibody classes that can bind alternate arrangements of glycans in the mannose-patch in the absence of N332 thereby limiting viral escape. This led us to further investigate viral resistance and escape mechanisms to the PGT125-131 bnAb family. Using an escape virus isolated from the PGT125-131 donor as a guide, we show that mutating both the V3 core protein epitope and repositioning critical N-linked glycosylation sites are required to restore neutralization sensitivity. Interestingly, neutralization sensitivity could be restored via different routes for the two distinct bnAb classes within the PGT125-131 family, which may have been important in generating the divergence in recognition. We demonstrate that the observed V3 mutations confer neutralization resistance in other virus strains through both gain-of-function and escape studies. Furthermore, we show that the V3 loop is important in facilitating promiscuous binding to glycans within the mannose-patch. These data highlight the importance of the V3 loop in the design of immunogens aimed at inducing broad and potent bnAbs that can bind promiscuously to the mannose-patch.