Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.

PubMed

Yadav, Rupali; Verma, Kuldeep; Chandra, Mintu; Mukherjee, Madhumita; Datta, Sunando

2016-09-01

Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Functional mechanics of the plant defensive Griffonia simplicifolia lectin II: resistance to proteolysis is independent of glycoconjugate binding in the insect gut.

PubMed

Zhu-Salzman, K; Salzman, R A

2001-10-01

Griffonia simplicifolia lectin II (GSII) is a plant defensive protein that significantly delays development of the cowpea bruchid Callosobruchus maculatus (F.). Previous structure/function analysis by site-directed mutagenesis indicated that carbohydrate binding and resistance to insect gut proteolysis are required for the anti-insect activity of this lectin. However, whether there is a causal link between carbohydrate binding and resistance to insect metabolism remains unknown. Two proteases principally responsible for digestive proteolysis in third and fourth instar larvae of C. maculatus were purified by activated thiol sepharose chromatography and resolved as cathepsin L-like proteases, based on N-terminal amino acid sequence analysis. Digestion of bacterially expressed recombinant GSII (rGSII) and its mutant protein variants with the purified gut proteases indicates that carbohydrate binding, presumably to a target ligand in insect gut, and proteolytic resistance are independent properties of rGSII, and that both facilitate its efficacy as a plant defensive molecule.

Cyborg lectins: novel leguminous lectins with unique specificities.

PubMed

Yamamoto, K; Maruyama, I N; Osawa, T

2000-01-01

Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.

Network analysis reveals the recognition mechanism for complex formation of mannose-binding lectins

NASA Astrophysics Data System (ADS)

Jian, Yiren; Zhao, Yunjie; Zeng, Chen

The specific carbohydrate binding of lectin makes the protein a powerful molecular tool for various applications including cancer cell detection due to its glycoprotein profile on the cell surface. Most biologically active lectins are dimeric. To understand the structure-function relation of lectin complex, it is essential to elucidate the short- and long-range driving forces behind the dimer formation. Here we report our molecular dynamics simulations and associated dynamical network analysis on a particular lectin, i.e., the mannose-binding lectin from garlic. Our results, further supported by sequence coevolution analysis, shed light on how different parts of the complex communicate with each other. We propose a general framework for deciphering the recognition mechanism underlying protein-protein interactions that may have potential applications in signaling pathways.

Structural analysis, molecular docking and molecular dynamics of an edematogenic lectin from Centrolobium microchaete seeds.

PubMed

Neco, Antonio Hadson Bastos; Pinto-Junior, Vanir Reis; Araripe, David Alencar; Santiago, Mayara Queiroz; Osterne, Vinicius Jose Silva; Lossio, Claudia Figueiredo; Nobre, Clareane Avelino Simplicio; Oliveira, Messias Vital; Silva, Mayara Torquato Lima; Martins, Maria Gleiciane Queiroz; Cajazeiras, Joao Batista; Marques, Gabriela Fernandes Oliveira; Costa, Diego Rabelo; Nascimento, Kyria Santiago; Assreuy, Ana Maria Sampaio; Cavada, Benildo Sousa

2018-05-24

Lectins represent a class of proteins or glycoproteins capable of reversibly binding to carbohydrates. Seed lectins from the Dalbergieae tribe (Leguminosae) have structural variability, carbohydrate specificity, and biological effects, such as inflammation, vasorelaxation and cancer antigen binding. To comprehensively address these factors, the present work aimed to establish and characterize the three-dimensional structure of Centrolobium microchaete lectin (CML) by homology modeling, investigate protein-carbohydrate interactions and evaluate its inflammatory effect on mice. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and N-glycans. Two dimannosides, methyl mannose-1,3-α-D-mannose (MDM) and mannose-1,3-α-D-mannose (M13), were used in molecular dynamics (MD) simulations to study the behavior of the carbohydrate-recognition domain (CRD) over time. Results showed an expanded domain within which hydrophobic interactions with the methyl group in the MDM molecule were established, thus revealing novel interactions for mannose-specific Dalbergieae lectins. To examine its biological activities, CML was purified in a single step by affinity chromatography on Sepharose-mannose matrix. The lectin demonstrated inflammatory response in the paw edema model and stimulated leukocyte migration to the animal peritoneal cavities, an effect elicited by CRD. For the first time, this work reports the molecular dynamics of a lectin from the Dalbergieae tribe. Copyright © 2018 Elsevier B.V. All rights reserved.

High glucose disrupts oligosaccharide recognition function via competitive inhibition: a potential mechanism for immune dysregulation in diabetes mellitus.

PubMed

Ilyas, Rebecca; Wallis, Russell; Soilleux, Elizabeth J; Townsend, Paul; Zehnder, Daniel; Tan, Bee K; Sim, Robert B; Lehnert, Hendrik; Randeva, Harpal S; Mitchell, Daniel A

2011-01-01

Diabetic complications include infection and cardiovascular disease. Within the immune system, host-pathogen and regulatory host-host interactions operate through binding of oligosaccharides by C-type lectin. A number of C-type lectins recognise oligosaccharides rich in mannose and fucose - sugars with similar structures to glucose. This raises the possibility that high glucose conditions in diabetes affect protein-oligosaccharide interactions via competitive inhibition. Mannose-binding lectin, soluble DC-SIGN and DC-SIGNR, and surfactant protein D, were tested for carbohydrate binding in the presence of glucose concentrations typical of diabetes, via surface plasmon resonance and affinity chromatography. Complement activation assays were performed in high glucose. DC-SIGN and DC-SIGNR expression in adipose tissues was examined via immunohistochemistry. High glucose inhibited C-type lectin binding to high-mannose glycoprotein and binding of DC-SIGN to fucosylated ligand (blood group B) was abrogated in high glucose. Complement activation via the lectin pathway was inhibited in high glucose and also in high trehalose - a nonreducing sugar with glucoside stereochemistry. DC-SIGN staining was seen on cells with DC morphology within omental and subcutaneous adipose tissues. We conclude that high glucose disrupts C-type lectin function, potentially illuminating new perspectives on susceptibility to infectious and inflammatory disease in diabetes. Mechanisms involve competitive inhibition of carbohydrate binding within sets of defined proteins, in contrast to broadly indiscriminate, irreversible glycation of proteins. Copyright © 2010 Elsevier GmbH. All rights reserved.

Alteration of the carbohydrate-binding specificity of a C-type lectin CEL-I mutant with an EPN carbohydrate-binding motif.

PubMed

Hatakeyama, Tomomitsu; Ishimine, Tomohiro; Baba, Tomohiro; Kimura, Masanari; Unno, Hideaki; Goda, Shuichiro

2013-07-01

CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins.

Surface Glycosylation Profiles of Urine Extracellular Vesicles

PubMed Central

Gerlach, Jared Q.; Krüger, Anja; Gallogly, Susan; Hanley, Shirley A.; Hogan, Marie C.; Ward, Christopher J.

2013-01-01

Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications. PMID:24069349

Microfibrillar associated protein 4 mfap4 genes in catfish play a novel role in innate immune responses

USDA-ARS?s Scientific Manuscript database

The lectin pathway of the complement system is characterized by two groups of soluble pattern recognition molecules, mannose-binding lectins (MBLs) and ficolins. These molecules recognize and bind carbohydrates in pathogens and activate complement leading to opsonization, leukocyte activation, and d...

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.

Symbiotic Bacteria Direct Expression of an Intestinal Bactericidal Lectin

PubMed Central

Cash, Heather L.; Whitham, Cecilia V.; Behrendt, Cassie L.; Hooper, Lora V.

2009-01-01

The mammalian intestine harbors complex societies of beneficial bacteria that are maintained in the lumen with minimal penetration of mucosal surfaces. Microbial colonization of germ-free mice triggers epithelial expression of RegIIIγ, a secreted C-type lectin. RegIIIγ binds intestinal bacteria but lacks the complement recruitment domains present in other microbe-binding mammalian C-type lectins. We show that RegIIIγ and its human counterpart, HIP/PAP, are directly antimicrobial proteins that bind their bacterial targets via interactions with peptidoglycan carbohydrate. We propose that these proteins represent an evolutionarily primitive form of lectin-mediated innate immunity, and that they reveal intestinal strategies for maintaining symbiotic host-microbial relationships. PMID:16931762

Non-Carbohydrate Glycomimetics and Glycoprotein Surrogates as DC-SIGN Antagonists and Agonists

PubMed Central

Prost, Lynne R.; Grim, Joseph C.; Tonelli, Marco; Kiessling, Laura L.

2012-01-01

An understanding of the biological roles of lectins will be advanced by ligands that can inhibit or even recruit lectin function. To this end, glycomimetics, non-carbohydrate ligands that function analogously to endogenous carbohydrates, are being sought. The advantage of having such ligands is illustrated by the many roles of the protein DC-SIGN. DC-SIGN is a C-type lectin displayed on dendritic cells, where it binds to mannosides and fucosides to mediate interactions with other host cells or bacterial or viral pathogens. DC-SIGN engagement can modulate host immune responses (e.g., suppress autoimmunity) or benefit pathogens (e.g., promote HIV dissemination). DC-SIGN can bind to glycoconjugates, internalize glycosylated cargo for antigen processing, and transduce signals. DC-SIGN ligands can serve as inhibitors as well as probes of the lectin’s function, so they are especially valuable for elucidating and controlling DC-SIGN’s roles in immunity. We previously reported a small molecule that embodies key features of the carbohydrates that bind DC-SIGN. Here, we demonstrate that this non-carbohydrate ligand acts as a true glycomimetic. Using NMR HSQC experiments, we found that the compound mimics saccharide ligands: It occupies the same carbohydrate-binding site and interacts with the same side chain residues on DC-SIGN. The glycomimetic also is functional. It had been shown previously to antagonize DC-SIGN function but here we use it to generate DC-SIGN agonists. Specifically, appending this glycomimetic to a protein scaffold affords a conjugate that elicits key cellular signaling responses. Thus, the glycomimetic can give rise to functional glycoprotein surrogates that elicit lectin-mediated signaling. PMID:22747463

Network Analysis Reveals the Recognition Mechanism for Mannose-binding Lectins

NASA Astrophysics Data System (ADS)

Zhao, Yunjie; Jian, Yiren; Zeng, Chen; Computational Biophysics Lab Team

The specific carbohydrate binding of mannose-binding lectin (MBL) protein in plants makes it a very useful molecular tool for cancer cell detection and other applications. The biological states of most MBL proteins are dimeric. Using dynamics network analysis on molecular dynamics (MD) simulations on the model protein of MBL, we elucidate the short- and long-range driving forces behind the dimer formation. The results are further supported by sequence coevolution analysis. We propose a general framework for deciphering the recognition mechanism underlying protein-protein interactions that may have potential applications in signaling pathways.

Diversity in recognition of glycans by F-type lectins and galectins: molecular, structural, and biophysical aspects

PubMed Central

Vasta, Gerardo R.; Ahmed, Hafiz; Bianchet, Mario A.; Fernández-Robledo, José A.; Amzel, L. Mario

2013-01-01

Although lectins are “hard-wired” in the germline, the presence of tandemly arrayed carbohydrate recognition domains (CRDs), of chimeric structures displaying distinct CRDs, of polymorphic genes resulting in multiple isoforms, and in some cases, of a considerable recognition plasticity of their carbohydrate binding sites, significantly expand the lectin ligand-recognition spectrum and lectin functional diversification. Analysis of structural/functional aspects of galectins and F-lectins—the most recently identified lectin family characterized by a unique CRD sequence motif (a distinctive structural fold) and nominal specificity for l-Fuc—has led to a greater understanding of self/nonself recognition by proteins with tandemly arrayed CRDs. For lectins with a single CRD, however, recognition of self and nonself glycans can only be rationalized in terms of protein oligomerization and ligand clustering and presentation. Spatial and temporal changes in lectin expression, secretion, and local concentrations in extracellular microenvironments, as well as structural diversity and spatial display of their carbohydrate ligands on the host or microbial cell surface, are suggestive of a dynamic interplay of their recognition and effector functions in development and immunity. PMID:22973821

Influence of Trp flipping on carbohydrate binding in lectins. An example on Aleuria aurantia lectin AAL.

PubMed

Houser, Josef; Kozmon, Stanislav; Mishra, Deepti; Mishra, Sushil K; Romano, Patrick R; Wimmerová, Michaela; Koča, Jaroslav

2017-01-01

Protein-carbohydrate interactions are very often mediated by the stacking CH-π interactions involving the side chains of aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) or phenylalanine (Phe). Especially suitable for stacking is the Trp residue. Analysis of the PDB database shows Trp stacking for 265 carbohydrate or carbohydrate like ligands in 5 208 Trp containing motives. An appropriate model system to study such an interaction is the AAL lectin family where the stacking interactions play a crucial role and are thought to be a driving force for carbohydrate binding. In this study we present data showing a novel finding in the stacking interaction of the AAL Trp side chain with the carbohydrate. High resolution X-ray structure of the AAL lectin from Aleuria aurantia with α-methyl-l-fucoside ligand shows two possible Trp side chain conformations with the same occupation in electron density. The in silico data shows that the conformation of the Trp side chain does not influence the interaction energy despite the fact that each conformation creates interactions with different carbohydrate CH groups. Moreover, the PDB data search shows that the conformations are almost equally distributed across all Trp-carbohydrate complexes, which would suggest no substantial preference for one conformation over another.

Structural Investigation of a Novel N-Acetyl Glucosamine Binding Chi-Lectin Which Reveals Evolutionary Relationship with Class III Chitinases

PubMed Central

Patil, Dipak N.; Datta, Manali; Dev, Aditya; Dhindwal, Sonali; Singh, Nirpendra; Dasauni, Pushpanjali; Kundu, Suman; Sharma, Ashwani K.; Tomar, Shailly; Kumar, Pravindra

2013-01-01

The glycosyl hydrolase 18 (GH18) family consists of active chitinases as well as chitinase like lectins/proteins (CLPs). The CLPs share significant sequence and structural similarities with active chitinases, however, do not display chitinase activity. Some of these proteins are reported to have specific functions and carbohydrate binding property. In the present study, we report a novel chitinase like lectin (TCLL) from Tamarindus indica. The crystal structures of native TCLL and its complex with N-acetyl glucosamine were determined. Similar to the other CLPs of the GH18 members, TCLL lacks chitinase activity due to mutations of key active site residues. Comparison of TCLL with chitinases and other chitin binding CLPs shows that TCLL has substitution of some chitin binding site residues and more open binding cleft due to major differences in the loop region. Interestingly, the biochemical studies suggest that TCLL is an N-acetyl glucosamine specific chi-lectin, which is further confirmed by the complex structure of TCLL with N-acetyl glucosamine complex. TCLL has two distinct N-acetyl glucosamine binding sites S1 and S2 that contain similar polar residues, although interaction pattern with N-acetyl glucosamine varies extensively among them. Moreover, TCLL structure depicts that how plants utilize existing structural scaffolds ingenuously to attain new functions. To date, this is the first structural investigation of a chi-lectin from plants that explore novel carbohydrate binding sites other than chitin binding groove observed in GH18 family members. Consequently, TCLL structure confers evidence for evolutionary link of lectins with chitinases. PMID:23717482

cDNA cloning, molecular modeling and docking calculations of L-type lectins from Swartzia simplex var. grandiflora (Leguminosae, Papilionoideae), a member of the tribe Swartzieae.

PubMed

Maranhão, Paulo A C; Teixeira, Claudener S; Sousa, Bruno L; Barroso-Neto, Ito L; Monteiro-Júnior, José E; Fernandes, Andreia V; Ramos, Marcio V; Vasconcelos, Ilka M; Gonçalves, José F C; Rocha, Bruno A M; Freire, Valder N; Grangeiro, Thalles B

2017-07-01

The genus Swartzia is a member of the tribe Swartzieae, whose genera constitute the living descendants of one of the early branches of the papilionoid legumes. Legume lectins comprise one of the main families of structurally and evolutionarily related carbohydrate-binding proteins of plant origin. However, these proteins have been poorly investigated in Swartzia and to date, only the lectin from S. laevicarpa seeds (SLL) has been purified. Moreover, no sequence information is known from lectins of any member of the tribe Swartzieae. In the present study, partial cDNA sequences encoding L-type lectins were obtained from developing seeds of S. simplex var. grandiflora. The amino acid sequences of the S. simplex grandiflora lectins (SSGLs) were only averagely related to the known primary structures of legume lectins, with sequence identities not greater than 50-52%. The SSGL sequences were more related to amino acid sequences of papilionoid lectins from members of the tribes Sophoreae and Dalbergieae and from the Cladratis and Vataireoid clades, which constitute with other taxa, the first branching lineages of the subfamily Papilionoideae. The three-dimensional structures of 2 representative SSGLs (SSGL-A and SSGL-E) were predicted by homology modeling using templates that exhibit the characteristic β-sandwich fold of the L-type lectins. Molecular docking calculations predicted that SSGL-A is able to interact with D-galactose, N-acetyl-D-galactosamine and α-lactose, whereas SSGL-E is probably a non-functional lectin due to 2 mutations in the carbohydrate-binding site. Using molecular dynamics simulations followed by density functional theory calculations, the binding free energies of the interaction of SSGL-A with GalNAc and α-lactose were estimated as -31.7 and -47.5 kcal/mol, respectively. These findings gave insights about the carbohydrate-binding specificity of SLL, which binds to immobilized lactose but is not retained in a matrix containing D-GalNAc as ligand. Copyright © 2017 Elsevier Ltd. All rights reserved.

Insights into animal and plant lectins with antimicrobial activities.

PubMed

Dias, Renata de Oliveira; Machado, Leandro Dos Santos; Migliolo, Ludovico; Franco, Octavio Luiz

2015-01-05

Lectins are multivalent proteins with the ability to recognize and bind diverse carbohydrate structures. The glyco -binding and diverse molecular structures observed in these protein classes make them a large and heterogeneous group with a wide range of biological activities in microorganisms, animals and plants. Lectins from plants and animals are commonly used in direct defense against pathogens and in immune regulation. This review focuses on sources of animal and plant lectins, describing their functional classification and tridimensional structures, relating these properties with biotechnological purposes, including antimicrobial activities. In summary, this work focuses on structural-functional elucidation of diverse lectin groups, shedding some light on host-pathogen interactions; it also examines their emergence as biotechnological tools through gene manipulation and development of new drugs.

Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

PubMed Central

Chang, Wei-Chieh; Liu, Kai-Lun; Hsu, Fang-Ciao; Jeng, Shih-Tong; Cheng, Yi-Sheng

2012-01-01

Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (KA) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. PMID:22808208

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

DOE PAGES

Ng, Simon; Lin, Edith; Kitov, Pavel I.; ...

2015-04-10

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

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

Ng, Simon; Lin, Edith; Kitov, Pavel I.

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Fungal lectin MpL enables entry of protein drugs into cancer cells and their subcellular targeting.

PubMed

Å Urga, Simon; Nanut, Milica Perišić; Kos, Janko; Sabotič, Jerica

2017-04-18

Lectins have been recognized as promising carrier molecules for targeted drug delivery. They specifically bind carbohydrate moieties on cell membranes and trigger cell internalization. Fungal lectin MpL (Macrolepiota procera lectin) does not provoke cancer cell cytotoxicity but is able to bind aminopeptidase N (CD13) and integrin α3β1, two glycoproteins that are overexpressed on the membrane of tumor cells. Upon binding, MpL is endocytosed in a clathrin-dependent manner and accumulates initially in the Golgi apparatus and, finally, in the lysosomes. For effective binding and internalization a functional binding site on the α-repeat is needed. To test the potential of MpL as a carrier for delivering protein drugs to cancer cells we constructed fusion proteins consisting of MpL and the cysteine peptidase inhibitors cystatin C and clitocypin. The fused proteins followed the same endocytic route as the unlinked MpL. Peptidase inhibitor-MpL fusions impaired both the intracellular degradation of extracellular matrix and the invasiveness of cancer cells. MpL is thus shown in vitro to be a lectin that can enable protein drugs to enter cancer cells, enhance their internalization and sort them to lysosomes and the Golgi apparatus.

Cytotoxicity of Crude Lectins from Red Macroalgae from the Southern Coast of Java Island, Gunung Kidul Regency, Yogyakarta, Indonesia

NASA Astrophysics Data System (ADS)

Anam, C.; Chasanah, E.; Perdhana, B. P.; Fajarningsih, ND; Yusro, N. F.; Sari, A. M.; Nursiwi, A.; Praseptiangga, D.; Yunus, A.

2017-04-01

Lectins or carbohydrate-binding proteins, are widely distributed in nature, including in marine algae. It may have been considered that binding specificity of lectins to some carbohydrates provokes to produce many unique biological activities, including cell agglutination, mitogenic activity, and antitumor activity. The aim of this study was to determine the cytotoxicity of crude lectins from red macroalgae collected from the southern coast of Java Island, Gunung Kidul Regency, Yogyakarta against MCF-7 and HeLa cancer cells. In vitro MTT assay was used in this study. The results showed that less than 50% of MCF-7 and HeLa cancer cells growth were inhibited by the crude lectins from five species of red macro algae used in this study. The highest inhibition ability shown in the red alga A. nana was able to kill 47.68% of HeLa cervical cancer cells.

Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin.

PubMed

Palaniyar, Nades; Nadesalingam, Jeya; Clark, Howard; Shih, Michael J; Dodds, Alister W; Reid, Kenneth B M

2004-07-30

Collectins are a family of innate immune proteins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). The CRDs of these proteins recognize various microbial surface-specific carbohydrate patterns, particularly hexoses. We hypothesized that collectins, such as pulmonary surfactant proteins (SPs) SP-A and SP-D and serum protein mannose-binding lectin, could recognize nucleic acids, pentose-based anionic phosphate polymers. Here we show that collectins bind DNA from a variety of origins, including bacteria, mice, and synthetic oligonucleotides. Pentoses, such as arabinose, ribose, and deoxyribose, inhibit the interaction between SP-D and mannan, one of the well-studied hexose ligands for SP-D, and biologically relevant d-forms of the pentoses are better competitors than the l-forms. In addition, DNA and RNA polymer-related compounds, such as nucleotide diphosphates and triphosphates, also inhibit the carbohydrate binding ability of SP-D, or approximately 60 kDa trimeric recombinant fragments of SP-D that are composed of the alpha-helical coiled-coil neck region and three CRDs (SP-D(n/CRD)) or SP-D(n/CRD) with eight GXY repeats (SPD(GXY)(8)(n/CRD)). Direct binding and competition studies suggest that collectins bind nucleic acid via their CRDs as well as by their collagen-like regions, and that SP-D binds DNA more effectively than do SP-A and mannose-binding lectin at physiological salt conditions. Furthermore, the SP-D(GXY)(8)(n/CRD) fragments co-localize with DNA, and the protein competes the interaction between propidium iodide, a DNA-binding dye, and apoptotic cells. In conclusion, we show that collectins are a new class of proteins that bind free DNA and the DNA present on apoptotic cells by both their globular CRDs and collagen-like regions. Collectins may therefore play an important role in decreasing the inflammation caused by DNA in lungs and other tissues.

Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

PubMed Central

Basu, S; Mandal, C; Allen, A K

1988-01-01

A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin. Images Fig. 3. PMID:3140796

A single-molecule force spectroscopy study of the interactions between lectins and carbohydrates on cancer and normal cells

NASA Astrophysics Data System (ADS)

Zhao, Weidong; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

2013-03-01

The interaction forces between carbohydrates and lectins were investigated by single-molecule force spectroscopy on both cancer and normal cells. The binding kinetics was also studied, which shows that the carbohydrate-lectin complex on cancer cells is less stable than that on normal cells.The interaction forces between carbohydrates and lectins were investigated by single-molecule force spectroscopy on both cancer and normal cells. The binding kinetics was also studied, which shows that the carbohydrate-lectin complex on cancer cells is less stable than that on normal cells. Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c3nr00553d

Histochemical analysis of glycoconjugates in the skin of a catfish (arius tenuispinis, day).

PubMed

Al-Banaw, A; Kenngott, R; Al-Hassan, J M; Mehana, N; Sinowatz, F

2010-02-01

A histochemical study using conventional carbohydrate histochemistry (periodic-acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC-labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose-binding lectins LCA and PSA; the galactosamine-binding lectins DBA, SBA and GLS I; the glucosamine-binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose-binding lectin UEA and the sialic acid-specific lectin SNA. In addition, the galactose-binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N-acetylgalactosamine and N-acetylglucosamine residues.

The functional characterization and comparison of two single CRD containing C-type lectins with novel and typical key motifs from Portunus trituberculatus.

PubMed

Huang, Mengmeng; Mu, Changkao; Wu, Yuehong; Ye, Fei; Wang, Dan; Sun, Cong; Lv, Zhengbing; Han, Bingnan; Wang, Chunlin; Xu, Xue-Wei

2017-11-01

C-type lectins are a superfamily of Ca 2+ -dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including nonself-recognition and pathogen elimination. In the present study, two single-CRD containing C-type lectins were identified from swimming crab Portunus trituberculatus (designated as PtCTL-2 and PtCTL-3). The open reading frame (ORF) of PtCTL-2 encoded polypeptides of 485 amino acids with a signal peptide and a single carbohydrate-recognition domain (CRD), while PtCTL-3's ORF encoded polypeptides of 241 amino acids with a coiled-coil region and a single-CRD. The key motifs determining carbohydrate binding specificity in PtCTL-2 and PtCTL-3 were EPR (Glu-Pro-Arg) and QPD (Gln-Pro-Asp). EPR is a motif being identified for the first time, whereas QPD is a typical motif in C-type lectins. Different PAMPs binding features of the two recombinant proteins - PtCTL-2 (rPtCTL-2) and PtCTL-3 (rPtCTL-3) have been observed in our experiments. rPtCTL-2 could bind three pathogen-associated molecular patterns (PAMPs) with relatively high affinity, including glucan, lipopolysaccharide (LPS) and peptidoglycan (PGN), while rPtCTL-3 could barely bind any of them. However, rPtCTL-2 could bind seven kinds of microbes and rPtCTL-3 could bind six kinds in microbe binding assay. Moreover, rPtCTL-2 and rPtCTL-3 exhibited similar agglutination activity against Gram-positive bacteria, Gram-negative bacteria and fungi in agglutination assay. All these results illustrated that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) with broad nonself-recognition spectrum involved in immune defense against invaders. In addition, the results of carbohydrate binding specificity showed that PtCTL-2 with novel key motif had broad carbohydrate binding specificity, while PtCTL-3 with typical key motif possessed different carbohydrate binding specificity from the classical binding rule. Furthermore, PtCTL-2 and PtCTL-3 could also function as opsonin to enhance encapsulation of hemocytes against Ni-NTA beads. Copyright © 2017 Elsevier Ltd. All rights reserved.

Calculating binding free energies for protein-carbohydrate complexes.

PubMed

Hadden, Jodi A; Tessier, Matthew B; Fadda, Elisa; Woods, Robert J

2015-01-01

A variety of computational techniques may be applied to compute theoretical binding free energies for protein-carbohydrate complexes. Elucidation of the intermolecular interactions, as well as the thermodynamic effects, that contribute to the relative strength of receptor binding can shed light on biomolecular recognition, and the resulting initiation or inhibition of a biological process. Three types of free energy methods are discussed here, including MM-PB/GBSA, thermodynamic integration, and a non-equilibrium alternative utilizing SMD. Throughout this chapter, the well-known concanavalin A lectin is employed as a model system to demonstrate the application of these methods to the special case of carbohydrate binding.

Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif.

PubMed

Alenton, Rod Russel R; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo

2017-04-04

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation.

Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif

PubMed Central

Alenton, Rod Russel R.; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo

2017-01-01

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation. PMID:28374848

Protein Adsorption and Its Role in Bacterial Film Development

DTIC Science & Technology

1989-06-27

only the secondary antibody conjugated to alkaline phosphatase was used. Combined Amino Acids as Measured by HPLC We are interested in a simple, direct...specific assay for chitin that relies on the lectin, wheat germ agglutinin (WGA). Lectins are a general class of proteins that bind to carbohydrates. The...protein; 2) a new method for measuring combined amino acids (includes proteins) in seawater was shown to measure higher concentration than the old

Mannobiose Binding Induces Changes in Hydrogen Bonding and Protonation States of Acidic Residues in Concanavalin A As Revealed by Neutron Crystallography

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

Gerlits, Oksana O.; Coates, Leighton; Woods, Robert J.

Plant lectins are carbohydrate-binding proteins with various biomedical applications. Concanavalin A (Con A) holds promise in treating cancerous tumors. To better understand the Con A carbohydrate binding specificity, we obtained a room-temperature neutron structure of this legume lectin in complex with a disaccharide Manα1–2Man, mannobiose. The neutron structure afforded direct visualization of the hydrogen bonding between the protein and ligand, showing that the ligand is able to alter both protonation states and interactions for residues located close to and distant from the binding site. An unprecedented low-barrier hydrogen bond was observed forming between the carboxylic side chains of Asp28 andmore » Glu8, with the D atom positioned equidistant from the oxygen atoms having an O···D···O angle of 101.5°.« less

Fungal lectin MpL enables entry of protein drugs into cancer cells and their subcellular targeting

PubMed Central

Kos, Janko; Sabotič, Jerica

2017-01-01

Lectins have been recognized as promising carrier molecules for targeted drug delivery. They specifically bind carbohydrate moieties on cell membranes and trigger cell internalization. Fungal lectin MpL (Macrolepiota procera lectin) does not provoke cancer cell cytotoxicity but is able to bind aminopeptidase N (CD13) and integrin α3β1, two glycoproteins that are overexpressed on the membrane of tumor cells. Upon binding, MpL is endocytosed in a clathrin-dependent manner and accumulates initially in the Golgi apparatus and, finally, in the lysosomes. For effective binding and internalization a functional binding site on the α-repeat is needed. To test the potential of MpL as a carrier for delivering protein drugs to cancer cells we constructed fusion proteins consisting of MpL and the cysteine peptidase inhibitors cystatin C and clitocypin. The fused proteins followed the same endocytic route as the unlinked MpL. Peptidase inhibitor-MpL fusions impaired both the intracellular degradation of extracellular matrix and the invasiveness of cancer cells. MpL is thus shown in vitro to be a lectin that can enable protein drugs to enter cancer cells, enhance their internalization and sort them to lysosomes and the Golgi apparatus. PMID:28460472

Spermadhesin AQN1 is a candidate receptor molecule involved in the formation of the oviductal sperm reservoir in the pig.

PubMed

Ekhlasi-Hundrieser, Mahnaz; Gohr, Katrin; Wagner, Andrea; Tsolova, Miroslava; Petrunkina, Anna; Töpfer-Petersen, Edda

2005-09-01

Sperm are stored in the isthmic region of the oviduct under conditions that maintain viability and suppress early capacitation steps until ovulation occurs. The initial contact between sperm and oviductal epithelium is mediated by carbohydrate-protein interactions. In the pig, the carbohydrate recognition system has been shown to involve oligomannosyl structures. The spermadhesins AWN and AQN1 are the dominant porcine carbohydrate-binding sperm proteins. The objective of this study was to demonstrate that AQN1 contributes to sperm binding to the oviductal epithelium. AQN1 showed a broad carbohydrate-binding pattern as it recognizes both alpha- and beta-linked galactose as well as Manalpha1-3(Manalpha1-6)Man structures, whereas AWN bound only the galactose species. Binding of ejaculated sperm to oviductal epithelium was inhibited by addition of AQN1 but not by AWN. Mannose-binding sites were localized over the rostral region of the sperm head. Flow cytometry showed that, under capacitating conditions, the population of live sperm was shifted within 30 min toward an increase in the proportion of cells with low mannose- and high galactose-binding. The loss of mannose-binding sites was accompanied by the loss of AQN1 in sperm extracts and the significant reduction in the sperm-oviduct binding. The oviductal epithelium was shown by GNA-lectin histochemistry and by SDS-PAGE and lectin blotting of the apical membrane fraction to express mannose components that could be recognized by AQN1. These results demonstrate that the sperm lectin AQN1 fulfils the criteria for an oviduct receptor in the pig and may play a role in the formation of the oviductal sperm reservoir.

Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

USGS Publications Warehouse

Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

2004-01-01

Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

The sequence and structure of snake gourd (Trichosanthes anguina) seed lectin, a three-chain nontoxic homologue of type II RIPs.

PubMed

Sharma, Alok; Pohlentz, Gottfried; Bobbili, Kishore Babu; Jeyaprakash, A Arockia; Chandran, Thyageshwar; Mormann, Michael; Swamy, Musti J; Vijayan, M

2013-08-01

The sequence and structure of snake gourd seed lectin (SGSL), a nontoxic homologue of type II ribosome-inactivating proteins (RIPs), have been determined by mass spectrometry and X-ray crystallography, respectively. As in type II RIPs, the molecule consists of a lectin chain made up of two β-trefoil domains. The catalytic chain, which is connected through a disulfide bridge to the lectin chain in type II RIPs, is cleaved into two in SGSL. However, the integrity of the three-dimensional structure of the catalytic component of the molecule is preserved. This is the first time that a three-chain RIP or RIP homologue has been observed. A thorough examination of the sequence and structure of the protein and of its interactions with the bound methyl-α-galactose indicate that the nontoxicity of SGSL results from a combination of changes in the catalytic and the carbohydrate-binding sites. Detailed analyses of the sequences of type II RIPs of known structure and their homologues with unknown structure provide valuable insights into the evolution of this class of proteins. They also indicate some variability in carbohydrate-binding sites, which appears to contribute to the different levels of toxicity exhibited by lectins from various sources.

Photogenerated Lectin Sensors Produced by Thiol-Ene/Yne Photo-Click Chemistry in Aqueous Solution

PubMed Central

Norberg, Oscar; Lee, Irene H.; Aastrup, Teodor; Yan, Mingdi; Ramström, Olof

2012-01-01

The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance flow-through system with recurring injections of selected lectins with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study. PMID:22341757

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.

Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity

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

Moulaei, Tinoush; Shenoy, Shilpa R.; Giomarelli, Barbara

2010-10-28

Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviralmore » activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.« less

Computer simulation of protein—carbohydrate complexes: application to arabinose-binding protein and pea lectin

NASA Astrophysics Data System (ADS)

Rao, V. S. R.; Biswas, Margaret; Mukhopadhyay, Chaitali; Balaji, P. V.

1989-03-01

The CCEM method (Contact Criteria and Energy Minimisation) has been developed and applied to study protein-carbohydrate interactions. The method uses available X-ray data even on the native protein at low resolution (above 2.4 Å) to generate realistic models of a variety of proteins with various ligands. The two examples discussed in this paper are arabinose-binding protein (ABP) and pea lectin. The X-ray crystal structure data reported on ABP-β- L-arabinose complex at 2.8, 2.4 and 1.7 Å resolution differ drastically in predicting the nature of the interactions between the protein and ligand. It is shown that, using the data at 2.4 Å resolution, the CCEM method generates complexes which are as good as the higher (1.7 Å) resolution data. The CCEM method predicts some of the important hydrogen bonds between the ligand and the protein which are missing in the interpretation of the X-ray data at 2.4 Å resolution. The theoretically predicted hydrogen bonds are in good agreement with those reported at 1.7 Å resolution. Pea lectin has been solved only in the native form at 3 Å resolution. Application of the CCEM method also enables us to generate complexes of pea lectin with methyl-α- D-glucopyranoside and methyl-2,3-dimethyl-α- D-glucopyranoside which explain well the available experimental data in solution.

Isolation and Characterization of a Sex-Specific Lectin in a Marine Red Alga, Aglaothamnion oosumiense Itono

PubMed Central

Han, Jong Won; Klochkova, Tatyana A.; Shim, Jun Bo; Yoon, Kangsup

2012-01-01

In red algae, spermatial binding to female trichogynes is mediated by a lectin-carbohydrate complementary system. Aglaothamnion oosumiense is a microscopic filamentous red alga. The gamete recognition and binding occur at the surface of the hairlike trichogyne on the female carpogonium. Male spermatia are nonmotile. Previous studies suggested the presence of a lectin responsible for gamete recognition on the surface of female trychogynes. A novel N-acetyl-d-galactosamine-specific protein was isolated from female plants of A. oosumiense by affinity chromatography and named AOL1. The lectin was monomeric and did not agglutinate horse blood or human erythrocytes. The N-terminal amino acid sequence of the protein was analyzed, and degenerate primers were designed. A full-length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends-PCR (RACE-PCR). The cDNA was 1,095 bp in length and coded for a protein of 259 amino acids with a deduced molecular mass of 21.4 kDa, which agreed well with the protein data. PCR analysis using genomic DNA showed that both male and female plants have this gene. However, Northern blotting and two-dimensional electrophoresis showed that this protein was expressed 12 to 15 times more in female plants. The lectin inhibited spermatial binding to the trichogynes when preincubated with spermatia, suggesting its involvement in gamete binding. PMID:22865077

Lectin activity in mycelial extracts of Fusarium species.

PubMed

Bhari, Ranjeeta; Kaur, Bhawanpreet; Singh, Ram S

2016-01-01

Lectins are non-immunogenic carbohydrate-recognizing proteins that bind to glycoproteins, glycolipids, or polysaccharides with high affinity and exhibit remarkable ability to agglutinate erythrocytes and other cells. In the present study, ten Fusarium species previously not explored for lectins were screened for the presence of lectin activity. Mycelial extracts of F. fujikuroi, F. beomiformii, F. begoniae, F. nisikadoi, F. anthophilum, F. incarnatum, and F. tabacinum manifested agglutination of rabbit erythrocytes. Neuraminidase treatment of rabbit erythrocytes increased lectin titers of F. nisikadoi and F. tabacinum extracts, whereas the protease treatment resulted in a significant decline in agglutination by most of the lectins. Results of hapten inhibition studies demonstrated unique carbohydrate specificity of Fusarium lectins toward O-acetyl sialic acids. Activity of the majority of Fusarium lectins exhibited binding affinity to d-ribose, l-fucose, d-glucose, l-arabinose, d-mannitol, d-galactosamine hydrochloride, d-galacturonic acid, N-acetyl-d-galactosamine, N-acetyl-neuraminic acid, 2-deoxy-d-ribose, fetuin, asialofetuin, and bovine submaxillary mucin. Melibiose and N-glycolyl neuraminic acid did not inhibit the activity of any of the Fusarium lectins. Mycelial extracts of F. begoniae, F. nisikadoi, F. anthophilum, and F. incarnatum interacted with most of the carbohydrates tested. F. fujikuroi and F. anthophilum extracts displayed strong interaction with starch. The expression of lectin activity as a function of culture age was investigated. Most species displayed lectin activity on the 7th day of cultivation, and it varied with progressing of culture age. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Lectin-Binding Specificity of the Fertilization-Relevant Protein PDC-109 by Means of Surface Plasmon Resonance and Carbohydrate REcognition Domain EXcision-Mass Spectrometry.

PubMed

Defaus, Sira; Avilés, Manuel; Andreu, David; Gutiérrez-Gallego, Ricardo

2018-04-04

Seminal plasma proteins are relevant for sperm functionality and some appear responsible for establishing sperm interactions with the various environments along the female genital tract towards the oocyte. In recent years, research has focused on characterizing the role of these proteins in the context of reproductive biology, fertility diagnostics and treatment of related problems. Herein, we focus on the main protein of bovine seminal plasma, PDC-109 (BSP-A1/-A2), which by virtue of its lectin properties is involved in fertilization. By means of surface plasmon resonance, the interaction of PDC-109 with a panel of the most relevant glycosidic epitopes of mammals has been qualitatively and quantitatively characterized, and a higher affinity for carbohydrates containing fucose has been observed, in line with previous studies. Additionally, using the orthogonal technique of Carbohydrate REcognition Domain EXcision-Mass Spectrometry (CREDEX-MS), the recognition domain of the interaction complexes between PDC-109 and all fucosylated disaccharides [(Fuc-α1,(3,4,6)-GlcNAc)] has been defined, revealing the specific glycotope and the peptide domain likely to act as the PDC-109 carbohydrate binding site.

Genome-wide analysis of lectin receptor-like kinases in Populus

DOE PAGES

Yang, Yongil; Labbé, Jessy; Muchero, Wellington; ...

2016-09-01

Receptor-like kinases (RLKs) belong to a large protein family with over 600 members in Arabidopsis and over 1000 in rice. Among RLKs, the lectin receptor-like kinases (LecRLKs) possess a characteristic extracellular carbohydrate-binding lectin domain and play important roles in plant development and innate immunity. In addition, there are 75 and 173 LecRLKs in Arabidopsis and rice, respectively. However, little is known about LecRLKs in perennial woody plants.

A simple and accessible synthetic lectin for glucose recognition and sensing

NASA Astrophysics Data System (ADS)

Ke, Chenfeng; Destecroix, Harry; Crump, Matthew P.; Davis, Anthony P.

2012-09-01

Binding carbohydrates from water is a difficult task, even for the natural carbohydrate-binding proteins known as lectins. The design of synthetic lectin mimics is correspondingly challenging, especially if good selectivities are required. In previous work we showed that success is possible, but only for complex polycyclic architectures that require lengthy and low-yielding syntheses; for example, one glucose-selective system was made in 21 steps and only 0.1% overall yield. Here we report the discovery of a simple monocyclic host that matches the earlier designs, but is far more accessible as it is prepared in just five steps and 23% overall yield. The new synthetic lectin binds glucose with excellent selectivity versus other common monosaccharides (for example, ∼50:1 versus galactose) and sufficient affinity for glucose sensing at the concentrations found in blood. It also features a built-in signalling system in the form of strong and guest-dependent fluorescence emission. The effectiveness and simplicity of this molecule suggests the potential for development into a new methodology for practical glucose monitoring.

Development of Seaweed-based Biopolymers for Edible Films and Lectins

NASA Astrophysics Data System (ADS)

Praseptiangga, D.

2017-04-01

Marine macroalgae (seaweeds) as one of important groups of biopolymers play an important role in human life. Biopolymers have been studied regarding their film-forming properties to produce edible films intended as food packaging and active ingredient carriers. Edible film, a thin layer or which is an integral part of food and can be eaten together with, have been used to avoid food quality deterioration due to physico-chemical changes, texture changes, or chemical reactions. Film-forming materials can be utilized individually or as mixed composite blends. Proteins and polysaccharides used for their mechanical and structural properties, and hydrophobic substances (lipids, essential oils, and emulsifiers) to provide good moisture barrier properties. In addition, bioactive substances from marine natural products, including seaweeds, have been explored for being used in the fields of medicine, food science, pharmaceutical science, biochemistry, and glycobiology. Among them, lectins or carbohydrate-binding proteins from seaweeds have recently been remarked. Lectins (hemagglutinins) are widely distributed in nature and also good candidates in such prospecting of seaweeds. They are useful as convenient tools to discriminate differences in carbohydrate structures and reveal various biological activities through binding and interacting to carbohydrates, suggesting that they are promising candidates for medicinal and clinical application.

Plant lectins: the ties that bind in root symbiosis and plant defense.

PubMed

De Hoff, Peter L; Brill, Laurence M; Hirsch, Ann M

2009-07-01

Lectins are a diverse group of carbohydrate-binding proteins that are found within and associated with organisms from all kingdoms of life. Several different classes of plant lectins serve a diverse array of functions. The most prominent of these include participation in plant defense against predators and pathogens and involvement in symbiotic interactions between host plants and symbiotic microbes, including mycorrhizal fungi and nitrogen-fixing rhizobia. Extensive biological, biochemical, and molecular studies have shed light on the functions of plant lectins, and a plethora of uncharacterized lectin genes are being revealed at the genomic scale, suggesting unexplored and novel diversity in plant lectin structure and function. Integration of the results from these different types of research is beginning to yield a more detailed understanding of the function of lectins in symbiosis, defense, and plant biology in general.

Structure prediction and functional analysis of a non-permutated lectin from Dioclea grandiflora.

PubMed

de Sousa, Bruno Lopes; Nagano, Celso Shiniti; Simões, Rafael da Conceição; Silva-Filho, José Caetano; Cunha, Rodrigo Maranguape da Silva; Cajazeiras, João Batista; do Nascimento, Kyria Santiago; Cavada, Benildo Sousa

2016-12-01

Legume lectins have been widely studied and applied for many purposes in the last few decades, but many of their physiological aspects remain elusive. The Diocleinae legume subtribe, which includes intensively explored lectins, such as ConA, presents an unusual and extensive post-translational process which results in minor alterations in protein structure, in turn making its function elusive. Despite previous reports about Diocleinae precursor activity, no structural or functional analyses have ever been carried out to understand the impacts of post-translational processing relative to lectin structure and binding specificity. Here we analyzed the functionality of a non glycosylated, recombinantly expressed lectin precursor from Dioclea grandiflora through inhibition assays, corroborating the experimental data with structural information generated by molecular modeling, docking calculations and molecular dynamics simulations. We demonstrate that Diocleinae precursors are active and share the same carbohydrate specificity as mature lectins. At the same time, however, subtle structural alterations were detected and mostly result in an "incomplete" functionality of the precursor, as consequence of an immature binding site and an unstructured tetramer interface, affecting carbohydrate binding and oligomer formation, respectively. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Lectins: production and practical applications

PubMed Central

2010-01-01

Lectins are proteins found in a diversity of organisms. They possess the ability to agglutinate erythrocytes with known carbohydrate specificity since they have at least one non-catalytic domain that binds reversibly to specific monosaccharides or oligosaccharides. This articles aims to review the production and practical applications of lectins. Lectins are isolated from their natural sources by chromatographic procedures or produced by recombinant DNA technology. The yields of animal lectins are usually low compared with the yields of plant lectins such as legume lectins. Lectins manifest a diversity of activities including antitumor, immunomodulatory, antifungal, HIV-1 reverse transcriptase inhibitory, and anti-insect activities, which may find practical applications. A small number of lectins demonstrate antibacterial and anti-nematode activities. PMID:20890754

Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus.

PubMed

Gardères, Johan; Domart-Coulon, Isabelle; Marie, Arul; Hamer, Bojan; Batel, Renato; Müller, Werner E G; Bourguet-Kondracki, Marie-Lise

2016-10-01

Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine-agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino-acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemagglutination assay, and its activity was found to be calcium dependent. Clathrilectin, a protein complex of 3200kDa estimated by gel filtration, is composed of monomers with apparent molecular masses of 208 and 180kDa estimated on 10% SDS-PAGE. Nine internal peptides were identified using proteomic analyses, and compared to protein libraries from the demosponge Amphimedon queenslandica and a calcareous sponge Sycon sp. from the Adriatic Sea. The clathrilectin is the first lectin isolated from a calcareous sponge and displays homologies with predicted sponge proteins potentially involved in cell aggregation and interaction with bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

Purification and Characterization of a Mucin Specific Mycelial Lectin from Aspergillus gorakhpurensis: Application for Mitogenic and Antimicrobial Activity

PubMed Central

Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Background Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Methods Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Results Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5–9.5, while optimum temperature for lectin activity was 20–30°C. Lectin was stable within a pH range of 7.0–10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. Conclusion This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin. PMID:25286160

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

PubMed

Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin.

Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis.

PubMed

Moriuchi, Hiromi; Unno, Hideaki; Goda, Shuichiro; Tateno, Hiroaki; Hirabayashi, Jun; Hatakeyama, Tomomitsu

2015-07-01

CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

A lectin of a non-invasive apple snail as an egg defense against predation alters the rat gut morphophysiology.

PubMed

Ituarte, Santiago; Brola, Tabata Romina; Fernández, Patricia Elena; Mu, Huawei; Qiu, Jian-Wen; Heras, Horacio; Dreon, Marcos Sebastián

2018-01-01

The eggs of the freshwater Pomacea apple snails develop above the water level, exposed to varied physical and biological stressors. Their high hatching success seems to be linked to their proteins or perivitellins, which surround the developing embryo providing nutrients, sunscreens and varied defenses. The defensive mechanism has been unveiled in P. canaliculata and P. maculata eggs, where their major perivitellins are pigmented, non-digestible and provide a warning coloration while another perivitellin acts as a toxin. In P. scalaris, a species sympatric to the former, the defense strategy seems different, since no toxin was found and the major perivitellin, PsSC, while also colored and non-digestible, is a carbohydrate-binding protein. In this study we examine the structure and function of PsSC by sequencing its subunits, characterizing its carbohydrate binding profile and evaluating its effect on gut cells. Whereas cDNA sequencing and database search showed no lectin domain, glycan array carbohydrate binding profile revealed a strong specificity for glycosphingolipids and ABO group antigens. Moreover, PsSC agglutinated bacteria in a dose-dependent manner. Inspired on the defensive properties of seed lectins we evaluated the effects of PsSC on intestinal cells both in vitro (Caco-2 and IEC-6 cells) and in the gastrointestinal tract of rats. PsSC binds to Caco-2 cell membranes without reducing its viability, while a PsSC-containing diet temporarily induces large epithelium alterations and an increased absorptive surface. Based on these results, we propose that PsSC is involved in embryo defenses by altering the gut morphophysiology of potential predators, a convergent role to plant defensive lectins.

Carbohydrate binding properties of the stinging nettle (Urtica dioica) rhizome lectin.

PubMed

Shibuya, N; Goldstein, I J; Shafer, J A; Peumans, W J; Broekaert, W F

1986-08-15

The interaction of the stinging nettle rhizome lectin (UDA) with carbohydrates was studied by using the techniques of quantitative precipitation, hapten inhibition, equilibrium dialysis, and uv difference spectroscopy. The Carbohydrate binding site of UDA was determined to be complementary to an N,N',N"-triacetylchitotriose unit and proposed to consist of three subsites, each of which has a slightly different binding specificity. UDA also has a hydrophobic interacting region adjacent to the carbohydrate binding site. Equilibrium dialysis and uv difference spectroscopy revealed that UDA has two carbohydrate binding sites per molecule consisting of a single polypeptide chain. These binding sites either have intrinsically different affinities for ligand molecules, or they may display negative cooperativity toward ligand binding.

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.

Plant Lectins and Lectin Receptor-Like Kinases: How Do They Sense the Outside?

PubMed Central

Bellande, Kevin; Bono, Jean-Jacques; Savelli, Bruno; Jamet, Elisabeth; Canut, Hervé

2017-01-01

Lectins are fundamental to plant life and have important roles in cell-to-cell communication; development and defence strategies. At the cell surface; lectins are present both as soluble proteins (LecPs) and as chimeric proteins: lectins are then the extracellular domains of receptor-like kinases (LecRLKs) and receptor-like proteins (LecRLPs). In this review; we first describe the domain architectures of proteins harbouring G-type; L-type; LysM and malectin carbohydrate-binding domains. We then focus on the functions of LecPs; LecRLKs and LecRLPs referring to the biological processes they are involved in and to the ligands they recognize. Together; LecPs; LecRLKs and LecRLPs constitute versatile recognition systems at the cell surface contributing to the detection of symbionts and pathogens; and/or involved in monitoring of the cell wall structure and cell growth. PMID:28561754

Plant Lectins and Lectin Receptor-Like Kinases: How Do They Sense the Outside?

PubMed

Bellande, Kevin; Bono, Jean-Jacques; Savelli, Bruno; Jamet, Elisabeth; Canut, Hervé

2017-05-31

Lectins are fundamental to plant life and have important roles in cell-to-cell communication; development and defence strategies. At the cell surface; lectins are present both as soluble proteins (LecPs) and as chimeric proteins: lectins are then the extracellular domains of receptor-like kinases (LecRLKs) and receptor-like proteins (LecRLPs). In this review; we first describe the domain architectures of proteins harbouring G-type; L-type; LysM and malectin carbohydrate-binding domains. We then focus on the functions of LecPs; LecRLKs and LecRLPs referring to the biological processes they are involved in and to the ligands they recognize. Together; LecPs; LecRLKs and LecRLPs constitute versatile recognition systems at the cell surface contributing to the detection of symbionts and pathogens; and/or involved in monitoring of the cell wall structure and cell growth.

A role for carbohydrate recognition in mammalian sperm-egg binding

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

Clark, Gary F., E-mail: clarkgf@health.missouri.edu

Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the eggmore » cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented.« less

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

Yang, Yongil; Labbé, Jessy; Muchero, Wellington

Receptor-like kinases (RLKs) belong to a large protein family with over 600 members in Arabidopsis and over 1000 in rice. Among RLKs, the lectin receptor-like kinases (LecRLKs) possess a characteristic extracellular carbohydrate-binding lectin domain and play important roles in plant development and innate immunity. In addition, there are 75 and 173 LecRLKs in Arabidopsis and rice, respectively. However, little is known about LecRLKs in perennial woody plants.

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.

Structure, properties and enhanced expression of galactose-binding C-type lectins in mucous cells of gills from freshwater Japanese eels (Anguilla japonica).

PubMed

Mistry, A C; Honda, S; Hirose, S

2001-11-15

Using a Japanese-eel (Anguilla japonica) gill cDNA subtraction library, two novel beta-d-galactose-binding lectins were identified that belong to group VII of the animal C-type lectin family. The eel C-type lectins, termed eCL-1 and eCL-2, are simple lectins composed of 163 amino acid residues, including a 22-residue signal peptide for secretion and a single carbohydrate-recognition domain (CRD) of approximately 130 residues typical of C-type lectins. The galactose specificity of the CRD was suggested by the presence of a QPD motif and confirmed by a competitive binding assay. Using Ruthenium Red staining, the lectins were shown to bind Ca(2+) ions. SDS/PAGE showed that native eCL-1 and eCL-2 have an SDS-resistant octameric structure (a tetramer of disulphide-linked dimers). Northern and Western blot analyses demonstrated high-level expression of eCL-1 and eCL-2 mRNAs and their protein products in gills from freshwater eels, which decreased markedly when the eels were transferred from freshwater to seawater. Immunohistochemistry showed that the eel lectins are localized in the exocrine mucous cells of the gill.

BEL β-trefoil: a novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms.

PubMed

Bovi, Michele; Cenci, Lucia; Perduca, Massimiliano; Capaldi, Stefano; Carrizo, Maria E; Civiero, Laura; Chiarelli, Laurent R; Galliano, Monica; Monaco, Hugo L

2013-05-01

A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

Plant lectins as defense proteins against phytophagous insects.

PubMed

Vandenborre, Gianni; Smagghe, Guy; Van Damme, Els J M

2011-09-01

One of the most important direct defense responses in plants against the attack by phytophagous insects is the production of insecticidal peptides or proteins. One particular class of entomotoxic proteins present in many plant species is the group of carbohydrate-binding proteins or lectins. During the last decade a lot of progress was made in the study of a few lectins that are expressed in response to herbivory by phytophagous insects and the insecticidal properties of plant lectins in general. This review gives an overview of lectins with high potential for the use in pest control strategies based on their activity towards pest insects. In addition, potential target sites for lectins inside the insect and the mode of action are discussed. In addition, the effect of plant lectins on non-target organisms such as beneficial insects as well as on human/animal consumers is discussed. It can be concluded that some insecticidal lectins are useful tools that can contribute to the development of integrated pest management strategies with minimal effect(s) on non-target organisms. Copyright © 2011 Elsevier Ltd. All rights reserved.

Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

PubMed Central

Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

2015-01-01

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis).

PubMed

Darville, Lancia N F; Merchant, Mark E; Maccha, Venkata; Siddavarapu, Vivekananda Reddy; Hasan, Azeem; Murray, Kermit K

2012-02-01

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35kDa protein was ~98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates. Copyright © 2011 Elsevier Inc. All rights reserved.

Lectins in human pathogenic fungi.

PubMed

Gallegos, Belém; Martínez, Ruth; Pérez, Laura; Del Socorro Pina, María; Perez, Eduardo; Hernández, Pedro

2014-01-01

Lectins are carbohydrate-binding proteins widely distributed in nature. They constitute a highly diverse group of proteins consisting of many different protein families that are, in general, structurally unrelated. In the last few years, mushroom and other fungal lectins have attracted wide attention due to their antitumour, antiproliferative and immunomodulatory activities. The present mini-review provides concise information about recent developments in understanding lectins from human pathogenic fungi. A bibliographic search was performed in the Science Direct and PubMed databases, using the following keywords "lectin", "fungi", "human" and "pathogenic". Lectins present in fungi have been classified; however, the role played by lectins derived from human pathogenic fungi in infectious processes remains uncertain; thus, this is a scientific field requiring more research. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Lectins in fish skin: do they play a role in host-monogenean interactions?

PubMed

Buchmann, K

2001-09-01

Mucus samples from rainbow trout skin with or without infections by Gyrodactylus derjavini were tested for the presence of lectins reacting with mannose, galactose and lactose. The samples inhibited the binding of biotinylated lectins (from Canavalia ensiformis, Artocarpus integrifolia and Erythrina corallodendron, respectively) to microtitre plates with covalently bound carbohydrates (mannopyranoside, galactopyranoside and lactose, respectively). However, the inhibition of C. ensiformis and A. integrifolia lectins was slightly greater when mucus from infected (but recovering) fish was used, suggesting an increase of mannose and galactose binding lectins in fish skin exposed to parasites. As mannose, galactose and lactose are present on the glycocalyx of Gyrodactylus derjavini, it is suggested that lectins could play a dual role in interactions between fish hosts and their monogenean parasites. Thus, recognition between parasite and host and also host responses towards parasite infections could both, at least partly, involve carbohydrate-lectin binding.

Differential Lectin Binding Patterns Identify Distinct Heart Regions in Giant Danio (Devario aequipinnatus) and Zebrafish (Danio rerio) Hearts

PubMed Central

Manalo, Trina; May, Adam; Quinn, Joshua; Lafontant, Dominique S.; Shifatu, Olubusola; He, Wei; Gonzalez-Rosa, Juan M.; Burns, Geoffrey C.; Burns, Caroline E.; Burns, Alan R.; Lafontant, Pascal J.

2016-01-01

Lectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish (Danio rerio) and giant danio (Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins—wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)—in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins’ ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts. PMID:27680670

Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

PubMed Central

Venable, Alison; Mitalipova, Maisam; Lyons, Ian; Jones, Karen; Shin, Soojung; Pierce, Michael; Stice, Steven

2005-01-01

Background Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent state of hESCs because binding percentages and binding localization of these lectins are similar to those of SSEA-4. Non-binding lectins, DBA and LTL, may identify differentiated cell types; however, we did not find these lectins to bind to pluripotent SSEA-4 positive hESCs. This work represents a fundamental base to systematically classify pluripotent hESCs, and in future studies these lectins may be used to distinguish differentiated hESC types based on glycan presentation that accompanies differentiation. PMID:16033656

Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins.

PubMed

Gray, Christopher J; Sánchez-Ruíz, Antonio; Šardzíková, Ivana; Ahmed, Yassir A; Miller, Rebecca L; Reyes Martinez, Juana E; Pallister, Edward; Huang, Kun; Both, Peter; Hartmann, Mirja; Roberts, Hannah N; Šardzík, Robert; Mandal, Santanu; Turnbull, Jerry E; Eyers, Claire E; Flitsch, Sabine L

2017-04-18

The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.

The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. Homologies with mammalian and insect lectins.

PubMed

Giga, Y; Ikai, A; Takahashi, K

1987-05-05

The complete amino acid sequence of echinoidin, the proposed name for a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina, has been determined by sequencing the peptides obtained from tryptic, Staphylococcus aureus V8 protease, chymotryptic, and thermolysin digestions. Echinoidin is a multimeric protein (Giga, Y., Sutoh, K., and Ikai, A. (1985) Biochemistry 24, 4461-4467) whose subunit consists of a total of 147 amino acid residues and one carbohydrate chain attached to Ser38. The molecular weight of the polypeptide without carbohydrate was calculated to be 16,671. Each polypeptide chain contains seven half-cystines, and six of them form three disulfide bonds in the single polypeptide chain (Cys3-Cys14, Cys31-Cys141, and Cys116-Cys132), while Cys2 is involved in an interpolypeptide disulfide linkage. From secondary structure prediction by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222) the protein appears to be rich in beta-sheet and beta-turn structures and poor in alpha-helical structure. The sequence of the COOH-terminal half of echinoidin is highly homologous to those of the COOH-terminal carbohydrate recognition portions of rat liver mannose-binding protein and several other hepatic lectins. This COOH-terminal region of echinoidin is also homologous to the central portion of the lectin from the flesh fly Sarcophaga peregrina. Moreover, echinoidin contains an Arg-Gly-Asp sequence which has been proposed to be a basic functional unit in cellular recognition proteins.

Structure, properties and enhanced expression of galactose-binding C-type lectins in mucous cells of gills from freshwater Japanese eels (Anguilla japonica).

PubMed Central

Mistry, A C; Honda, S; Hirose, S

2001-01-01

Using a Japanese-eel (Anguilla japonica) gill cDNA subtraction library, two novel beta-d-galactose-binding lectins were identified that belong to group VII of the animal C-type lectin family. The eel C-type lectins, termed eCL-1 and eCL-2, are simple lectins composed of 163 amino acid residues, including a 22-residue signal peptide for secretion and a single carbohydrate-recognition domain (CRD) of approximately 130 residues typical of C-type lectins. The galactose specificity of the CRD was suggested by the presence of a QPD motif and confirmed by a competitive binding assay. Using Ruthenium Red staining, the lectins were shown to bind Ca(2+) ions. SDS/PAGE showed that native eCL-1 and eCL-2 have an SDS-resistant octameric structure (a tetramer of disulphide-linked dimers). Northern and Western blot analyses demonstrated high-level expression of eCL-1 and eCL-2 mRNAs and their protein products in gills from freshwater eels, which decreased markedly when the eels were transferred from freshwater to seawater. Immunohistochemistry showed that the eel lectins are localized in the exocrine mucous cells of the gill. PMID:11695997

Lectindb: a plant lectin database.

PubMed

Chandra, Nagasuma R; Kumar, Nirmal; Jeyakani, Justin; Singh, Desh Deepak; Gowda, Sharan B; Prathima, M N

2006-10-01

Lectins, a class of carbohydrate-binding proteins, are now widely recognized to play a range of crucial roles in many cell-cell recognition events triggering several important cellular processes. They encompass different members that are diverse in their sequences, structures, binding site architectures, quaternary structures, carbohydrate affinities, and specificities as well as their larger biological roles and potential applications. It is not surprising, therefore, that the vast amount of experimental data on lectins available in the literature is so diverse, that it becomes difficult and time consuming, if not impossible to comprehend the advances in various areas and obtain the maximum benefit. To achieve an effective use of all the data toward understanding the function and their possible applications, an organization of these seemingly independent data into a common framework is essential. An integrated knowledge base ( Lectindb, http://nscdb.bic.physics.iisc.ernet.in ) together with appropriate analytical tools has therefore been developed initially for plant lectins by collating and integrating diverse data. The database has been implemented using MySQL on a Linux platform and web-enabled using PERL-CGI and Java tools. Data for each lectin pertain to taxonomic, biochemical, domain architecture, molecular sequence, and structural details as well as carbohydrate and hence blood group specificities. Extensive links have also been provided for relevant bioinformatics resources and analytical tools. Availability of diverse data integrated into a common framework is expected to be of high value not only for basic studies in lectin biology but also for basic studies in pursuing several applications in biotechnology, immunology, and clinical practice, using these molecules.

Quantitative assessment of the multivalent protein-carbohydrate interactions on silicon.

PubMed

Yang, Jie; Chazalviel, Jean-Noël; Siriwardena, Aloysius; Boukherroub, Rabah; Ozanam, François; Szunerits, Sabine; Gouget-Laemmel, Anne Chantal

2014-10-21

A key challenge in the development of glycan arrays is that the sensing interface be fabricated reliably so as to ensure the sensitive and accurate analysis of the protein-carbohydrate interaction of interest, reproducibly. These goals are complicated in the case of glycan arrays as surface sugar density can influence dramatically the strength and mode of interaction of the sugar ligand at any interface with lectin partners. In this Article, we describe the preparation of carboxydecyl-terminated crystalline silicon (111) surfaces onto which are grafted either mannosyl moieties or a mixture of mannose and spacer alcohol molecules to provide "diluted" surfaces. The fabrication of the silicon surfaces was achieved efficiently through a strategy implicating a "click" coupling step. The interactions of these newly fabricated glycan interfaces with the lectin, Lens culinaris, have been characterized using quantitative infrared (IR) spectroscopy in the attenuated total geometry (ATR). The density of mannose probes and lectin targets was precisely determined for the first time by the aid of special IR calibration experiments, thus allowing for the interpretation of the distribution of mannose and its multivalent binding with lectins. These experimental findings were accounted for by numerical simulations of lectin adsorption.

Expression and localization in the developing cerebellum of the carbohydrate epitopes revealed by Elec-39, an IgM monoclonal antibody related to HNK-1.

PubMed

Kuchler, S; Zanetta, J P; Bon, S; Zaepfel, M; Massoulie, J; Vincendon, G

1991-01-01

The immunochemical and immunocytochemical reactivity of an anti-carbohydrate monoclonal antibody (Elec-39), obtained against acetylcholinesterase from Electrophorus electricus electric organ, was followed during the postnatal development of the rat cerebellum. The specificity of this antibody resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1 and NSP-4, as well as IgMs that occur in some human neuropathies. As revealed by immunoblotting techniques, the reactivity of Elec-39 is maximum around postnatal days 10-12. At this age, the antibody reveals eight major proteins of mol. wt ranging between 14 and 150 kDa. Some of them (with mol. wts of 14, 18, 28 and 31 kDa) are transiently expressed. They correspond to previously identified glycoproteins binding to the plant lectin concanavalin A and binding also to the endogenous mannose-binding lectin CSL and endogenous membrane-bound mannose-binding lectin. In young animals, an important staining with the Elec-39 antibody can be observed on postmitotic precursors of granule cells, on astrocyte processes in the external granular layer, on newly formed parallel fibres and on unmyelinated axons of the white matter. In adult animals, the labelling is localized essentially in myelin and also in the cytoplasm of astrocytes. These results are discussed in relation to ontogenetic phenomena occurring during cerebellar development and the potential role of the carbohydrate epitope revealed with Elec-39 as a determinant in cell adhesion processes.

Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

PubMed

Hatakeyama, Tomomitsu; Ichise, Ayaka; Unno, Hideaki; Goda, Shuichiro; Oda, Tatsuya; Tateno, Hiroaki; Hirabayashi, Jun; Sakai, Hitomi; Nakagawa, Hideyuki

2017-08-01

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure. © 2017 The Protein Society.

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

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycoclusters to lectins and tetanus toxin C-fragment

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Minoura, Norihiko

2011-03-01

We develop a fluorescent ruthenium metalloglycocluster for use as a powerful molecular probe in evaluating the binding between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analyses. Changes in the FE and FP of these metalloglycoclusters are measured following the addition of lectin [peanut agglutinin (PNA), Ricinus communis agglutinin 120, Concanavalin A (ConA), or wheat germ agglutinin] or tetanus toxin c-fragment (TCF). After the addition of PNA, the FE spectrum of [Ru(bpy-2Gal)3] shows a new emission peak and the FP value of [Ru(bpy-2Gal)3] increases. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] shows a new emission peak and the FP value increases on addition of ConA. Because other combinations of metalloglycoclusters and lectins show little change, specific binding of galactose to PNA and that of glucose to ConA are confirmed by the FE and FP measurements. Resulting dissociation constants (Kd) prove that the metalloglycoclusters with highly clustered carbohydrates show higher affinity for the respective lectins than those with less clustered carbohydrates. Furthermore, specific binding of [Ru(bpy-2Gal)3] to TCF was confirmed by the FP measurement.

Fluorescent carbohydrate probes for cell lectins

NASA Astrophysics Data System (ADS)

Galanina, Oxana; Feofanov, Alexei; Tuzikov, Alexander B.; Rapoport, Evgenia; Crocker, Paul R.; Grichine, Alexei; Egret-Charlier, Marguerite; Vigny, Paul; Le Pendu, Jacques; Bovin, Nicolai V.

2001-09-01

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe x-PAA-flu, Sia 2-PAA-flu, GlcNAc 2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe x-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia 2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe x-PAA- 3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe x-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

A novel L-ficolin/mannose-binding lectin chimeric molecule with enhanced activity against Ebola virus.

PubMed

Michelow, Ian C; Dong, Mingdong; Mungall, Bruce A; Yantosca, L Michael; Lear, Calli; Ji, Xin; Karpel, Marshall; Rootes, Christina L; Brudner, Matthew; Houen, Gunnar; Eisen, Damon P; Kinane, T Bernard; Takahashi, Kazue; Stahl, Gregory L; Olinger, Gene G; Spear, Gregory T; Ezekowitz, R Alan B; Schmidt, Emmett V

2010-08-06

Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.

Glycodendritic structures based on Boltorn hyperbranched polymers and their interactions with Lens culinaris lectin.

PubMed

Arce, Eva; Nieto, Pedro M; Díaz, Vicente; Castro, Rossana García; Bernad, Antonio; Rojo, Javier

2003-01-01

Multivalent scaffolds bearing carbohydrates have been prepared to mediate biological processes where carbohydrates are involved. These systems consist of dendritic structures based on Boltorn H20 and H30 hyperbranched polymers to which carbohydrates are linked through a convenient spacer. Mannose has been chosen as a sugar unit to test the viability of this strategy. These glycodendritic compounds have been prepared in a few steps with good yields, showing a high solubility in physiological media and low toxicity. The binding of these dendritic polymers to the mannose-binding lectin Lens culinaris (LCA) was studied using STD-NMR experiments and quantitative precipitation assays. The results demonstrate the existence of a clear interaction between the mannose derivative systems and the Lens lectin where the dendritic scaffold does not have an important role in mannose binding but supplies the necessary multivalence for lectin cluster formation. These glycodendritic structures are able to interact with a receptor, and therefore they can be considered as promising tools for biological studies.

The β-Prism Lectin Domain of Vibrio cholerae Hemolysin Promotes Self-assembly of the β-Pore-forming Toxin by a Carbohydrate-independent Mechanism*

PubMed Central

Ganguly, Sreerupa; Mukherjee, Amarshi; Mazumdar, Budhaditya; Ghosh, Amar N.; Banerjee, Kalyan K.

2014-01-01

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC50) without the lectin domain, and mutant VCCD617A with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 107 m−1. However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC50 was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer. PMID:24356964

Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis.

PubMed

Wei, Xiumei; Yang, Jianmin; Liu, Xiangquan; Yang, Dinglong; Xu, Jie; Fang, Jinghui; Wang, Weijun; Yang, Jialong

2012-08-01

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition. Copyright © 2012 Elsevier Ltd. All rights reserved.

Functional characterization of chitin-binding lectin from Solanum integrifolium containing anti-fungal and insecticidal activities.

PubMed

Chen, Chang-Shan; Chen, Chun-Yi; Ravinath, Divya Malathy; Bungahot, Agustina; Cheng, Chi-Ping; You, Ren-In

2018-01-03

Along with the rapid development of glycomic tools, the study of lectin-carbohydrate interactions has expanded, opening the way for applications in the fields of analytic, diagnostic, and drug delivery. Chitin-binding lectins (CBLs) play roles in immune defense against chitin-containing pathogens. CBLs from species of the Solanaceae family, such as tomato, potato and jimsonweed, display different binding specificities to sugar chains containing poly-N-acetyllactosamine. In this report, CBLs from Solanum integrifolium were isolated by ion exchange chromatography. The fractions showed hemagglutination activity (HA). The recombinant CBL in the 293F cell culture supernatant was able to inhibit the growth of Rhizoctonia solani and Colletotrichum gloeosporioide. Furthermore, the carbohydrate-binding property of CBLs was confirmed with the inhibition of HA. Binding of CBL to Spodoptera frugiperda (sf21) insect cells can partly be inhibited by N-Acetylglucosamine (GlcNAc), which is related to decrease mitochondrial membrane potential of sf21 cells. The results showed that CBL exhibited antifungal properties and inhibited insect cell growth, which is directly correlated to the lectin-carbohydrate interaction. Further identification and characterization of CBLs will help to broaden their scope of application in plant defense and in biomedical applications.

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.

Dual recognition activity of a rhamnose-binding lectin to pathogenic bacteria and zooxanthellae in stony coral Pocillopora damicornis.

PubMed

Zhou, Zhi; Yu, Xiaopeng; Tang, Jia; Zhu, Yunjie; Chen, Guangmei; Guo, Liping; Huang, Bo

2017-05-01

Rhamnose-binding lectin (RBL) is a type of Ca 2+ -independent lectin with tandem repeat carbohydrate-recognition domain, and is crucial for the innate immunity in many invertebrates. In this study, the cDNA sequence encoding RBL in coral Pocillopora damicornis (PdRBL-1) was cloned. The PdRBL-1 protein shared highest amino acid sequence similarity (55%) with the polyp of Hydra vulgaris, and contained a signal peptide and two tandem carbohydrate-recognition domains in which all cysteine residues were conserved. Surface plasmon resonance method revealed that the recombinant PdRBL-1 protein bound to LPS and Lipid A, but not to LTA, β-glucan, mannose and Poly (I:C). Results also showed that it bonded with zooxanthellae using western blotting method, and that the bound protein was detectable only at concentrations higher than 10 2 zooxanthellae cell mL -1 . When recombinant PdRBL-1 protein was preincubated with LPS, lower amounts of protein bound to zooxanthellae compared to cells not preincubated with LPS. Furthermore, PdRBL-1 mRNA expression increased significantly at 12 h, and declined to the baseline at 24 h after heat stress at 31 °C. These results collectively suggest that PdRBL-1 could recognize not only pathogenic bacteria but also symbiotic zooxanthellae, and that the recognition of zooxanthellae by PdRBL-1 could be repressed by pathogenic bacteria through competitive binding. This information allows us to gain new insights in the mechanisms influencing the establishment and maintenance of coral-zooxanthella symbiosis in coral P. damicornis. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Tn Antigen-Specific Lectin from Ground Ivy Is an Insecticidal Protein with an Unusual Physiology1

PubMed Central

Wang, Weifang; Hause, Bettina; Peumans, Willy J.; Smagghe, Guy; Mackie, Anne; Fraser, Robin; Van Damme, Els J.M.

2003-01-01

Leaves of ground ivy (Glechoma hederacea) contain a lectin (called Gleheda) that is structurally and evolutionary related to the classical legume lectins. Screening of a population of wild plants revealed that Gleheda accounts for more than one-third of the total leaf protein in some clones, whereas it cannot be detected in other clones growing in the same environment. Gleheda is predominantly expressed in the leaves where it accumulates during early leaf maturation. The lectin is not uniformly distributed over the leaves but exhibits a unique localization pattern characterized by an almost exclusive confinement to a single layer of palisade parenchyma cells. Insect feeding trials demonstrated that Gleheda is a potent insecticidal protein for larvae of the Colorado potato beetle (Leptinotarsa decemlineata). Because Gleheda is not cytotoxic, it is suggested that the insecticidal activity is linked to the carbohydrate-binding specificity of the lectin, which as could be demonstrated by agglutination assays with different types of polyagglutinable human erythrocytes is specifically directed against the Tn antigen structure (N-acetylgalactosamine O-linked to serine or threonine residues of proteins). PMID:12857814

Binding of Human GII.4 Norovirus Virus-Like Particles to Carbohydrates of Romaine Lettuce Leaf Cell Wall Materials

PubMed Central

Esseili, Malak A.

2012-01-01

Norovirus (NoV) genogroup II genotype 4 (GII.4) strains are the dominant cause of the majority of food-borne outbreaks, including those that involve leafy greens, such as lettuce. Since human NoVs use carbohydrates of histo-blood group antigens as receptors/coreceptors, we examined the role of carbohydrates in the attachment of NoV to lettuce leaves by using virus-like particles (VLPs) of a human NoV/GII.4 strain. Immunofluorescence analysis showed that the VLPs attached to the leaf surface, especially to cut edges, stomata, and along minor veins. Binding was quantified using enzyme-linked immunosorbent assay (ELISA) performed on cell wall materials (CWM) from innermost younger leaves and outermost lamina of older leaves. The binding to CWM of older leaves was significantly (P < 0.05) higher (1.5- to 2-fold) than that to CWM of younger leaves. Disrupting the carbohydrates of CWM or porcine gastric mucin (PGM) (a carbohydrate control) using 100 mM sodium periodate (NaIO4) significantly decreased the binding an average of 17% in younger leaves, 43% in older leaves, and 92% for PGM. In addition, lectins recognizing GalNAc, GlcNAc, and sialic acid at 100 μg/ml significantly decreased the binding an average of 41%, 33%, and 20% on CWM of older leaves but had no effect on younger leaves. Lectins recognizing α-d-Gal, α-d-Man/α-d-Glc, and α-l-Fuc showed significant inhibition on CWM of older leaves as well as that of younger leaves. All lectins, except for the lectin recognizing α-d-Gal, significantly inhibited NoV VLP binding to PGM. Collectively, our results indicate that NoV VLPs bind to lettuce CWM by utilizing multiple carbohydrate moieties. This binding may enhance virus persistence on the leaf surface and prevent effective decontamination. PMID:22138991

Mannan-binding lectin of the sea urchin Strongylocentrotus nudus.

PubMed

Bulgakov, Aleksandr A; Eliseikina, Marina G; Kovalchuk, Svetlana N; Petrova, Irina Yu; Likhatskaya, Galina N; Shamshurina, Ekaterina V; Rasskazov, Valery A

2013-02-01

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/β-protein with eight β-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.

O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside.

PubMed

Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Imberty, Anne; Künzler, Markus; Titz, Alexander

2016-01-01

Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O -methylated selenoglycoside, specifically methyl 2- O -methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor . The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2- O -methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.

Histochemistry of lectin-binding sites in Halicryptus spinulosus (Priapulida).

PubMed

Busch, A; Schumacher, U; Storch, V

2001-02-01

Priapulida represent one of the phylogenetically oldest multicellular animal groups. In multicellular animals (Metazoa) cell-to-cell and cell-to-matrix interactions are often mediated by carbohydrate residues of glycoconjugates. To analyze the carbohydrate composition of a phylogenetically old species, lectin histochemistry was employed on 5 specimens of the priapulid Halicryptus spinulosus. Many lectins bound to the chitin-containing cuticle, including those specific for carbohydrates other than N-acetylglucosamine, the principle building block of chitin. The connective tissue of the animals contained both N-acetylglucosamine and N-acetylgalactosamine. Mannose residues were widely distributed with the exception of the cuticle, but complex type carbohydrates were not present in the entire animal. Sialic acid residues were only detected in the cuticle and brush border of the intestinal epithelium, while fucose was limited to the cuticle. Thus, the lectin-binding pattern indicated that sugars typical for the linking region of both N- and O-glycoproteins in mammals are also present in H. spinulosus. Carbohydrate residues that are typical for the complex type of N-linked glycans in vertebrates are not present as are carbohydrate residues typical for the termination of O-linked carbohydrate chains. Hence, a truncated form of both N- and O-linked glycosylation is present in H. spinulosus indicating that more complex patterns of glycosylation developed later during evolution.

Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms.

PubMed

Bovi, Michele; Carrizo, Maria E; Capaldi, Stefano; Perduca, Massimiliano; Chiarelli, Laurent R; Galliano, Monica; Monaco, Hugo L

2011-08-01

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.

Molecular cloning of a C-type lectin (LvLT) from the shrimp Litopenaeus vannamei: early gene down-regulation after WSSV infection.

PubMed

Ma, Tracy Hoi Tung; Tiu, Shirley Hiu Kwan; He, Jian-Guo; Chan, Siu-Ming

2007-08-01

C-type lectin is one of the pattern-recognition proteins of the non-self innate immune system in the invertebrates. In this study, a lectin-like cDNA (LvLT) of Litopenaeus vannamei was cloned and characterized. LvLT cDNA consists of 1035 nt encoding for a protein with 345 amino acid residues. The deduced LvLT consists of two putative carbohydrate-recognition domains (CRDs) as found in most C-type lectins. The first CRD consists of an amino acid motif (QPD) for the binding of galactose and the other CRDs consist of amino acid motifs (EPN) for the binding of mannose. Except for some conserved amino acid residues, the CRD of LvLT shared an overall low amino acid sequence identity with CRDs of other lectins. Unlike other shrimp lectins, LvLT is expressed only in the hepatopancreas but not in the hemocytes as revealed by RT-PCR. When juvenile shrimp were challenged with shrimp extracts containing white spot syndrome virus (WSSV), the expression levels of LvLT decreased initially in the first 2 h and then increased to a much higher level after 4 h. The results suggest that the initial reduction in LvLT transcript level may be related to the WSSV infection in shrimp.

Stability and Sugar Recognition Ability of Ricin-Like Carbohydrate Binding Domains

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

Yao, Jianzhuang; Nellas, Ricky B; Glover, Mary M

2011-01-01

Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two -trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained amore » detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1 and site 2 recognition.« less

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

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

Linear Precision Glycomacromolecules with Varying Interligand Spacing and Linker Functionalities Binding to Concanavalin A and the Bacterial Lectin FimH.

PubMed

Igde, Sinaida; Röblitz, Susanna; Müller, Anne; Kolbe, Katharina; Boden, Sophia; Fessele, Claudia; Lindhorst, Thisbe K; Weber, Marcus; Hartmann, Laura

2017-12-01

A series of precision glycomacromolecules is prepared following previously established solid phase synthesis allowing for controlled variations of interligand spacing and the overall number of carbohydrate ligands. In addition, now also different linkers are installed between the carbohydrate ligand and the macromolecular scaffold. The lectin binding behavior of these glycomacromolecules is then evaluated in isothermal titration calorimetry (ITC) and kinITC experiments using the lectin Concanavalin A (Con A) in its dimeric and tetrameric form. The results indicate that both sterical and statistical effects impact lectin binding of precision glycomacromolecules. Moreover, ITC results show that highest affinity toward Con A can be achieved with an ethyl phenyl linker, which parallels earlier findings with the bacterial lectin FimH. In this way, a first set of glycomacromolecule structures is selected for testing in a bacterial adhesion-inhibition study. Here, the findings point to a one-sugar binding mode mainly affected by sterical restraints of the nonbinding parts of the respective glycomacromolecule. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MBL-associated serine proteases (MASPs) and infectious diseases.

PubMed

Beltrame, Marcia H; Boldt, Angelica B W; Catarino, Sandra J; Mendes, Hellen C; Boschmann, Stefanie E; Goeldner, Isabela; Messias-Reason, Iara

2015-09-01

The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis.

PubMed

Liu, Peng; Stajich, Jason E

2015-04-01

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians. Copyright © 2015 Elsevier Inc. All rights reserved.

Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.

PubMed

Biswas, Himadri; Chattopadhyaya, Rajagopal

2016-04-01

Curcuma longa rhizome lectin, a mannose-binding protein of non-seed portions of turmeric, is known to have antifungal, antibacterial and α-glucosidase inhibitory activities. We studied the role of complex-type glycans attached to asparagine (Asn) 66 and Asn 110 to elucidate the role of carbohydrates in lectin activity and stability. Apart from the native lectin, the characteristics of a deglycosylated Escherichia coli expressed lectin, high-mannose oligosaccharides at both asparagines and its glycosylation mutants N66Q and N110Q expressed in Pichia pastoris, were compared to understand the relationship between glycosylation and activity. Far UV circular dichroism (CD) spectra, fluorescence emission maximum, hemagglutination assay show no change in secondary or tertiary structures or sugar-binding properties between wild-type and aforementioned recombinant lectins under physiological pH. But reduced agglutination activity and loss of tertiary structure are observed in the acidic pH range for the deglycosylated and the N110Q protein. In thermal and guanidine hydrochloride (GdnCl)-induced unfolding, the wild-type and high-mannose lectins possess higher stability compared with the deglycosylated recombinant lectin and both mutants, as measured by a higher Tm of denaturation or a greater free energy change, respectively. Reversibility experiments after thermal denaturation reveal that deglycosylated proteins tend to aggregate during thermal inactivation but the wild type shows a much greater recovery to the native state upon refolding. These results suggest that N-glycosylation in turmeric lectin is important for the maintenance of its proper folding upon changes in pH, and that the oligosaccharides help in maintaining the active conformation and prevent aggregation in unfolded or partially folded molecules. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

Wurzburg, Beth A.; Tarchevskaya, Svetlana S.; Jardetzky, Theodore S.

CD23, the low-affinity receptor for IgE (Fc{var_epsilon}RII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca{sup 2+}. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in humanmore » CD23. Conformational differences between the apo and Ca{sup 2+} bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.« less

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

PubMed Central

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-01-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding. PMID:10727405

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

PubMed

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-04-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

PubMed

Konami, Y; Yamamoto, K; Osawa, T

1991-02-01

A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

PubMed

Chandrasekaran, E V; Xue, Jun; Xia, Jie; Khaja, Siraj D; Piskorz, Conrad F; Locke, Robert D; Neelamegham, Sriram; Matta, Khushi L

2016-10-01

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucβ1-3 GalNAc and Fucα-1-2 D-Fucβ-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc β-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

DOE PAGES

Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; ...

2016-09-01

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecularmore » interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. Finally, the in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.« less

Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

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

Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecularmore » interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. Finally, the in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.« less

New structural insights into the molecular deciphering of mycobacterial lipoglycan binding to C-type lectins: lipoarabinomannan glycoform characterization and quantification by capillary electrophoresis at the subnanomole level.

PubMed

Nigou, J; Vercellone, A; Puzo, G

2000-06-23

Lipoarabinomannans are key molecules of the mycobacterial envelopes involved in many steps of tuberculosis immunopathogenesis. Several of the biological activities of lipoarabinomannans are mediated by their ability to bind human C-type lectins, such as the macrophage mannose receptor, the mannose-binding protein and the surfactant proteins A and D. The lipoarabinomannan mannooligosaccharide caps have been demonstrated to be involved in the binding to the lectin carbohydrate recognition domains. We report an original analytical approach, based on capillary electrophoresis monitored by laser-induced fluorescence, allowing the absolute quantification, in nanomole quantities of lipoarabinomannan, of the number of mannooligosaccharide units per lipoarabinomannan molecule. Moreover, this analytical approach was successful for the glycosidic linkage determination of the mannooligosaccharide motifs and has been applied to the comparative analysis of parietal and cellular lipoarabinomannans of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv, H37Ra and Erdman strains. Significant differences were observed in the amounts of the various mannooligosaccharide units between lipoarabinomannans of different strains and between parietal and cellular lipoarabinomannans of the same strain. Nevertheless, no relationship was found between the number of mannooligosaccharide caps and the virulence of the corresponding strain. The results of the present study should help us to gain more understanding of the molecular basis of lipoarabinomannan discrimination in the process of binding to C-type lectins. Copyright 2000 Academic Press.

Radiolabeled Peptide Scaffolds for PET/SPECT - Optical in Vivo Imaging of Carbohydrate-Lectin Interactions

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

Deutscher, Susan

2014-09-30

The objective of this research is to develop phage display-selected peptides into radio- and fluoresecently- labeled scaffolds for the multimodal imaging of carbohydrate-lectin interactions. While numerous protein and receptor systems are being explored for the development of targeted imaging agents, the targeting and analysis of carbohydrate-lectin complexes in vivo remains relatively unexplored. Antibodies, nanoparticles, and peptides are being developed that target carbohydrate-lectin complexes in living systems. However, antibodies and nanoparticles often suffer from slow clearance and toxicity problems. Peptides are attractive alternative vehicles for the specific delivery of radionuclides or fluorophores to sites of interest in vivo, although, because ofmore » their size, uptake and retention may be less than antibodies. We have selected high affinity peptides that bind a specific carbohydrate-lectin complex involved in cell-cell adhesion and cross-linking using bacteriophage (phage) display technologies (1,2). These peptides have allowed us to probe the role of these antigens in cell adhesion. Fluorescent versions of the peptides have been developed for optical imaging and radiolabeled versions have been used in single photon emission computed tomography (SPECT) and positron emission tomography (PET) in vivo imaging (3-6). A benefit in employing the radiolabeled peptides in SPECT and PET is that these imaging modalities are widely used in living systems and offer deep tissue sensitivity. Radiolabeled peptides, however, often exhibit poor stability and high kidney uptake in vivo. Conversely, optical imaging is sensitive and offers good spatial resolution, but is not useful for deep tissue penetration and is semi-quantitative. Thus, multimodality imaging that relies on the strengths of both radio- and optical- imaging is a current focus for development of new in vivo imaging agents. We propose a novel means to improve the efficacy of radiolabeled and fluorescently labeled peptides, including our lectin/carbohydrate- targeting peptides, by displaying the targeting epitopes on small ~29 amino acid cyclic plant protein scaffolds known as cyclotides. Cyclotides are extremely stable molecules with long serum half-lives and low kidney uptake (7). More than one copy of the peptide can be engineered into the cyclotide loops, thus increasing the avidity of the peptide construct for its target.« less

"Click" saccharide/beta-lactam hybrids for lectin inhibition.

PubMed

Palomo, Claudio; Aizpurua, Jesus M; Balentová, Eva; Azcune, Itxaso; Santos, J Ignacio; Jiménez-Barbero, Jesús; Cañada, Javier; Miranda, José Ignacio

2008-06-05

Hybrid glycopeptide beta-lactam mimetics designed to bind lectins or carbohydrate recognition domains in selectins have been prepared according to a "shape-modulating linker" design. This approach was implemented using the azide-alkyne "click" cycloaddition reaction, and as shown by NMR/MD experiments, binding of the resulting mimetics to Ulex Europaeus Lectin-1 (UEL-1) occurred after a "bent-to-extended" conformational change around a partially rotatable triazolylmethylene moiety.

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)

Lectin-Like Bacteriocins from Pseudomonas spp. Utilise D-Rhamnose Containing Lipopolysaccharide as a Cellular Receptor

PubMed Central

Josts, Inokentijs; Roszak, Aleksander W.; Waløen, Kai I.; Cogdell, Richard J.; Milner, Joel; Evans, Tom; Kelly, Sharon; Tucker, Nicholas P.; Byron, Olwyn; Smith, Brian; Walker, Daniel

2014-01-01

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins. PMID:24516380

Impact of human galectin-1 binding to saccharide ligands on dimer dissociation kinetics and structure.

PubMed

Romero, Juan M; Trujillo, Madia; Estrin, Darío A; Rabinovich, Gabriel A; Di Lella, Santiago

2016-12-01

Endogenous lectins can control critical biological responses, including cell communication, signaling, angiogenesis and immunity by decoding glycan-containing information on a variety of cellular receptors and the extracellular matrix. Galectin-1 (Gal-1), a prototype member of the galectin family, displays only one carbohydrate recognition domain and occurs in a subtle homodimerization equilibrium at physiologic concentrations. Such equilibrium critically governs the function of this lectin signaling by allowing tunable interactions with a preferential set of glycosylated receptors. Here, we used a combination of experimental and computational approaches to analyze the kinetics and mechanisms connecting Gal-1 ligand unbinding and dimer dissociation processes. Kinetic constants of both processes were found to differ by an order of magnitude. By means of steered molecular dynamics simulations, the ligand unbinding process was followed monitoring water occupancy changes. By determining the water sites in a carbohydrate binding place during the unbinding process, we found that rupture of ligand-protein interactions induces an increase in energy barrier while ligand unbinding process takes place, whereas the entry of water molecules to the binding groove and further occupation of their corresponding water sites contributes to lowering of the energy barrier. Moreover, our findings suggested local asymmetries between the two subunits in the dimer structure detected at a nanosecond timescale. Thus, integration of experimental and computational data allowed a more complete understanding of lectin ligand binding and dimerization processes, suggesting new insights into the relationship between Gal-1 structure and function and renewing the discussion on the biophysics and biochemistry of lectin-ligand lattices. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Immobilization of sugars in supermacroporous cryogels for the purification of lectins by affinity chromatography.

PubMed

Gonçalves, Gabriel Ramos Ferreira; Gandolfi, Olga Reinert Ramos; Santos, Leandro Soares; Bonomo, Renata Cristina Ferreira; Veloso, Cristiane Martins; Veríssimo, Lizzy Ayra Alcântara; Fontan, Rafael da Costa Ilhéu

2017-11-15

Lectins are glycoproteins that bind to carbohydrates or glycoconjugates by specific interactions. The specificity of lectins to various carbohydrates is a determinant factor in the choice of ligand for the chromatographic matrix when using chromatography as a lectin purification technique. In this work, the immobilization of three different aminated carbohydrates on the surface of macroporous polymeric cryogels was evaluated. Carbohydrates were immobilized on cryogel surfaces via the glutaraldehyde method to create spacer arms, reducing steric hindrance. The immobilized N-acetyl-d-glucosamine and N-acetyl-d-mannosamine concentrations contained approximately 130mg of carbohydrate/g dehydrated cryogel, while the N-acetyl-d-galactosamine contained 105mg of carbohydrate/g dehydrated cryogel. Scanning electron microscopy showed that the physical structure and porosity of the chromatographic columns were not affected by the immobilization process, maintaining an elevated hydration capacity and the macroporous structure of the cryogels. Adsorption of concanavalin A on cryogels functionalized with N-acetyl-d-glucosamine (cryo-d-GlcNAc) was tested, as well as its reuse capability. After 5 cycles of use, cryo-d-GlcNAc was shown to be stable, with an adsorptive capacity of around 50mg/g. Carbohydrate immobilization in polyacrylamide cryogels was satisfactory, with promise for applications in lectin purification processes. Copyright © 2017 Elsevier B.V. All rights reserved.

PmLT, a C-type lectin specific to hepatopancreas is involved in the innate defense of the shrimp Penaeus monodon.

PubMed

Ma, Tracy Hoi-Tung; Benzie, John A H; He, Jian-Guo; Chan, Siu-Ming

2008-11-01

A diverse class of proteins called lectins plays a major role in shrimp innate immunity. In this study, the cDNA encoding a C-type lectin of Penaeus monodon (PmLT) was cloned, and its potential role examined. Despite the low overall amino acid sequence identity with other animal lectins, PmLT includes conserved carbohydrate recognition domains (CRDs) characteristic of animal C-type lectins. Unlike the other two P. monodon lectin-like proteins described to date that have one CRD, PmLT has two CRDs. The first CRD contains a QPD motif with specificity for binding galactose, while the second CRD contains a EPN motif for binding mannose. PmLT transcripts can be detected in the hepatopancreas but not in other tissues. Expression studies showed that PmLT mRNA transcript level decreased initially and then gradually increased after whole shrimp or hepatopancreas tissue fragments were treated with white spot syndrome virus (WSSV) extract but were not affected by bacteria. Using anti-rPmLT antibody, PmLT was detected only in the hepatopancreas specific F cells (Hpf). In vitro encapsulation assay showed that agarose beads coated with rPmLT were encapsulated by hemocytes indicating a role in innate immune response. In summary, PmLT is produced in the hepatopancreas and may act as a pattern recognition protein for viral pathogens and also activates the innate immune responses of the shrimp to bacteria. The dual-CRD structure of PmLT may assist the recognition of diverse pathogens.

Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth.

PubMed

Lubkowski, Jacek; Durbin, Sarah V; Silva, Mariana C C; Farnsworth, David; Gildersleeve, Jeffrey C; Oliva, Maria Luiza V; Wlodawer, Alexander

2017-02-01

Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in solution and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have especially strong binding (apparent K d  = 0.5-1.0 nm) to the tumor-associated Tn antigen. High-resolution crystal structures were determined for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive analysis of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the molecular recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. Structural data are available in the PDB under the accession numbers 5T50, 5T52, 5T55, 5T54, 5T5L, 5T5J, 5T5P, and 5T5O. © 2016 Federation of European Biochemical Societies.

Deterrent activity of plant lectins on cowpea weevil Callosobruchus maculatus (F.) oviposition.

PubMed

Sadeghi, Amin; Van Damme, Els J M; Peumans, Willy J; Smagghe, Guy

2006-09-01

A set of 14 plant lectins was screened in a binary choice bioassay for inhibitory activity on cowpea weevil Callosobruchus maculatus (F.) oviposition. Coating of chickpea seeds (Cicer arietinum L.) with a 0.05% (w/v) solution of plant lectins caused a significant reduction in egg laying. Control experiments with heat inactivated lectin and BSA indicated that the observed deterrent effects are specific and require carbohydrate-binding activity. However, no clear correlation could be established between deterrent activity and sugar-binding specificity/molecular structure of the lectins. Increasing the insect density reduced the inhibitory effect of the lectins confirming that female insects are capable of adjusting their oviposition rates as a function of host availability.

NMR and MD Investigations of Human Galectin-1/Oligosaccharide Complexes

PubMed Central

Meynier, Christophe; Feracci, Mikael; Espeli, Marion; Chaspoul, Florence; Gallice, Philippe; Schiff, Claudine; Guerlesquin, Françoise; Roche, Philippe

2009-01-01

Abstract The specific recognition of carbohydrates by lectins plays a major role in many cellular processes. Galectin-1 belongs to a family of 15 structurally related β-galactoside binding proteins that are able to control a variety of cellular events, including cell cycle regulation, adhesion, proliferation, and apoptosis. The three-dimensional structure of galectin-1 has been solved by x-ray crystallography in the free form and in complex with various carbohydrate ligands. In this work, we used a combination of two-dimensional NMR titration experiments and molecular-dynamics simulations with explicit solvent to study the mode of interaction between human galectin-1 and five galactose-containing ligands. Isothermal titration calorimetry measurements were performed to determine their affinities for galectin-1. The contribution of the different hexopyranose units in the protein-carbohydrate interaction was given particular consideration. Although the galactose moiety of each oligosaccharide is necessary for binding, it is not sufficient by itself. The nature of both the reducing sugar in the disaccharide and the interglycosidic linkage play essential roles in the binding to human galectin-1. PMID:20006954

Fabrication of Carbohydrate Microarrays by Boronate Formation.

PubMed

Adak, Avijit K; Lin, Ting-Wei; Li, Ben-Yuan; Lin, Chun-Cheng

2017-01-01

The interactions between soluble carbohydrates and/or surface displayed glycans and protein receptors are essential to many biological processes and cellular recognition events. Carbohydrate microarrays provide opportunities for high-throughput quantitative analysis of carbohydrate-protein interactions. Over the past decade, various techniques have been implemented for immobilizing glycans on solid surfaces in a microarray format. Herein, we describe a detailed protocol for fabricating carbohydrate microarrays that capitalizes on the intrinsic reactivity of boronic acid toward carbohydrates to form stable boronate diesters. A large variety of unprotected carbohydrates ranging in structure from simple disaccharides and trisaccharides to considerably more complex human milk and blood group (oligo)saccharides have been covalently immobilized in a single step on glass slides, which were derivatized with high-affinity boronic acid ligands. The immobilized ligands in these microarrays maintain the receptor-binding activities including those of lectins and antibodies according to the structures of their pendant carbohydrates for rapid analysis of a number of carbohydrate-recognition events within 30 h. This method facilitates the direct construction of otherwise difficult to obtain carbohydrate microarrays from underivatized glycans.

Molecular Simulations of Carbohydrates with a Fucose-Binding Burkholderia ambifaria Lectin Suggest Modulation by Surface Residues Outside the Fucose-Binding Pocket

PubMed Central

Dingjan, Tamir; Imberty, Anne; Pérez, Serge; Yuriev, Elizabeth; Ramsland, Paul A.

2017-01-01

Burkholderia ambifaria is an opportunistic respiratory pathogen belonging to the Burkholderia cepacia complex, a collection of species responsible for the rapidly fatal cepacia syndrome in cystic fibrosis patients. A fucose-binding lectin identified in the B. ambifaria genome, BambL, is able to adhere to lung tissue, and may play a role in respiratory infection. X-ray crystallography has revealed the bound complex structures for four fucosylated human blood group epitopes (blood group B, H type 1, H type 2, and Lex determinants). The present study employed computational approaches, including docking and molecular dynamics (MD), to extend the structural analysis of BambL-oligosaccharide complexes to include four additional blood group saccharides (A, Lea, Leb, and Ley) and a library of blood-group-related carbohydrates. Carbohydrate recognition is dominated by interactions with fucose via a hydrogen-bonding network involving Arg15, Glu26, Ala38, and Trp79 and a stacking interaction with Trp74. Additional hydrogen bonds to non-fucose residues are formed with Asp30, Tyr35, Thr36, and Trp74. BambL recognition is dominated by interactions with fucose, but also features interactions with other parts of the ligands that may modulate specificity or affinity. The detailed computational characterization of the BambL carbohydrate-binding site provides guidelines for the future design of lectin inhibitors. PMID:28680402

Characterization of the carbohydrate components of Taenia solium oncosphere proteins and their role in the antigenicity.

PubMed

Arana, Yanina; Verastegui, Manuela; Tuero, Iskra; Grandjean, Louis; Garcia, Hector H; Gilman, Robert H

2013-10-01

This study examines the carbohydrate composition of Taenia solium whole oncosphere antigens (WOAs), in order to improve the understanding of the antigenicity of the T. solium. Better knowledge of oncosphere antigens is crucial to accurately diagnose previous exposure to T. solium eggs and thus predict the development of neurocysticercosis. A set of seven lectins conjugates with wide carbohydrate specificity were used on parasite fixations and somatic extracts. Lectin fluorescence revealed that D-mannose, D-glucose, D-galactose and N-acetyl-D-galactosamine residues were the most abundant constituents of carbohydrate chains on the surface of T. solium oncosphere. Lectin blotting showed that posttranslational modification with N-glycosylation was abundant while little evidence of O-linked carbohydrates was observed. Chemical oxidation and enzymatic deglycosylation in situ were performed to investigate the immunoreactivity of the carbohydrate moieties. Linearizing or removing the carbohydrate moieties from the protein backbones did not diminish the immunoreactivity of these antigens, suggesting that a substantial part of the host immune response against T. solium oncosphere is directed against the peptide epitopes on the parasite antigens. Finally, using carbohydrate probes, we demonstrated for the first time that the presence of several lectins on the surface of the oncosphere was specific to carbohydrates found in intestinal mucus, suggesting a possible role in initial attachment of the parasite to host cells.

CHARACTERIZATION OF THE CARBOHYDRATE COMPONENTS OF Taenia solium ONCOSPHERE PROTEINS AND THEIR ROLE IN THE ANTIGENICITY

PubMed Central

Arana, Yanina; Verastegui, Manuela; Tuero, Iskra; Grandjean, Louis; Garcia, Hector H.; Gilman, Robert H.

2015-01-01

This study examines the carbohydrate composition of Taenia solium whole oncosphere antigens (WOAs), in order to improve the understanding of the antigenicity of the T. solium. Better knowledge of oncosphere antigens is crucial to accurately diagnose previous exposure to T. solium eggs and thus predict the development of neurocysticercosis. A set of seven lectins conjugates with wide carbohydrate specificity were used on parasite fixations and somatic extracts. Lectin fluorescence revealed that D-mannose, D-glucose, D-galactose and N-acetyl-D-galactosamine residues were the most abundant constituents of carbohydrate chains on the surface of T. solium oncosphere. Lectin blotting showed that post-translational modification with N-glycosylation was abundant while little evidence of O-linked carbohydrates was observed. Chemical oxidation and enzymatic deglycosylation in situ were performed to investigate the immunoreactivity of the carbohydrate moieties. Linearizing or removing the carbohydrate moieties from the protein backbones did not diminish the immunoreactivity of these antigens, suggesting that a substantial part of the host immune response against T. solium oncosphere is directed against the peptide epitopes on the parasite antigens. Finally, using carbohydrate probes, we demonstrated for the first time that the presence of several lectins on the surface of the oncosphere was specific to carbohydrates found in intestinal mucus, suggesting a possible role in initial attachment of the parasite to host cells. PMID:23982308

A new C-type lectin (FcLec5) from the Chinese white shrimp Fenneropenaeus chinensis.

PubMed

Xu, Wen-Teng; Wang, Xian-Wei; Zhang, Xiao-Wen; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing

2010-11-01

C-type lectins are one family of pattern recognition receptors (PRRs) that play important roles in innate immunity. In this work, cDNA and genomic sequences for a new C-type lectin (FcLec5) were obtained from the Chinese white shrimp Fenneropenaeus chinensis. FcLec5 cDNA contains an open reading frame of 1,008 bp and its genomic sequence is 1,137 bp with 4 exons and 3 introns. The predicted FcLec5 protein contains a signal peptide and two carbohydrate recognition domains (CRDs). The N-terminal CRD of FcLec5 has a predicted carbohydrate recognition motif of Gln-Pro-Asp (QPD), while the C-terminal CRD contains a motif of Glu-Pro-Gln (EPQ). Northern blot analysis showed that FcLec5 mRNA was specifically expressed in hepatopancreas. FcLec5 protein was expressed in hepatopancreas and secreted into hemolymph. Real-time PCR showed that FcLec5 transcript exhibited different expression profiles after immune-challenged with Vibrio anguillarum or White Spot Syndrome Virus (WSSV). Recombinant FcLec5 and its two individual CRDs could agglutinate most bacteria tested, and the agglutinating activity was Ca2+-dependent. Besides, the agglutinating activity to gram-negative bacteria is higher than that to gram-positive bacteria. Direct binding assay showed that recombinant FcLec5 could bind to all microorganisms tested (five gram-positive and four gram-negative bacteria, as well as yeast) in a Ca2+-independent manner. Recombinant FcLec5 also directly bound to bacterial peptidoglycan, lipopolysaccharide and lipoteichoic acids. These results suggest that FcLec5 may act as a PRR for bacteria via binding to bacterial cell wall polysaccharides in Chinese white shrimp.

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.

Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis.

PubMed

Skjoedt, Mikkel-Ole; Palarasah, Yaseelan; Rasmussen, Karina; Vitved, Lars; Salomonsen, Jan; Kliem, Anette; Hansen, Soren; Koch, Claus; Skjodt, Karsten

2010-01-01

The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intestinalis. The CioMBLs display similarities with vertebrate MBLs and comprise a collagen-like region, alpha-helical coiled-coils and a carbohydrate recognition domain (CRD) with conserved residues involved in calcium and carbohydrate binding. Structural analysis revealed an oligomerization through interchain disulphide bridges between N-terminal cysteine residues and cysteines located between the neck region and the CRD. RT-PCR showed a tissue specific expression of CioMBL in the gut and by immunohistochemistry analysis we also demonstrated that CioMBL co-localize with an MBL-associated serine protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway.

Lectin histochemistry reveals SNA as a prognostic carbohydrate-dependent probe for invasive ductal carcinoma of the breast: a clinicopathological and immunohistochemical auxiliary tool

PubMed Central

dos-Santos, Petra B; Zanetti, Juliana S; Vieira-de-Mello, Gabriela S; Rêgo, Moacyr BM; A, Alfredo Ribeiro-Silva; Beltrão, Eduardo Isidoro Carneiro

2014-01-01

Increased sialylation and β1,6-branched oligosaccharides has been associated with a variety of structural changes in cell surface carbohydrates, most notably in tumorigenesis. Lectins are defined as proteins that preferentially recognize and bind carbohydrate complexes protruding from glycolipids and glycoproteins. This interaction with carbohydrates can be as specific as the interaction between antigen and antibody. Due to this type of interaction lectins have been used as experimental auxiliary tools in histopathological diagnosis of cancer. This study was designed to evaluate the differential expression of sialic acids and β1,6-N-acetylglucosaminyltransferase V (MGAT5) in invasive (IDC) and in situ (DCIS) ductal carcinoma of the breast and its possible application as prognostic biomarkers. A possible transition between pre-malign and malign lesions was evaluated using DCIS samples. Biopsies were analyzed regarding the expression of MUC1, p53, Ki-67, estrogen receptor, progesterone receptor, HER-2 and MGAT5. α2,6-linked sialic acids residues recognized by SNA lectin was overexpressed in 33.3% of IDC samples and it was related with Ki-67 (p=0.042), PR (p=0.029), lymphnodes status (p=0.017) and death (p=0.011). Regarding survival analysis SNA was the only lectin able to correlate with specific-disease survival and disease-free survival (p=0.024 and p=0.041, respectively), besides, it presents itself as an independent variable by Cox Regression analysis (p= 0.004). Comparing IDC and DCIS cases, only SNA showed different staining pattern (p=0.034). The presence of sialic acids on tumor cell surface can be an indicative of poor prognosis and our study provides further evidence that SNA lectin can be used as a prognostic probe in IDC and DCIS patients. PMID:24966944

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.

The synthesis and host-guest applications of synthetic receptor molecules

NASA Astrophysics Data System (ADS)

Osner, Zachary R.

2011-12-01

Host-guest chemistry involves the complimentary binding between two molecules. Host molecules have been synthesized to bind negative, positive, and neutral molecules such as proteins and enzymes, and have been used as optical sensors, electrochemical sensors, supramolecular catalysts, and in the pharmaceutical industry as anti-cancer agents.1 The field of nanoscience has exploited guest-host interactions to create optical sensors with colloidal gold and Dip-Pen nanolithography technologies. Gold nanoparticles, have been functionalized with DNA, and have been developed as a selective colorimetric detection system, that upon binding turns the solution from a red to blue in color.2 Cyclotriveratrylene (CTV) 1 is a common supramolecular scaffold that has been previously employed in guest-host chemistry, and the construction of CTV involves the cyclic trimerization of veratryl alcohol via the veratryl cation.3 Due to the rigid bowl shaped structure of CTV, CTV has been shown to act as a host molecule for fullerene-C60.4 Lectin binding receptor proteins are a specific class of proteins found in bacteria, viruses, plants, and animals that can bind to complimentary carbohydrates. It is these lectins that are believed to be responsible for cell-cell interactions and the formation of biofilms in pathenogenic bacteria.5 P. aeruginosa is a pathenogenic bacterium, shown to have a high resistance to many antibiotics, which can form biofilms in human lung tissue, causing respiratory tract infections in patients with compromised immune systems. 5 I will exploit guest-host interactions to create synthetic supramolecular and carbohydrate receptor molecules to that will be of use as biological sensing devices via self-assembled monolayers on solid surfaces and nanoparticle technologies. *Please refer to dissertation for references/footnotes.

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

Docking, synthesis, and NMR studies of mannosyl trisaccharide ligands for DC-SIGN lectin.

PubMed

Reina, José J; Díaz, Irene; Nieto, Pedro M; Campillo, Nuria E; Páez, Juan A; Tabarani, Georges; Fieschi, Franck; Rojo, Javier

2008-08-07

DC-SIGN, a lectin, which presents at the surface of immature dendritic cells, constitutes nowadays a promising target for the design of new antiviral drugs. This lectin recognizes highly glycosylated proteins present at the surface of several pathogens such as HIV, Ebola virus, Candida albicans, Mycobacterium tuberculosis, etc. Understanding the binding mode of this lectin is a topic of tremendous interest and will permit a rational design of new and more selective ligands. Here, we present computational and experimental tools to study the interaction of di- and trisaccharides with DC-SIGN. Docking analysis of complexes involving mannosyl di- and trisaccharides and the carbohydrate recognition domain (CRD) of DC-SIGN have been performed. Trisaccharides Manalpha1,2[Manalpha1,6]Man 1 and Manalpha1,3[Manalpha1,6]Man 2 were synthesized from an orthogonally protected mannose as a common intermediate. Using these ligands and the soluble extracellular domain (ECD) of DC-SIGN, NMR experiments based on STD and transfer-NOE were performed providing additional information. Conformational analysis of the mannosyl ligands in the free and bound states was done. These studies have demonstrated that terminal mannoses at positions 2 or 3 in the trisaccharides are the most important moiety and present the strongest contact with the binding site of the lectin. Multiple binding modes could be proposed and therefore should be considered in the design of new ligands.

The influence of surface carbohydrates during in vitro infection of mammalian cells by the dermatophyte Trichophyton rubrum.

PubMed

Esquenazi, Daniele; Alviano, Celuta S; de Souza, Wanderley; Rozental, Sonia

2004-04-01

In order to better understand the role played by surface glycoconjugates during host cell adhesion and endocytosis of Trichophyton rubrum, we looked for the presence of carbohydrate-binding adhesins on the microconidia surface and their role on cellular interaction with epithelial and macrophages cells. The interaction of T. rubrum with chinese hamster ovary epithelial cells and their glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, that express mannose and galactose, respectively. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates to the interaction medium, pretreatment with lectins and with sodium periodate decreased the adhesion and endocytic index for all mutants. The ability of the fungus to penetrate into mammalian cells was confirmed in experiments using macrophages treated with cytochalasin D. Flow cytometric analysis showed that this fungus recognizes mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 than at 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature for the microconidia adhesins. The presence of lectin-like molecules in fungus cell could be observed by scanning electron microscopy of the fungus incubated with colloidal-gold labeled neoglycoproteins. Our results suggest that T. rubrum has the ability to invade mammalian cells and expresses carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. These adhesins may play an important role on the adhesion and invasion of the fungus during the infectious process of dermatophytosis.

Glycoconjugate distribution in early human notochord and axial mesenchyme.

PubMed

Götz, W; Quondamatteo, F

2001-02-01

Glycosylation patterns of cells and tissues give insights into spatially and temporally regulated developmental processes and can be detected histochemically using plant lectins with specific affinities for sugar moieties. The early development of the vertebral column in man is a process which has never been investigated by lectin histochemistry. Therefore, we studied binding of several lectins (AIA, Con A, GSA II, LFA, LTA, PNA, RCA I, SBA, SNA, WGA) in formaldehyde-fixed sections of the axial mesenchyme of 5 human embryos in Carnegie stages 12-15. During these developmental stages, an unsegmented mesenchyme covers the notochord. Staining patterns did not show striking temporal variations except for SBA which stained the cranial axial mesenchyme only in the early stage 12 embryo and for PNA, of which the staining intensity in the mesenchyme decreased with age. The notochord appeared as a highly glycosylated tissue. Carbohydrates detected may correspond to adhesion molecules or to secreted substances like proteoglycans or proteins which could play an inductive role, for example, for the neural tube. The axial perinotochordal unsegmented mesenchyme showed strong PNA binding. Therefore, its function as a PNA-positive "barrier" tissue is discussed. The endoderm of the primitive gut showed a lectin-binding pattern that was similar to that of the notochord, which may correlate with interactions between these tissues during earlier developmental stages.

Structural characterization of coagulant Moringa oleifera Lectin and its effect on hemostatic parameters.

PubMed

Luz, Luciana de Andrade; Silva, Mariana Cristina Cabral; Ferreira, Rodrigo da Silva; Santana, Lucimeire Aparecida; Silva-Lucca, Rosemeire Aparecida; Mentele, Reinhard; Oliva, Maria Luiza Vilela; Paiva, Patricia Maria Guedes; Coelho, Luana Cassandra Breitenbach Barroso

2013-07-01

Lectins are carbohydrate recognition proteins. cMoL, a coagulant Moringa oleifera Lectin, was isolated from seeds of the plant. Structural studies revealed a heat-stable and pH resistant protein with 101 amino acids, 11.67 theoretical pI and 81% similarity with a M. oleifera flocculent protein. Secondary structure content was estimated as 46% α-helix, 12% β-sheets, 17% β-turns and 25% unordered structures belonging to the α/β tertiary structure class. cMoL significantly prolonged the time required for blood coagulation, activated partial thromboplastin (aPTT) and prothrombin times (PT), but was not so effective in prolonging aPTT in asialofetuin presence. cMoL acted as an anticoagulant protein on in vitro blood coagulation parameters and at least on aPTT, the lectin interacted through the carbohydrate recognition domain. Copyright © 2013 Elsevier B.V. All rights reserved.

Purification and characterization of a novel thermostable mycelial lectin from Aspergillus terricola.

PubMed

Singh, Ram Sarup; Bhari, Ranjeeta; Kaur, Hemant Preet; Vig, Monika

2010-11-01

Lectin has been isolated from mycelia of Aspergillus terricola by single step purification on porcine stomach mucin-Sepharose 4B affinity column. Lectin could be effectively purified with 75% recovery and 4.47-fold increase in specific activity. Lectin migrated as a single band on SDS-PAGE with an apparent molecular mass of 32.5 kDa. Sugar inhibition assay revealed that the lectin did not strongly interact with most carbohydrates and their derivatives tested while strong binding affinity to D-glucose, D-sucrose, N-acetyl-D-galactosamine, asialofetuin, porcine stomach mucin, and bovine submaxillary mucin was indicated. Neuraminidase and protease treatment to erythrocytes enhanced lectin titre. Lectin activity was stable within the pH range of 7.0-10.5. A. terricola lectin displayed remarkable thermostability and remained unaffected upon incubation at 70 degrees C for 2.5 h. Lectin did not require metal ions for its activity. Incubation with denaturants (urea, thiourea, and guanidine-HCl) substantially reduced lectin activity. Carbohydrate analysis revealed that it is a glycoprotein with 9.76% total sugars.

Selective binding of lectins to normal and neoplastic urothelium in rat and mouse bladder carcinogenesis models.

PubMed

Zupančič, Daša; Kreft, Mateja Erdani; Romih, Rok

2014-01-01

Bladder cancer adjuvant intravesical therapy could be optimized by more selective targeting of neoplastic tissue via specific binding of lectins to plasma membrane carbohydrates. Our aim was to establish rat and mouse models of bladder carcinogenesis to investigate in vivo and ex vivo binding of selected lectins to the luminal surface of normal and neoplastic urothelium. Male rats and mice were treated with 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water and used for ex vivo and in vivo lectin binding experiments. Urinary bladder samples were also used for paraffin embedding, scanning electron microscopy and immunofluorescence labelling of uroplakins. During carcinogenesis, the structure of the urinary bladder luminal surface changed from microridges to microvilli and ropy ridges and the expression of urothelial-specific glycoproteins uroplakins was decreased. Ex vivo and in vivo lectin binding experiments gave comparable results. Jacalin (lectin from Artocarpus integrifolia) exhibited the highest selectivity for neoplastic compared to normal urothelium of rats and mice. The binding of lectin from Amaranthus caudatus decreased in rat model and increased in mouse carcinogenesis model, indicating interspecies variations of plasma membrane glycosylation. Lectin from Datura stramonium showed higher affinity for neoplastic urothelium compared to the normal in rat and mouse model. The BBN-induced animal models of bladder carcinogenesis offer a promising approach for lectin binding experiments and further lectin-mediated targeted drug delivery research. Moreover, in vivo lectin binding experiments are comparable to ex vivo experiments, which should be considered when planning and optimizing future research.

Transmission-blocking antibodies against mosquito C-type lectins for dengue prevention.

PubMed

Liu, Yang; Zhang, Fuchun; Liu, Jianying; Xiao, Xiaoping; Zhang, Siyin; Qin, Chengfeng; Xiang, Ye; Wang, Penghua; Cheng, Gong

2014-02-01

C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1), facilitating the attachment of West Nile virus (WNV) on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E) protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden.

Effect of the lectin of Bauhinia variegata and its recombinant isoform on surgically induced skin wounds in a murine model.

PubMed

Neto, Luiz Gonzaga do Nascimento; Pinto, Luciano da Silva; Bastos, Rafaela Mesquita; Evaristo, Francisco Flávio Vasconcelos; Vasconcelos, Mayron Alves de; Carneiro, Victor Alves; Arruda, Francisco Vassiliepe Sousa; Porto, Ana Lúcia Figueiredo; Leal, Rodrigo Bainy; Júnior, Valdemiro Amaro da Silva; Cavada, Benildo Sousa; Teixeira, Edson Holanda

2011-11-07

Lectins are a structurally heterogeneous group of highly specific carbohydrate-binding proteins. Due to their great biotechnological potential, lectins are widely used in biomedical research. The purpose of the present study was to evaluate the healing potential of the lectin of Bauhinia variegata (nBVL) and its recombinant isoform (rBVL-1). Following surgical creation of dorsal skin wounds, seven groups of mice were submitted to topical treatment for 12 days with lectin, D-galactose, BSA and saline. The animals were anesthetized and euthanized on POD 2, 7 and 12 in order to evaluate the healing potential of each treatment. The parameters considered included wound size, contraction rate, epithelialization rate and histopathological findings. Wound closure was fastest in animals treated with rBVL-1 (POD 7). nBVL was more effective than the controls. All skin layers were reconstructed and keratin deposition increased. Our findings indicate that the lectin of Bauhinia variegata possesses pro-healing properties and may be employed in the treatment of acute skin wounds.

Human lung surfactant protein A exists in several different oligomeric states: oligomer size distribution varies between patient groups.

PubMed Central

Hickling, T. P.; Malhotra, R.; Sim, R. B.

1998-01-01

BACKGROUND: Lung surfactant protein A (SP-A) is a complex molecule composed of up to 18 polypeptide chains. In vivo, SP-A probably binds to a wide range of inhaled materials via the interaction of surface carbohydrates with the lectin domains of SP-A and mediates their interaction with cells as part of a natural defense system. Multiplicity of lectin domains gives high-affinity binding to carbohydrate-bearing surfaces. MATERIALS AND METHODS: Gel filtration analyses were performed on bronchoalveolar lavage (BAL) fluid samples from three patient groups: pulmonary alveolar proteinosis (n = 12), birch pollen allergy (n = 11), and healthy volunteers (n = 4). Sucrose density gradient centrifugation was employed to determine molecular weights of SP-A oligomers. SP-A was solubilized from the lipid phase to compare oligomeric state with that of water soluble SP-A. RESULTS: SP-A exists as fully assembled complexes with 18 polypeptide chains, but it is also consistently found in smaller oligomeric forms. This is true for both the water- and lipid-soluble fractions of SP-A. CONCLUSION: The three patient groups analyzed show a shift towards lower oligomeric forms of SP-A in the following sequence: healthy-pulmonary alveolar proteinosis-pollen allergy. Depolymerization would be expected to lead to loss of binding affinity for carbohydrate-rich surfaces, with loss or alteration of biological function. While there are many complex factors involved in the establishment of an allergy, it is possible that reduced participation of SP-A in clearing a potential allergen from the lungs could be an early step in the chain of events. Images Fig. 4 FIG. 6 Fig. 7 Fig. 8 PMID:9606179

A lectin histochemical study on carbohydrate moieties of the gonadotropin-like substance in the epithelial cells of Hatschek's pit of Branchiostoma belcheri

NASA Astrophysics Data System (ADS)

Fang, Y. Q.; Welsch, U.

1997-03-01

The present light microscopic lectin, histochemical study suggests for the first time that the vertebrate gonadotropin-like substance in the basal part of the epithelial cells of Hatschek's pit is a sialic acid-containing glycoprotein. The binding intensity of the epithelial cells in Hatschek's pit to 6 lectins ( Limulus polyphemus agglutinin (LPA), Wheat germ agglutinin (WGA), Helix pomatia agglutinin (HPA), Concanavalin A (Con A), Ulex europaeus agglutinin I (UEA I) and Ricinus communis agglutinin I (RCA I)) indicate that the carbohydrate composition of the gonadotrophic glycoprotein is similar to that of mammals and fish, and that N-acetyl-D-galactosamine, sialic acid, glucosamine, D-mannose and L-fucose are components of the carbohydrate portion.

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

Investigation on interaction of Achatinin, a 9-O-acetyl sialic acid-binding lectin, with lipopolysaccharide in the innate immunity of Achatina fulica snails.

PubMed

Biswas, C; Sinha, D; Mandal, C

2000-01-01

Achatinin, a 9-O-acetyl sialic acid (9-O-AcSA) binding lectin, has been demonstrated to be synthesized in amoebocytes of Achatina fulica snails. This lectin was affinity-purified from Achatina amoebocytes lysate (AAL); it appeared as a single band on native polyacrylamide gel electrophoresis (PAGE) and showed 16 identical subunits of M.W. 15 kDa on sodium dodecyl sulphate (SDS)-PAGE. It was found to be homologous with an earlier reported lectin, Achatinin-H, derived from hemolymph of A. fulica snails (Sen, G., Mandal, C., 1995. The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achantia fulica. Carbohydr. Res., 268, 115-125). Homology between both lectins was confirmed by their similar electrophoretic mobilities, carbohydrate specificity and cross reactivity on immunodiffusion. Achatinin showed in vitro calcium dependent binding to two 9-O-acetylated sialoglyoconjugates (9-O-AcSG) on lipopolysaccharide (LPS) (Escherichia coli 055: B5) of M.W. 40 kDa and 27.5 kDa, which was abolished following de-O-acetylation. Based on the previously defined narrow sugar specificity of Achatinin towards 9-O-AcSAalpha2-->6GalNAc [Sen, G., Mandal, C., 1995. The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achatina fulica. Carbohydr. Res., 268, 115-125], we conclude that LPS contains this lectinogenic epitope at the terminal sugar moiety. The Achatinin-mediated hemagglutination inhibition of rabbit erythrocytes by LPS further confirmed it. The lectin exhibited bacteriostatic effect on Gram-negative bacteria E. coli, DH5alpha and C600. AAL was earlier reported to undergo coagulation in presence of pg level of LPS (Biswas, C., Mandal, C., 1999. The role of amoebocytes in the endotoxin-mediated coagulation in the innate immunity of Achatina fulica snail, Scand. J. Immunol. 49, 131-138). We now demonstrate that Achatinin participates in LPS-mediated coagulation of AAL as indicated by enhanced release of Achatinin from the LPS stimulated amoebocytes and most importantly, by exhibiting a 77% decline in the coagulation of AAL when depleted of Achatinin. Level of Achatinin sharply declined (17-fold) following injection of LPS (20 microg per snail) to the snails, which was reversible by simultaneous injection of LPS and leupeptin implying the presence of LPS-mediated serine protease activity in Achatinin. This was substantiated when purified Achatinin in vitro showed serine protease activity in the presence of LPS followed by its complete blockage in the presence of leupeptin and phenyl methyl sulphonyl fluoride. Therefore, Achatinin, an abundantly available lectin at multiple sites of A. fulica, by virtue of its interaction with LPS, essentially plays a crucial role in the innate immune protection of A. fulica snails.

A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein.

PubMed

Zhang, Xiao-Wen; Xu, Wen-Teng; Wang, Xian-Wei; Mu, Yi; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing

2009-05-01

Lectins are regarded as potential immune recognition proteins. In this study, a novel C-type lectin (Fc-Lec2) was cloned from the hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-Lec2 is 1219 bp with an open reading frame (ORF) of 1002 bp that encodes a protein of 333 amino acids. Fc-Lec2 contains a signal peptide and two different carbohydrate recognition domains (CRDs) arranged in tandem. The first CRD contains a QPD (Gln-Pro-Asp) motif that has a predicted binding specificity for galactose and the second CRD contains a EPN (Glu-Pro-Asn) motif for mannose. Fc-Lec2 was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated in the hepatopancreas of shrimp challenged with bacteria or viruses. Recombinant mature Fc-Lec2 and its two individual CRDs (CRD1 and 2) did not have hemagglutinating activity against animal red blood cells, but agglutinated some gram-positive and gram-negative bacteria in a calcium-dependent manner. The three recombinant proteins also bound to bacteria in the absence of calcium. Fc-Lec2 seems to have broader specificity and higher affinity for bacteria and polysaccharides (peptidoglycan, lipoteichoic acid and lipopolysaccharide) than each of the two individual CRDs. These data suggest that the two CRDs have synergistic effect, and the intact lectin may be more effective in response to bacterial infection, the Fc-Lec2 performs its pattern recognition function by binding to polysaccharides of pathogen cells.

Development of gastrointestinal surface. VIII. Lectin identification of carbohydrate differences

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

Pang, K.Y.; Bresson, J.L.; Walker, W.A.

Binding of microvillus membranes (MVM) from newborn and adult rats by concanavalin A (Con A), Ulex europaeus (UEA I), Dolichos bifluorus (DBA), and Triticum vulgaris (WGA) was examined to determine the availability of carbohydrate-containing sites for these lectins on the intestinal surface during development. Consistent patterns of differences in the reaction of MVM with these lectins were found. Con A and UEA had much higher reactivities to MVM of adult than newborn rats. /sup 125/I-labeled-UEA gel overlay experiments revealed the abundance of UEA-binding sites in MVM of adult rat in contrast to the two binding sites in MVM of amore » newborn rat. DBA bound only to MVM of the adults, and very few binding sites were found in immature MVM. In contrast to these lectins, WGA binding was much higher in MVM of the newborns and decreased with maturation. Additional experiments on the age dependence of UEA and DBA reactivities revealed that the most striking changes occur in animals from 2 to 2 wk of age. In MVM from 2-wk-old rats, there were only 13.9% and < 0.2% of the adult binding capacities for UEA and DBA, respectively. By the time the animals were 4 wk old, the binding capacity for UEA had attained close to the level of the adults, whereas for DBA it reached 71.3% of the adult value. These results provide definite evidence of changes in the intestinal surface during perinatal development.« less

Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

PubMed

Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

2015-02-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Carbohydrate-Dependent Binding of Langerin to SodC, a Cell Wall Glycoprotein of Mycobacterium leprae

PubMed Central

Kim, Hee Jin; Brennan, Patrick J.; Heaslip, Darragh; Udey, Mark C.; Modlin, Robert L.

2014-01-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. PMID:25422308

Eutirucallin, a RIP-2 Type Lectin from the Latex of Euphorbia tirucalli L. Presents Proinflammatory Properties

PubMed Central

Santana, Sanzio Silva; Gennari-Cardoso, Margareth Leitão; Carvalho, Fernanda Caroline; Roque-Barreira, Maria Cristina; Santiago, André da Silva; Alvim, Fátima Cerqueira; Pirovani, Carlos Priminho

2014-01-01

Lectins are carbohydrate-binding proteins that recognize and modulate physiological activities and have been used as a toll for detection and identification of biomolecules, and therapy of diseases. In this study we have isolated a lectin present in the latex of Euphorbia tirucalli, and named it Eutirucallin. The latex protein extract was subjected to ion exchange chromatography and showed two peaks with haemagglutinating activity. Polypeptides of 32 kDa protein extract strongly interacted with immobilized galactose (α-lactose > D-N-acetylgalactosamine). The Eutirucallin was obtained with a yield of 5.6% using the α-lactose column. The lectin domain has 32 kDa subunits and at least two of which are joined by disulfide bridges. The agglutinating capacity for human erythrocytes A+, B+ and O+ is inhibited by D-galactose. The haemagglutinating activity of Eutirucallin was independent of Ca2+ and maintained until the temperature of 55°C. Eutirucallin presented biological activities such as neutrophils recruitment and cytokine prodution by macrophages. The analysis of the trypsin-digested Eutirucallin by ms/ms in ESI-Q-TOFF resulted in nine peptides similar to type 2 ribosome-inactivating protein (type-2 RIP). It's partial sequence showed a similarity of 67.4 – 83.1% for the lectin domain of type-2 RIP [Ricin and Abrin (83.1%), Viscumin, Ebulin, Pulchellin, Cinnamomin, Volkensin and type-2 RIP Iris hollandica]. Our data suggest that Eutirucallin is a new member of type 2 ribosome-inactivating protein and presents biotechnological potential. PMID:24558388

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

PubMed

Wu, A M; Lin, S R; Chin, L K; Chow, L P; Lin, J Y

1992-09-25

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.

My career as an immunoglycobiologist.

PubMed

Marcus, Donald M

2013-01-01

The research program of my laboratory included three major topics: the structures and immunology of human carbohydrate blood group and glycosphingolipid antigens; the tissue distribution, subcellular localization and biosynthesis of glycosphingolipids; and the structural basis of the binding of carbohydrates by antibodies and lectins.

Hexameric supramolecular scaffold orients carbohydrates to sense bacteria.

PubMed

Grünstein, Dan; Maglinao, Maha; Kikkeri, Raghavendra; Collot, Mayeul; Barylyuk, Konstantin; Lepenies, Bernd; Kamena, Faustin; Zenobi, Renato; Seeberger, Peter H

2011-09-07

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.

Carbohydrate recognition by the antiviral lectin cyanovirin-N

PubMed Central

Fujimoto, Yukiji K.; Green, David F.

2012-01-01

Cyanovirin-N is a cyanobacterial lectin with potent antiviral activity, and has been the focus of extensive pre-clinical investigation as a potential prophylactic for the prevention of the sexual transmission of the human immunodeficiency virus (HIV). Here we present a detailed analysis of carbohydrate recognition by this important protein, using a combination of computational methods, including extensive molecular dynamics simulations and Molecular-Mechanics/ Poisson–Boltzmann/Surface-Area (MM/PBSA) energetic analysis. The simulation results strongly suggest that the observed tendency of wildtype CVN to form domain-swapped dimers is the result of a previously unidentified cis-peptide bond present in the monomeric state. The energetic analysis additionally indicates that the highest-affinity ligand for CVN characterized to date (α-Man-(1,2)-α-Man-(1,2)-α-Man) is recognized asymmetrically by the two binding sites. Finally, we are able to provide a detailed map of the role of all binding site functional groups (both backbone and side chain) to various aspects of molecular recognition: general affinity for cognate ligands, specificity for distinct oligosaccharide targets and the asymmetric recognition of α-Man-(1,2)-α-Man-(1,2)-α-Man. Taken as a whole, these results complement past experimental characterization (both structural and thermodynamic) to provide the most complete understanding of carbohydrate recognition by CVN to date. The results also provide strong support for the application of similar approaches to the understanding of other protein–carbohydrate complexes. PMID:23057413

Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy.

PubMed

Peláez, Jesús; Long, Julie A

2007-01-01

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycocluster to lectin and tetanus toxin c-fragment

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Minoura, Norihiko

2010-02-01

We have developed a fluorescent ruthenium metalloglycocluster as a powerful molecular probe for evaluating a binding event between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analysis. The fluorescent ruthenium metalloglycoclusters, [Ru(bpy-2Gal)3] and [Ru(bpy-2Glc)3], possess clustered galactose and glucose surrounding the ruthenium center. Changes in FE and FP of these metalloglycoclusters were measured by adding each lectin (Peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), Concanavalin A (ConA), or Wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA, the FE spectrum of [Ru(bpy- 2Gal)3] showed new emission peak and the FP value of [Ru(bpy-2Gal)3] increased. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] showed new emission peak and the FP value increased following the addition of ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were proved by the FE and FP measurement. From nonlinear least-squares fitting, dissociation constants (Kd) of [Ru(bpy-2Gal)3] to PNA was 6.1 μM, while the Kd values of [Ru(bpy)2(bpy-2Gal)] to PNA was ca. 10-4 M. Therefore, the clustered carbohydrates were proved to increase affinity to lectins. Furthermore, the FP measurements proved specific binding of [Ru(bpy-2Gal)3] to TCF.

Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor

PubMed Central

1990-01-01

Considerable evidence implicates gp90MEL as a lymphocyte homing receptor mediating lymphocyte attachment to high endothelial venules of lymph nodes in mouse. The protein appears to function as a calcium- dependent, lectin-like receptor as inferred primarily by the ability of specific carbohydrates to block its function and by the presence of a calcium-type lectin domain in its primary sequence. An ELISA assay is described which provides the first demonstration that the isolated protein has lectin activity and allows a further definition of its carbohydrate specificity. In addition to the monosaccharides mannose-6- phosphate and fructose-1-phosphate, ligand activity is shown for the sulfated glycolipid, sulfatide, and for two sulfated fucose-containing polysaccharides (fucoidin and egg jelly coat) from nonmammalian sources. PMID:2202735

A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

PubMed

Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd

2014-02-10

Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties. Copyright © 2013 Elsevier B.V. All rights reserved.

Deficiency in Mannose-Binding Lectin-Associated Serine Protease-2 Does Not Increase Susceptibility to Trypanosoma cruzi Infection

PubMed Central

Ribeiro, Carolina H.; Lynch, Nicholas J.; Stover, Cordula M.; Ali, Youssif M.; Valck, Carolina; Noya-Leal, Francisca; Schwaeble, Wilhelm J.; Ferreira, Arturo

2015-01-01

Trypanosoma cruzi is the causative agent of Chagas' disease, a chronic illness affecting 10 million people around the world. The complement system plays an important role in fighting microbial infections. The recognition molecules of the lectin pathway of complement activation, mannose-binding lectin (MBL), ficolins, and CL-11, bind to specific carbohydrates on pathogens, triggering complement activation through MBL-associated serine protease-2 (MASP-2). Previous in vitro work showed that human MBL and ficolins contribute to T. cruzi lysis. However, MBL-deficient mice are only moderately compromised in their defense against the parasite, as they may still activate the lectin pathway through ficolins and CL-11. Here, we assessed MASP-2-deficient mice, the only presently available mouse line with total lectin pathway deficiency, for a phenotype in T. cruzi infection. Total absence of lectin pathway functional activity did not confer higher susceptibility to T. cruzi infection, suggesting that it plays a minor role in the immune response against this parasite. PMID:25548381

Lectin I from Bauhinia variegata (BVL-I) expressed by Pichia pastoris inhibits initial adhesion of oral bacteria in vitro.

PubMed

Klafke, Gabriel Baracy; Moreira, Gustavo Marçal Schmidt Garcia; Pereira, Juliano Lacava; Oliveira, Patrícia Diaz; Conceição, Fabricio Rochedo; Lund, Rafael Guerra; Grassmann, André Alex; Dellagostin, Odir Antonio; da Silva Pinto, Luciano

2016-12-01

Lectins are non-immune proteins that reversibly bind to carbohydrates in a specific manner. Bauhinia variegata lectin I (BVL-I) is a Gal/GalNAc-specific, single-chain lectin isolated from Bauhinia variegata seeds that has been implicated in the inhibition of bacterial adhesion and the healing of damaged skin. Since the source of the native protein (nBVL) is limited, this study aimed to produce recombinant BVL-I in Pichia pastoris (rBVL-Ip). The coding sequence for BVL-I containing preferential codons for P. pastoris was cloned into the pPICZαB plasmid. A single expressing clone was selected and fermented, resulting in the secretion and glycosylation of the protein. Fed-batch fermentation in 7L-scale was performed, and the recombinant lectin was purified from culture supernatant, resulting in a yield of 1.5mg/L culture. Further, rBVL-Ip was compared to nBVL and its recombinant version expressed in Escherichia coli BL21 (DE3) (rBVL-Ie). Although it was expressed as a monomer, rBVL-Ip retained its biological activity since it was able to impair the initial adhesion of Streptococcus mutans and S. sanguinis in an in vitro model of biofilm formation and bacterial adhesion. In summary, rBVL-Ip produced in Pichia pastoris represents a viable alternative to large-scale production, encouraging further biological application studies with this lectin. Copyright © 2016 Elsevier B.V. All rights reserved.

Lectins and their application to clinical microbiology.

PubMed Central

Slifkin, M; Doyle, R J

1990-01-01

Lectins are generally associated with plant or animal components, selectively bind carbohydrates, and interact with procaryotic and eucaryotic cells. Lectins have various specificities that are associated with their ability to interact with acetylaminocarbohydrates, aminocarbohydrates, sialic acids, hexoses, pentoses, and as other carbohydrates. Microbial surfaces generally contain many of the sugar residues that react with lectins. Lectins are presently used in the clinical laboratory to type blood cells and are used in a wide spectrum of applications, including, in part, as carriers of chemotherapeutic agents, as mitogens, for fractionation of animal cells, and for investigations of cellular surfaces. Numerous studies have shown that lectins can be used to identify rapidly certain microorganisms isolated from a clinical specimen or directly in a clinical specimen. Lectins have been demonstrated to be important diagnostic reagents in the major realms of clinical microbiology. Thus, they have been applied in bacteriology, mycology, mycobacteriology, and virology for the identification and/or differentiation of various microorganisms. Lectins have been used successfully as epidemiologic as well as taxonomic markers of specific microorganisms. Lectins provide the clinical microbiologist with cost-effective and potential diagnostic reagents. This review describes the applications of lectins in clinical microbiology. Images PMID:2200603

Determination of the glycosylation-pattern of the middle ear mucosa in guinea pigs.

PubMed

Engleder, Elisabeth; Demmerer, Elisabeth; Wang, Xueyan; Honeder, Clemens; Zhu, Chengjing; Studenik, Christian; Wirth, Michael; Arnoldner, Christoph; Gabor, Franz

2015-04-30

In the present study the glycosylation pattern of the middle ear mucosa (MEM) of guinea pigs, an approved model for middle ear research, was characterized with the purpose to identify bioadhesive ligands which might prolong the contact time of drug delivery systems with the middle ear mucosa (MEM). To assess the utility of five fluorescein labeled plant lectins with different carbohydrate specificities as bioadhesive ligands, viable MEM specimens were incubated at 4°C and the lectin binding capacities were calculated from the MEM-associated relative fluorescence intensities. Among all lectins under investigation, fluorescein-labeled wheat germ agglutinin (F-WGA) emerged as the highest bioadhesive lectin. In general, the accessibility of carbohydrate moieties of the MEM followed the order: sialic acid and N-acetyl-d-glucosamine (WGA)>mannose and galactosamine (Lensculinaris agglutinin)>N-acetyl-d-glucosamine (Solanumtuberosum agglutinin)>fucose (Ulexeuropaeus isoagglutinin I)>terminal mannose α-(1,3)-mannose (Galanthusnivalis agglutinin). Competitive inhibition studies with the corresponding carbohydrate revealed that F-WGA-binding was inhibited up to 90% confirming specificity of the F-WGA-MEM interaction. The cilia of the MEM were identified as F-WGA binding sites by fluorescence imaging as well as a z-stack of overlays of transmission, F-WGA- and nuclei-stained images of the MEM. Additionally, co-localisation experiments revealed that F-WGA bound to acidic mucopolysaccharides of the MEM. All in all, lectin-mediated bioadhesion to the MEM is proposed as a new concept for drug delivery to prolong the residence time of the drug in the tympanic cavity especially for successful therapy for difficult-to-treat diseases such as otitis media. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Sequence-based predictive modeling to identify cancerlectins

PubMed Central

Lai, Hong-Yan; Chen, Xin-Xin; Chen, Wei; Tang, Hua; Lin, Hao

2017-01-01

Lectins are a diverse type of glycoproteins or carbohydrate-binding proteins that have a wide distribution to various species. They can specially identify and exclusively bind to a certain kind of saccharide groups. Cancerlectins are a group of lectins that are closely related to cancer and play a major role in the initiation, survival, growth, metastasis and spread of tumor. Several computational methods have emerged to discriminate cancerlectins from non-cancerlectins, which promote the study on pathogenic mechanisms and clinical treatment of cancer. However, the predictive accuracies of most of these techniques are very limited. In this work, by constructing a benchmark dataset based on the CancerLectinDB database, a new amino acid sequence-based strategy for feature description was developed, and then the binomial distribution was applied to screen the optimal feature set. Ultimately, an SVM-based predictor was performed to distinguish cancerlectins from non-cancerlectins, and achieved an accuracy of 77.48% with AUC of 85.52% in jackknife cross-validation. The results revealed that our prediction model could perform better comparing with published predictive tools. PMID:28423655

Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Acα2-6Galβ1-4GlcNAc human-type influenza receptor

PubMed Central

Kadirvelraj, Renuka; Grant, Oliver C; Goldstein, Irwin J; Winter, Harry C; Tateno, Hiroaki; Fadda, Elisa; Woods, Robert J

2011-01-01

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7 Å) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding. PMID:21436237

Molecular cloning of a C-type lectin with two CRD domains from the banana shrimp Fenneropenaeus merguiensis: early gene up-regulation after Vibrio harveyi infection.

PubMed

Rattanaporn, Onnicha; Utarabhand, Prapaporn

2011-02-01

A diverse class of pattern-recognition proteins called lectins play important roles in shrimp innate immunity. A novel C-type lectin gene (FmLC) was cloned from the hepatopancreas of banana shrimp Fenneropenaeus merguiensis by means of PCR and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length cDNA consists of 1118 bp with one 1002 bp open reading frame, encoding 333 amino acids. Its deduced amino acid sequence contains a putative signal peptide of 20 amino acids. FmLC contains two carbohydrate recognition domains, CRD1 and CRD2, that share only 30% identity with each other. The first CRD comprises a QPD motif with specificity for binding galactose and a single Ca(2+) binding site, while the second CRD consists of an EPN motif for a mannose-specific binding site. FmLC had a close evolutionary relationship to other dual-CRD lectins of penaeid shrimp. Expression results showed that transcripts of FmLC were detected only in the hepatopancreas, none was found in other tissues. After challenging either whole shrimp or hepatopancreas tissue fragments with Vibrioharveyi, the expression of FmLC was up-regulated. This indicates that FmLC is inducible and may be involved in a shrimp immune response to recognize potential bacterial pathogens. Copyright © 2010 Elsevier Inc. All rights reserved.

Crystal structure of Pisum arvense seed lectin (PAL) and characterization of its interaction with carbohydrates by molecular docking and dynamics.

PubMed

Pinto-Junior, Vanir Reis; Santiago, Mayara Queiroz; Nobre, Camila Bezerra; Osterne, Vinicius Jose Silva; Leal, Rodrigo Bainy; Cajazeiras, Joao Batista; Lossio, Claudia Figueiredo; Rocha, Bruno Anderson Matias; Martins, Maria Gleiciane Queiroz; Nobre, Clareane Avelino Simplicio; Silva, Mayara Torquato Lima; Nascimento, Kyria Santiago; Cavada, Benildo Sousa

2017-09-15

The Pisum arvense lectin (PAL), a legume protein belonging to the Vicieae tribe, is capable of specific recognition of mannose, glucose and its derivatives without altering its structure. In this work, the three-dimensional structure of PAL was determined by X-ray crystallography and studied in detail by a combination of molecular docking and molecular dynamics (MD). Crystals belonging to monoclinic space group P2 1 were grown by the vapor diffusion method at 293 K. The structure was solved at 2.16 Å and was similar to that of other Vicieae lectins. The structure presented R factor and R free of 17.04% and 22.08%, respectively, with all acceptable geometric parameters. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and high-mannose N-glycans. PAL demonstrated different affinities on carbohydrates, depending on bond orientation and glycosidic linkage present in ligands. Furthermore, the lectin interacted with representative N-glycans in a manner consistent with the biological effects described for Vicieae lectins. Carbohydrate-recognition domain (CRD) in-depth analysis was performed by MD, describing the behavior of CRD residues in complex with ligand, stability, flexibility of the protein over time, CRD volume and topology. This is a first report of its kind for a lectin of the Vicieae tribe. Copyright © 2017 Elsevier Inc. All rights reserved.

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).

Identification and Characterization of C-type Lectins in Ostrinia furnacalis (Lepidoptera: Pyralidae)

PubMed Central

Shen, Dongxu; Wang, Lei; Ji, Jiayue; Liu, Qizhi; An, Chunju

2018-01-01

Abstract C-type lectins (CTLs) are a large family of calcium-dependent carbohydrate-binding proteins. They function primarily in cell adhesion and immunity by recognizing various glycoconjugates. We identified 14 transcripts encoding proteins with one or two CTL domains from the transcriptome from Asian corn borer, Ostrinia furnacalis (Guenée; Lepidoptera: Pyralidae). Among them, five (OfCTL-S1 through S5) only contain one CTL domain, the remaining nine (OfIML-1 through 9) have two tandem CTL domains. Five CTL-Ss and six OfIMLs have a signal peptide are likely extracellular while another two OfIMLs might be cytoplasmic. Phylogenetic analysis indicated that OfCTL-Ss had 1:1 orthologs in Lepidoptera, Diptera, Coleoptera and Hymenoptera species, but OfIMLs only clustered with immulectins (IMLs) from Lepidopteran. Structural modeling revealed that the 22 CTL domains adopt a similar double-loop fold consisting of β-sheets and α-helices. The key residues for calcium-dependent or independent binding of specific carbohydrates by CTL domains were predicted with homology modeling. Expression profiles assay showed distinct expression pattern of 14 CTLs: the expression and induction were related to the developmental stages and infected microorganisms. Overall, our work including the gene identification, sequence alignment, phylogenetic analysis, structural modeling, and expression profile assay would provide a valuable basis for the further functional studies of O. furnacalis CTLs. PMID:29718486

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

PubMed

Hoover, G J; el-Mowafi, A; Simko, E; Kocal, T E; Ferguson, H W; Hayes, M A

1998-07-01

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Protein-Carbohydrate Interactions Studied by NMR: From Molecular Recognition to Drug Design

PubMed Central

Fernández-Alonso, María del Carmen; Díaz, Dolores; Berbis, Manuel Álvaro; Marcelo, Filipa; Cañada, Javier; Jiménez-Barbero, Jesús

2012-01-01

Diseases that result from infection are, in general, a consequence of specific interactions between a pathogenic organism and the cells. The study of host-pathogen interactions has provided insights for the design of drugs with therapeutic properties. One area that has proved to be promising for such studies is the constituted by carbohydrates which participate in biological processes of paramount importance. On the one hand, carbohydrates have shown to be information carriers with similar, if not higher, importance than traditionally considered carriers as amino acids and nucleic acids. On the other hand, the knowledge on molecular recognition of sugars by lectins and other carbohydrate-binding proteins has been employed for the development of new biomedical strategies. Biophysical techniques such as X-Ray crystallography and NMR spectroscopy lead currently the investigation on this field. In this review, a description of traditional and novel NMR methodologies employed in the study of sugar-protein interactions is briefly presented in combination with a palette of NMR-based studies related to biologically and/or pharmaceutically relevant applications. PMID:23305367

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

A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3+, CD4+, and CD8+ populations of human PBMCs.

PubMed

Gavrovic-Jankulovic, Marija; Poulsen, Knud; Brckalo, Tamara; Bobic, Sonja; Lindner, Buko; Petersen, Arnd

2008-01-01

Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal alpha1,3 linkages as well as beta1,3 linkages at the reducing termini. The immunomodulatory potential of natural BanLec was recognized by a strong immunoglobulin G4 antibody response and T cell mitogen activity in humans. To explore its applicability in glycoproteomics and its modulatory potential, the gene of banana lectin was cloned, sequenced and a recombinant protein was produced in Escherichia coli. The obtained cDNA revealed a novel banana lectin isoform, with an open reading frame of 426 nucleotides, encoding a cytoplasmatic protein of 141 amino acids. The molecular mass of rBanLec determined by ESI FT-MS and N-terminal sequencing confirmed the cDNA at the protein level. The specificity of rBanLec for detection glycan structures was the same as for natural BanLec as examined with five protein extracts rich in glycoprotein content, as well as with horseradish peroxidase glycoprotein. Besides, the immunomodulatory potential of rBanLec and nBanLec were comparable as assessed by an inhibition assay and a human T cell proliferation assay where they induced a strong proliferation response in CD3+, CD4+, and CD8+ populations of human PBMCs. This recombinant BanLec is a useful reagent for glycoproteomics and lectin microarrays, with a potential for modulation of the immune response.

Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization.

PubMed

Ji, Xin; Olinger, Gene G; Aris, Sheena; Chen, Ying; Gewurz, Henry; Spear, Gregory T

2005-09-01

Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

Carbohydrate-protein interactions: molecular modeling insights.

PubMed

Pérez, Serge; Tvaroška, Igor

2014-01-01

The article reviews the significant contributions to, and the present status of, applications of computational methods for the characterization and prediction of protein-carbohydrate interactions. After a presentation of the specific features of carbohydrate modeling, along with a brief description of the experimental data and general features of carbohydrate-protein interactions, the survey provides a thorough coverage of the available computational methods and tools. At the quantum-mechanical level, the use of both molecular orbitals and density-functional theory is critically assessed. These are followed by a presentation and critical evaluation of the applications of semiempirical and empirical methods: QM/MM, molecular dynamics, free-energy calculations, metadynamics, molecular robotics, and others. The usefulness of molecular docking in structural glycobiology is evaluated by considering recent docking- validation studies on a range of protein targets. The range of applications of these theoretical methods provides insights into the structural, energetic, and mechanistic facets that occur in the course of the recognition processes. Selected examples are provided to exemplify the usefulness and the present limitations of these computational methods in their ability to assist in elucidation of the structural basis underlying the diverse function and biological roles of carbohydrates in their dialogue with proteins. These test cases cover the field of both carbohydrate biosynthesis and glycosyltransferases, as well as glycoside hydrolases. The phenomenon of (macro)molecular recognition is illustrated for the interactions of carbohydrates with such proteins as lectins, monoclonal antibodies, GAG-binding proteins, porins, and viruses. © 2014 Elsevier Inc. All rights reserved.

The pepper GNA-related lectin and PAN domain protein gene, CaGLP1, is required for plant cell death and defense signaling during bacterial infection.

PubMed

Kim, Nak Hyun; Lee, Dong Hyuk; Choi, Du Seok; Hwang, Byung Kook

2015-12-01

Carbohydrate-binding proteins, commonly referred to as lectins or agglutinins, function in defense responses to microbial pathogens. Pepper (Capsicum annuum) GNA-related lectin and PAN-domain protein gene CaGLP1 was isolated and functionally characterized from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaGLP1 contained an amine-terminus prokaryotic membrane lipoprotein lipid attachment site, a Galanthus nivalis agglutinin (GNA)-related lectin domain responsible for the recognition of high-mannose N-glycans, and a carboxyl-terminus PAN/apple domain. RNA gel blot and immunoblot analyses determined that CaGLP1 was strongly induced in pepper by compatible and incompatible Xcv infection. CaGLP1 protein localized primarily to the plasma membrane and exhibited mannose-binding specificity. CaGLP1-silenced pepper plants were more susceptible to compatible or incompatible Xcv infection compared with that of non-silenced control plants. CaGLP1 silencing in pepper leaves did not accumulate H2O2 and induce cell death during incompatible Xcv infection. Defense-related CaDEF1 (defensin) gene expression was significantly reduced in CaGLP1-silenced pepper plants. CaGLP1-overexpression in Arabidopsis thaliana enhanced resistance to Pseudomonas syringae pv. tomato. Defense-related AtPDF1.2 expression was elevated in CaGLP1-overexpression lines. Together, these results suggest that CaGLP1 is required for plant cell death and defense responses through the reactive oxygen species burst and downstream defense-related gene expression in response to bacterial pathogen challenge. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Influence of the state of phase of lipid bilayer on the exposure of glucose residues on the surface of liposomes.

PubMed

Villalva, Denise Gradella; Giansanti, Luisa; Mauceri, Alessandro; Ceccacci, Francesca; Mancini, Giovanna

2017-11-01

The presence of carbohydrate-binding proteins (i.e. lectins) on the surface of various bacterial strains and their overexpression in some tumor tissues makes the use of glycosylated liposomes a promising approach for the specific drug delivery in antibacterial and anti-cancer therapies. However, the functionalization of liposome surface with sugar moieties by glycosylated amphiphiles does not ensure the binding of sugar-coated vesicles with lectins. In fact, the composition and properties of lipid bilayer play a pivotal role in the exposure of sugar residues and in the interaction with lectins. The influence of the length of the hydrophilic spacer that links the sugar to liposome surface and of the presence of saturated or unsaturated phospholipids in the lipid bilayer on the ability of glucosylated liposomes to interact with a model lectin, Concanavalin A, was investigated. Our results demonstrate that both the chain length and the prensece of unsaturation, parameters that strongly affect the fluidity of the lipid bilayer, affect agglutination. In particular, agglutination is favored when liposomes are in the gel phase within a defined range of temperature. Moreover, the obtained results confirm that the length of the PEG spacer, that influences both lipid organization and the exposure of sugar moieties to the bulk, plays a crucial role in liposome/lectin interaction. Copyright © 2017 Elsevier B.V. All rights reserved.

Mannose glycoconjugates functionalized at positions 1 and 6. Binding analysis to DC-SIGN using biosensors.

PubMed

Reina, José J; Maldonado, Olivia S; Tabarani, Georges; Fieschi, Franck; Rojo, Javier

2007-01-01

The design of glycoconjugates to allow the generation of multivalent ligands capable of interacting with the receptor DC-SIGN is a topic of high interest due to the role played by this lectin in pathogen infections. Mannose, a ligand of this lectin, could be conjugated at two different positions, 1 and 6, not implicated in the binding process. We have prepared mannose conjugates at these two positions with a long spacer to allow their attachment to a biosensor chip surface. Analysis of the interaction between these surfaces and the tetravalent extracellular domain (ECD) of DC-SIGN by SPR biosensor has demonstrated that both positions are available for this conjugation without affecting the protein binding process. These results emphasize the possibility to conjugate mannose at position 6, allowing the incorporation of hydrophobic groups at the anomeric position to interact with hydrophobic residues in the carbohydrate recognition domain of DC-SIGN, increasing binding affinities. This fact is relevant for the future design of new ligands and the corresponding multivalent systems for DC-SIGN.

Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin.

PubMed

Sarabia-Sainz, Andre-i; Ramos-Clamont, Gabriela; Candia-Plata, Ma María del Carmen; Vázquez-Moreno, Luz

2009-03-01

Escherichia coli (E. coli) that expresses galactose-reactive lectins, like K88 adhesin, causes high mortality among piglets. Carbohydrates that compete for adhesion could serve as an alternative for disease prevention. Porcine serum albumin (PSA) was modified by non-enzymatic glycation with lactose to produce PSA-Lac or PSA-Glc beta (1-4) Gal, as confirmed by reduction of available free amino groups, increased molecular mass and by Ricinus communis lectin recognition. E. coli K88 binds to PSA-Lac treatments containing three and four lactoses, respectively. In addition, PSA-Lac partially inhibited K88 strain adherence to mucins. These results suggest that neoglycoconjugates obtained by non-enzymatic glycation of proteins may serve in the prophylaxis of piglets' diarrhea.

Diagnosis of myocardial infarction based on lectin-induced erythrocyte agglutination: a feasibility study

NASA Astrophysics Data System (ADS)

Bocsi, József; Nieschke, Kathleen; Mittag, Anja; Reichert, Thomas; Laffers, Wiebke; Marecka, Monika; Pierzchalski, Arkadiusz; Piltz, Joachim; Esche, Hans-Jürgen; Wolf, Günther; Dähnert, Ingo; Baumgartner, Adolf; Tarnok, Attila

2014-03-01

Myocardial infarction (MI) is an acute life-threatening disease with a high incidence worldwide. Aim of this study was to test lectin-carbohydrate binding-induced red blood cell (RBC) agglutination as an innovative tool for fast, precise and cost effective diagnosis of MI. Five lectins (Ricinus communis agglutinin (RCA), Phaseolus vulgaris erythroagglutinin (PHA), Datura stramonium agglutinin (DSA), Artocarpus agglutinin (ArA), Triticum agglutinin (TA)) were tested for ability to differentiate between agglutination characteristics in patients with MI (n = 101) or angina pectoris without MI (AP) (n = 34) and healthy volunteers (HV) as control (n =68) . RBC agglutination was analyzed by light absorbance of a stirred RBC suspension in the green to red light spectrum in an agglutimeter (amtec, Leipzig, Germany) for 15 min after lectin addition. Mean cell count in aggregates was estimated from light absorbance by a mathematical model. Each lectin induced RBC agglutination. RCA led to the strongest RBC agglutination (~500 RBCs/aggregate), while the others induced substantially slower agglutination and lead to smaller aggregate sizes (5-150 RBCs/aggregate). For all analyzed lectins the lectin-induced RBC agglutination of MI or AP patients was generally higher than for HV. However, only PHA induced agglutination that clearly distinguished MI from HV. Variance analysis showed that aggregate size after 15 min. agglutination induced by PHA was significantly higher in the MI group (143 RBCs/ aggregate) than in the HV (29 RBC-s/aggregate, p = 0.000). We hypothesize that pathological changes during MI induce modification of the carbohydrate composition on the RBC membrane and thus modify RBC agglutination. Occurrence of carbohydrate-lectin binding sites on RBC membranes provides evidence about MI. Due to significant difference in the rate of agglutination between MI > HV the differentiation between these groups is possible based on PHA-induced RBC-agglutination. This novel assay could serve as a rapid, cost effective valuable new tool for diagnosis of MI.

Interspecific and host-related gene expression patterns in nematode-trapping fungi.

PubMed

Andersson, Karl-Magnus; Kumar, Dharmendra; Bentzer, Johan; Friman, Eva; Ahrén, Dag; Tunlid, Anders

2014-11-11

Nematode-trapping fungi are soil-living fungi that capture and kill nematodes using special hyphal structures called traps. They display a large diversity of trapping mechanisms and differ in their host preferences. To provide insights into the genetic basis for this variation, we compared the transcriptome expressed by three species of nematode-trapping fungi (Arthrobotrys oligospora, Monacrosporium cionopagum and Arthrobotrys dactyloides, which use adhesive nets, adhesive branches or constricting rings, respectively, to trap nematodes) during infection of two different plant-pathogenic nematode hosts (the root knot nematode Meloidogyne hapla and the sugar beet cyst nematode Heterodera schachtii). The divergence in gene expression between the fungi was significantly larger than that related to the nematode species being infected. Transcripts predicted to encode secreted proteins and proteins with unknown function (orphans) were overrepresented among the highly expressed transcripts in all fungi. Genes that were highly expressed in all fungi encoded endopeptidases, such as subtilisins and aspartic proteases; cell-surface proteins containing the carbohydrate-binding domain WSC; stress response proteins; membrane transporters; transcription factors; and transcripts containing the Ricin-B lectin domain. Differentially expressed transcripts among the fungal species encoded various lectins, such as the fungal fruit-body lectin and the D-mannose binding lectin; transcription factors; cell-signaling components; proteins containing a WSC domain; and proteins containing a DUF3129 domain. A small set of transcripts were differentially expressed in infections of different host nematodes, including peptidases, WSC domain proteins, tyrosinases, and small secreted proteins with unknown function. This is the first study on the variation of infection-related gene expression patterns in nematode-trapping fungi infecting different host species. A better understanding of these patterns will facilitate the improvements of these fungi in biological control programs, by providing molecular markers for screening programs and candidates for genetic manipulations of virulence and host preferences.

The homologue of mannose-binding lectin in the carp family Cyprinidae is expressed at high level in spleen, and the deduced primary structure predicts affinity for galactose.

PubMed

Vitved, L; Holmskov, U; Koch, C; Teisner, B; Hansen, S; Salomonsen, J; Skjødt, K

2000-09-01

Mannose-binding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. We isolated and characterized cDNA transcripts encoding an MBL homologue from three members of the carp family Cyprinidae, the zebrafish Danio rerio, the goldfish Carassius auratus, and the carp Cyprinus carpio. The carp and zebrafish transcripts contain two polyadenylation sites and RT-PCR on mRNA from carp tissues revealed the carp transcript to be most prominently expressed in the spleen. The deduced mature proteins contain 228 or 233 amino acids with a short N-terminal segment containing a single conserved cysteine expected to form interchain disulfide bridges, a collagen domain interrupted by four amino acids between two glycine residues, a neck region predicted to form an alpha-helical coiled-coil structure, and a C-terminal carbohydrate recognition domain (CRD). Several of the structurally important residues in the CRD are conserved, but the residues known to interact with the calcium ion and hydroxyl groups of the carbohydrate ligand are different. The amino acid motif EPN, important for mannose specificity, was QPD in the Cyprinidae homologue, suggesting specificity for galactose instead. The identity between the deduced amino acid sequences is more than 90% between the carp and the goldfish and 68% and 65% between these two species, respectively, and the zebrafish. The identity with bird and mammalian MBLs ranges from 28 to 33%.

Purification and characterization of liver lectins from a lizard, Sceloporus spinosus.

PubMed

Fenton, N Bertha; Arreguín, L Barbarin; Méndez, C Fausto; Arreguín, E Roberto

2004-05-01

This study discusses the purification of soluble beta-galactose lectins obtained from the lizard liver of Sceloporus spinosus. The first lectin named lizard hepatic lectin-1 (LHL-1) presented a molecular weight of 31,750, with an isoelectric point of 4.25. The highest specific hemagglutinating activity was achieved using human blood type A1: N-acetylgalactosamine (GalNAc)-galactose (Gal)-fucose (Fuc). Carbohydrate inhibition assays indicated a higher lectin specificity for GalNAc. For LHL-2 the molecular weight obtained was 23,850 with an isoelectric point of 3.25. The highest carbohydrate specificity was observed for Gal. These lizard hepatic lectins are similar to the mammal hepatic lectins previously reported. However, it is different from the alligator hepatic lectin (AHL). The homology analyses of LHL-1 resulted in 100% identity with the Steroidogenic acute regulatory protein (StAR), while LHL-2 was similar to adenylate kinase (75% identity). We suggest that these liver lectins are related to the inherent functions of liver previously reported.

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus.

PubMed

Wei, Xiumei; Liu, Xiangquan; Yang, Jianmin; Fang, Jinghui; Qiao, Hongjin; Zhang, Ying; Yang, Jialong

2012-01-01

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca(2+) binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln(122)-Pro(123)-Asn(124)) in LvLectin-1, but QPD (Gln(128)-Pro(129)-Asp(130)) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P<0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P<0.01) and 12 h (P<0.05), but the expression level of LvLectin-1 down-regulated significantly (P<0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco.

PubMed

Ke, F; Zhang, H B; Wang, Y; Hou, L F; Dong, H J; Wang, Z F; Pan, G W; Cao, X Y

2016-09-01

This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR). © 2016 The Fisheries Society of the British Isles.

Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

PubMed

Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

2016-07-01

Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis.

PubMed

Liu, Yi-Chen; Li, Fu-Hua; Dong, Bo; Wang, Bing; Luan, Wei; Zhang, Xiao-Jun; Zhang, Liu-Suo; Xiang, Jian-Hai

2007-01-01

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have 11 amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site 1 are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control.

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

Expression of Ulex europaeus agglutinin I lectin-binding sites in squamous cell carcinomas and their absence in basal cell carcinomas. Indicator of tumor type and differentiation.

PubMed

Heng, M C; Fallon-Friedlander, S; Bennett, R

1992-06-01

Lectins bind tightly to carbohydrate moieties on cell surfaces. Alterations in lectin binding have been reported to accompany epidermal cell differentiation, marking alterations in membrane sugars during this process. The presence of UEA I (Ulex europaeus agglutinin I) L-fucose-specific lectin-binding sites has been used as a marker for terminally differentiated (committed) keratinocytes. In this article, we report the presence of UEA-I-binding sites on squamous keratinocytes of well-differentiated squamous cell carcinomas, with patchy loss of UEA I positivity on poorly differentiated cells of squamous cell carcinomas, suggesting a possible use for this technique in the rapid assessment of less differentiated areas within the squamous cell tumor. The absence of UEA-I-binding sites on basal cell carcinomas may be related to an inability of cells comprising this tumor to convert the L-D-pyranosyl moiety on basal cells to the L-fucose moiety, resulting in an inability of basal cell carcinoma cell to undergo terminal differentiation into a committed keratinocyte.

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold.

PubMed Central

Rosa, J. C.; De Oliveira, P. S.; Garratt, R.; Beltramini, L.; Resing, K.; Roque-Barreira, M. C.; Greene, L. J.

1999-01-01

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature. PMID:10210179

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

PubMed

Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

1985-05-01

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

Insecticidal activity of plant lectins and potential application in crop protection.

PubMed

Macedo, Maria Lígia R; Oliveira, Caio F R; Oliveira, Carolina T

2015-01-27

Lectins constitute a complex group of proteins found in different organisms. These proteins constitute an important field for research, as their structural diversity and affinity for several carbohydrates makes them suitable for numerous biological applications. This review addresses the classification and insecticidal activities of plant lectins, providing an overview of the applicability of these proteins in crop protection. The likely target sites in insect tissues, the mode of action of these proteins, as well as the use of lectins as biotechnological tools for pest control are also described. The use of initial bioassays employing artificial diets has led to the most recent advances in this field, such as plant breeding and the construction of fusion proteins, using lectins for targeting the delivery of toxins and to potentiate expected insecticide effects. Based on the data presented, we emphasize the contribution that plant lectins may make as tools for the development of integrated insect pest control strategies.

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

PubMed

Endo, T; Ohbayashi, H; Kanazawa, K; Kochibe, N; Kobata, A

1992-01-15

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.

Molecular cloning of rat sperm galactosyl receptor, a C-type lectin with in vitro egg binding activity.

PubMed

Rivkin, E; Tres, L L; Kaplan-Kraicer, R; Shalgi, R; Kierszenbaum, A L

2000-07-01

Rat sperm galactosyl receptor is a member of the C-type animal lectin family showing preferential binding to N-acetylgalactosamine compared to galactose. Binding is mediated by a Ca(2+)-dependent carbohydrate-recognition domain (CRD) identical to that of the minor variant of rat hepatic lectin receptor 2/3 (RHL-2/3). The molecular organization of the genomic DNA, cDNA, and derived amino acid sequence of rat testis galactosyl receptor have been determined and in vitro fertilization studies were conducted to ascertain its role. We have determined that the rat testis galactosyl receptor gene generates two mRNA species: one species, designated liver-type, is identical to RHL-2/3; the other, designated testis-type, contains one unspliced intron (86 nt) which alters the reading frame and changes the amino acid sequence of the carboxyl terminus. As a result, the CRD (glutamine-proline-aspartic acid/QPD) and flanked Ca(2+)-binding amino acid sequences were not present in the testis-type protein. Northern and Southern blots demonstrated presence of transcripts with unspliced intron in rat sperm but not liver. Similarly, antibody, raised against a synthetic 12-amino acid peptide (p12) encoded by the unspliced intron, recognized in immunoblots a 54 kDa receptor protein in protein extracts from testis but not from liver. Immunofluorescence and immunogold electron microscopy studies demonstrated that both protein species localized on the plasma membrane surface of the head and tail of rat sperm. Furthermore, capacitated rat sperm preincubated with polyclonal antisera to RHL-2/3 or to the CRD of the liver-type galactosyl receptor showed a statistically significant decrease in the in vitro fertilization rate. We conclude that rat sperm galactosyl receptor may play a role in egg binding and that an undetermined molecular mechanism operates to generate two proteins with identical intracellular amino terminal domain but only one of them displays a CRD and associated Ca(2+)-binding sites at the carboxyl terminal extracellular domain. Copyright 2000 Wiley-Liss, Inc.

Glycosaminoglycans are interactants of Langerin: comparison with gp120 highlights an unexpected calcium-independent binding mode.

PubMed

Chabrol, Eric; Nurisso, Alessandra; Daina, Antoine; Vassal-Stermann, Emilie; Thepaut, Michel; Girard, Eric; Vivès, Romain R; Fieschi, Franck

2012-01-01

Langerin is a C-type lectin specifically expressed in Langerhans cells. As recently shown for HIV, Langerin is thought to capture pathogens and mediate their internalisation into Birbeck Granules for elimination. However, the precise functions of Langerin remain elusive, mostly because of the lack of information on its binding properties and physiological ligands. Based on recent reports that Langerin binds to sulfated sugars, we conducted here a comparative analysis of Langerin interaction with mannose-rich HIV glycoprotein gp120 and glycosaminoglycan (GAGs), a family of sulfated polysaccharides expressed at the surface of most mammalian cells. Our results first revealed that Langerin bound to these different glycans through very distinct mechanisms and led to the identification of a novel, GAG-specific binding mode within Langerin. In contrast to the canonical lectin domain, this new binding site showed no Ca(2+)-dependency, and could only be detected in entire, trimeric extracellular domains of Langerin. Interestingly binding to GAGs, did not simply rely on a net charge effect, but rather on more discrete saccharide features, such as 6-O-sulfation, or iduronic acid content. Using molecular modelling simulations, we proposed a model of Langerin/heparin complex, which located the GAG binding site at the interface of two of the three Carbohydrate-recognition domains of the protein, at the edge of the a-helix coiled-coil. To our knowledge, the binding properties that we have highlighted here for Langerin, have never been reported for C-type lectins before. These findings provide new insights towards the understanding of Langerin biological functions.

Changes in cell surface structure by viral transformation studied by binding of lectins differing in sugar specificity.

PubMed

Tsuda, M; Kurokawa, T; Takeuchi, M; Sugino, Y

1975-10-01

Changes in cell surface structure by viral transformation were studied by examining changes in the binding of various lectins differing in carbohydrate specificities. Binding of lectins was assayed directly using cells grown in coverslips. The following 125I-lectins were used: Concanavalin-A (specific for glucose and mannose), wheat germ agglutinin (specific for N-acetylglucosamine), castor bean agglutinin (specific for galactose), Wistaria floribunda agglutinin (specific for N-acetylgalactosamine), and soybean agglutinin (specific for N-acetyl-galactosamine). Cells for a clone, SS7, transformed by bovine adenovirus type-3, were found to bind 5 to 6 times more Wistaria floribunda agglutinin than the normal counterpart cells (clone C31, from C3H mouse kidney). In contrast, the binding of soybean agglutinin, which has a sugar specificity similar to Wistaria floribunda agglutinin, to normal and transformed cells was similar. The binding of wheat germ agglutinin and castor bean agglutinin, respectively, to normal and transformed cells was also similar. However, normal cells bound twice as much concanavalin-A as transformed cells. Only half as much Wistaria floribunda agglutinin was bound to transformed cells when they had been dispersed with EDTA. These changes in the number of lectin binding sites on transformation are thought to reflect alteration of the cell surface structure. The amount of lectins bound per cell decreased with increase in cell density, especially in the case of binding of Wistaria floribunda agglutinin to normal cells.

RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides*

PubMed Central

Abdian, Patricia L.; Caramelo, Julio J.; Ausmees, Nora; Zorreguieta, Angeles

2013-01-01

In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. PMID:23235153

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination.

PubMed

Mu, Changkao; Song, Xiaoyan; Zhao, Jianmin; Wang, Lingling; Qiu, Limei; Zhang, Huan; Zhou, Zhi; Wang, Mengqiang; Song, Linsheng; Wang, Chunlin

2012-05-01

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin. Copyright © 2012 Elsevier Ltd. All rights reserved.

Survey of immune-related, mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities

PubMed Central

Lee, Reiko T; Hsu, Tsui-Ling; Huang, Shau Ku; Hsieh, Shie-Liang; Wong, Chi-Huey; Lee, Yuan C

2011-01-01

C-type lectins (CTLs) are proteins that contain one or more carbohydrate-recognition domains (CRDs) that require calcium for sugar binding and share high degree of sequence homology and tertiary structure. CTLs whose CRD contain EPN (Glu-Pro-Asn) tripeptide motifs have potential to bind mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc) and l-fucose (Fuc), whereas those with QPD (Glu-Pro-Asp) tripeptide motifs bind galactose (Gal) and N-acetylgalactosamine (GalNAc). We report here for the first time a direct comparison of monosaccharide (and some di- and trisaccharides)-binding characteristics of 11 EPX-containing (X = N, S or D) immune-related CTLs using a competition assay and an enzyme-linked immunosorbent assay, and neoglycoproteins as ligand. The EPX CTLs studied are DC-SIGN, L-SIGN, mSIGNR1, human and mouse mannose receptors, Langerin, BDCA-2, DCIR, dectin-2, MCL and MINCLE. We found that: (1) they all bound Man and Fuc; (2) binding of Glc and GlcNAc varied considerably among these lectins, but was always less than Man and Fuc; (3) in general, Gal and GalNAc were not bound. However, dectin-2, DCIR and MINCLE showed ability to bind Gal/GalNAc; (4) DC-SIGN, L-SIGN, mSIGNR1 and Langerin showed enhanced binding of Manα2Man over Man, whereas all others showed no enhancement; (5) DC-SIGN bound Lex trisaccharide structure, which has terminal Gal and Fuc residues, more avidly than Fuc, whereas L-SIGN, mSIGNR1, DCIR and MINCLE bound Lex less avidly than Fuc. BDCA-2, dectin-2, Langerin, MCL and mannose receptor did not bind Lex at all. PMID:21112966

Survey of immune-related, mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities.

PubMed

Lee, Reiko T; Hsu, Tsui-Ling; Huang, Shau Ku; Hsieh, Shie-Liang; Wong, Chi-Huey; Lee, Yuan C

2011-04-01

C-type lectins (CTLs) are proteins that contain one or more carbohydrate-recognition domains (CRDs) that require calcium for sugar binding and share high degree of sequence homology and tertiary structure. CTLs whose CRD contain EPN (Glu-Pro-Asn) tripeptide motifs have potential to bind mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc) and l-fucose (Fuc), whereas those with QPD (Glu-Pro-Asp) tripeptide motifs bind galactose (Gal) and N-acetylgalactosamine (GalNAc). We report here for the first time a direct comparison of monosaccharide (and some di- and trisaccharides)-binding characteristics of 11 EPX-containing (X = N, S or D) immune-related CTLs using a competition assay and an enzyme-linked immunosorbent assay, and neoglycoproteins as ligand. The EPX CTLs studied are DC-SIGN, L-SIGN, mSIGNR1, human and mouse mannose receptors, Langerin, BDCA-2, DCIR, dectin-2, MCL and MINCLE. We found that: (1) they all bound Man and Fuc; (2) binding of Glc and GlcNAc varied considerably among these lectins, but was always less than Man and Fuc; (3) in general, Gal and GalNAc were not bound. However, dectin-2, DCIR and MINCLE showed ability to bind Gal/GalNAc; (4) DC-SIGN, L-SIGN, mSIGNR1 and Langerin showed enhanced binding of Manα2Man over Man, whereas all others showed no enhancement; (5) DC-SIGN bound Le(x) trisaccharide structure, which has terminal Gal and Fuc residues, more avidly than Fuc, whereas L-SIGN, mSIGNR1, DCIR and MINCLE bound Le(x) less avidly than Fuc. BDCA-2, dectin-2, Langerin, MCL and mannose receptor did not bind Le(x) at all.

Knowledge-based modeling of a legume lectin and docking of the carbohydrate ligand: the Ulex europaeus lectin I and its interaction with fucose.

PubMed

Gohier, A; Espinosa, J F; Jimenez-Barbero, J; Carrupt, P A; Pérez, S; Imberty, A

1996-12-01

Ulex europaeus isolectin I is specific for fucose-containing oligosaccharide such as H type 2 trisaccharide alpha-L-Fuc (1-->2) beta-D-Gal (1-->4) beta-D-GlcNAc. Several legume lectins have been crystallized and modeled, but no structural data are available concerning such fucose-binding lectin. The three-dimensional structure of Ulex europaeus isolectin I has been constructed using seven legume lectins for which high-resolution crystal structures were available. Some conserved water molecules, as well as the structural cations, were taken into account for building the model. In the predicted binding site, the most probable locations of the secondary hydroxyl groups were determined using the GRID method. Several possible orientations could be determined for a fucose residue. All of the four possible conformations compatible with energy calculations display several hydrogen bonds with Asp-87 and Ser-132 and a stacking interaction with Tyr-220 and Phe-136. In two orientations, the O-3 and O-4 hydroxyl groups of fucose are the most buried ones, whereas two other, the O-2 and O-3 hydroxyl groups are at the bottom of the site. Possible docking modes are also studied by analysis of the hydrophobic and hydrophilic surfaces for both the ligand and the protein. The SCORE method allows for a quantitative evaluation of the complementarity of these surfaces, on the basis of molecular lipophilicity calculations. The predictions presented here are compared with known biochemical data.

The distribution of lectin receptor sites in human breast lesions.

PubMed

Skutelsky, E; Hoenig, S; Griffel, B; Alroy, J

1988-08-01

Conflicting data regarding the status of A, B, H and T antigens in epithelium of normal, mastopathies, fibroadenomas and carcinomas of the breast stimulated us to re-examine the carbohydrate residues in these condition. Currently, we extended the number of carbohydrate residues studied by using ten different biotinylated lectins as probes and avidin-biotin-peroxidase complex (ABC) as a visualant. In addition, the pattern of lectin staining of cancerous cells in primary and metastatic sites was compared. In primary and metastatic breast carcinomas, lectin receptor sites were stained more intensely with Concanavalia ensiformi agglutinin (*Con A), Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA), than in normal breast, in mastopathies or in fibroadenomas. Cryptic receptor sites for peanut agglutinin (PNA) were stained in all cases of breast carcinomas, while free PNA sites stained only in a few cases of well-differentiated carcinomas. Receptors sites for Ulex europaeus agglutinin-I (UEA-I) stained non-malignant epithelium of patients with blood group H but did not stain malignant cells. The results show significant differences in lectin-binding patterns and staining intensities between normal and non-malignant, and malignant epithelial breast cells. Furthermore, these results indicate that in malignant cells, there is an increased content of sialic acid-rich carbohydrates but not of asialylated glycoconjugates.

Folding and Homodimerization of Wheat Germ Agglutinin

PubMed Central

Portillo-Téllez, María del Carmen; Bello, Martiniano; Salcedo, Guillermo; Gutiérrez, Gabriel; Gómez-Vidales, Virginia; García-Hernández, Enrique

2011-01-01

Wheat germ agglutinin (WGA) is emblematic of proteins that specialize in the recognition of carbohydrates. It was the first lectin reported to have a capacity for discriminating between normal and malignant cells. Since then, it has become a preferred model for basic research and is frequently considered in the development of biomedical and biotechnological applications. However, the molecular basis for the structural stability of this homodimeric lectin remains largely unknown, a situation that limits the rational manipulation and modification of its function. In this work we performed a thermodynamic characterization of WGA folding and self-association processes as a function of pH and temperature by using differential scanning and isothermal dilution calorimetry. WGA is monomeric at pH 2, and one of its four hevein-like domains is unfolded at room temperature. Under such conditions, the agglutinin exhibits a fully reversible thermal unfolding that consists of three two-state transitions. At higher pH values, the protein forms weak, nonobligate dimers. This behavior contrasts with that observed for the other plant lectins studied thus far, which form strong, obligate oligomers, indicating a distinctly different molecular basis for WGA function. For dimer formation, the four domains must be properly folded. Nevertheless, depending on the solution conditions, self-association may be coupled with folding of the labile domain. Therefore, dimerization may proceed as a rigid-body-like association or a folding-by-binding event. This hybrid behavior is not seen in other plant lectins. The emerging molecular picture for the WGA assembly highlights the need for a reexamination of existing ligand-binding data in the literature. PMID:21943423

The Cysteine-Rich Domain of the Macrophage Mannose Receptor Is a Multispecific Lectin That Recognizes Chondroitin Sulfates a and B and Sulfated Oligosaccharides of Blood Group Lewisa and Lewisx Types in Addition to the Sulfated N-Glycans of Lutropin

PubMed Central

Leteux, Christine; Chai, Wengang; Loveless, R. Wendy; Yuen, Chun-Ting; Uhlin-Hansen, Lars; Combarnous, Yves; Jankovic, Mila; Maric, Svetlana C.; Misulovin, Ziva; Nussenzweig, Michel C.; Ten Feizi

2000-01-01

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells. PMID:10748230

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum.

PubMed

Molchanova, Valentina; Chikalovets, Irina; Li, Wei; Kobelev, Stanislav; Kozyrevskaya, Svetlana; Bogdanovich, Raisa; Howard, Eric; Belogortseva, Natalia

2005-05-25

Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their alpha- but not beta-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic alpha-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.

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.

Galectin-3 as a Potential Target to Prevent Cancer Metastasis

PubMed Central

Ahmed, Hafiz; AlSadek, Dina M. M.

2015-01-01

Interactions between two cells or between cell and extracellular matrix mediated by protein–carbohydrate interactions play pivotal roles in modulating various biological processes such as growth regulation, immune function, cancer metastasis, and apoptosis. Galectin-3, a member of the β-galactoside-binding lectin family, is involved in fibrosis as well as cancer progression and metastasis, but the detailed mechanisms of its functions remain elusive. This review discusses its structure, carbohydrate-binding properties, and involvement in various aspects of tumorigenesis and some potential carbohydrate ligands that are currently investigated to block galectin-3 activity. PMID:26640395

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.

The Interaction of Pneumocystis with the C-Type Lectin Receptor Mincle Exerts a Significant Role in Host Defense Against Infection

PubMed Central

Kottom, Theodore J.; Hebrink, Deanne M.; Jenson, Paige E.; Nandakumar, Vijayalakshmi; Wüthrich, Marcel; Wang, Huafeng; Klein, Bruce; Yamasaki, Sho; Lepenies, Bernd; Limper, Andrew H.

2017-01-01

Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality within immunocompromised patients. In this study, we examined the potential role of Mincle (Macrophage inducible C-type lectin) for host defense against Pneumocystis. Binding assays implementing soluble Mincle Carbohydrate Recognition Domain (CRD) fusion proteins demonstrated binding to intact Pneumocystis carinii (Pc) as well as to organism homogenates, and purified major surface glycoprotein/glycoprotein A derived from the organism. Additional experiments showed that rats with Pneumocystis pneumonia (PCP) expressed increased Mincle mRNA levels. Mouse macrophages over-expressing Mincle displayed increased binding to Pc life forms and enhanced protein tyrosine phosphorylation. The binding of Pc to Mincle resulted in activation of Fc receptor γ (FcRγ) mediated cell signaling. RNA silencing of Mincle in mouse macrophages resulted in decreased activation of Syk kinase after Pc challenge, critical in downstream inflammatory signaling. Mincle deficient CD-4 depleted (Mincle−/−) mice showing a significant defect in organism clearance from the lungs with higher organism burdens and altered lung cytokine responses during Pneumocystis murina (Pm) pneumonia. Interestingly, Mincle−/− did not demonstrate worsened survival during PCP compared to wild type mice, despite the markedly increased organism burdens. This may be related to increased expression of anti-inflammatory factors such as IL-1Ra during infection in the Mincle−/− mice. Of note, the Pm infected Mincle−/− mice demonstrated increased expression of known C-type lectin receptors Dectin-1, Dectin-2, and MCL compared to infected wild type mice. Taken together, these data support a significant role for Mincle in Pneumocystis modulating host defense during infection. PMID:28298521

Regional differences in lectin binding patterns of vestibular hair cells

NASA Technical Reports Server (NTRS)

Baird, Richard A.; Schuff, N. R.; Bancroft, J.

1994-01-01

Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not stain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type 1 hair cells while labeling, as in the bullfrog, Type 2 hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

A convenient method for synthesis of glyconanoparticles for colorimetric measuring carbohydrate-protein interactions

PubMed Central

Chuang, Yen-Jun; Zhou, Xichun; Pan, Zhengwei; Turchi, Craig

2009-01-01

Carbohydrate functionalized nanoparticles, i.e., the glyconanoparticles, have wide application ranging from studies of carbohydrate-protein interactions, in vivo cell imaging, biolabeling, etc. Currently reported methods for preparation of glyconanoaprticles require multi-step modifications of carbohydrates moieties to conjugate to nanoparticle surface. However, the required synthetic manipulations are difficult and time consuming. We report herewith a simple and versatile method for preparing glyconanoparticles. This method is based on the utilization of clean and convenient microwave irradiation energy for one-step, site-specific conjugation of unmodified carbohydrates onto hydrazide-functionalized Au nanoparticles. A colorimetric assay that utilizes the ensemble of gold glyconanoparticles and Concanavalin A (ConA) was also presented. This feasible assay system was developed to analyze multivalent interactions and to determine the dissociation constant (Kd) for five kind of Au glyconanoparticles with lectin. Surface plasmon changes of the Au glyconanparticles as a function of lectin-carbohydrate interactions were measured and the dissociation constants were determined based on non-linear curve fitting. The strength of the interaction of carbohydrates with ConA was found to be as follows: Maltose > Mannose > Glucose > Lactose > MAN5. PMID:19698698

Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica.

PubMed

Sultan, Nabil Ali Mohammed; Kenoth, Roopa; Swamy, Musti J

2004-12-15

A new galactose-specific lectin has been purified from the extracts of Trichosanthes dioica seeds by affinity chromatography on cross-linked guar gum. The purified lectin (T. dioica seed lectin, TDSL) moved as a single symmetrical peak on gel filtration on Superose-12 in the presence of 0.1 M lactose with an M(r) of 55 kDa. In the absence of ligand, the movement was retarded, indicating a possible interaction of the lectin with the column matrix. In SDS-PAGE, in the presence of beta-mercaptoethanol, two non-identical bands of M(r) 24 and 37 kDa were observed, whereas in the absence of beta-mercaptoethanol, the lectin yielded a single band corresponding to approximately 55,000 Da, indicating that the two subunits of TDSL are connected by one or more disulfide bridges. TDSL is a glycoprotein with about 4.9% covalently bound neutral sugar. Analysis of near-UV CD spectrum by three different methods (CDSSTR, CONTINLL, and SELCON3) shows that TDSL contains 13.3% alpha-helix, 36.7% beta-sheet, 19.4% beta-turns, and 31.6% unordered structure. Among a battery of sugars investigated, TDSL was inhibited strongly by beta-d-galactopyranosides, with 4-methylumbelliferyl-beta-d-galactopyranoside being the best ligand. Chemical modification studies indicate that tyrosine residues are important for the carbohydrate-binding and hemagglutinating activities of the lectin. A partial protection was observed when the tyrosine modification was performed in the presence of 0.2 M lactose. The tryptophan residues of TDSL appear to be buried in the protein interior as they could not be modified under native conditions, whereas upon denaturation with 8 M urea two Trp residues could be selectively modified by N-bromosuccinimide. The subunit composition and size, secondary structure, and sugar specificity of this lectin are similar to those of type-2 ribosome inactivating proteins, suggesting that TDSL may belong to this protein family.

Structural comparisons of two allelic variants of human placental alkaline phosphatase.

PubMed

Millán, J L; Stigbrand, T; Jörnvall, H

1985-01-01

A simple immunosorbent purification scheme based on monoclonal antibodies has been devised for human placental alkaline phosphatase. The two most common allelic variants, S and F, have similar amino acid compositions with identical N-terminal amino acid sequences through the first 13 residues. Both variants have identical lectin binding properties towards concanavalin A, lentil-lectin, wheat germ agglutinin, phytohemagglutinin and soybean agglutinin, and identical carbohydrate contents as revealed by methylation analysis. CNBr fragments of the variants demonstrate identical high performance liquid chromatography patterns. The carbohydrate containing fragment is different from the 32P-labeled active site fragment and the N-terminal fragment.

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

Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of amore » large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.« less

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

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

Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding sitemore » has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.« less

Photoswitchable Janus glycodendrimer micelles as multivalent inhibitors of LecA and LecB from Pseudomonas aeruginosa.

PubMed

Hu, Yingxue; Beshr, Ghamdan; Garvey, Christopher J; Tabor, Rico F; Titz, Alexander; Wilkinson, Brendan L

2017-11-01

The first example of the self-assembly and lectin binding properties of photoswitchable glycodendrimer micelles is reported. Light-addressable micelles were assembled from a library of 12 amphiphilic Janus glycodendrimers composed of variable carbohydrate head groups and hydrophobic tail groups linked to an azobenzene core. Spontaneous association in water gave cylindrical micelles with uniform size distribution as determined by dynamic light scattering (DLS) and small angle neutron scattering (SANS). Trans-cis photoisomerization of the azobenzene dendrimer core was used to probe the self-assembly behaviour and lectin binding properties of cylindrical micelles, revealing moderate-to-potent inhibition of lectins LecA and LecB from Pseudomonas aeruginosa. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases.

PubMed

Naidu, Rayapati A; Ingle, Caroline J; Deom, Carl M; Sherwood, John L

2004-02-05

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G(N) (N-terminal) and G(C) (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G(N) and G(C). The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G(C) showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G(N) was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G(C), with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G(N). None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G(C) or G(N). Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G(C) to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G(N). These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G(C) and possibly high mannose-type on G(N). Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G(C) is likely to be more heavily N-glycosylated than G(N). No evidence was observed for the presence of O-linked oligosaccharides on G(N) or G(C).

Carbohydrate conjugation through microwave-assisted functionalization of single-walled carbon nanotubes using perfluorophenyl azides.

PubMed

Kong, Na; Shimpi, Manishkumar R; Ramström, Olof; Yan, Mingdi

2015-03-20

Carbohydrate-functionalized single-walled carbon nanotubes (SWNTs) were synthesized using microwave-assisted reaction of perfluorophenyl azide with the nanotubes. The results showed that microwave radiation provides a rapid and effective means to covalently attach carbohydrates to SWNTs, producing carbohydrate-SWNT conjugates for biorecognition. The carbohydrate-functionalized SWNTs were furthermore shown to interact specifically with cognate carbohydrate-specific proteins (lectins), resulting in predicted recognition patterns. The carbohydrate-presenting SWNTs constitute a new platform for sensitive protein- or cell recognition, which pave the way for glycoconjugated carbon nanomaterials in biorecognition applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structures of parasite calreticulins provide insights into their flexibility and dual carbohydrate/peptide-binding properties.

PubMed

Moreau, Christophe; Cioci, Gianluca; Iannello, Marina; Laffly, Emmanuelle; Chouquet, Anne; Ferreira, Arturo; Thielens, Nicole M; Gaboriaud, Christine

2016-11-01

Calreticulin (CRT) is a multifaceted protein, initially discovered as an endoplasmic reticulum (ER) chaperone protein, that is essential in calcium metabolism. Various implications in cancer, early development and immunology have been discovered more recently for CRT, as well as its role as a dominant 'eat-me' prophagocytic signal. Intriguingly, cell-surface exposure/secretion of CRT is among the infective strategies used by parasites such as Trypanosoma cruzi , Entamoeba histolytica , Taenia solium , Leishmania donovani and Schistosoma mansoni . Because of the inherent flexibility of CRTs, their analysis by X-ray crystallography requires the design of recombinant constructs suitable for crystallization, and thus only the structures of two very similar mammalian CRT lectin domains are known. With the X-ray structures of two distant parasite CRTs, insights into species structural determinants that might be harnessed to fight against the parasites without affecting the functions of the host CRT are now provided. Moreover, although the hypothesis that CRT can exhibit both open and closed conformations has been proposed in relation to its chaperone function, only the open conformation has so far been observed in crystal structures. The first evidence is now provided of a complex conformational transition with the junction reoriented towards P-domain closure. SAXS experiments also provided additional information about the flexibility of T. cruzi CRT in solution, thus complementing crystallographic data on the open conformation. Finally, regarding the conserved lectin-domain structure and chaperone function, evidence is provided of its dual carbohydrate/protein specificity and a new scheme is proposed to interpret such unusual substrate-binding properties. These fascinating features are fully consistent with previous experimental observations, as discussed considering the broad spectrum of CRT sequence conservations and differences.

Sorafenib induced alteration of protein glycosylation in hepatocellular carcinoma cells

PubMed Central

Liu, Tianhua; Liu, Riqiang; Zhang, Shu; Guo, Kun; Zhang, Qinle; Li, Wei; Liu, Yinkun

2017-01-01

Sorafenib is a multikinase inhibitor and is effective in treating hepatocellular carcinoma (HCC). However, it remains unknown whether sorafenib induces the alteration of protein glycosylation. The present study treated HCC MHCC97L and MHCC97H cells with a 50% inhibitory concentration of sorafenib. Following this treatment, alteration of protein glycosylation was detected using a lectin microarray. Compared with the controls, the binding capacity of glycoproteins extracted from sorafenib-treated HCC cells to the lectins Bauhinia purpurea lectin, Dolichos biflorus agglutinin, Euonymus europaeus lectin, Helix aspersa lectin, Helix pomatia lectin, Jacalin, Maclura pomifera lectin and Vicia villosa lectin were enhanced; while, the binding capacities to the lectins Caragana arborescens lectin, Lycopersicon esculentum lectin, Limulus polyphemus lectin, Maackia amurensis lecin I, Phaseolus vulgaris leucoagglutinin, Ricinus communis agglutinin 60, Sambucus nigra lectin and Solanum tuberosum lectin were reduced (spot intensity median/background intensity median ≥2, P<0.05). This difference in glycoprotein binding capacity indicates that cells treated with sorafenib could increase α-1,3GalNAc/Gal, β-1,3 Gal, GalNAcα-Ser/Thr(Tn) and α-GalNAc structures and decrease GlcNAc, sialic acid, tetra-antennary complex-type N-glycan and β-1,4Gal structures. These results were additionally confirmed by lectin blotting. Expression levels of signaling molecules including erythroblastosis 26–1 (Ets-1), extracellular signal-related kinases (ERK) and phosphorylated-ERK were measured by western blotting. There was a reduction in the expression of Ets-1 and ERK phosphorylation in sorafenib or 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene treated cells suggesting that sorafenib may reduce the expression levels of Ets-1 by blocking the Ras/Raf/mitogen activated protein kinase signaling pathway. In the present study, it was clear that sorafenib could inhibit the proliferation of HCC cells and alter protein glycosylation. The findings of this study may lead to providing a novel way of designing new anti-HCC drugs. PMID:28693200

C-Glycosyl Analogs of Oligosaccharides

NASA Astrophysics Data System (ADS)

Vauzeilles, Boris; Urban, Dominique; Doisneau, Gilles; Beau, Jean-Marie

This chapter covers the synthesis of a large collection of "C-oligosaccharides ", synthetic analogs of naturally occurring oligosaccharides in which a carbon atom replaces the anomeric, interglycosidic oxygen atom. These non-natural constructs are stable to chemical and enzymatic degradation, and are primarily devised to probe carbohydrate-based biological processes. These mainly target carbohydrate-protein interactions such as the modulation of glycoenzyme (glycosylhydrolases and transferases) activities or the design of ligands for lectin Carbohydrate Recognition Domains. The discussion is based on the key carbon-carbon bond assembling steps on carbohydrate templates: ionic (anionic and cationic chemistries, sigmatropic rearrangements) or radical assemblage, and olefin metathesis. Synthetic schemes in which at least one of the monosaccharide units is constructed by total synthesis or by cyclization of acyclic chiral chains are presented separately in a "partial de novo synthesis" section. The review also provides comments, when they are known, on the conformational and binding properties of these synthetic analogs, as well as their biological behavior when tested.

Technical Report for DE-FG02-03ER46029 Sugar-Coated PPEs, Novel Nanomaterials and Sensing Modules for Disease and Bioterrorism Related Threats

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

Uwe Bunz

2003-08-27

The detection and sensing of biological warfare agents (Ricin, Anthrax toxin), of disease agents (cholera, botulinum and tetanus toxins, influenza virus etc) and of biologically active species is important for national security and disease control. A premiere goal would be the simple colorimetric or fluorimetric detection of such toxins by a dipstick test. It would be desirable to sense 5,000-10,000 toxin molecules, i.e. 10-100 fg of a toxin contained 1-5 mL of sample. Fluorescent conjugated polymers should be particularly interesting in this regard, because they can carry multiple identical and/or different recognition units. Such an approach is particularly valuable formore » the detection of lectin toxins, because these bind to oligomeric carbohydrate displays. Lectins bind multivalently to sugars, i.e. several covalently connected sugar moieties have to be exposed to the lectin at the same time to obtain binding. The requirement of multivalency of the lectin-sugar interactions should allow a very sensitive detection of lectins with sugar coated conjugated polymers in an agglutination type assay, where the fluorescence of the PPEs disappears upon binding to the lectins. High molecular weights of the used PPEs would mean high sensitivity. Herein we present our progress towards that goal up to date.« less

Carbohydrate-binding specificities of potential probiotic Lactobacillus strains in porcine jejunal (IPEC-J2) cells and porcine mucin.

PubMed

Valeriano, Valerie Diane; Bagon, Bernadette B; Balolong, Marilen P; Kang, Dae-Kyung

2016-07-01

Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen-probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

PubMed Central

Sato, Yuichiro; Morimoto, Kinjiro; Kubo, Takanori; Sakaguchi, Takemasa; Nishizono, Akira; Hirayama, Makoto; Hori, Kanji

2015-01-01

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells. PMID:26035023

Human Milk Blocks DC-SIGN–Pathogen Interaction via MUC1

PubMed Central

Koning, Nathalie; Kessen, Sabine F. M.; Van Der Voorn, J. Patrick; Appelmelk, Ben J.; Jeurink, Prescilla V.; Knippels, Leon M. J.; Garssen, Johan; Van Kooyk, Yvette

2015-01-01

Beneficial effects of breastfeeding are well-recognized and include both immediate neonatal protection against pathogens and long-term protection against allergies and autoimmune diseases. Although several proteins have been identified to have anti-viral or anti-bacterial effects like secretory IgA or lactoferrin, the mechanisms of immune modulation are not fully understood. Recent studies identified important beneficial effects of glycans in human milk, such as those expressed in oligosaccharides or on glycoproteins. Glycans are recognized by the carbohydrate receptors C-type lectins on dendritic cell (DC) and specific tissue macrophages, which exert important functions in immune modulation and immune homeostasis. A well-characterized C-type lectin is dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), which binds terminal fucose. The present study shows that in human milk, MUC1 is the major milk glycoprotein that binds to the lectin domain of DC-SIGN and prevents pathogen interaction through the presence of Lewis x-type oligosaccharides. Surprisingly, this was specific for human milk, as formula, bovine or camel milk did not show any presence of proteins that interacted with DC-SIGN. The expression of DC-SIGN is found in young infants along the entire gastrointestinal tract. Our data thus suggest the importance of human milk glycoproteins for blocking pathogen interaction to DC in young children. Moreover, a potential benefit of human milk later in life in shaping the infants immune system through DC-SIGN cannot be ruled out. PMID:25821450

Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

DOE PAGES

da Silva, Roberta Peres; Heiss, Christian; Black, Ian; ...

2015-09-21

Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) andmore » α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.« less

Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

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

da Silva, Roberta Peres; Heiss, Christian; Black, Ian

Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) andmore » α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.« less

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus).

PubMed

Yu, Shanshan; Yang, Hui; Chai, Yingmei; Liu, Yingying; Zhang, Qiuxia; Ding, Xinbiao; Zhu, Qian

2013-02-01

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5' untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3' UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys(61)-Cys(158), Cys(134)-Cys(150)) and a Ca(2+)/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca(2+)-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca(2+)-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR. Copyright © 2012 Elsevier Ltd. All rights reserved.

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

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

James, W.M.; Emerick, M.C.; Agnew, W.S.

1989-07-11

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated bindingmore » and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.« less

Lactose binding to galectin-1 modulates structural dynamics, increases conformational entropy, and occurs with apparent negative cooperativity.

PubMed

Nesmelova, Irina V; Ermakova, Elena; Daragan, Vladimir A; Pang, Mabel; Menéndez, Margarita; Lagartera, Laura; Solís, Dolores; Baum, Linda G; Mayo, Kevin H

2010-04-16

Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with beta-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the beta-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K(1)=21+/-6 x 10(3) M(-1)) than the second (K(2)=4+/-2 x 10(3) M(-1)). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K(1)=20+/-10 x 10(3) M(-1) and K(2)=1.67+/-0.07 x 10(3) M(-1). Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the beta-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general. Copyright (c) 2010. Published by Elsevier Ltd.

Prediction of Carbohydrate Binding Sites on Protein Surfaces with 3-Dimensional Probability Density Distributions of Interacting Atoms

PubMed Central

Tsai, Keng-Chang; Jian, Jhih-Wei; Yang, Ei-Wen; Hsu, Po-Chiang; Peng, Hung-Pin; Chen, Ching-Tai; Chen, Jun-Bo; Chang, Jeng-Yih; Hsu, Wen-Lian; Yang, An-Suei

2012-01-01

Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall prediction MCC was 0.49. Independent tests on anti-carbohydrate antibodies showed that the carbohydrate antigen binding sites were predicted with comparable accuracy. These results demonstrate that the predictors are among the best in carbohydrate binding site predictions to date. PMID:22848404

A novel C-type lectin from the sea cucumber Apostichopus japonicus (AjCTL-2) with preferential binding of d-galactose.

PubMed

Wang, Hui; Xue, Zhuang; Liu, Zhaoqun; Wang, Weilin; Wang, Feifei; Wang, Ying; Wang, Lingling; Song, Linsheng

2018-05-15

C-type lectins (CTLs) are Ca 2+ dependent carbohydrate-binding proteins that share structural homology in their carbohydrate-recognition domains (CRDs). In the present study, a novel CTL was identified from sea cucumber Apostichopus japonicus (named as AjCTL-2). The deduced amino acid sequence of AjCTL-2 was homologous to CTLs from other animals with the identities ranging from 33% to 40%. It contained a canonical signal peptide at the N-terminus, a low density lipoprotein receptor class A (LDLa), a C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB), and a CRD with two motifs Glu-Pro-Asn (EPN) and Trp-Asn-Asp (WND) in Ca 2+ binding site 2. The mRNA transcripts of AjCTL-2 were extensively expressed in all the tested tissues including respiratory tree, muscle, gut, coelomocyte, tube-foot, body wall and gonad, and the highest expression level of AjCTL-2 in coelomocyte was about 4.2-fold (p < 0.05) of that in body wall. The mRNA expression level of AjCTL-2 in coelomocyte increased significantly after Vibrio splendidus stimulation, and dramatically peaked at 12 h, which was 206.4-fold (p < 0.05) of that in control group. AjCTL-2 protein was mainly detected in cytoplasm of coelomocyte by immunofluorescence. The recombinant AjCTL-2 (rAjCTL-2) displayed binding activity to d-galactose independent of Ca 2+ , while the binding activity to other tested pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS), peptidoglycan (PGN), and mannose (Man) could not be detected. Surface plasmon resonance (SPR) analysis further revealed the high binding specificity and moderate binding affinity of rAjCTL-2 to d-galactose (KD = 4.093 × 10 -6  M). After rAjCTL-2 was blocked by its polyclonal antibody, the binding activity to d-galactose could not be detected by using a blocking ELISA (B-ELISA). Moreover, rAjCTL-2 could bind various microorganisms including V. splendidus, V. anguillarum, Staphylococcus aureus, Bifidobacterium breve and Yarrowia lipolytica with the strongest binding activity to B. breve. These results collectively suggested that AjCTL-2 was a member of CTL superfamily (CTLs) with preferential binding of d-galactose and participated in the immune response of sea cucumber. Copyright © 2018. Published by Elsevier Ltd.

Effects of lectins on calcification by vesicles isolated from aortas of cholesterol-fed rabbits.

PubMed

Hsu, H H; Tawfik, O; Sun, F

2000-04-05

Advanced vascular calcification in atherosclerosis weakens arterial walls, thereby imposing a serious rupturing effect. However, the mechanism of dystrophic calcification remains unknown. Although accumulating morphological and biochemical evidence reveals a role for calcifiable vesicles in plaque calcification, the mechanism of vesicle-mediated calcification has not been fully explored. To study whether vesicles' membrane components, such as carbohydrates, may have a role in vesicle-mediated calcification, the effect of sugar-binding lectins on calcification was investigated. Atherosclerosis was developed by feeding rabbits with a diet supplemented with 0.5% cholesterol and 2% peanut oil for 4 months. Calcifiable vesicles were then isolated from thoracic aortas by collagenase digestion. The histological examination of aortas with hematoxylin counter-staining indicated abnormal formation of large plaques enriched with macrophage-derived foam cells. Fourier transform spectroscopy revealed mild calcification in aortas indicating that advanced stages of heavy calcification have yet to be reached. However, vesicles isolated from the aortas were capable of calcification in the presence of physiological levels of Ca(2+), Pi, and ATP. Thus, at this stage of atherosclerosis, aortas may start to produce calcifiable vesicles, but at a level insufficient for substantial formation of mineral in aortas. The assessments by FT-IR analysis and Alizarin red staining indicated that concanavalin A (Con A) substantially increased mineral formation by isolated vesicles. Con A also exerted a marked stimulatory effect on (45)Ca and (32)Pi deposition in a dose-dependent fashion with a half-maximal effect at 6-10 microg/ml. Either alpha-methylmannoside or alpha-methylglucoside, but not mannitol, at 10 mM abolished the stimulation. Con A stimulation was abolished after Con A was removed from calcifying media, suggesting that covalent binding may not be involved in the effect. Galactosides appear to also be implicated in (45)Ca and (32)Pi deposition since Abrus precartorius agglutinin, which specifically binds galactosides, enhanced the deposition. Neither wheat-germ agglutinin that binds N-acetylglucoside nor N-acetylgalactoside-specific Helix pomatia agglutinin was effective, suggesting that the acetylated forms of carbohydrate moieties are either absent in vesicles or may not be involved in calcification. None of these lectins exerted an effect on ATPase. Thus, the effects of lectins appeared to be mediated through interactions with carbohydrate moieties of calcifiable vesicles. Whether stimulation of vesicle-calcification by lectins is of pathological significance in atherosclerotic calcification requires further investigation.

Regional differences in lectin binding patterns of vestibular hair cells

NASA Technical Reports Server (NTRS)

Baird, R. A.; Schuff, N. R.; Bancroft, J.

1993-01-01

Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylglucosamine (WGA), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not strain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type I hair cells while labeling, as in the bullfrog, Type II hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p.

PubMed

Satoh, Tadashi; Sato, Ken; Kanoh, Akira; Yamashita, Katsuko; Yamada, Yusuke; Igarashi, Noriyuki; Kato, Ryuichi; Nakano, Akihiko; Wakatsuki, Soichi

2006-04-14

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

Wzi is an outer membrane lectin that underpins group 1 capsule assembly in Escherichia coli.

PubMed

Bushell, Simon R; Mainprize, Iain L; Wear, Martin A; Lou, Hubing; Whitfield, Chris; Naismith, James H

2013-05-07

Many pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded β-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides. Copyright © 2013 Elsevier Ltd. All rights reserved.

Purification, characterization and biological effect of lectin from the marine sponge Stylissa flexibilis (Lévi, 1961).

PubMed

Hung, Le Dinh; Ly, Bui Minh; Hao, Vo Thi; Trung, Dinh Thanh; Trang, Vo Thi Dieu; Trinh, Phan Thi Hoai; Ngoc, Ngo Thi Duy; Quang, Thai Minh

2018-02-01

SFL, a lectin from the marine sponge Stylissa flexibilis was purified by cold ethanol precipitation followed by ion exchange chromatography on DEAE Sepharose column and Sephacryl S-200 gel filtration. SFL is a dimeric glycoprotein of 32kDa subunits linked by a disulfide bridge with a molecular mass of 64kDa by SDS-PAGE and 65kDa by Sephacryl S-200 gel filtration. SFL preferentially agglutinated enzyme treated human A erythrocytes. The activity of lectin was strongly inhibited by monosaccharide d-galactose and glycoproteins asialo-porcine stomach mucin and asialo-fetuin. The lectin was Ca 2+ dependent, stable over a range of pH from 5 to 8, and up to 60°C for 30min. The N-terminal amino acid sequence of SFL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with lectins from the marine sponge Spheciospongia vesparia. SFL caused agglutination of Vibrio alginolyticus and V. parahaemolyticus in a dose dependent manner and inhibited the growth rates of the virulent bacterial strains. Growth inhibition of V. alginolyticus and V. parahaemolyticus with SFL was not observed in the presence of d-galactose or asialo-porcine stomach mucin, suggesting that the lectin caused the agglutination through binding to the target receptor(s) on the surface of Vibrios. Thus, the marine sponge S. flexibilis could promise to be a good source of a lectin(s) that may be useful as a carbohydrate probe and an antibacterial reagent. Copyright © 2017 Elsevier Inc. All rights reserved.

Multi-specificity of a Psathyrella velutina mushroom lectin: heparin/pectin binding occurs at a site different from the N-acetylglucosamine/N-acetylneuraminic acid-specific site.

PubMed

Ueda, H; Saitoh, T; Kojima, K; Ogawa, H

1999-09-01

An N-acetylglucosamine (GlcNAc)/N-acetylneuraminic acid-specific lectin from the fruiting body of Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GlcNAc5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GlcNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GlcNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc, N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycoconjugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms.

Nanoporous Gold as a Solid Support for Protein Immobilization for the Development of Immunoassays, and for Biomolecular Interaction Studies

NASA Astrophysics Data System (ADS)

Pandey, Binod Prasad

Nanoporous gold (NPG) is a versatile material of high surface area to volume ratio that can be readily modified with self-assembled monolayers of alkanethiols to which biomolecules can be linked. NPG presents new opportunities for the development of immunoassays, and for the development of carbohydrate based assays. This thesis explores the use of NPG as a support for self-assembled monolayers, their linkage to antibody-enzyme conjugates for immunoassay development, and for the study and application of carbohydrate-protein interactions. Direct kinetic electrochemical immunoassays were developed on NPG for prostate specific antigen (PSA) and carcinoembryonic antigen (CEA). The decrease in enzymatic conversion of p-aminophenylphosphate to p-aminophenol, by alkaline phosphatase conjugated to an antibody, due to steric hindrance caused by the presence of antigen on antibody, was observed as a drop in peak current in square-wave voltammetry. Detection limit of these assays was 0.075 ng mL -1 and 0.015 ng mL-1 for PSA and CEA, respectively. Similarly, the linear range of determination of these biomarkers extended up to 30 ng mL-1 and 10 ng mL-1 for PSA and CEA, respectively. Minimal interference was observed using newborn calf serum as a substitute for the human serum matrix. A rapid and sensitive enzyme linked lectinsorbant assay was also developed for the study of glycoprotein-lectin interactions on the NPG surface. Self-assembled monolayers of alkanethiols on NPG were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Similarly, the applicability of this surface for the formation of carbohydrate monolayers and its application for lectin carbohydrate interactions was also studied. Pure and mixed SAMs of 8-mercaptooctyl β-D-mannopyranoside (αMan-C8-SH) and α-D-Gal-(1→4)-β-D-Gal-(1α)-D-Glc-1-O-mercaptooctane (Gb3-C8-SH) with alkanethiols having varying tail groups were prepared. Binding affinity and binding kinetics of concanavalin A to mannoside and soybean agglutinin to galactose in these SAMs were found to be different on NPG than on flat polycrystalline gold, and was also sensitive to the chemical composition of the modified surfaces.

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

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells.

PubMed

Silva, Mariana C C; de Paula, Cláudia A A; Ferreira, Joana G; Paredes-Gamero, Edgar J; Vaz, Angela M S F; Sampaio, Misako U; Correia, Maria Tereza S; Oliva, Maria Luiza V

2014-07-01

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity. MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting. BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α1, α6 and β1 integrin subunit expression, and increases α5 subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21. BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells. Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

PubMed

Goh, Boon Chong; Wu, Huixing; Rynkiewicz, Michael J; Schulten, Klaus; Seaton, Barbara A; McCormack, Francis X

2016-07-05

Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

PubMed

Lerner, D R; Raikhel, N V

1992-06-05

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.

Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL.

PubMed

Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

2017-01-01

Site-directed mutagenesis is a powerful technique which is used to understand the basis of interactions between proteins and their binding partners, as well as to modify these interactions. Methods of rational design that are based on detailed knowledge of the structure of a protein of interest are often used for preliminary investigations of the possible outcomes which can result from the practical application of site-directed mutagenesis. Also, random mutagenesis can be used in tandem with site-directed mutagenesis for an examination of amino acid "hotspots."Lectins are sugar-binding proteins which, among other functions, mediate the recognition of host cells by a pathogen and its adhesion to the host cell surface. Hence, lectins and their binding properties are studied and engineered using site-directed mutagenesis.In this chapter, we describe a site-directed mutagenesis method used for investigating the sugar binding pattern of the PA-IIL lectin from the pathogenic bacterium Pseudomonas aeruginosa. Moreover, procedures for the production and purification of PA-IIL mutants are described, and several basic methods for characterizing the mutants are discussed.

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.

Maximizing Immune Response to Carbohydrate Antigens on Breast Tumors

DTIC Science & Technology

2004-08-01

selectin binding. In vitro phenotyping of the tumor cells suggests that both KM93-Neg and Pos cells grow at the same rate; however, KM-93Neg cells are...and Figure 2. ABL ( Agaricus bisporus, mushroom, lectin) and ACA (Amaranthus caudatus, lectin) do not react with either the KM93-Pos or KM93-Neg...is different (Figure 6B). The KM93-Pos variant, which is similar to the original 4T1, tends to grow in clusters with high densities. While clusters are

Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain.

PubMed

Huang, Huey-Chun; Shi, Guey-Yueh; Jiang, Shinn-Jong; Shi, Chung-Sheng; Wu, Chun-Mei; Yang, Hsi-Yuan; Wu, Hua-Lin

2003-11-21

Thrombomodulin (TM) is an integral membrane glycoprotein that is a potent anticoagulant factor. TM may also possess functions distinct from its anticoagulant activity. Here the influence of TM on cell adhesion was studied in TM-negative melanoma A2058 cells transfected with green fluorescent protein-tagged TM (TMG) or lectin domain-deleted TM (TMG(DeltaL)). Confocal microscopy demonstrated that both TMG and TMG(DeltaL) were distributed in the plasma membrane. TMG-expressed cells grew as closely clustered colonies, with TM localized prominently in the intercellular boundaries. TMG(DeltaL)-expressed cells grew singly. Overexpression of TMG, but not TMG(DeltaL), decreased monolayer permeability in vitro and tumor growth in vivo. The cell-to-cell adhesion in TMG-expressed cells was Ca2+-dependent and was inhibited by monoclonal antibody against the lectin-like domain of TM. The effects of TM-mediated cell adhesion were abolished by the addition of mannose, chondroitin sulfate A, or chondroitin sulfate C. In addition, anti-lectin-like domain antibody disrupted the close clustering of the endogenous TM-expressed keratinocyte HaCaT cell line derived from normal human epidermis. Double-labeling immunofluorescence staining revealed similar distributions of TM and actin filament in the cortex region of the TMG-expressed cells. Thus, TM can function as a Ca2+-dependent cell-to-cell adhesion molecule. Binding of specific carbohydrates to the lectin-like domain is essential for this specific function.

The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

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

Bradshaw, William J.; Public Health England, Porton Down, Salisbury SP4 0JG; Kirby, Jonathan M.

2014-07-01

The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA)more » into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.« less

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein.

PubMed

Favier, Anne-Laure; Gout, Evelyne; Reynard, Olivier; Ferraris, Olivier; Kleman, Jean-Philippe; Volchkov, Viktor; Peyrefitte, Christophe; Thielens, Nicole M

2016-06-01

Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. A specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the balance toward its elimination. An interaction between the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of Ebola virus GP occurred. In this model, the enhancement of infection was shown to be independent of the serum complement. The facilitation of EBOV entry into target host cells by the interaction with ficolin-1 and other host lectins shunts virus elimination, which likely facilitates the survival of the virus in infected host cells and contributes to the virus strategy to subvert the innate immune response. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The role of surface carbohydrates on the interaction of microconidia of Trichophyton mentagrophytes with epithelial cells.

PubMed

Esquenazi, Daniele; de Souza, Wanderley; Alviano, Celuta Sales; Rozental, Sonia

2003-03-20

The presence of carbohydrate-binding adhesins on the microconidia of Trichophyton mentagrophytes surface and their role on cellular interactions were investigated. Flow cytometry showed that this fungus recognizes the sugars mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 degrees C than 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature of the microconidia adhesin. The interaction of the fungus to Chinese hamster ovary epithelial cells and its glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, which express mannose and galactose, respectively, as the terminal carbohydrate on the cell surface. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside to the interaction medium, pretreatment of Lec1 and Lec2 cells with lectins Concanavalina A and Arachis hypogaea and pretreatment with sodium periodate decreased the adhesion and the endocytic index. Examination of thin section by transmission electron microscopy showed that after fungal ingestion by Lec2 cells the fungi are enclosed in a 'loose'-type vacuole while the other cells are found within a 'tight'-type membrane-bound cytoplasmic vacuole. Our results suggest the occurrence of carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. This may have a role in the adhesion process during the infectious process of dermatophytosis.

Antibacterial activity of lactose-binding lectins from Bufo arenarum skin.

PubMed

Sánchez Riera, Alicia; Daud, Adriana; Gallo, Adriana; Genta, Susana; Aybar, Manuel; Sánchez, Sara

2003-04-01

Amphibians respond to microbial infection through cellular and humoral defense mechanisms such as antimicrobial protein secretion. Most humoral defense proteins are synthetized in the skin. In this study we isolated two beta-galactoside-binding lectins with molecular weights of 50 and 56 KDa from the skin of Bufo arenarum. These lectins have significant hemagglutination activity against trypsinized rabbit erythrocytes, which was inhibited by galactose-containing saccharides. They are water-soluble and independent of the presence of calcium. The antimicrobial analysis for each lectin was performed. At mumolar concentration lectins show strong bacteriostatic activity against Gram negative bacteria (Escherichia coli K12 4100 and wild strains of Escherichia coli and Proteus morganii) and Gram positive bacteria (Enterococcus faecalis). The antibacterial activity of these lectins may provide an effective defense against invading microbes in the amphibian Bufo arenarum.

Agglutination of Helicobacter pylori coccoids by lectins

PubMed Central

Khin, Mar Mar; Hua, Jie Song; Ng, Han Cong; Wadström, Torkel; Ho, Bow

2000-01-01

AIM: To study the agglutination pattern of Helicobacter pylori coccoid and spiral forms. METHODS: Assays of agglutination and agglutination inhibition were applied using fifteen commercial lectins. RESULTS: Strong agglutination was observed with mannose-specific Concanavalin A (Con A), fucose-specific Tetragonolobus purpureas (Lotus A) and N-acetyl glucosamine-specific Triticum vulgaris (WGA) lectins. Mannose and fucose specific lectins were reactive with all strains of H. pylori coccoids as compared to the spirals. Specific carbohydrates, glycoproteins and mucin were shown to inhibit H. pylori lectin-agglutination reactions. Pre-treatment of the bacterial cells with formalin and sulphuric acid did not alter the agglutination patterns with lectins. However, sodium periodate treatment of bacterial cells were shown to inhibit agglutination reaction with Con A, Lotus A and WGA lectins. On the contrary, enzymatic treatment of coccoids and spirals did not show marked inhibition of H. pylori lectin agglutination. Interes tingly, heating of H. pylori cells at 60 °C for 1 h was shown to augment the agglutination with all of the lectins tested. CONCLUSION: The considerable differences in lectin agglutination patterns seen among the two differentiated forms of H. pylori might be attributable to the structural changes during the events of morphological transformation, resulting in exposing or masking some of the sugar residues on the cell surface. Possibility of various sugar residues on the cell wall of the coccoids may allow them to bind to different carbohydrate receptors on gastric mucus and epithelial cells. The coccoids with adherence characteristics like the spirals could aid in the pathogenic process of Helicobacter infection. This may probably lead to different clinical outcome of H. pylori associated gastroduodenal disease. PMID:11819557

Design and syntheses of mono and multivalent mannosyl-lipoconjugates for targeted liposomal drug delivery.

PubMed

Štimac, Adela; Cvitaš, Jelena TrmĿiĿ; Frkanec, Leo; Vugrek, Oliver; Frkanec, Ruža

2016-09-10

Multivalent mannosyl-lipoconjugates may be of interest for glycosylation of liposomes and targeted drug delivery because the mannose specifically binds to C-type lectin receptors on the particular cells. In this paper syntheses of two types of novel O-mannosides are presented. Conjugates 1 and 2 with a COOH- and NH2-functionalized spacer and the connection to a lysine and FmocNH-PEG-COOH, are described. The coupling reactions of prepared intermediates 6 and 4 with a PEGylated-DSPE or palmitic acid, respectively, are presented. Compounds 5, mono-, 8, di- and 12, tetravalent mannosyl-lipoconjugates, were synthesized. The synthesized compounds were incorporated into liposomes and liposomal preparations featuring exposed mannose units were characterized. Carbohydrate liposomal quartz crystal microbalance based assay has been established for studying carbohydrate-lectin binding. It was demonstrated that liposomes with incorporated mannosyl-lipoconjugates were effectively recognized by Con A and have great potential to be used for targeted liposomal drug delivery systems. Copyright © 2016 Elsevier B.V. All rights reserved.

Chitovibrin: a chitin-binding lectin from Vibrio parahemolyticus.

PubMed

Gildemeister, O S; Zhu, B C; Laine, R A

1994-12-01

A novel 134 kDa, calcium-independent chitin-binding lectin, 'chitovibrin', is secreted by the marine bacterium Vibrio parahemolyticus, inducible with chitin or chitin-oligomers. Chitovibrin shows no apparent enzymatic activity but exhibits a strong affinity for chitin and chito-oligomers > dp9. The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0-4 M NaCl. Chitovibrin appears to be completely different from other reported Vibrio lectins and may function to bind V. parahemolyticus to chitin substrates, or to capture or sequester chito-oligomers. It may be a member of a large group of recently described proteins in Vibrios related to a complex chitinoclastic (chitinivorous) system.

CancerLectinDB: a database of lectins relevant to cancer.

PubMed

Damodaran, Deepa; Jeyakani, Justin; Chauhan, Alok; Kumar, Nirmal; Chandra, Nagasuma R; Surolia, Avadhesha

2008-04-01

The role of lectins in mediating cancer metastasis, apoptosis as well as various other signaling events has been well established in the past few years. Data on various aspects of the role of lectins in cancer is being accumulated at a rapid pace. The data on lectins available in the literature is so diverse, that it becomes difficult and time-consuming, if not impossible to comprehend the advances in various areas and obtain the maximum benefit. Not only do the lectins vary significantly in their individual functional roles, but they are also diverse in their sequences, structures, binding site architectures, quaternary structures, carbohydrate affinities and specificities as well as their potential applications. An organization of these seemingly independent data into a common framework is essential in order to achieve effective use of all the data towards understanding the roles of different lectins in different aspects of cancer and any resulting applications. An integrated knowledge base (CancerLectinDB) together with appropriate analytical tools has therefore been developed for lectins relevant for any aspect of cancer, by collating and integrating diverse data. This database is unique in terms of providing sequence, structural, and functional annotations for lectins from all known sources in cancer and is expected to be a useful addition to the number of glycan related resources now available to the community. The database has been implemented using MySQL on a Linux platform and web-enabled using Perl-CGI and Java tools. Data for individual lectins pertain to taxonomic, biochemical, domain architecture, molecular sequence and structural details as well as carbohydrate specificities. Extensive links have also been provided for relevant bioinformatics resources and analytical tools. Availability of diverse data integrated into a common framework is expected to be of high value for various studies on lectin cancer biology. CancerLectinDB can be accessed through http://proline.physics.iisc.ernet.in/cancerdb .

Investigation of Carbohydrate Recognition via Computer Simulation

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

Johnson, Quentin R.; Lindsay, Richard J.; Petridis, Loukas

Carbohydrate recognition by proteins, such as lectins and other (bio)molecules, can be essential for many biological functions. Interest has arisen due to potential protein and drug design and future bioengineering applications. A quantitative measurement of carbohydrate-protein interaction is thus important for the full characterization of sugar recognition. Here, we focus on the aspect of utilizing computer simulations and biophysical models to evaluate the strength and specificity of carbohydrate recognition in this review. With increasing computational resources, better algorithms and refined modeling parameters, using state-of-the-art supercomputers to calculate the strength of the interaction between molecules has become increasingly mainstream. We reviewmore » the current state of this technique and its successful applications for studying protein-sugar interactions in recent years.« less

Investigation of Carbohydrate Recognition via Computer Simulation

DOE PAGES

Johnson, Quentin R.; Lindsay, Richard J.; Petridis, Loukas; ...

2015-04-28

Carbohydrate recognition by proteins, such as lectins and other (bio)molecules, can be essential for many biological functions. Interest has arisen due to potential protein and drug design and future bioengineering applications. A quantitative measurement of carbohydrate-protein interaction is thus important for the full characterization of sugar recognition. Here, we focus on the aspect of utilizing computer simulations and biophysical models to evaluate the strength and specificity of carbohydrate recognition in this review. With increasing computational resources, better algorithms and refined modeling parameters, using state-of-the-art supercomputers to calculate the strength of the interaction between molecules has become increasingly mainstream. We reviewmore » the current state of this technique and its successful applications for studying protein-sugar interactions in recent years.« less

The consequence of low mannose-binding lectin plasma concentration in relation to susceptibility to Salmonella Infantis in chickens.

PubMed

Ulrich-Lynge, Sofie L; Dalgaard, Tina S; Norup, Liselotte R; Kjærup, Rikke M; Olsen, John E; Sørensen, Poul; Juul-Madsen, Helle R

2015-01-15

Mannose-binding lectin (MBL) is a key protein in innate immunity. MBL binds to carbohydrates on the surface of pathogens, where it initiates complement activation via the lectin-dependent pathway or facilitates opsonophagocytosis. In vitro studies have shown that human MBL is able to bind to Salmonella, but knowledge in relation to chicken MBL and Salmonella is lacking. In order to study this relation day-old chickens from two selected lines L10H and L10L, differing in MBL serum concentration, were either orally infected with S. Infantis (S.123443) or kept as non-infected controls. The differences between healthy L10H and L10L chicken sublines were more profound than differences caused by the S. Infantis infection. The average daily body weight was higher for L10H than for L10L, regardless of infection, indicating beneficial effects of MBL selection on growth. Salmonella was detected in cloacal swabs and the number of Salmonella positive chickens during the experiment was significantly higher in L10L than L10H, indicating that MBL may affect the magnitude of Salmonella colonisation in day-old chickens. MBL expression was determined in ceca tissue by real-time RT-PCR. L10H chickens showed a significantly higher relative expression than L10L at days 1 and 41 pi, regardless of infection. Finally, flow cytometric analysis of whole blood from infected chickens showed that L10H had a significantly higher count of all assessed leucocyte subsets on day 5 pi, and also a higher count of monocytes on day 12 pi than L10L. No difference was observed between infected and non-infected L10L chicken. Copyright © 2014 Elsevier B.V. All rights reserved.

Lectin histochemistry of goblet cell sugar residues in the gut of the chick embryo and of the newborn.

PubMed

Bryk, S G; Sgambati, E; Gheri Bryk, G

1999-04-01

The anlage of duodenum, ileum and colon were removed from chick embryos of day 8-21 of incubation and from 1-day-old chicks. A battery of seven different horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, Con A, WGA, LTA and UEAI) was used to study the carbohydrate residues of the glycoconjugates in the goblet cells of the three parts of the intestine. The main results can be summarized as follows: differences in lectin binding were absent in the proximal and distal parts of the duodenum, ileum and colon. Lectin histochemistry showed differences among the three intestinal segments for the time of appearance of the oligosaccharides in the goblet mucus. In the colonic goblet cells of 1-day-old chicks, LTA and UEAI lectins showed two different types of linkage of alpha-L-fucose. This is the first demonstration of UEAI reactive sites in Gallus domesticus.

Innate immune response of channel catfish (Ictalurus punctatus) mannose-binding lectin to channel catfish virus

USDA-ARS?s Scientific Manuscript database

The channel catfish virus (CCV) is a pathogenic herpesvirus that infects channel catfish (Ictalurus punctatus) in pond aquaculture in the Southeast USA. The innate immune protein mannose-binding lectin (MBL) could play an important role in the innate response of channel catfish by binding to the CC...

Localization of binding sites of Ulex europaeus I, Helix pomatia and Griffonia simplicifolia I-B4 lectins and analysis of their backbone structures by several glycosidases and poly-N-acetyllactosamine-specific lectins in human breast carcinomas.

PubMed

Ito, N; Imai, S; Haga, S; Nagaike, C; Morimura, Y; Hatake, K

1996-09-01

Several studies have shown the deletion of blood group A or B antigens and the accumulation of H antigens in human breast carcinomas. Other studies have independently demonstrated that the binding sites of lectins such as Helix pomatia agglutinin (HPA) and Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) are highly expressed in these cells. In order to clarify the molecular mechanisms of malignant transformation and metastasis of carcinoma cells, it is important to understand the relationship between such phenotypically distinct events. For this purpose, we examined whether the binding sites of these lectins and Ulex europaeus agglutinin I (UEA-I) are expressed concomitantly in the same carcinoma cells and analyzed their backbone structures. The expression of the binding sites of these lectins was observed independently of the blood group (ABO) of the patients and was not affected by the histological type of the carcinomas. Observation of serial sections stained with these lectins revealed that the distribution of HPA binding sites was almost identical to that of GSAI-B4 in most cases. Furthermore, in some cases, UEA-I binding patterns were similar to those of HPA and GSAI-B4 but in other cases, mosaic staining patterns with these lectins were also observed, i.e., some cell clusters were stained with both HPA and GSAI-B4 but not with UEA-I and adjacent cell clusters were stained only with UEA-I. Digestion with endo-beta-galactosidase or N-glycosidase F markedly reduced the staining intensity of these lectins. Together with the reduction of staining by these lectins, reactivity with Griffonia simplicifolia agglutinin II appeared in carcinoma cells following endo-beta-galactosidase digestion. Among the lectins specific to poly-N-acetyllactosamine, Lycopersicon esculentum agglutinin (LEA) most vividly and consistently stained the cancer cells. Next to LEA, pokeweed mitogen agglutinin was also effective in staining these cells. Carcinoma cells reactive with these lectins corresponded well to those stained with both HPA and GSAI-B4, and in some cases, with UEA-I. These results demonstrate that the binding sites of UEA-I, HPA, and GSAI-B4 are expressed concomitantly in the same carcinoma cells and all carry linear and branched poly-N-acetyllactosamine on N-glycans, suggesting that the synthesis of this complex carbohydrate is one of the most important and basic processes leading to the malignant transformation of cells, invasion, and metastasis of carcinoma cells.

Functional evaluation of carbohydrate-centred glycoclusters by enzyme-linked lectin assay: ligands for concanavalin A.

PubMed

Köhn, Maja; Benito, Juan M; Ortiz Mellet, Carmen; Lindhorst, Thisbe K; García Fernández, José M

2004-06-07

The affinities of the mannose-specific lectin concanavalin A (Con A) towards D-glucose-centred mannosyl clusters differing in the anomeric configuration of the monosaccharide core, nature of the bridging functional groups and valency, have been measured by a competitive enzyme-linked lectin assay. Pentavalent thioether-linked ligands (5 and 7) were prepared by radical addition of 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranose to the corresponding penta-O-allyl-alpha- or -beta-D-glucopyranose, followed by deacetylation. The distinct reactivity of the anomeric position in the D-glucose scaffold was exploited in the preparation of a tetravalent cluster (10) that keeps a reactive aglyconic group for further manipulation, including incorporation of a reporter group or attachment to a solid support. Hydroboration of the double bonds in the penta-O-allyl-alpha-D-glucopyranose derivative and replacement of the hydroxy groups with amine moieties gave a suitable precursor for the preparation of pentavalent and 15-valent mannosides through the thiourea-bridging reaction (17 and 20, respectively). The diastereomeric 1-thiomannose-coated clusters 5 and 7 were demonstrated to be potent ligands for Con A, with IC(50) values for the inhibition of the Con A-yeast mannan association indicative of 6.4- and 5.5-fold increases in binding affinity (valency-corrected values), respectively, relative to the value for methyl alpha-D-mannopyranoside. The tetravalent cluster 10 exhibited a valency-corrected relative lectin-binding potency virtually identical to that of the homologous pentavalent mannoside 7. In sharp contrast, replacement of the 1-thiomannose wedges of 5 with alpha-D-mannopyranosylthioureido units (17) virtually abolished any multivalent or statistic effects, with a dramatic decrease of binding affinity. The 15-valent ligand 20, possessing classical O-glycosidic linkages, exhibited a twofold increase in lectin affinity relative to the penta-O-(thioglycoside) 5; it is less efficient based on the number of mannose units. The results illustrate the potential of carbohydrates as polyfunctional platforms for glycocluster construction and underline the importance of careful design of the overall architecture in optimising glycocluster recognition by specific lectins.

Lectin-carbohydrate recognition mechanism of Plasmodium berghei in the midgut of malaria vector Anopheles stephensi using quantum dot as a new approach.

PubMed

Basseri, Hamid R; Javazm, Mahdi Salari; Farivar, Leila; Abai, Mohammad R

2016-04-01

Potential targets of Plasmodium ookinetes at the mosquito midgut walls were investigated in relation to interfering malarial transmission. In this study, the essential application of Quantum Dots (QDs) was used to examine the interaction between Plasmodium berghei ookinetes and the Anopheles stephensi midgut, based on lectin-carbohydrate recognition. Two significant lectins were utilized to determine this interaction. Two QDs, cadmium telluride (CdTe)/CdS and cadmium selenide (CdSe)/CdS, were employed in staining Plasmodium ookinete to study its interaction in the midgut of the mosquito vector in vivo. Concurrently, two lectins, wheat germ agglutinin (WGA) and concanavalin A (Con A), were inadvertently exploited to mask lectin binding sites between ookinetes and mosquito midgut cells. The numbers of ookinetes in both lumen and epithelial cells were eventually counted, following adequate preparation of wax sections extracted from whole midgut, and subsequent examination using a differential interference contrast a fluorescence microscopic technique. Interestingly, we detected that neither of the QDs mutated ookinete invasion into the midgut cells of the investigated mosquitoes. QD staining of ookinetes remained permanent despite the effective embedding procedure. The massive binding potency of ookinetes to midgut cells of the cross-examined mosquitoes undoubtedly revealed that Con A did not interrupt ookinete penetration into the midgut wall. In contrast, WGA inhibited ookinete invasion into the midgut cells. The results proved that QD nanoparticles are biocompatible, non-toxic to P. berghei and stable to photobleaching. The QDs staining, which was successfully implemented for ookinete labelling, is a simple and effective tool which plays a crucial role in bioimaging including the study of parasite-vector interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Nictaba Homologs from Arabidopsis thaliana Are Involved in Plant Stress Responses

PubMed Central

Eggermont, Lore; Stefanowicz, Karolina; Van Damme, Els J. M.

2018-01-01

Plants are constantly exposed to a wide range of environmental stresses, but evolved complicated adaptive and defense mechanisms which allow them to survive in unfavorable conditions. These mechanisms protect and defend plants by using different immune receptors located either at the cell surface or in the cytoplasmic compartment. Lectins or carbohydrate-binding proteins are widespread in the plant kingdom and constitute an important part of these immune receptors. In the past years, lectin research has focused on the stress-inducible lectins. The Nicotiana tabacum agglutinin, abbreviated as Nictaba, served as a model for one family of stress-related lectins. Here we focus on three non-chimeric Nictaba homologs from Arabidopsis thaliana, referred to as AN3, AN4, and AN5. Confocal microscopy of ArathNictaba enhanced green fluorescent protein (EGFP) fusion constructs transiently expressed in N. benthamiana or stably expressed in A. thaliana yielded fluorescence for AN4 and AN5 in the nucleus and the cytoplasm of the plant cell, while fluorescence for AN3 was only detected in the cytoplasm. RT-qPCR analysis revealed low expression for all three ArathNictabas in different tissues throughout plant development. Stress application altered the expression levels, but all three ArathNictabas showed a different expression pattern. Pseudomonas syringae infection experiments with AN4 and AN5 overexpression lines demonstrated a significantly higher tolerance of several transgenic lines to P. syringae compared to wild type plants. Finally, AN4 was shown to interact with two enzymes involved in plant defense, namely TGG1 and BGLU23. Taken together, our data suggest that the ArathNictabas represent stress-regulated proteins with a possible role in plant stress responses. On the long term this research can contribute to the development of more stress-resistant plants. PMID:29375596

Purification and partial characterization of a new mannose/glucose-specific lectin from Dialium guineense Willd seeds that exhibits toxic effect.

PubMed

Bari, Alfa U; Silva, Helton C; Silva, Mayara T L; Pereira Júnior, Francisco N; Cajazeiras, João B; Sampaio, Alexandre H; Leal, Rodrigo B; Teixeira, Edson H; Rocha, Bruno A M; Nascimento, Kyria S; Nagano, Celso S; Cavada, Benildo S

2013-08-01

A new mannose/glucose-specific lectin, named DigL, was purified from seeds of Dialium guineense by a single step using a Sepharose 4b-Mannose affinity chromatography column. DigL strongly agglutinated rabbit erythrocytes and was inhibited by d-mannose, d-glucose, and derived sugars, especially α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine. DigL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with EDTA. DigL is a glycoprotein composite by approximately 2.9% of carbohydrates by weight. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, the purified DigL exhibited an electrophoretic profile consisting of a broad band of 28-30 kDa. Analysis using electrospray ionization mass spectrometry indicated that purified DigL possesses a molecular average mass of 28 452 ± 2 Da and shows the presence of possible glycoforms. In addition, DigL exhibited an intermediary toxic effect on Artemia sp. nauplii, and this effect was both dependent on native structure and mediated by a carbohydrate-binding site. Copyright © 2013 John Wiley & Sons, Ltd.

Antiviral Lectins: Selective Inhibitors of Viral Entry

PubMed Central

Mitchell, Carter A.; Ramessar, Koreen; O’Keefe, Barry R.

2017-01-01

Many natural lectins have been reported to have antiviral activity. As some of these have been put forward as potential development candidates for preventing or treating viral infections, we have set out in this review to survey the literature on antiviral lectins. The review groups lectins by structural class and class of source organism we also detail their carbohydrate specificity and their reported antiviral activities. The review concludes with a brief discussion of several of the pertinent hurdles that heterologous proteins must clear to be useful clinical candidates and cites examples where such studies have been reported for antiviral lectins. Though the clearest path currently being followed is the use of antiviral lectins as anti-HIV microbicides via topical mucosal administration, some investigators have also found systemic efficacy against acute infections following subcutaneous administration. PMID:28322922

Expression of Lectins in Heterologous Systems

PubMed Central

Martínez-Alarcón, Dania; Blanco-Labra, Alejandro

2018-01-01

Lectins are proteins that have the ability to recognize and bind in a reversible and specific way to free carbohydrates or glycoconjugates of cell membranes. For these reasons, they have been extensively used in a wide range of industrial and pharmacological applications. Currently, there is great interest in their production on a large scale. Unfortunately, conventional techniques do not provide the appropriate platform for this purpose and therefore, the heterologous production of lectins in different organisms has become the preferred method in many cases. Such systems have the advantage of providing better yields as well as more homogeneous and better-defined properties for the resultant products. However, an inappropriate choice of the expression system can cause important structural alterations that have repercussions on their biological activity since the specificity may lay in their post-translational processing, which depends largely on the producing organism. The present review aims to examine the most representative studies in the area, exposing the four most frequently used systems (bacteria, yeasts, plants and animal cells), with the intention of providing the necessary information to determine the strategy to follow in each case as well as their respective advantages and disadvantages. PMID:29466298

A Novel Functional Role of Collagen Glycosylation

PubMed Central

Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels

2011-01-01

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090

Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approach.

PubMed

Pei, Zhichao; Saint-Guirons, Julien; Käck, Camilla; Ingemarsson, Björn; Aastrup, Teodor

2012-05-15

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

A label-free fluorescence biosensor for highly sensitive detection of lectin based on carboxymethyl chitosan-quantum dots and gold nanoparticles.

PubMed

Liu, Ziping; Liu, Hua; Wang, Lei; Su, Xingguang

2016-08-17

In this work, we report a novel label-free fluorescence "turn off-on" biosensor for lectin detection. The highly sensitive and selective sensing system is based on the integration of carboxymethyl chitosan (CM-CHIT), CuInS2 quantum dots (QDs) and Au nanoparticles (NPs). Firstly, CuInS2 QDs featuring carboxyl groups were directly synthesized via a hydrothermal synthesis method. Then, the carboxyl groups on the CuInS2 QDs surface were interacted with the amino groups (NH2), carboxyl groups (COOH) and hydroxyl groups (OH) within CM-CHIT polymeric chains via electrostatic interactions and hydrogen bonding to form CM-CHIT-QDs assemblies. Introduction of Au NPs could quench the fluorescence of CM-CHIT-QDs through electron and energy transfer. In the presence of lectin, lectin could bind exclusively with CM-CHIT-QDs by means of specific multivalent carbohydrate-protein interaction. Thus, the electron and energy transfer process between CM-CHIT-QDs and Au NPs was inhibited, and as a result, the fluorescence of CM-CHIT-QDs was effectively "turned on". Under the optimum conditions, there was a good linear relationship between the fluorescence intensity ratio I/I0 (I and I0 were the fluorescence intensity of CM-CHIT-QDs-Au NPs in the presence and absence of lectin, respectively) and lectin concentration in the range of 0.2-192.5 nmol L(-1), And the detection limit could be down to 0.08 nmol L(-1). Furthermore, the proposed biosensor was employed for the determination of lectin in fetal bovine serum samples with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

Plant Lectins: Wheat Defense Strategy Against Hessian Fly

USDA-ARS?s Scientific Manuscript database

Plants produce a variety of defense proteins, including lectins in response to attack by phytophagous insects. Ultrastructural studies reveal that binding to insect gut structures and resistance to proteolytic degradation by insect digestive enzymes are the two main prerequisites for the lectins to...

The Formation and Stability of DC-SIGN Microdomains Require its Extracellular Moiety

PubMed Central

Liu, Ping; Wang, Xiang; Itano, Michelle S.; Neumann, Aaron K.; Jacobson, Ken; Thompson, Nancy L.

2012-01-01

DC-SIGN (Dendritic cell-specific ICAM-3-grabbing non-integrin) is a Ca2+-dependent transmembrane lectin that binds a large variety of pathogens and facilitates their uptake for subsequent antigen presentation. This receptor is present in cell surface microdomains, but factors involved in microdomain formation and their exceptional stability are not clear. To determine which domain/motif of DC-SIGN facilitates its presence in microdomains, we studied mutations at key locations including truncation of the cytoplasmic tail, and ectodomain mutations that resulted in removal of the N-linked glycosylation site, the tandem repeats and the carbohydrate recognition domain (CRD) as well as modification of the calcium sites in the CRD required for carbohydrate binding. Confocal imaging and FRAP measurements showed that the cytoplasmic domain and N-linked glycosylation site do not affect the ability of DC-SIGN to form stable microdomains. However, truncation of the CRD results in complete loss of visible microdomains and subsequent lateral diffusion of the mutants. Apart from cell adhesions, membrane domains are thought to be localized primarily via the cytoskeleton. By contrast, we propose that interactions between the CRD of DC-SIGN and the extracellular matrix and/or cis interactions with transmembrane scaffolding protein(s) play an essential role in organizing these microdomains. PMID:22292921

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

Binding of navy bean (Phaseolus vulgaris) lectin to the intestinal cells of the rat and its effect on the absorption of glucose

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

Donatucci, D.A.; Liener, I.E.; Gross, C.J.

The main objectives of this investigation were to study the binding of a lectin from navy beans with the epithelial cells of the rat intestine and to assess the effect of such binding on the ability of the intestine to absorb glucose. A Scatchard plot, based on the binding of /sup 125/I-labeled lectin to isolated intestinal epithelial cells, was used to calculate an association constant (Ka) of 15 x 10(6)M-1 and the number of binding sites per cell, 12 x 10(6). Metabolic studies were conducted over a period of 5 d on groups of rats fed raw or autoclaved navymore » bean flour and casein with or without the purified lectin. Growth, protein digestibility, biological value and net protein utilization were significantly lower in animals that had been fed raw navy bean flour or casein plus lectin than in control groups fed diets containing autoclaved navy bean flour or casein alone. Vascular perfusion was used to measure the rate of uptake of glucose by the intestines of rats that had received the various dietary treatments. The rate of absorption of (/sup 14/C)glucose by intestines from rats fed raw navy bean flour or casein plus lectin was approximately one-half that of their counterparts fed the autoclaved flour or casein alone. These results provide evidence that the lectin, by virtue of its interference with intestinal absorption, is responsible, at least in part, for the nutritional inferiority of raw navy beans.« less

Medicinal Properties of the Genus Clitocybe and of Lectins from the Clouded Funnel Cap Mushroom, C. nebularis (Agaricomycetes): A Review.

PubMed

Pohleven, Jure; Kos, Janko; Sabotic, Jerica

2016-01-01

Current knowledge of the medicinal properties of Basidiomycetes mushroom species of the genus Clitocybe and of the biological activity of C. nebularis fruiting bodies is reviewed. The main focus is the therapeutic potential of lectins from C. nebularis. Species of the genus Clitocybe, including C. nebularis, have not been traditionally considered as medicinal mushrooms; however, recent studies have demonstrated their antitumor, immunomodulatory, and antioxidative properties, their antimicrobial (antiviral, antibacterial, and antifungal) activities against various bacteria and fungi, as well as their potential use in therapy for alcoholism and as psychotropic agents. These activities have been shown to be due to various compounds, either isolated or in extracts, mainly polysaccharides but also phenols, ribonucleosides, and proteins. These include laccase, protease inhibitors, and lectins. C. nebularis has been shown to be rich in a variety of lectins and isolectins with distinct carbohydrate-binding specificities, showing versatile biological activities. They exhibit immunostimulatory and adhesion-/phagocytosis-promoting properties, as well as toxicity in various invertebrates. Mushroom species of the genus Clitocybe, including C. nebularis, thus constitute a valuable source of compounds showing diverse biological activities with a broad potential for applications in biomedicine or biotechnology. On the basis of such evidence reviewed here, we propose that C. nebularis and other Clitocybe species can be considered to be medicinal mushrooms.

Lectin staining of epithelia lining the uterovaginal junction and sperm-storage tubules in chicken hens

USDA-ARS?s Scientific Manuscript database

In most mammals sperm are subject to a transient storage period in the caudal region of the oviduct during which they undergo cellular and molecular modifications associated with capacitation. During this storage period sperm bind to a terminal carbohydrate moiety associated with a glycoconjugate o...

A novel C-type lectin with triple carbohydrate recognition domains has critical roles for the hard tick Haemaphysalis longicornis against Gram-negative bacteria.

PubMed

Maeda, Hiroki; Miyata, Takeshi; Kusakisako, Kodai; Galay, Remil Linggatong; Talactac, Melbourne Rio; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

2016-04-01

C-type lectins (CLecs) play an important role in innate immunity against invaders. In this study, a novel CLec was identified from Haemaphysalis longicornis ticks (HlCLec). HlCLec contains a signal peptide and a transmembrane region. Interestingly, HlCLec possesses three dissimilar carbohydrate recognition domains (CRDs). Each CRD contains the mutated motif of Ca(2+)-binding site 2. HlCLec mRNA was up-regulated during blood feeding, and had highest expression in the midgut and ovary. HlCLec localization was also confirmed by immunofluorescent antibody test (IFAT). HlCLec was found on the cell membrane and basal lamina of midgut and ovary. In addition, the recombinant HlCLec and individual CRDs demonstrated direct binding activity to Escherichia coli and Staphylococcus aureus; however, no growth inhibition activity was observed. Furthermore, E. coli injection after silencing of HlCLec caused drastic reduction in survival rate of ticks. These results strongly suggest the key role of HlCLec in tick innate immunity against Gram-negative bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

Legume Lectins: Proteins with Diverse Applications

PubMed Central

Lagarda-Diaz, Irlanda; Guzman-Partida, Ana Maria; Vazquez-Moreno, Luz

2017-01-01

Lectins are a diverse class of proteins distributed extensively in nature. Among these proteins; legume lectins display a variety of interesting features including antimicrobial; insecticidal and antitumor activities. Because lectins recognize and bind to specific glycoconjugates present on the surface of cells and intracellular structures; they can serve as potential target molecules for developing practical applications in the fields of food; agriculture; health and pharmaceutical research. This review presents the current knowledge of the main structural characteristics of legume lectins and the relationship of structure to the exhibited specificities; provides an overview of their particular antimicrobial; insecticidal and antitumor biological activities and describes possible applications based on the pattern of recognized glyco-targets. PMID:28604616

Plant Lectins as Medical Tools against Digestive System Cancers.

PubMed

Estrada-Martínez, Laura Elena; Moreno-Celis, Ulisses; Cervantes-Jiménez, Ricardo; Ferriz-Martínez, Roberto Augusto; Blanco-Labra, Alejandro; García-Gasca, Teresa

2017-07-03

Digestive system cancers-those of the esophagus, stomach, small intestine, colon-rectum, liver, and pancreas-are highly related to genetics and lifestyle. Most are considered highly mortal due to the frequency of late diagnosis, usually in advanced stages, caused by the absence of symptoms or masked by other pathologies. Different tools are being investigated in the search of a more precise diagnosis and treatment. Plant lectins have been studied because of their ability to recognize and bind to carbohydrates, exerting a variety of biological activities on animal cells, including anticancer activities. The present report integrates existing information on the activity of plant lectins on various types of digestive system cancers, and surveys the current state of research into their properties for diagnosis and selective treatment.

Structural characterization of human galectin-4 C-terminal domain: elucidating the molecular basis for recognition of glycosphingolipids, sulfated saccharides and blood group antigens.

PubMed

Bum-Erdene, Khuchtumur; Leffler, Hakon; Nilsson, Ulf J; Blanchard, Helen

2015-09-01

Human galectin-4 is a lectin that is expressed mainly in the gastrointestinal tract and exhibits metastasis-promoting roles in some cancers. Its tandem-repeat nature exhibits two distinct carbohydrate recognition domains allowing crosslinking by simultaneous binding to sulfated and non-sulfated (but not sialylated) glycosphingolipids and glycoproteins, facilitating stabilization of lipid rafts. Critically, galectin-4 exerts favourable or unfavourable effects depending upon the cancer. Here we report the first X-ray crystallographic structural information on human galectin-4, specifically the C-terminal carbohydrate recognition domain of human (galectin-4C) in complex with lactose, lactose-3'-sulfate, 2'-fucosyllactose, lacto-N-tetraose and lacto-N-neotetraose. These structures enable elucidation of galectin-4C binding fine-specificity towards sulfated and non-sulfated lacto- and neolacto-series sphingolipids as well as to human blood group antigens. Analysis of the lactose-3'-sulfate complex structure shows that galectin-4C does not recognize the sulfate group using any specific amino acid, but binds the ligand nonetheless. Complex structures with lacto-N-tetraose and lacto-N-neotetraose displayed differences in binding interactions exhibited by the non-reducing-end galactose. That of lacto-N-tetraose points outward from the protein surface whereas that of lacto-N-neotetraose interacts directly with the protein. Recognition patterns of human galectin-4C towards lacto- and neolacto-series glycosphingolipids are similar to those of human galectin-3; however, detailed scrutiny revealed differences stemming from the extended binding site that offer distinction in ligand profiles of these two galectins. Structural characterization of the complex with 2'-fucosyllactose, a carbohydrate with similarity to the H antigen, and molecular dynamics studies highlight structural features that allow specific recognition of A and B antigens, whilst a lack of interaction with the 2'-fucose of blood group antigens was revealed. 4YLZ, 4YM0, 4YM1, 4YM2, 4YM3. © 2015 FEBS.

Dectin-2 Is a C-Type Lectin Receptor that Recognizes Pneumocystis and Participates in Innate Immune Responses.

PubMed

Kottom, Theodore J; Hebrink, Deanne M; Jenson, Paige E; Marsolek, Paige L; Wüthrich, Marcel; Wang, Huafeng; Klein, Bruce; Yamasaki, Sho; Limper, Andrew H

2018-02-01

Pneumocystis is an important fungal pathogen that causes life-threatening pneumonia in patients with AIDS and malignancy. Lung fungal pathogens are recognized by C-type lectin receptors (CLRs), which bind specific ligands and stimulate innate immune responses. The CLR Dectin-1 was previously shown to mediate immune responses to Pneumocystis spp. For this reason, we investigated a potential role for Dectin-2. Rats with Pneumocystis pneumonia (PCP) exhibited elevated Dectin-2 mRNA levels. Soluble Dectin-2 carbohydrate-recognition domain fusion protein showed binding to intact Pneumocystis carinii (Pc) and to native Pneumocystis major surface glycoprotein/glycoprotein A (Msg/gpA). RAW macrophage cells expressing V5-tagged Dectin-2 displayed enhanced binding to Pc and increased protein tyrosine phosphorylation. Furthermore, the binding of Pc to Dectin-2 resulted in Fc receptor-γ-mediated intracellular signaling. Alveolar macrophages from Dectin-2-deficient mice (Dectin-2 -/- ) showed significant decreases in phospho-Syk activation after challenge with Pc cell wall components. Stimulation of Dectin-2 -/- alveolar macrophages with Pc components showed significant decreases in the proinflammatory cytokines IL-6 and TNF-α. Finally, during infection with Pneumocystis murina, Dectin-2 -/- mice displayed downregulated mRNA expression profiles of other CLRs implicated in fungal immunity. Although Dectin-2 -/- alveolar macrophages had reduced proinflammatory cytokine release in vitro, Dectin-2 -/- deficiency did not reduce the overall resistance of these mice in the PCP model, and organism burdens were statistically similar in the long-term immunocompromised and short-term immunocompetent PCP models. These results suggest that Dectin-2 participates in the initial innate immune signaling response to Pneumocystis, but its deficiency does not impair resistance to the organism.

Lectins as endocytic ligands: an assessment of lectin binding and uptake to rabbit conjunctival epithelial cells.

PubMed

Qaddoumi, Mohamed; Lee, Vincent H L

2004-07-01

To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.

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

Isolation of a Hemagglutinin with Potent Antiproliferative Activity and a Large Antifungal Defensin from Phaseolus vulgaris cv. Hokkaido Large Pinto Beans.

PubMed

Yin, Cuiming; Wong, Jack Ho; Ng, Tzi Bun

2015-06-10

Lectins (hemagglutinins) are defined as sugar-binding proteins or glycoproteins with various biological activities. A 60 kDa dimeric hemagglutinin with a blocked N-terminus was isolated in large yield (190 mg/60 g) from the common edible bean Phaseolus vulgaris cv. Hokkaido large pinto bean. Its hemagglutinating, antifungal, and antitumor activities as well as the effects of carbohydrate and metal ions on its hemagglutinating activity were examined. It inhibited the proliferation of nasopharyngeal carcinoma (CNE2), human breast cancer (MCF7), and hepatoma (HepG2) cells. The IC50 values toward HepG2, MCF7, and CNE2 cells after treatment for 48 h were 8.1, 6.07, and 7.49 μM, respectively, which were relatively low among lectins of different P. vulgaris cultivars. From the pinto beans, a 10888 Da antifungal peptide with similarity to plant defensins as revealed by mass spectroscopic analysis was also isolated with a yield of 3.2 mg of proteins from 60 g of beans. The large defensin was capable of inhibiting mycelial growth in Mycosphaerella arachidicola, Setosphaeria turcica, Bipolaris maydis, and Fusarium oxysporum but not in Valsa mali.

Electronic Detection of Lectins Using Carbohydrate Functionalized Nanostructures: Graphene versus Carbon Nanotubes

PubMed Central

Chen, Yanan; Vedala, Harindra; Kotchey, Gregg P.; Audfray, Aymeric; Cecioni, Samy; Imberty, Anne; Vidal, Sébastien; Star, Alexander

2012-01-01

Here we investigated the interactions between lectins and carbohydrates using field-effect transistor (FET) devices comprised of chemically converted graphene (CCG) and single-walled carbon nanotubes (SWNTs). Pyrene- and porphyrin-based glycoconjugates were functionalized noncovalently on the surface of CCG-FET and SWNT-FET devices, which were then treated with 2 µM of nonspecific and specific lectins. In particular, three different lectins (PA-IL, PA-IIL and ConA) and three carbohydrate epitopes (galactose, fucose and mannose) were tested. The responses of 36 different devices were compared and rationalized using computer-aided models of carbon nanostructure/glycoconjugate interactions. Glycoconjugates surface coverage in addition to one-dimensional structures of SWNTs resulted in optimal lectin detection. Additionally, lectin titration data of SWNT- and CCG-based biosensors were used to calculate lectin dissociation constants (Kd) and compare them to the values obtained from the isothermal titration microcalorimetry (ITC) technique. PMID:22136380

The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties.

PubMed

Souza, Maria A; Carvalho, Fernanda C; Ruas, Luciana P; Ricci-Azevedo, Rafael; Roque-Barreira, Maria Cristina

2013-10-01

Advances in the glycobiology and immunology fields have provided many insights into the role of carbohydrate-protein interactions in the immune system. We aim to present a comprehensive review of the effects that some plant lectins exert as immunomodulatory agents, showing that they are able to positively modify the immune response to certain pathological conditions, such as cancer and infections. The present review comprises four main themes: (1) an overview of plant lectins that exert immunomodulatory effects and the mechanisms accounting for these activities; (2) general characteristics of the immunomodulatory lectin ArtinM from the seeds of Artocarpus heterophyllus; (3) activation of innate immunity cells by ArtinM and consequent induction of Th1 immunity; (4) resistance conferred by ArtinM administration in infections with intracellular pathogens, such as Leishmania (Leishmania) major, Leishmania (Leishmania) amazonensis, and Paracoccidioides brasiliensis. We believe that this review will be a valuable resource for more studies in this relatively neglected area of research, which has the potential to reveal carbohydrate targets for novel prophylactic and therapeutic strategies.

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

Microglial Lectins in Health and Neurological Diseases

PubMed Central

Siew, Jian Jing; Chern, Yijuang

2018-01-01

Microglia are the innate sentinels of the central nervous system (CNS) and are responsible for the homeostasis and immune defense of the CNS. Under the influence of the local environment and cell-cell interaction, microglia exhibit a multidimensional and context-dependent phenotypes that can be cytotoxic and neuroprotective. Recent studies suggest that microglia express multitudinous types of lectins, including galectins, Siglecs, mannose-binding lectins (MBLs) and other glycan binding proteins. Because most studies that examine lectins focus on the peripheral system, the functions of lectins have not been critically investigated in the CNS. In addition, the types of brain cells that contribute to the altered levels of lectins present in diseases are often unclear. In this review, we will discuss how galectins, Siglecs, selectins and MBLs contribute to the dynamic functions of microglia. The interacting ligands of these lectins are complex glycoconjugates, which consist of glycoproteins and glycolipids that are expressed on microglia or surrounding cells. The current understanding of the heterogeneity and functions of glycans in the brain is limited. Galectins are a group of pleotropic proteins that recognize both β-galactoside-containing glycans and non- β-galactoside-containing proteins. The function and regulation of galectins have been implicated in immunomodulation, neuroinflammation, apoptosis, phagocytosis and oxidative bursts. Most Siglecs are expressed at a low level on the plasma membrane and bind to sialic acid residues for immunosurveillance and cell-cell communication. Siglecs are classified based on their inhibitory and activatory downstream signaling properties. Inhibitory Siglecs negatively regulate microglia activation upon recognizing the intact sialic acid patterns and vice versa. MBLs are expressed upon infection in cytoplasm and can be secreted in order to recognize molecules containing terminal mannose as an innate immune defense machinery. Most importantly, multiple studies have reported dysregulation of lectins in neurological disorders. Here, we reviewed recent studies on microglial lectins and their functions in CNS health and disease, and suggest that these lectin families are novel, potent therapeutic targets for neurological diseases. PMID:29867350

The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

PubMed Central

Frisardi, Marta; Ghosh, Sudip K.; Field, Jessica; Van Dellen, Katrina; Rogers, Rick; Robbins, Phillips; Samuelson, John

2000-01-01

The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study an extraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional protein gels of cyst walls purified from Entamoeba invadens. Jacob, which was acidic and had an apparent molecular mass of ∼100 kDa, contained sugars that bound to concanavalin A and ricin. The jacob gene encoded a 45-kDa protein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but not homologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins that surround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecyl sulfate-treated cyst walls. Conversely, the E. histolytica plasma membrane Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob. In the presence of galactose, E. invadens formed wall-less cysts, which were quadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles. A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria and mucin-coated beads. These results suggest that the E. invadens cyst wall forms when the plasma membrane galactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains. PMID:10858239

High entomotoxicity and mechanism of the fungal GalNAc/Gal-specific Rhizoctonia solani lectin in pest insects.

PubMed

Hamshou, Mohamad; Van Damme, Els J M; Caccia, Silvia; Cappelle, Kaat; Vandenborre, Gianni; Ghesquière, Bart; Gevaert, Kris; Smagghe, Guy

2013-03-01

Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leaf-worm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10 mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8 days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3 days with 9 μM of RSA in the liquid diet. Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells. In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3 μM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC-MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA. Copyright © 2012 Elsevier Ltd. All rights reserved.

Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests.

PubMed

Boddupally, Dayakar; Tamirisa, Srinath; Gundra, Sivakrishna Rao; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2018-05-31

To evolve rice varieties resistant to different groups of insect pests a fusion gene, comprising DI and DII domains of Bt Cry1Ac and carbohydrate binding domain of garlic lectin (ASAL), was constructed. Transgenic rice lines were generated and evaluated to assess the efficacy of Cry1Ac::ASAL fusion protein against three major pests, viz., yellow stem borer (YSB), leaf folder (LF) and brown planthopper (BPH). Molecular analyses of transgenic plants revealed stable integration and expression of the fusion gene. In planta insect bioassays on transgenics disclosed enhanced levels of resistance compared to the control plants. High insect mortality of YSB, LF and BPH was observed on transgenics compared to that of control plants. Furthermore, honeydew assays revealed significant decreases in the feeding ability of BPH on transgenic plants as compared to the controls. Ligand blot analysis, using BPH insects fed on cry1Ac::asal transgenic rice plants, revealed a modified receptor protein-binding pattern owing to its ability to bind to additional receptors in insects. The overall results authenticate that Cry1Ac::ASAL protein is endowed with remarkable entomotoxic effects against major lepidopteran and hemipteran insects. As such, the fusion gene appears promising and can be introduced into various other crops to control multiple insect pests.

Visualization of melanoma tumor with lectin-conjugated rare-earth doped fluoride nanocrystals

PubMed Central

Dumych, Tetiana; Lutsyk, Maxym; Banski, Mateusz; Yashchenko, Antonina; Sojka, Bartlomiej; Horbay, Rostyslav; Lutsyk, Alexander; Stoika, Rostyslav; Misiewicz, Jan; Podhorodecki, Artur; Bilyy, Rostyslav

2014-01-01

Aim To develop specific fluorescent markers for melanoma tumor visualization, which would provide high selectivity and reversible binding pattern, by the use of carbohydrate-recognizing proteins, lectins, combined with the physical ability for imaging deep in the living tissues by utilizing red and near infrared fluorescent properties of specific rare-earth doped nanocrystals (NC). Methods B10F16 melanoma cells were inoculated to C57BL/6 mice for inducing experimental melanoma tumor. Tumors were removed and analyzed by lectin-histochemistry using LABA, PFA, PNA, HPA, SNA, GNA, and NPL lectins and stained with hematoxylin and eosin. NPL lectin was conjugated to fluorescent NaGdF4:Eu3+-COOH nanoparticles (5 nm) via zero length cross-linking reaction, and the conjugates were purified from unbound substances and then used for further visualization of histological samples. Fluorescent microscopy was used to visualize NPL-NaGdF4:Eu3+ with the fluorescent emission at 600-720 nm range. Results NPL lectin selectively recognized regions of undifferentiated melanoblasts surrounding neoangiogenic foci inside melanoma tumor, PNA lectin recognized differentiated melanoblasts, and LCA and WGA were bound to tumor stroma regions. NPL-NaGdF4:Eu3+ conjugated NC were efficiently detecting newly formed regions of melanoma tumor, confirmed by fluorescent microscopy in visible and near infrared mode. These conjugates possessed high photostability and were compatible with convenient xylene-based mounting systems and preserved intensive fluorescent signal at samples storage for at least 6 months. Conclusion NPL lectin-NaGdF4:Eu3+ conjugated NC permitted distinct identification of contours of the melanoma tissue on histological sections using red excitation at 590-610 nm and near infrared emission of 700-720 nm. These data are of potential practical significance for development of glycans-conjugated nanoparticles to be used for in vivo visualization of melanoma tumor. PMID:24891277

The galactose-binding lectin isolated from Bauhinia bauhinioides Mart seeds inhibits neutrophil rolling and adhesion via primary cytokines.

PubMed

Girão, Deysen Kerlla Fernandes Bezerra; Cavada, Benildo Sousa; de Freitas Pires, Alana; Martins, Timna Varela; Franco, Álvaro Xavier; Morais, Cecília Mendes; Santiago do Nascimento, Kyria; Delatorre, Plinio; da Silva, Helton Colares; Nagano, Celso Shiniti; Assreuy, Ana Maria Sampaio; Soares, Pedro Marcos Gomes

2015-05-01

In this study, the amino acid sequence and anti-inflammatory effect of Bauhinia bauhinioides (BBL) lectin were evaluated. Tandem mass spectrometry revealed that BBL possesses 86 amino acid residues. BBL (1 mg/kg) intravenously injected in rats 30 min prior to inflammatory stimuli inhibited the cellular edema induced by carrageenan in only the second phase (21% - 3 h, 19% - 4 h) and did not alter the osmotic edema induced by dextran. BBL also inhibited carrageenan peritoneal neutrophil migration (51%), leukocyte rolling (58%) and adhesion (68%) and the neutrophil migration induced by TNF-α (64%). These effects were reversed by the association of BBL with galactose, demonstrating that the carbohydrate-binding domain is essential for lectin activity. In addition, BBL reduced myeloperoxidase activity (84%) and TNF-α (68%) and IL1-β (47%) levels. In conclusion, the present investigation demonstrated that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti-inflammatory activity by inhibiting neutrophil migration by reducing TNF-α and IL1-β levels via the lectin domain. Copyright © 2015 John Wiley & Sons, Ltd.

Morus alba Leaf Lectin (MLL) Sensitizes MCF-7 Cells to Anoikis by Inhibiting Fibronectin Mediated Integrin-FAK Signaling through Ras and Activation of P38 MAPK

PubMed Central

Saranya, Jayaram; Shilpa, Ganesan; Raghu, Kozhiparambil G.; Priya, Sulochana

2017-01-01

Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK. PMID:28223935

Identification of Peroxiredoxin-5 in Bovine Cauda Epididymal Sperm

PubMed Central

Nagdas, Subir K; Buchanan, Teresa; Raychoudhury, Samir

2013-01-01

Developing spermatozoa require a series of post-testicular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5kDa Wheat Germ Agglutinin (WGA) binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA stained bands, the presence of a 17.5kDa WGA binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide. PMID:24186847

G₂/M cell cycle arrest by an N-acetyl-D-glucosamine specific lectin from Psathyrella asperospora.

PubMed

Rouf, Razina; Stephens, Alexandre S; Spaan, Lina; Arndt, Nadia X; Day, Christopher J; May, Tom W; Tiralongo, Evelin; Tiralongo, Joe

2014-01-01

A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6-10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC50 0.48 μM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G2/M phase in a manner dependent on its ability to bind GlcNAc.

A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine

PubMed Central

Jiang, Shuai; Chen, Yijie; Wang, Man; Yin, Yalin; Pan, Yongfu; Gu, Bianli; Yu, Guojun; Li, Yamu; Wong, Barry Hon Cheung; Liang, Yi; Sun, Hui

2012-01-01

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo. PMID:22268569

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains.

PubMed

Chigira, Yuko; Oka, Takuji; Okajima, Tetsuya; Jigami, Yoshifumi

2008-04-01

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

Plant Lectins as Medical Tools against Digestive System Cancers

PubMed Central

Estrada-Martínez, Laura Elena; Cervantes-Jiménez, Ricardo; Ferriz-Martínez, Roberto Augusto; Blanco-Labra, Alejandro

2017-01-01

Digestive system cancers—those of the esophagus, stomach, small intestine, colon-rectum, liver, and pancreas—are highly related to genetics and lifestyle. Most are considered highly mortal due to the frequency of late diagnosis, usually in advanced stages, caused by the absence of symptoms or masked by other pathologies. Different tools are being investigated in the search of a more precise diagnosis and treatment. Plant lectins have been studied because of their ability to recognize and bind to carbohydrates, exerting a variety of biological activities on animal cells, including anticancer activities. The present report integrates existing information on the activity of plant lectins on various types of digestive system cancers, and surveys the current state of research into their properties for diagnosis and selective treatment. PMID:28671623

Rational design of adjuvants targeting the C-type lectin Mincle.

PubMed

Decout, Alexiane; Silva-Gomes, Sandro; Drocourt, Daniel; Barbe, Sophie; André, Isabelle; Cueto, Francisco J; Lioux, Thierry; Sancho, David; Pérouzel, Eric; Vercellone, Alain; Prandi, Jacques; Gilleron, Martine; Tiraby, Gérard; Nigou, Jérôme

2017-03-07

The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cell-mediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6'-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O -6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties.

Atomic Force Microscopy for Investigation of Ribosome-inactivating Proteins' Type II Tetramerization

NASA Astrophysics Data System (ADS)

Savvateev, M.; Kozlovskaya, N.; Moisenovich, M.; Tonevitsky, A.; Agapov, I.; Maluchenko, N.; Bykov, V.; Kirpichnikov, M.

2003-12-01

Biology of the toxins violently depends on their carbohydrate-binding centres' organization. Toxin tetramerization can lead to both increasing of lectin-binding centres' number and changes in their structural organization. A number and three-dimensional localization of such centres per one molecule strongly influence on toxins' biological properties. Ricin was used to obtain the AFM images of natural dimeric RIPsII structures as far as ricinus agglutinin was used for achievement of AFM images of natural tetrameric RIPsII forms. It is well-known that viscumin (60 kDa) has a property to form tetrameric structures dependently on ambient conditions and its concentration. Usage of the model dimer-tetramer based on ricin-agglutinin allowed to identify viscumin tetramers in AFM scans and to differ them from dimeric viscumin structures. Quantification analysis produced with the NT-MDT software allowed to estimate the geometrical parameters of ricin, ricinus agglutinin and viscumin molecules.

Complex thiolated mannose/quinone film modified on EQCM/Au electrode for recognizing specific carbohydrate-proteins.

PubMed

Zeng, Hongjuan; Yu, Junsheng; Jiang, Yadong; Zeng, Xiangqun

2014-05-15

A complex thiolated mannose (TM)/quinone functionalised polythiophene (QFPT) thin film was modified on EQCM/Au electrode for recognition of specific carbohydrate-proteins. Different lectins such as those from Sambucus nigra (elder berry), Arachis hypogaea (peanut), Ulex europaeus (gorse, furze), Triticum vulgaris and Concanavalin A (ConA) was used for probes to evaluate bio-sensing performance of the TM/QFPT film. A specific response was observed for ConA from lectins when using the TM/QFPT film as sensing material and employing either elelctrochemical or the QCM method. No response was detected between thiolated mannose and other lectins. The linear relationship between current and ConA concentration is in the range of 0.5-17.5 nM by the elelctrochemical method and the linear relationship between frequency change and ConA concentration is in the range of 0.5-4.5 nM by the QCM method. This shows that the TM/QFPT-modified EQCM biosensor presents a paralleled determination by using electrochemical and the QCM method. The elelctrochemical method of the biosensor can be applicable in a large concentration range and its frequency change can be more precise. © 2013 Published by Elsevier B.V.

Differential release and phagocytosis of tegument glycoconjugates in neurocysticercosis: implications for immune evasion strategies.

PubMed

Alvarez, Jorge I; Rivera, Jennifer; Teale, Judy M

2008-04-09

Neurocysticercosis (NCC) is an infection of the central nervous system (CNS) by the metacestode of the helminth Taenia solium. The severity of the symptoms is associated with the intensity of the immune response. First, there is a long asymptomatic period where host immunity seems incapable of resolving the infection, followed by a chronic hypersensitivity reaction. Since little is known about the initial response to this infection, a murine model using the cestode Mesocestoides corti (syn. Mesocestoides vogae) was employed to analyze morphological changes in the parasite early in the infection. It was found that M. corti material is released from the tegument making close contact with the nervous tissue. These results were confirmed by infecting murine CNS with ex vivo-labeled parasites. Because more than 95% of NCC patients exhibit humoral responses against carbohydrate-based antigens, and the tegument is known to be rich in glycoconjugates (GCs), the expression of these types of molecules was analyzed in human, porcine, and murine NCC specimens. To determine the GCs present in the tegument, fluorochrome-labeled hydrazides as well as fluorochrome-labeled lectins with specificity to different carbohydrates were used. All the lectins utilized labeled the tegument. GCs bound by isolectinB4 were shed in the first days of infection and not resynthesized by the parasite, whereas GCs bound by wheat germ agglutinin and concavalinA were continuously released throughout the infectious process. GCs bound by these three lectins were taken up by host cells. Peanut lectin-binding GCs, in contrast, remained on the parasite and were not detected in host cells. The parasitic origin of the lectin-binding GCs found in host cells was confirmed using antibodies against T. solium and M. corti. We propose that both the rapid and persistent release of tegumental GCs plays a key role in the well-known immunomodulatory effects of helminths, including immune evasion and life-long inflammatory sequelae seen in many NCC patients.

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.

Cancer biomarker discovery for cholangiocarcinoma: the high-throughput approaches

PubMed Central

Silsirivanit, Atit; Sawanyawisuth, Kanlayanee; Riggins, Gregory J.; Wongkham, Chaisiri

2015-01-01

Cholangiocarcinoma (CCA) is difficult to diagnose at an early stage and most tumors are detected at late stage where surgery or other therapy is ineffective. Many advanced techniques are applied to diagnose CCA; however, most are expensive and have varying degrees of accuracy. A less invasive and simpler procedure such as serum markers would be of substantial clinical benefit for diagnosis, monitoring, and predicting outcome for CCA patients. Recent advances in “Omics” technologies offer remarkable opportunities for establishment of biomarker-related to diseases. In this review, the potential biomarkers obtained from proteomics and glycomic studies are evaluated. Several protein markers were discovered from patient specimen, using two dimensional-differential gel electrophoresis couple with liquid chromatography tandem mass spectrometry (2D-DIGE/LC-MS-MS), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), surface enhanced laser desorption/ionization (SELDI)-TOF-MS and capillary electrophoresis (CE)-MS, etc. Newly reported CCA-associated glycobiomarkers were identified using lectin-assisted, monoclonal antibody-assisted or specific-target strategies. The combination between carbohydrate binding-lectin and core protein-binding mAb significantly increased the values for detection of the glyco-biomarkers for CCA. Searching for specific and sensitive molecular markers to be used for population screening is worth being evaluated. This could lead to earlier diagnosis and improve outcome. Further investigation of those biomarker functions is also of value in order to better understand the tumor biology and use them as targets for future therapeutic agents. PMID:24616382

Intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein.

PubMed

Liu, Li; Bastien, Nathalie; Li, Yan

2007-12-01

The biosynthesis and posttranslational processing of human metapneumovirus attachment G glycoprotein were investigated. After pulse-labeling, the G protein accumulated as three species with molecular weights of 45,000, 50,000, and 53,000 (45K, 50K, and 53K, respectively). N-Glycosidase digestion indicated that these forms represent the unglycosylated precursor and N-glycosylated intermediate products, respectively. After an appropriate chase, these three naive forms were further processed to a mature 97K form. The presence of O-linked sugars in mature G protein was confirmed by O-glycanase digestion and lectin-binding assay using Arachis hypogaea (peanut agglutinin), an O-glycan-specific lectin. In addition, in the O-glycosylation-deficient cell line (CHO ldlD cell), the G protein could not be processed to the mature form unless the exogenous Gal and GalNAc were supplemented, which provided added evidence supporting the O-linked glycosylation of G protein. The maturation of G was completely blocked by monensin but was partially sensitive to brefeldin A (BFA), suggesting the O-linked glycosylation of G initiated in the trans-Golgi compartment and terminated in the trans-Golgi network. Enzymatic deglycosylation analysis confirmed that the BFA-G was a partial mature form containing N-linked oligosaccharides and various amounts of O-linked carbohydrate side chains. The expression of G protein at the cell surface could be detected by indirect immunofluorescence staining assay. Furthermore, cell surface immunoprecipitation displayed an efficient intracellular transport of G protein.

Glycophenotype evaluation in cutaneous tumors using lectins labeled with acridinium ester.

PubMed

Lima, Luiza Rayanna Amorim; Bezerra, Matheus Filgueira; Almeida, Sinara Mônica Vitalino; Silva, Lúcia Patrícia Bezerra Gomes; Beltrão, Eduardo Isidoro Carneiro; Carvalho Júnior, Luiz Bezerra

2013-01-01

Tumor cells show alterations in their glycosylation patterns when compared to normal cells. Lectins can be used to evaluate these glycocode changes. Chemiluminescence assay is an effective technique for quantitative analysis of proteins, nucleic acids, and carbohydrates due to its high sensitivity, specificity, and rapid testing. To use histochemiluminescence based on lectin conjugated to acridinium ester (AE) for the investigation of glycophenotype changes in cutaneous tumors. Concanavalin A (Con A), Peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I), and Maackia amurensis agglutinin (MAA) were conjugated to acridinium ester. Biopsies of cutaneous tumors and normal skin were incubated with the lectins-AE, and chemiluminescence was quantified and expressed as Relative Light Units (RLU). Results. Actinic keratosis (AK), keratoacanthoma (KA), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) showed lower expression of α -D-glucose/mannose and α -L-fucose residues compared to normal tissue. Cutaneous tumors displayed higher expression of Gal- β (1-3)-GalNAc residues than normal tissue. AK and SCC exhibited higher expression of Neu5Ac- α (2,3)Gal residues than normal epidermis. KA and BCC showed equivalent RLU values compared to normal tissue. Lectin histochemiluminescence allowed quantitative assessment of the carbohydrate expression in cutaneous tissues, contributing to eliminate the subjectivity of conventional techniques used in the histopathological diagnosis.

Nanostructured disposable impedimetric sensors as tools for specific biomolecular interactions: sensitive recognition of concanavalin A.

PubMed

Loaiza, Oscar A; Lamas-Ardisana, Pedro J; Jubete, Elena; Ochoteco, Estibalitz; Loinaz, Iraida; Cabañero, Germán; García, Isabel; Penadés, Soledad

2011-04-15

The development of sensors to detect specific weak biological interactions is still today a challenging topic. Characteristics of carbohydrate-protein (lectin) interactions include high specificity and low affinity. This work describes the development of nanostructured impedimetric sensors for the detection of concanavalin A (Con A) binding to immobilized thiolated carbohydrate derivatives (D-mannose or D-glucose) onto screen-printed carbon electrodes (SPCEs) modified with gold nanoparticles. Thiolated D-galactose derivative was employed as negative control to evaluate the selectivity of the proposed methodology. After binding the thiolated carbohydrate to the nanostructured SPCEs, different functionalized thiols were employed to form mixed self-assembled monolayers (SAM). Electrochemical impedance spectroscopy (EIS) was employed as a technique to evaluate the binding of Con A to selected carbohydrates through the increase of electron transfer resistance of the ferri/ferrocyanide redox probe at the differently SAM modified electrodes. Different variables of the assay protocol were studied in order to optimize the sensor performance. Selective Con A determinations were only achieved by the formation of mixed SAMs with adequate functionalized thiols. Important differences were obtained depending on the chain lengths and functional groups of these thiols. For the 3-mercapto-1-propanesulfonate mixed SAMs, the electron transfer resistance varied linearly with the Con A concentration in the 2.2-40.0 μg mL(-1) range for D-mannose and D-glucose modified sensors. Low detection limits (0.099 and 0.078 pmol) and good reproducibility (6.9 and 6.1%, n=10) were obtained for the D-glucose and D-mannose modified sensors, respectively, without any amplification strategy. © 2011 American Chemical Society

Parkia pendula lectin as histochemistry marker for meningothelial tumour.

PubMed

Beltrão, E I C; Medeiros, P L; Rodrigues, O G; Figueredo-Silva, J; Valença, M M; Coelho, L C B B; Carvalho, L B

2003-01-01

Lectins have been intensively used in histochemical techniques for cell surface characterization. These proteins are involved in several biological processes and their use as histochemical markers have been evaluated since they can indicate differences in cell surfaces. Parkia pendula lectin (PpeL) was evaluated as histochemical marker for meningothelial meningioma biopsies. Tissue slices were incubated with PpeL conjugated to horseradish peroxidase (PpeL-HRP) and Concanavalin A-HRP (ConA-HPR) and the binding visualized with diaminobenzidine and hydrogen peroxide. The lectin-tissue binding was inhibited with D-glucose. PpeL showed to be a useful tool for the characterization of meningothelial tumour and clinico-pathological diagnosis.

Aptamer-recognized carbohydrates on the cell membrane revealed by super-resolution microscopy.

PubMed

Jing, Yingying; Cai, Mingjun; Xu, Haijiao; Zhou, Lulu; Yan, Qiuyan; Gao, Jing; Wang, Hongda

2018-04-26

Carbohydrates are one of the most important components on the cell membrane, which participate in various physiological activities, and their aberrant expression is a consequence of pathological changes. In previous studies, carbohydrate analysis basically relied on lectins. However, discrimination between lectins still exists due to their multivalent character. Furthermore, the structures obtained by carbohydrate-lectin crosslinking confuse our direct observation to some extent. Fortunately, the emergence of aptamers, which are smaller and more flexible, has provided us an unprecedented choice. Herein, an aptamer recognition method with high precise localization was developed for imaging membrane-bound N-acetylgalactosamine (GalNAc). By using direct stochastic optical reconstruction microscopy (dSTORM), we compared this aptamer recognition method with the lectin recognition method for visualizing the detailed structure of GalNAc at the nanometer scale. The results indicated that GalNAc forms irregular clusters on the cell membrane with a resolution of 23 ± 7 nm by aptamer recognition. Additionally, when treated with N-acetylgalactosidase, the aptamer-recognized GalNAc shows a more significant decrease in cluster size and localization density, thus verifying better specificity of aptamers than lectins. Collectively, our study suggests that aptamers can act as perfect substitutes for lectins in carbohydrate labeling, which will be of great potential value in the field of super-resolution fluorescence imaging.

Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition.

PubMed

Neth, O; Jack, D L; Dodds, A W; Holzel, H; Klein, N J; Turner, M W

2000-02-01

Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. Diverse Candida species, Aspergillus fumigatus, Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereas Escherichia coli, Klebsiella species, and Haemophilus influenzae type b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci, Streptococcus pneumoniae, and Staphylococcus epidermidis showed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.

Exploring the free-energy landscape of carbohydrate-protein complexes: development and validation of scoring functions considering the binding-site topology

NASA Astrophysics Data System (ADS)

Eid, Sameh; Saleh, Noureldin; Zalewski, Adam; Vedani, Angelo

2014-12-01

Carbohydrates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the structure-based design of carbohydrate-based ligands. We assembled a diverse data set comprising 273 carbohydrate-protein crystal structures with known binding affinity and evaluated the prediction accuracy of a large collection of well-established scoring and free-energy functions, as well as combinations thereof. Unfortunately, the tested functions were not capable of reproducing binding affinities in the studied complexes. To simplify the complex free-energy surface of carbohydrate-protein systems, we classified the studied proteins according to the topology and solvent exposure of the carbohydrate-binding site into five distinct categories. A free-energy model based on the proposed classification scheme reproduced binding affinities in the carbohydrate data set with an r 2 of 0.71 and root-mean-squared-error of 1.25 kcal/mol ( N = 236). The improvement in model performance underlines the significance of the differences in the local micro-environments of carbohydrate-binding sites and demonstrates the usefulness of calibrating free-energy functions individually according to binding-site topology and solvent exposure.

Mechanism of allosteric propagation across a β-sheet structure investigated by molecular dynamics simulations.

PubMed

Interlandi, Gianluca; Thomas, Wendy E

2016-07-01

The bacterial adhesin FimH consists of an allosterically regulated mannose-binding lectin domain and a covalently linked inhibitory pilin domain. Under normal conditions, the two domains are bound to each other, and FimH interacts weakly with mannose. However, under tensile force, the domains separate and the lectin domain undergoes conformational changes that strengthen its bond with mannose. Comparison of the crystallographic structures of the low and the high affinity state of the lectin domain reveals conformational changes mainly in the regulatory inter-domain region, the mannose binding site and a large β sheet that connects the two distally located regions. Here, molecular dynamics simulations investigated how conformational changes are propagated within and between different regions of the lectin domain. It was found that the inter-domain region moves towards the high affinity conformation as it becomes more compact and buries exposed hydrophobic surface after separation of the pilin domain. The mannose binding site was more rigid in the high affinity state, which prevented water penetration into the pocket. The large central β sheet demonstrated a soft spring-like twisting. Its twisting motion was moderately correlated to fluctuations in both the regulatory and the binding region, whereas a weak correlation was seen in a direct comparison of these two distal sites. The results suggest a so called "population shift" model whereby binding of the lectin domain to either the pilin domain or mannose locks the β sheet in a rather twisted or flat conformation, stabilizing the low or the high affinity state, respectively. Proteins 2016; 84:990-1008. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Alterations in protein glycosylation in PMA-differentiated U-937 cells exposed to mineral particles.

PubMed Central

Trabelsi, N; Greffard, A; Pairon, J C; Bignon, J; Zanetti, G; Fubini, B; Pilatte, Y

1997-01-01

Carbohydrate moieties of cell glycoconjugates play a pivotal role in molecular recognition phenomena involved in the regulation of most biological systems and the changes observed in cell surface carbohydrates during cell activation or differentiation frequently modulate certain cell functions. Consequently, some aspects of macrophage response to particle exposure might conceivably result from alterations in glycosylation. Therefore, the effect of mineral particles on protein glycosylation was investigated in phorbol myristate acetate (PMA)-differentiated U-937. Jacalin, a lectin specific for O-glycosylated structures, showed a global increase in O-glycosylation in particle-treated cells. In contrast, no significant modifications were observed with concanavalin A, a lectin that recognizes certain N-glycosylated structures. The sialic acid-specific lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin and the galactose-specific lectin Ricinus communis agglutinin revealed a complex pattern of alterations in glycoprotein glycosylation after crystalline silica or manganese dioxide treatments. Expression of sialyl Lewis(x), a glycosylated structure implicated in leukocyte trafficking, could not be detected in control or treated cells. This finding was consistent with the decrease in sialyl Lewis(x) expression observed during PMA-induced differentiation. In conclusion, various treatments used in this study induced quantitative as well as qualitative changes in protein glycosylation. Whether these changes are due to glycosidase release or to an alteration in glycosyltransferase expression remains to be determined. The potential functional implications of these changes are currently under investigation. Images Figure 1. A Figure 1. B Figure 2. A Figure 2. B Figure 3. A Figure 3. B Figure 3. C Figure 4. PMID:9400716

Mushroom Lectins: Specificity, Structure and Bioactivity Relevant to Human Disease

PubMed Central

Hassan, Mohamed Ali Abol; Rouf, Razina; Tiralongo, Evelin; May, Tom W.; Tiralongo, Joe

2015-01-01

Lectins are non-immunoglobulin proteins that bind diverse sugar structures with a high degree of selectivity. Lectins play crucial role in various biological processes such as cellular signaling, scavenging of glycoproteins from the circulatory system, cell–cell interactions in the immune system, differentiation and protein targeting to cellular compartments, as well as in host defence mechanisms, inflammation, and cancer. Among all the sources of lectins, plants have been most extensively studied. However, more recently fungal lectins have attracted considerable attention due to their antitumor, antiproliferative and immunomodulatory activities. Given that only 10% of mushroom species are known and have been taxonomically classified, mushrooms represent an enormous unexplored source of potentially useful and novel lectins. In this review we provide an up-to-date summary on the biochemical, molecular and structural properties of mushroom lectins, as well as their versatile applications specifically focusing on mushroom lectin bioactivity. PMID:25856678

The 2.2 A resolution structure of the O(H) blood-group-specific lectin I from Ulex europaeus.

PubMed

Audette, G F; Vandonselaar, M; Delbaere, L T

2000-12-01

The tertiary and quaternary structure of the lectin I from Ulex europaeus (UE-I) has been determined to 2.2 A resolution. UE-I is a dimeric metalloglycoprotein that binds the H-type 2 human blood group determinant [alpha-L-Fucalpha(1-->2)-beta-D-Galbeta(1-->4)-beta-D-Glc NAcalpha-]. Nine changes from the published amino acid sequence were necessary to account for the electron density. The quaternary structural organization of UE-I is that of the most commonly occurring legume lectin dimer. The tertiary structure of the monomeric subunits is similar to that in the conventional lectin subunit; however, some structural differences are noted. These differences include a four-stranded anti-parallel "S" sheet in UE-I versus the five-stranded S sheet in other lectin monomers. The Ala residue of the Ala-Asp cis-peptide bond present in the carbohydrate-binding site of the conventional lectin monomer is replaced with a Thr in the UE-I structure. Also, a novel disulfide bridge linking Cys115 and Cys150 is present. There are two metallic ions, one calcium and the other manganese, per subunit. N-linked oligosaccharides are at residues 23 and 111 of each subunit. One molecule of R-2-methyl-2, 4-pentanediol (R-MPD) is present in a shallow depression on the surface of each subunit. In order to examine the binding of the H-type 2 blood group determinant by UE-I, its beta-methyl glycoside (H-type 2-OMe) was docked into the binding site of R-MPD. The epitope previously identified for H-type 2-OMe by chemical mapping proved, with only minor adjustment of amino acid residues, to be complementary to the shallow cavity occupied by R-MPD in the structure. Several key interactions have been proposed between the H-type 2-OMe and UE-I. Copyright 2000 Academic Press.

Efficient and accelerated growth of multifunctional dendrimers using orthogonal thiol-ene and SN2 reactions.

PubMed

Kottari, Naresh; Chabre, Yoann M; Shiao, Tze Chieh; Rej, Rabindra; Roy, René

2014-02-25

An orthogonal coupling strategy was developed by combining thiol-ene and SN2 reactions, which was subsequently applied to the accelerated synthesis of multifunctional dendrimers using carbohydrate building blocks. In surface plasmon resonance (SPR) studies, the β-d-galactopyranoside-coated dendrimer exhibited nM binding affinity with the bacterial LecA lectin extracted from Pseudomonas aeruginosa.

Study of surface carbohydrates in Galba truncatula tissues before and after infection with Fasciola hepatica

PubMed Central

Georgieva, Katya; Georgieva, Liliya; Mizinska-Boevska, Yana; Stoitsova, Stoyanka R

2016-01-01

The presence and distribution of surface carbohydrates in the tissues of Galba truncatula snails uninfected or after infection with Fasciola hepatica as well as on the surface of the snail-pathogenic larval stages of the parasite were studied by lectin labelling assay. This is an attempt to find similarities that indicate possible mimicry, utilised by the parasite as an evasion strategy in this snail-trematode system. Different binding patterns were identified on head-foot-mantle, hepatopancreas, genital glands, renopericardial complex of the host as well as of the snail-pathogenic larval stages of F. hepatica. The infection with F. hepatica leads to changes of labelling with Glycine max in the head-mantle cells and Arachis hypogaea in the tubular epithelium of the hepatopancreas. The lectin binding on the other snail tissues is not changed by the development of the larvae. Our data clearly demonstrated the similarity in labelling of G. truncatula tissues and the surface of the snail-pathogenic larval stages of F. hepatica. The role of glycosylation of the contact surfaces of both organisms in relation to the host-parasite interactions is also discussed. PMID:27384082

Study of surface carbohydrates in Galba truncatula tissues before and after infection with Fasciola hepatica.

PubMed

Georgieva, Katya; Georgieva, Liliya; Mizinska-Boevska, Yana; Stoitsova, Stoyanka R

2016-07-04

The presence and distribution of surface carbohydrates in the tissues of Galba truncatula snails uninfected or after infection with Fasciola hepatica as well as on the surface of the snail-pathogenic larval stages of the parasite were studied by lectin labelling assay. This is an attempt to find similarities that indicate possible mimicry, utilised by the parasite as an evasion strategy in this snail-trematode system. Different binding patterns were identified on head-foot-mantle, hepatopancreas, genital glands, renopericardial complex of the host as well as of the snail-pathogenic larval stages of F. hepatica. The infection with F. hepatica leads to changes of labelling with Glycine max in the head-mantle cells and Arachis hypogaea in the tubular epithelium of the hepatopancreas. The lectin binding on the other snail tissues is not changed by the development of the larvae. Our data clearly demonstrated the similarity in labelling of G. truncatula tissues and the surface of the snail-pathogenic larval stages of F. hepatica. The role of glycosylation of the contact surfaces of both organisms in relation to the host-parasite interactions is also discussed.

Structural, functional, and evolutionary aspects of galectins in aquatic mollusks: From a sweet tooth to the Trojan horse

PubMed Central

Vasta, GR; Feng, C; Bianchet, MA; Bachvaroff, TR; Tasumi, S

2015-01-01

Galectins constitute a conserved and widely distributed lectin family characterized by their binding affinity for β-galactosides and a unique binding site sequence motif in the carbohydrate recognition domain (CRD). In spite of their structural conservation, galectins display a remarkable functional diversity, by participating in developmental processes, cell adhesion and motility, regulation of immune homeostasis, and recognition of glycans on the surface of viruses, bacteria and protozoan parasites. In contrast with mammals, and other vertebrate and invertebrate taxa, the identification and characterization of bona fide galectins in aquatic mollusks has been relatively recent. Most of the studies have focused on the identification and domain organization of galectin-like transcripts or proteins in diverse tissues and cell types, including hemocytes, and their expression upon environmental or infectious challenge. Lectins from the eastern oyster Crassostrea virginica, however, have been characterized in their molecular, structural and functional aspects and some notable features have become apparent in the galectin repertoire of aquatic mollusks. These including less diversified galectin repertoires and different domain organizations relative to those observed in vertebrates, carbohydrate specificity for blood group oligosaccharides, and up regulation of galectin expression by infectious challenge, a feature that supports their proposed role(s) in innate immune responses. Although galectins from some aquatic mollusks have been shown to recognize microbial pathogens and parasites and promote their phagocytosis, they can also selectively bind to phytoplankton components, suggesting that they also participate in uptake and intracellular digestion of microalgae. In addition, the experimental evidence suggests that the protozoan parasite Perkinsus marinus has co-evolved with the oyster host to be selectively recognized by the oyster hemocyte galectins over algal food or bacterial pathogens, thereby subverting the oyster’s innate immune/feeding recognition mechanisms to gain entry into the host cells. PMID:25982395

A Lectin-Like Receptor is Involved in Invasion of Erythrocytes by Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Jungery, M.; Pasvol, G.; Newbold, C. I.; Weatherall, D. J.

1983-02-01

Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.

Carbohydrate-Aromatic Interactions in Proteins.

PubMed

Hudson, Kieran L; Bartlett, Gail J; Diehl, Roger C; Agirre, Jon; Gallagher, Timothy; Kiessling, Laura L; Woolfson, Derek N

2015-12-09

Protein-carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C-H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C-H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C-H bonds engage more often in CH-π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate-aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C-H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein-carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein-carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites.

Purification and Characterization of Two Major Lectins from Araucaria brasiliensis syn. Araucaria angustifolia Seeds (Pinhão) 1

PubMed Central

Datta, Pradip K.; Figueroa, Maria O. D. C. R.; Lajolo, Franco M.

1991-01-01

Two major lectins (lectin I and lectin II) were purified to homogeneity from the seeds of Araucaria brasiliensis (Gymnospermae). The purity of the lectins was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and high performance liquid chromatography. They are glycoproteins in nature containing 6.3 and 2.9%, respectively, of neutral sugar and have absorption coefficients of 3.8 and 4.7, respectively, at 280 nanometers. The molecular weights of both lectins obtained by gel filtration on Sephacryl S-400 were equal: 200,000. After dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular weights were 20,000 and 34,000, respectively, for lectin I and lectin II, suggesting they are decameric and hexameric in nature. The amino acid composition of both lectins showed little difference, but both had high amounts of acidic amino acids and lacked methionine in their molecule. The carbohydrate binding specificity of lectins was directed towards mannose, glucose, and their oligomers. High inhibitory activity was also found with thyroglobulin. The erythroagglutinating activity of the lectins was enhanced in the presence of high-molecular-weight substances both at 37 and 4°C. Divalent cations do not appear to be essential for activity. They maintained their agglutinating activity over a broad but different range of pH: 5.5 to 7.5 and 6.5 to 7.5, respectively. Both lectins agglutinated erythrocytes of human ABO blood types equally well. ImagesFigure 2Figure 3 PMID:16668523

Defined presentation of carbohydrates on a duplex DNA scaffold.

PubMed

Schlegel, Mark K; Hütter, Julia; Eriksson, Magdalena; Lepenies, Bernd; Seeberger, Peter H

2011-12-16

A new method for the spatially defined alignment of carbohydrates on a duplex DNA scaffold is presented. The use of an N-hydroxysuccinimide (NHS)-ester phosphoramidite along with carbohydrates containing an alkylamine linker allows for on-column labeling during solid-phase oligonucleotide synthesis. This modification method during solid-phase synthesis only requires the use of minimal amounts of complex carbohydrates. The covalently attached carbohydrates are presented in the major groove of the B-form duplex DNA as potential substrates for murine type II C-type lectin receptors mMGL1 and mMGL2. CD spectroscopy and thermal melting revealed only minimal disturbance of the overall helical structure. Surface plasmon resonance and cellular uptake studies with bone-marrow-derived dendritic cells were used to assess the capability of these carbohydrate-modified duplexes to bind to mMGL receptors. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

O-GlcNAc-mediated interaction between VER2 and TaGRP2 elicits TaVRN1 mRNA accumulation during vernalization in winter wheat

PubMed Central

Xiao, Jun; Xu, Shujuan; Li, Chunhua; Xu, Yunyuan; Xing, Lijing; Niu, Yuda; Huan, Qing; Tang, Yimiao; Zhao, Changping; Wagner, Doris; Gao, Caixia; Chong, Kang

2014-01-01

Vernalization, sensing of prolonged cold, is important for seasonal flowering in eudicots and monocots. While vernalization silences a repressor (FLC, MADS-box transcription factor) in eudicots, it induces an activator (TaVRN1, an AP1 clade MADS-box transcription factor) in monocots. The mechanism for TaVRN1 induction during vernalization is not well understood. Here we reveal a novel mechanism for controlling TaVRN1 mRNA accumulation in response to prolonged cold sensing in wheat. The carbohydrate-binding protein VER2, a jacalin lectin, promotes TaVRN1 upregulation by physically interacting with the RNA-binding protein TaGRP2. TaGRP2 binds to TaVRN1 pre-mRNA and inhibits TaVRN1 mRNA accumulation. The physical interaction between VER2 and TaGRP2 is controlled by TaGRP2 O-GlcNAc modification, which gradually increases during vernalization. The interaction between VER2 and O-GlcNAc-TaGRP2 reduces TaGRP2 protein accumulation in the nucleus and/or promotes TaGRP2 dissociation from TaVRN1, leading to TaVRN1 mRNA accumulation. Our data reveal a new mechanism for sensing prolonged cold in temperate cereals. PMID:25091017

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

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.

Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

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

Mikeska, Ruth; Wacker, Roland; Arni, Raghuvir

2005-01-01

The structures of mistletoe lectin I in complex with lactose and galactose reveal differences in binding by the two known sites in subdomains α1 and γ2 and suggest the presence of a third low-affinity site in subdomain β1. The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (R{sub free} = 23.6%) and 20.9 (R{sub free} = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chainmore » has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound.« less

Identification and characterization of two novel C-type lectins from the larvae of housefly, Musca domestica L.

PubMed

Zhou, Jing; Fang, Nai-Nai; Zheng, Ya; Liu, Kai-Yu; Mao, Bin; Kong, Li-Na; Chen, Ya; Ai, Hui

2018-04-20

Lectins and antimicrobial peptides (AMPs) are widely distributed in various insects and play crucial roles in primary host defense against pathogenic microorganisms. Two AMPs (cecropin and attacin) have been identified and characterized in the larvae of housefly. In this study, two novel C-type lectins (CTLs) were obtained from Musca domestica, while their agglutinating and antiviral properties were evaluated. Real-time PCR analysis showed that the mRNA levels of four immune genes (MdCTL1, MdCTL2, Cecropin, and Attacin) from M. domestica were significantly upregulated after injection with killed Gram-negative Escherichia coli. Moreover, purified MdCTL1-2 proteins can agglutinate E. coli and Staphylococcus aureus in the presence of calcium ions, suggesting their immune function is Ca 2+ dependent. Sequence analysis indicated that typical WND and QPD motifs were found in the Ca 2+ -binding site 2 of carbohydrate recognition domain from MdCTL1-2, which was consistent with their agglutinating activities. Subsequently, antiviral experiments indicated that MdCTL1-2 proteins could significantly reduce the infection rate of Spodoptera frugiperda 9 cells by the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, indicating they might play important roles in insect innate immunity against microbial pathogens. In addition, MdCTL1-2 proteins could effectively inhibit the replication of influenza H 1 N 1 virus, which was similar to the effect of ribavirin. These results suggested that two novel CTLs could be considered a promising drug candidate for the treatment of influenza. Moreover, it is believed that the discovery of the CTLs with antiviral effects in M. domestica will improve our understanding of the molecular mechanism of insect immune response against viruses. © 2018 Wiley Periodicals, Inc.

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator

PubMed Central

Chang, Xiu-bao; Mengos, April; Hou, Yue-xian; Cui, Liying; Jensen, Timothy J.; Aleksandrov, Andrei; Riordan, John R.; Gentzsch, Martina

2009-01-01

Summary The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated Δ F508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway. PMID:18682497

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.

PubMed

Chang, Xiu-Bao; Mengos, April; Hou, Yue-Xian; Cui, Liying; Jensen, Timothy J; Aleksandrov, Andrei; Riordan, John R; Gentzsch, Martina

2008-09-01

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity.

PubMed

Swanson, Michael D; Boudreaux, Daniel M; Salmon, Loïc; Chugh, Jeetender; Winter, Harry C; Meagher, Jennifer L; André, Sabine; Murphy, Paul V; Oscarson, Stefan; Roy, René; King, Steven; Kaplan, Mark H; Goldstein, Irwin J; Tarbet, E Bart; Hurst, Brett L; Smee, Donald F; de la Fuente, Cynthia; Hoffmann, Hans-Heinrich; Xue, Yi; Rice, Charles M; Schols, Dominique; Garcia, J Victor; Stuckey, Jeanne A; Gabius, Hans-Joachim; Al-Hashimi, Hashim M; Markovitz, David M

2015-10-22

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code. Copyright © 2015 Elsevier Inc. All rights reserved.

Engineering a Therapeutic Lectin by Uncoupling Mitogenicity from Antiviral Activity

PubMed Central

Swanson, Michael D.; Boudreaux, Daniel M.; Salmon, Loïc; Chugh, Jeetender; Winter, Harry C.; Meagher, Jennifer L.; André, Sabine; Murphy, Paul V.; Oscarson, Stefan; Roy, René; King, Steven; Kaplan, Mark H.; Goldstein, Irwin J.; Tarbet, E. Bart; Hurst, Brett L.; Smee, Donald F.; de la Fuente, Cynthia; Hoffmann, Hans-Heinrich; Xue, Yi; Rice, Charles M.; Schols, Dominique; Garcia, J. Victor; Stuckey, Jeanne A.; Gabius, Hans-Joachim; Al-Hashimi, Hashim M.; Markovitz, David M.

2015-01-01

Summary A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code. PMID:26496612

Integrated Microfluidic Lectin Barcode Platform for High-Performance Focused Glycomic Profiling

NASA Astrophysics Data System (ADS)

Shang, Yuqin; Zeng, Yun; Zeng, Yong

2016-02-01

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges in analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and speed of lectin microarray. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, CA125 protein purified from ovarian cancer cell line and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated “sample-to-answer” microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glyco-biomarker development.

Integrated Microfluidic Lectin Barcode Platform for High-Performance Focused Glycomic Profiling

PubMed Central

Shang, Yuqin; Zeng, Yun; Zeng, Yong

2016-01-01

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges in analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and speed of lectin microarray. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, CA125 protein purified from ovarian cancer cell line and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated “sample-to-answer” microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glyco-biomarker development. PMID:26831207

Synthesis and optimization of lectin functionalized nanoprobes for the selective recovery of glycoproteins from human body fluids.

PubMed

Ferreira, José A; Daniel-da-Silva, Ana Luísa; Alves, Renato M P; Duarte, Daniel; Vieira, Igor; Santos, Lúcio Lara; Vitorino, Rui; Amado, Francisco

2011-09-15

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein magnetic nanoprobes (MNP), after being coated with three broad-spectrum lectins-concanavalin A (ConA), wheat germ agglutinin (WGA), and Maackia amurensis lectin (MA)-were utilized to selectively capture glycoproteins from human body fluids. Additionally, a new methodology, based on protection of the lectins with their target sugars prior to coupling with MNPs, was proposed to overcome the nonspecific nature of conjugation. This approach contributed to preserve lectin conformation, increasing by 40% and 90% the affinity of ConA and MA for glycoproteins in relation to synthesis with nonprotected lectins. Optimal operating conditions (temperature, time) and maximum binding capacities were further determined for each lectin by use of fetuin as a reference. The enhanced performance of lectin-based nanoplatforms was demonstrated by comparing MNP@ConA with conventional Sepharose@ConA. These experiments have shown that ConA immobilized on MNP exhibited 5 times higher affinity for fetuin and ovalbumin when compared with Sepharose@ConA with the same amount of immobilized lectin. MNP@Lectins were then applied to human serum, saliva, and urine and the recovered proteins were digested with trypsin and analyzed by nano-HPLC MALDI-TOF/TOF. This allowed the identification of 180 proteins, 90% of which were found to be glycosylated by use of bioinformatics tools, therefore revealing low levels of unspecific binding. Thus, MNP@lectins have proved to be a valuable tool for glycoproteomic studies, particularly when dealing with minute amounts of material.

Crystallization and preliminary X-ray study of the common edible mushroom (Agaricus bisporus) lectin.

PubMed

Carrizo, Maria E; Irazoqui, Fernando J; Lardone, Ricardo D; Nores, Gustavo A; Curtino, Juan A; Capaldi, Stefano; Perduca, Massimiliano; Monaco, Hugo L

2004-04-01

The lectin from the common edible mushroom Agaricus bisporus (ABL) belongs to the group of proteins that have the property of binding the Thomsen-Friedenreich antigen (T-antigen) selectively and with high affinity, but does not show any sequence similarity to the other proteins that share this property. The ABL sequence is instead similar to those of members of the saline-soluble fungal lectins, a protein family with pesticidal properties. The presence of different isoforms has been reported. It has been found that in order to be able to grow diffraction-quality crystals of the lectin, it is essential to separate the isoforms, which was performed by preparative isoelectric focusing. Using standard procedures, it was possible to crystallize the most basic of the forms by either vapour diffusion or equilibrium dialysis, but attempts to grow crystals of the other more acidic forms were unsuccessful. The ABL crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 93.06, b = 98.16, c = 76.38 A, and diffract to a resolution of 2.2 A on a conventional source at room temperature. It is expected that the solution of this structure will yield further valuable information on the differences in the T-antigen-binding folds and will perhaps help to clarify the details of the ligand binding to the protein.

Transcriptional switches in the control of macronutrient metabolism.

PubMed

Wise, Alan

2008-06-01

This review shows how some transcription factors respond to alterations in macronutrients. Carbohydrates induce enzymes for their metabolism and fatty acid synthesis. Fatty acids reduce carbohydrate processing, induce enzymes for their metabolism, and increase both gluconeogenesis and storage of fat. Fat stores help control carbohydrate uptake by other cells. The following main transcription factors are discussed: carbohydrate response element-binding protein; sterol regulatory element-binding protein-1c, cyclic AMP response element-binding protein, peroxisome proliferator-activated receptor-alpha, and peroxisome proliferator-activated receptor-gamma.

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas.

PubMed

Willemer, S; Köhler, H; Naumann, R; Kern, H F; Adler, G

1990-01-01

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.

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

Glycophenotype Evaluation in Cutaneous Tumors Using Lectins Labeled with Acridinium Ester

PubMed Central

Lima, Luiza Rayanna Amorim; Almeida, Sinara Mônica Vitalino; Silva, Lúcia Patrícia Bezerra Gomes; Beltrão, Eduardo Isidoro Carneiro; Carvalho Júnior, Luiz Bezerra

2013-01-01

Background. Tumor cells show alterations in their glycosylation patterns when compared to normal cells. Lectins can be used to evaluate these glycocode changes. Chemiluminescence assay is an effective technique for quantitative analysis of proteins, nucleic acids, and carbohydrates due to its high sensitivity, specificity, and rapid testing. Objective. To use histochemiluminescence based on lectin conjugated to acridinium ester (AE) for the investigation of glycophenotype changes in cutaneous tumors. Methods. Concanavalin A (Con A), Peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I), and Maackia amurensis agglutinin (MAA) were conjugated to acridinium ester. Biopsies of cutaneous tumors and normal skin were incubated with the lectins-AE, and chemiluminescence was quantified and expressed as Relative Light Units (RLU). Results. Actinic keratosis (AK), keratoacanthoma (KA), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) showed lower expression of α-D-glucose/mannose and α-L-fucose residues compared to normal tissue. Cutaneous tumors displayed higher expression of Gal-β(1-3)-GalNAc residues than normal tissue. AK and SCC exhibited higher expression of Neu5Ac-α(2,3)Gal residues than normal epidermis. KA and BCC showed equivalent RLU values compared to normal tissue. Conclusions. Lectin histochemiluminescence allowed quantitative assessment of the carbohydrate expression in cutaneous tissues, contributing to eliminate the subjectivity of conventional techniques used in the histopathological diagnosis. PMID:24167360

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer.

PubMed

Peiris, Diluka; Ossondo, Marlène; Fry, Simon; Loizidou, Marilena; Smith-Ravin, Juliette; Dwek, Miriam V

2015-01-01

Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer.

Galectin-3 in angiogenesis and metastasis

PubMed Central

Funasaka, Tatsuyoshi; Raz, Avraham; Nangia-Makker, Pratima

2014-01-01

Galectin-3 is a member of the family of β-galactoside-binding lectins characterized by evolutionarily conserved sequences defined by structural similarities in their carbohydrate-recognition domains. Galectin-3 is a unique, chimeric protein consisting of three distinct structural motifs: (i) a short NH2 terminal domain containing a serine phosphorylation site; (ii) a repetitive proline-rich collagen-α-like sequence cleavable by matrix metalloproteases; and (iii) a globular COOH-terminal domain containing a carbohydrate-binding motif and an NWGR anti-death motif. It is ubiquitously expressed and has diverse biological functions depending on its subcellular localization. Galectin-3 is mainly found in the cytoplasm, also seen in the nucleus and can be secreted by non-classical, secretory pathways. In general, secreted galectin-3 mediates cell migration, cell adhesion and cell–cell interactions through the binding with high affinity to galactose-containing glycoproteins on the cell surface. Cytoplasmic galectin-3 exhibits anti-apoptotic activity and regulates several signal transduction pathways, whereas nuclear galectin-3 has been associated with pre-mRNA splicing and gene expression. Its unique chimeric structure enables it to interact with a plethora of ligands and modulate diverse functions such as cell growth, adhesion, migration, invasion, angiogenesis, immune function, apoptosis and endocytosis emphasizing its significance in the process of tumor progression. In this review, we have focused on the role of galectin-3 in tumor metastasis with special emphasis on angiogenesis. PMID:25138305

Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

PubMed

Torres-Tirado, David; Knabb, Maureen; Castaño, Irene; Patrón-Soberano, Araceli; De Las Peñas, Alejandro; Rubio, Rafael

2016-01-01

Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses. Copyright © 2016 the American Physiological Society.

Temperature effect on affinity chromatography of two lectins from the seeds of Ricinus communis

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

Hsu, H.W.; Davis, D.S.; Wei, C.H.

1976-06-01

Specific adsorption capacity of Sepharose 4B in affinity chromatography for two purified galactose-binding lectins, designated as III/sub L/ and III/sub H/, from the seed of Ricinus communis (castor bean) was measured from 7 to 24/sup 0/C. The adsorption coefficients for these two protein fractions as a function of temperature were also obtained. It was found that there is a characteristic transition of adsorption coefficient at 18/sup 0/C for both lectins. Adsorption coefficients between Sepharose 4B and these two lectins were also expressed in terms of ..delta..G, ..delta..H, and ..delta..S. It is suggested that the difference in the temperature dependence ofmore » the binding energy of these two lectins may be used for their separation at selected temperature.« less

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus

PubMed Central

Gao, Xiang; Esseili, Malak A.; Lu, Zhongyan; Saif, Linda J.

2016-01-01

ABSTRACT Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. IMPORTANCE Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination. PMID:26969699

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus.

PubMed

Gao, Xiang; Esseili, Malak A; Lu, Zhongyan; Saif, Linda J; Wang, Qiuhong

2016-05-15

Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Identification of Cell Surface Molecules Involved in Dystroglycan-Independent Lassa Virus Cell Entry

PubMed Central

Ströher, Ute; Ebihara, Hideki; Feldmann, Heinz

2012-01-01

Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry. PMID:22156524

Glycoscience@Synchrotron: Synchrotron radiation applied to structural glycoscience

PubMed Central

de Sanctis, Daniele

2017-01-01

Synchrotron radiation is the most versatile way to explore biological materials in different states: monocrystalline, polycrystalline, solution, colloids and multiscale architectures. Steady improvements in instrumentation have made synchrotrons the most flexible intense X-ray source. The wide range of applications of synchrotron radiation is commensurate with the structural diversity and complexity of the molecules and macromolecules that form the collection of substrates investigated by glycoscience. The present review illustrates how synchrotron-based experiments have contributed to our understanding in the field of structural glycobiology. Structural characterization of protein–carbohydrate interactions of the families of most glycan-interacting proteins (including glycosyl transferases and hydrolases, lectins, antibodies and GAG-binding proteins) are presented. Examples concerned with glycolipids and colloids are also covered as well as some dealing with the structures and multiscale architectures of polysaccharides. Insights into the kinetics of catalytic events observed in the crystalline state are also presented as well as some aspects of structure determination of protein in solution. PMID:28684994

West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection.

PubMed

Davis, Carl W; Nguyen, Hai-Yen; Hanna, Sheri L; Sánchez, Melissa D; Doms, Robert W; Pierson, Theodore C

2006-02-01

The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.

Involvement of sulfates from cruzipain, a major antigen of Trypanosoma cruzi, in the interaction with immunomodulatory molecule Siglec-E.

PubMed

Ferrero, Maximiliano R; Heins, Anja M; Soprano, Luciana L; Acosta, Diana M; Esteva, Mónica I; Jacobs, Thomas; Duschak, Vilma G

2016-02-01

In order to investigate the involvement of sulfated groups in the Trypanosoma cruzi host-parasite relationship, we studied the interaction between the major cysteine proteinase of T. cruzi, cruzipain (Cz), a sulfate-containing sialylated molecule and the sialic acid-binding immunoglobulin like lectin-E (Siglec-E). To this aim, ELISA, indirect immunofluorescence assays and flow cytometry, using mouse Siglec-E-Fc fusion molecules and glycoproteins of parasites, were performed. Competition assays verified that the lectins, Maackia amurensis II (Mal II) and Siglec-E-Fc, compete for the same binding sites. Taking into account that Mal II binding remains unaltered by sulfation, we established this lectin as sialylation degree control. Proteins of an enriched microsomal fraction showed the highest binding to Siglec-E as compared with those from the other parasite subcellular fractions. ELISA assays and the affinity purification of Cz by a Siglec-E column confirmed the interaction between both molecules. The significant decrease in binding of Siglec-E-Fc to Cz and to its C-terminal domain (C-T) after desulfation of these molecules suggests that sulfates contribute to the interaction between Siglec-E-Fc and these glycoproteins. Competitive ELISA assays confirmed the involvement of sulfated epitopes in the affinity between Siglec-E and Cz, probably modified by natural protein environment. Interestingly, data from flow cytometry of untreated and chlorate-treated parasites suggested that sulfates are not primary receptors, but enhance the binding of Siglec-E to trypomastigotic forms. Altogether, our findings support the notion that sulfate-containing sialylated glycoproteins interact with Siglec-E, an ortholog protein of human Siglec-9, and might modulate the immune response of the host, favoring parasitemia and persistence of the parasite.

Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense

PubMed Central

Waespy, Mario; Gbem, Thaddeus T.; Elenschneider, Leroy; Jeck, André-Philippe; Day, Christopher J.; Hartley-Tassell, Lauren; Bovin, Nicolai; Tiralongo, Joe; Haselhorst, Thomas; Kelm, Sørge

2015-01-01

Fourteen different active Trypanosoma congolense trans-sialidases (TconTS), 11 variants of TconTS1 besides TconTS2, TconTS3 and TconTS4, have been described. Notably, the specific transfer and sialidase activities of these TconTS differ by orders of magnitude. Surprisingly, phylogenetic analysis of the catalytic domains (CD) grouped each of the highly active TconTS together with the less active enzymes. In contrast, when aligning lectin-like domains (LD), the highly active TconTS grouped together, leading to the hypothesis that the LD of TconTS modulates its enzymatic activity. So far, little is known about the function and ligand specificity of these LDs. To explore their carbohydrate-binding potential, glycan array analysis was performed on the LD of TconTS1, TconTS2, TconTS3 and TconTS4. In addition, Saturation Transfer Difference (STD) NMR experiments were done on TconTS2-LD for a more detailed analysis of its lectin activity. Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD. Interestingly, terminal mannose residues are not acceptor substrates for TconTS activity. This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself. Experimental evidence for such a scenario is presented. PMID:26474304

Characterization of Protein-Carbohydrate Interactions by NMR Spectroscopy.

PubMed

Grondin, Julie M; Langelaan, David N; Smith, Steven P

2017-01-01

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1 H- 15 N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate binding partners, to quantify the dissociation constant (K d ) of any identified interactions, and to map the carbohydrate binding site on the structure of the protein. Here, we describe the titration of a family 32 carbohydrate binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction, and map the GalNAc binding site onto the structure of CpCBM32.

Food proteins and maturation of small intestinal microvillus membranes (MVM). I. Binding characteristics of cow's milk proteins and concanavalin A to MVM from newborn and adult rats.

PubMed

Stern, M; Gellermann, B

1988-01-01

To study maturational changes of food protein and lectin binding to rat small intestinal microvillus membranes (MVM), MVM were prepared from newborn and adult animals by a modified CaCl2 precipitation technique. Radiolabeled cow's milk proteins [alpha-lactalbumin, alpha-casein, beta-lactoglobulin, bovine serum albumin (BSA)] and the lectin concanavalin A (Con A) were used for incubations. Binding assays were done using miniature ultracentrifugation for separation of unbound material. Binding of Con A to MVM from newborn and adult rats was strong, specific, and saturable. Binding of Con A was inhibited by cold Con A and by the sugar ligand polymer mannan. Adult MVM bound more Con A than newborn preparations. Unlike Con A, binding of cow's milk proteins by MVM was weak, nonspecific, and noninhibitable. Newborn MVM bound more cow's milk proteins than adult controls. This was true for all the proteins tested (p less than 0.001). Binding rose with decreased molecular weight of cow's milk proteins, but molecular weight was not the only determining factor for binding. Trypsin treatment of MVM caused a marked increase of BSA binding in adult but not in newborn preparations. This finding indicated the importance of protein components of MVM for cow's milk protein binding. Maturational changes in protein-lipid interactions and membrane fluidity possibly influence nonspecific cow's milk protein binding to MVM. Differences in binding between newborns and adults were not directly related to maturational shifts in membrane glycosylation that are indicated by differential Con A binding. Increased cow's milk protein binding in newborn individuals might increase the potential risk to develop an adverse reaction to food proteins.

Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells.

PubMed

de Andrade Luz, Luciana; Rossato, Franco Aparecido; Costa, Rute Alves Pereira E; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; Coelho, Luana Cassandra Breitenbach Barroso

2017-10-01

Moringa oleifera seeds are used in alternative medicine to treat inflammation, tumors and bacterial and protozoan infections, for example. The seeds contain lectins, which are carbohydrate-binding proteins with several biological properties including cytotoxicity to cancer cells. In this work, we examined the cytotoxicity of the coagulant M. oleifera lectin (cMoL) on B16-F10 murine melanoma cells. cMoL cytotoxic effects were evaluated through trypan blue assay and flow cytometry analysis. Mitochondrial superoxide levels and activation of caspases 3, 8 and 9 were measured. cMoL (1.5-16μM) reduced viability and caused cell death of B16-F10 cells with an IC 50 of 9.72μM. Flow cytometry analysis indicated induction of necrosis and suggested the presence of cells in late apoptosis. Specificity for tumor cells was observed since death of normal human fibroblasts (GN) was not higher than 20% in treatments with cMoL from 1.5 to 16μM. Microscopy images revealed rounded shape and reduction of volume in B16-F10 cells treated with cMoL. cMoL increased mitochondrial ROS production and promoted caspases 3, 8 and 9 activation in B16-F10 cells, indicating the activation of apoptosis-related pathway. In conclusion, this study demonstrates that cMoL is cytotoxic to B16-F10 cells, which stimulates more investigation on the anticancer potential of this lectin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Increased lectin binding capacity of trophoblastic cells of late day 5 rat blastocysts.

PubMed Central

Stein, B A; Shaw, T J; Turner, V F; Murphy, C R

1994-01-01

The binding of lectins to the trophoblast of rat blastocysts has been studied using quantitative ultrastructural cytochemistry. Rat blastocysts from early, mid and late d 5 of gestation were stained using biotinylated lectins (Phytolacca americana [Phy am], fucose binding protein [FBP] and soybean agglutinin [SBA]) and a sensitive avidin-ferritin cytochemical method. Electron micrographs of ferritin particles along the membrane were processed to produce images for which grey scale levels could be established and the ferritin particles automatically counted. The ferritin:membrane ratio was then calculated. Increased binding with Phy am (which detects short chain oligosaccharides) was found after midday of d 5, i.e. after hatching. Binding of FBP and SBA did not alter during the period studied. The increased concentration of oligosaccharides on the blastocyst surface membrane after hatching may have important implications for blastocyst attachment to the endometrium. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7649802

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

Cloning and the mRNA expression of a C-type lectin with one carbohydrate recognition domain from Fenneropenaeus merguiensis in response to pathogenic inoculation.

PubMed

Runsaeng, Phanthipha; Thepnarong, Supattra; Rattanaporn, Onnicha; Utarabhand, Prapaporn

2015-12-01

Crustaceans are deficient in an adaptive immune system and depend solely on their innate immunity. One kind of pattern recognition proteins which plays an important role in the shrimp immunity is lectin. A new C-type lectin called FmLC2 was cloned from the stomach of the banana shrimp Fenneropenaeus merguiensis by means of RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE). Its full-length cDNA contains 1098 bp with a single open reading frame of 738 bp, encoding a peptide of 245 amino acids. The deduced amino acid sequence of FmLC2 consists of a signal peptide of 17 amino acids with a molecular mass of 28,115 Da and an isoelectric point of 6.94. The primary structure of FmLC2 comprises a single carbohydrate recognition domain (CRD) with a QPD (Gln-Pro-Asp) motif and one Ca(2+) binding site. Like other C-type lectins, its CRD structure contains a double-loop characteristic being stabilized by two conserved disulfide linkages. The mRNA expression of FmLC2 was detected specifically in the stomach and gills, less was found in the hepatopancreas. Upon inoculation of shrimp with Vibrio harveyi or white spot syndrome virus (WSSV), the FmLC2 expression either in stomach or gills was higher than in the hepatopancreas. Besides, its expression in these tissues was up-regulated to reach the highest levels at 12 or 18 h for V. harveyi or WSSV stimulation, respectively. RNAi-based silencing of FmLC2 resulted in suppression of its expression, increases in mortality when the shrimp were challenged with V. harveyi or WSSV, and the median lethal time was reduced compared with controls. These results suggest that FmLC2 may serve as receptor molecules which recognize invading bacterial and viral pathogens and thus contribute a role in the shrimp immune response. Copyright © 2015 Elsevier Ltd. All rights reserved.

Suppression of proliferation and neurite extension of human neuroblastoma SH-SY5Y cells on immobilized Psathyrella velutina lectin.

PubMed

Kitamura, Noriaki; Ikekita, Masahiko; Hayakawa, Satoru; Funahashi, Hisayuki; Furukawa, Kiyoshi

2004-02-01

Glycoproteins from mammalian brain tissues contain unique N-linked oligosaccharides terminating with beta-N-acetylglucosamine residues. Lectin blot analysis of membrane glycoprotein samples from human neuroblastoma SH-SY5Y cells showed that several protein bands bind to Psathylera velutina lectin (PVL), which interacts with beta-N-acetylglucosamine-terminating oligosaccharides. No lectin positive bands were detected by digestion with jack bean beta-N-acetyl-hexosaminidase or N-glycanase before incubation with the lectin, indicating that the cells contain beta-N-acetylglucosamine-terminating N-linked oligosaccharides. When cells were cultured in dishes with different concentrations of PVL, the cell proliferation was inhibited in a dose-dependent manner. Similarly, the neurite extension, which was stimulated with nerve growth factor, was also inhibited in a manner dependent on the lectin dose. Cell proliferation and neurite extension were recovered by the addition of 10 mM N-acetylglucosamine into the medium. Immunoblot analysis of the activation of mitogen-activated protein (MAP) kinases and protein kinase C revealed that phosphorylation of 42-kDa and 44-kDa MAP kinases and 80-kDa protein kinase C are inhibited when SH-SY5Y cells are cultured in PVL-coated dishes, but are restored by the addition of the haptenic sugar into the medium, indicating that MAP kinase and protein kinase C pathways are inhibited by interaction with immobilized PVL. These results indicate that beta-N-acetylglucosamine-terminating N-linked oligosaccharides expressed on neural cells can induce intracellular signals upon binding to extracellular receptors, and are important for growth regulation of neural cells. Copyright 2003 Wiley-Liss, Inc.

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

Unterberger, Claudia; Hanson, Steven; Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 tomore » be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.« less

Distribution of binding sites for the plant lectin Ulex europaeus agglutinin I on primary sensory neurones in seven different mammalian species.

PubMed

Gerke, Michelle B; Plenderleith, Mark B

2002-01-01

There is an increasing body of evidence to suggest that different functional classes of neurones express characteristic cell-surface carbohydrates. Previous studies have shown that the plant lectin Ulex europaeus agglutinin-I (UEA) binds to a population of small to medium diameter primary sensory neurones in rabbits and humans. This suggests that a fucose-containing glycoconjugate may be expressed by nociceptive primary sensory neurones. In order to determine the extent to which this glycoconjugate is expressed by other species, in the current study, we have examined the distribution of UEA-binding sites on primary sensory neurones in seven different mammals. Binding sites for UEA were associated with the plasma membrane and cytoplasmic granules of small to medium dorsal root ganglion cells and their axon terminals in laminae I-III of the grey matter of the spinal cord, in the rabbit, cat and marmoset monkey. However, no binding was observed in either the dorsal root ganglia or spinal cord in the mouse, rat, guinea pig or flying fox. These results indicate an inter-species variation in the expression of cell-surface glycoconjugates on mammalian primary sensory neurones.

(15)N and (13)C group-selective techniques extend the scope of STD NMR detection of weak host-guest interactions and ligand screening.

PubMed

Kövér, Katalin E; Wéber, Edit; Martinek, Tamás A; Monostori, Eva; Batta, Gyula

2010-10-18

Saturation transfer difference (STD) is a valuable tool for studying the binding of small molecules to large biomolecules and for obtaining detailed information on the binding epitopes. Here, we demonstrate that the proposed (15)N/(13)C variants of group-selective, "GS-STD" experiments provide a powerful approach to mapping the binding epitope of a ligand even in the absence of efficient spin diffusion within the target protein. Therefore, these experimental variants broaden the scope of STD studies to smaller and/or more-dynamic targets. The STD spectra obtained in four different experimental setups (selective (1)H STD, (15)N GS-STD, (13)C(Ar) and (13)C(aliphatic) GS-STD approaches) revealed that the signal-intensity pattern of the difference spectra is affected by both the type and the spatial distribution of the excited "transmitter" atoms, as well as by the efficiency of the spin-diffusion-mediated magnetization transfer. The performance of the experiments is demonstrated on a system by using the lectin, galectin-1 and its carbohydrate ligand, lactose.

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

Association of Lectin Pathway Protein Levels and Genetic Variants Early after Injury with Outcomes after Severe Traumatic Brain Injury: A Prospective Cohort Study.

PubMed

Osthoff, Michael; Walder, Bernhard; Delhumeau, Cécile; Trendelenburg, Marten; Turck, Natacha

2017-09-01

The lectin pathway of the complement system has been implicated in secondary ischemic/inflammatory injury after traumatic brain injury (TBI). However, previous experimental studies have yielded conflicting results, and human studies are scarce. In this exploratory study, we investigated associations of several lectin pathway proteins early after injury and single-nucleotide polymorphisms (SNP) with outcomes after severe TBI (mortality at 14 days [primary outcome] and consciousness assessed with the Glasgow Coma Scale [GCS] at 14 days, disability assessed with the Glasgow Outcome Scale Extended [GOSE] at 90 days). Forty-four patients with severe TBI were included. Plasma levels of lectin pathway proteins were sampled at 6, 12, 24, and 48 h after injury and eight mannose-binding lectin (MBL) and ficolin (FCN)2 SNPs were analyzed by enzyme-linked immunosorbent assay (ELISA) and genotyping, respectively. Plasma protein levels were stable with only a slight increase in mannose-binding protein-associated serine protease (MASP)-2 and FCN2 levels after 48 h (p < 0.05), respectively. Neither lectin protein plasma levels (6 h or mean levels) nor MBL2 genotypes or FCN2 variant alleles were associated with 14 day mortality or 14 day consciousness. However, FCN2, FCN3, and MASP-2 levels were higher in patients with an unfavorable outcome (GOSE 1-4) at 90 days (p < 0.05), whereas there was no difference in MBL2 genotypes or FCN2 variant alleles. In particular, higher mean MASP-2 levels over 48 h were independently associated with a GOSE score < 4 at 90 days after adjustment (odds ratio 3.46 [95% confidence interval 1.12-10.68] per 100 ng/mL increase, p = 0.03). No association was observed between the lectin pathway of the complement system and 14 day mortality or 14 day consciousness. However, higher plasma FCN2, FCN3, and, in particular, MASP-2 levels early after injury were associated with an unfavorable outcome at 90 days (death, vegetative state, and severe disability) which may be related to an increased activation of the lectin pathway.

FmLC5, a putative galactose-binding C-type lectin with two QPD motifs from the hemocytes of Fenneropenaeus merguiensis participates in shrimp immune defense.

PubMed

Senghoi, Wilaiwan; Runsaeng, Phanthipha; Utarabhand, Prapaporn

2017-11-01

Crustaceans are deficient in adaptive immune system. They depend completely on an innate immunity to protect themselves from invading microorganisms. One kind of pattern recognition receptors that contribute roles in the innate immunity is lectin. A new C-type lectin gene designated as FmLC5 was isolated from Fenneropenaeus merguiensis. Its full-length cDNA is composed of 1526bp and one open reading frame of 852bp encoding a peptide of 284 amino acids. The deduced amino acid sequence of FmLC5 comprises a signal peptide of 20 contiguous amino acids with a molecular mass of 31.47kDa and an isoelectric point of 4.35. The primary structure of FmLC5 consists of two similar carbohydrate recognition domains (CRDs), each CRD contains a Ca 2+ binding site-2 and a QPD motif specific for galactose-binding. The FmLC5 transcripts were detected only in the hemocytes analyzed by RT-PCR and in situ hybridization. The FmLC5 expression was significantly up-regulated after challenge with Vibrio harveyi, white spot syndrome virus (WSSV) or lipopolysaccharide. RNAi-based silencing with co-injection by V. harveyi or WSSV resulted in critical suppression of the FmLC5 expression, increasing in mortality and reduction of the median lethal time. These results conclude that FmLC5 is unique putative galactose-binding C-type lectin in F. merguiensis that may contribute as receptor molecule in the immune response to defend the shrimp from pathogenic bacteria and viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

PubMed Central

Alegre, Ana Claudia Paiva; Oliveira, Aline Ferreira; Dos Reis Almeida, Fausto Bruno; Roque-Barreira, Maria Cristina; Hanna, Ebert Seixas

2014-01-01

Background Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. Methodology/principal findings The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. Conclusions/Significance Our results showed that the recombinant protein reproduced the biological properties described for the native protein—including binding to laminin in a manner that is dependent on carbohydrate recognition—showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The results indicated that paracoccin is the protein encoded by PADG-3347, and we propose that this gene and homologous proteins in other P. brasiliensis strains be called paracoccin. We also concluded that recombinant paracoccin confers resistance to murine P. brasiliensis infection by exerting immunomodulatory effects. PMID:24743161

Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

2016-01-01

The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

Interfering with Gal-1-mediated angiogenesis contributes to the pathogenesis of preeclampsia.

PubMed

Freitag, Nancy; Tirado-González, Irene; Barrientos, Gabriela; Herse, Florian; Thijssen, Victor L J L; Weedon-Fekjær, Susanne M; Schulz, Herbert; Wallukat, Gerd; Klapp, Burghard F; Nevers, Tania; Sharma, Surendra; Staff, Anne Cathrine; Dechend, Ralf; Blois, Sandra M

2013-07-09

Preeclampsia (PE) is a pregnancy-specific disorder characterized by sudden onset of hypertension and proteinuria in the second half of pregnancy (>20 wk). PE is strongly associated with abnormal placentation and an excessive maternal inflammatory response. Galectin-1 (Gal-1), a member of a family of carbohydrate-binding proteins, has been shown to modulate several processes associated with placentation and to promote maternal tolerance toward fetal antigens. Here, we show that Gal-1 exhibits proangiogenic functions during early stages of pregnancy, promoting decidual vascular expansion through VEGF receptor 2 signaling. Blocking Gal-1-mediated angiogenesis or lectin, galactoside-binding, soluble, 1 deficiency results in a spontaneous PE-like syndrome in mice, mainly by deregulating processes associated with good placentation and maternal spiral artery remodeling. Consistent with these findings, we observed a down-regulation of Gal-1 in patients suffering from early onset PE. Collectively, these results strengthen the notion that Gal-1 is required for healthy gestation and highlight Gal-1 as a valuable biomarker for early PE diagnosis.

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

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.

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.

Sweet Polymers: Poly(2-ethyl-2-oxazoline) Glycopolymers by Reductive Amination.

PubMed

Mees, Maarten A; Effenberg, Christiane; Appelhans, Dietmar; Hoogenboom, Richard

2016-12-12

Carbohydrates are important in signaling, energy storage, and metabolism. Depending on their function, carbohydrates can be part of larger structures, such as glycoproteins, glycolipids, or other functionalities (glycoside). To this end, polymers can act as carriers of carbohydrates in so-called glycopolymers, which mimic the multivalent carbohydrate functionalities. We chose a biocompatible poly(2-ethyl-2-oxazoline) (PEtOx) as the basis for making glycopolymers. Via the partial hydrolysis of PEtOx, a copolymer of PEtOx and polyethylenimine (PEI) was obtained; the subsequent reductive amination with the linear forms of glucose and maltose yielded the glycopolymers. The ratios of PEtOx and carbohydrates were varied systematically, and the solution behaviors of the resulting glycoconjugates are discussed. Dynamic light scattering (DLS) revealed that, depending on the carbohydrate ratio, the glycopolymers were either fully water-soluble or formed agglomerates in a temperature-dependent manner. Finally, these polymers were tested for their biological availability by studying their lectin binding ability with Concanavalin A.

Properties of the glycoprotein laccase immobilized by two methods.

PubMed

Froehner, S C; Eriksson, K

1975-01-01

Laccase (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) from Neurospora crassa has been immobilized by two different procedures: (1) Covalent attachment to Sepharose 4B activated with cyanogen bromide, and (2) Adsorption to Concanavalin A-Sepharose via the carbohydrate moiety. Except for small changes in the Michaelis-Menten constants, no differences were noted in the enzymological properties of the immobilized enzymes when compared to free enzyme. The carbohydrate moiety of laccase involved in the interaction with Concanavalin A does not appear to be closely associated with the active center since binding to the lectin has no effect on the enzymological parameters investigated.

C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein β-Glucuronidase on Dendritic Cells

PubMed Central

Mori, Daiki; Shibata, Kensuke; Yamasaki, Sho

2017-01-01

C-type lectin receptors (CLRs) recognize pathogen-derived ligands and abnormal self that trigger protective immune responses. However, the precise nature of self ligands recognized by CLRs remains to be determined. Here, we found that Dectin-2 recognizes bone marrow-derived dendritic cells (BMDCs) using Dectin-2-expressing reporter cells. This activity was inhibited by an excessive amount of mannose, and by the mutation of mannose-binding motif in Dectin-2. β-glucuronidase (Gusb) was identified as a protein bound to Dectin-2 and mutations of N-glycosylation sites in Gusb impaired the binding of Gusb to Dectin-2. Overexpression of Gusb in a macrophage cell line conferred an ability to stimulate Dectin-2-expressing reporter cells. Our study suggests that a glycosylated protein with mannose-related structure is recognized by Dectin-2. PMID:28046067

C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein β-Glucuronidase on Dendritic Cells.

PubMed

Mori, Daiki; Shibata, Kensuke; Yamasaki, Sho

2017-01-01

C-type lectin receptors (CLRs) recognize pathogen-derived ligands and abnormal self that trigger protective immune responses. However, the precise nature of self ligands recognized by CLRs remains to be determined. Here, we found that Dectin-2 recognizes bone marrow-derived dendritic cells (BMDCs) using Dectin-2-expressing reporter cells. This activity was inhibited by an excessive amount of mannose, and by the mutation of mannose-binding motif in Dectin-2. β-glucuronidase (Gusb) was identified as a protein bound to Dectin-2 and mutations of N-glycosylation sites in Gusb impaired the binding of Gusb to Dectin-2. Overexpression of Gusb in a macrophage cell line conferred an ability to stimulate Dectin-2-expressing reporter cells. Our study suggests that a glycosylated protein with mannose-related structure is recognized by Dectin-2.

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

Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA).

PubMed

Kumari, Amrita; Koyama, Tetsuo; Hatano, Ken; Matsuoka, Koji

2016-10-01

A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C6 linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λex 295nm and it was found to be very high Ka∼1.39×10(7) M(-1) in case of ABG complex as compared to GlcNAc only Ka∼1.01×10(4) M(-1) with the phenomenon proven to be due to glycocluster effect. Copyright © 2016 Elsevier Inc. All rights reserved.

Carbohydrate recognition: A minimalistic approach to binding

NASA Astrophysics Data System (ADS)

Kubik, Stefan

2012-09-01

Synthetic receptors with properties resembling those of carbohydrate-binding proteins are known, but they are structurally rather complex. Elaborate structures are, however, not always required to bind carbohydrates in water -- much simpler compounds can be just as effective.

Evolutionary analysis of the jacalin-related lectin family genes in 11 fishes.

PubMed

Cao, Jun; Lv, Yueqing

2016-09-01

Jacalin-related lectins are a type of carbohydrate-binding proteins, which are distributed across a wide variety of organisms and involved in some important biological processes. The evolution of this gene family in fishes is unknown. Here, 47 putative jacalin genes in 11 fish species were identified and divided into 4 groups through phylogenetic analysis. Conserved gene organization and motif distribution existed in each group, suggesting their functional conservation. Some fishes have eleven jacalin genes, while others have only one or zero gene in their genomes, suggesting dynamic changes in the number of jacalin genes during the evolution of fishes. Intragenic recombination played a key role in the evolution of jacalin genes. Synteny analyses of jacalin genes in some fishes implied conserved and dynamic evolution characteristics of this gene family and related genome segments. Moreover, a few functional divergence sites were identified within each group pairs. Divergent expression profiles of the zebra fish jacalin genes were further investigated in different stresses. The results provided a foundation for exploring the characterization of the jacalin genes in fishes and will offer insights for additional functional studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse.

PubMed

Schulte, B A; Spicer, S S

1983-12-01

Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal alpha-N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate beta-galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20-50% of these cells in all glands contained terminal N-acetylglucosamine residues. In contrast, terminal alpha-N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.

Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: A receptor for lectin-mediated interbacterial adherence

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

Abeygunawardana, C.; Bush, C.A.; Cisar, J.O.

1991-07-02

Lectin-carbohydrate binding is known to play an important role in a number of different cell-cell interactions including those between certain species of oral streptococci and actinomyces that colonize teeth. The cell wall polysaccharides of Streptococcus oralis ATCC 10557, S. oralis 34, and Streptococcus mitis J22, although not identical antigenically, each function as a receptor molecule for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii. Carbohydrate analysis of the receptor polysaccharide isolated from S. oralis ATCC 10557 shows galactose (3 mol), glucose (1 mol), GalNAc (1 mol), and rhamnose (1 mol). {sup 1}H NMR spectra ofmore » the polysaccharide show that is partially O-acetylated. Analysis of the {sup 1}H NMR spectrum of the de-O-acetylated polysaccharide shows that it is composed of repeating subunits containing six monosaccharides and that the subunits are joined by a phosphodiester linkage. The {sup 1}H and {sup 13}C NMR spectra were completely assigned by two-dimensional homonuclear correlation methods and by {sup 1}H-detected heteronuclear multiple-quantum correlation ({sup 1}H({sup 13}C)HMQC). The complete {sup 1}H and {sup 13}C assignment of the native polysaccharide was carried out by the same techniques augmented by a {sup 13}C-coupled hybrid HMQC-COSY method, which is shown to be especially useful for carbohydrates in which strong coupling and overlapping peaks in the {sup 1}H spectrum pose difficulties.« less

Structural Basis of Specific Recognition of Non-Reducing Terminal N-Acetylglucosamine by an Agrocybe aegerita Lectin

PubMed Central

Ren, Xiao-Ming; Li, De-Feng; Jiang, Shuai; Lan, Xian-Qing; Hu, Yonglin; Sun, Hui; Wang, Da-Cheng

2015-01-01

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently. PMID:26114302

Bauhinia variegata var. variegata lectin: isolation, characterization, and comparison.

PubMed

Chan, Yau Sang; Ng, Tzi Bun

2015-01-01

Bauhinia variegata var. variegata seeds are rich in proteins. Previously, one of the major storage proteins of the seeds was found to be a trypsin inhibitor that possessed various biological activities. By using another purification protocol, a glucoside- and galactoside-binding lectin that demonstrated some differences from the previously reported B. variegata lectin could be isolated from the seeds. It involved affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Q-Sepharose and Mono Q, and also size exclusion chromatography on Superdex 75. The lectin was not retained on Affi-gel blue gel but interacted with Q-Sepharose. The lectin was a 64-kDa protein with two 32-kDa subunits. It had low thermostability (stable up to 50 °C) and moderate pH stability (stable in pH 3-10). It exhibited anti-proliferative activity on nasopharyngeal carcinoma HONE1 cells with an IC50 of 12.8 μM after treatment for 48 h. It also slightly inhibited the growth of hepatoma HepG2 cells. The lectin may have potential in aiding cancer treatments.

Study of Small Ligands Which Bind Specifically to Breast Cancer Cells

DTIC Science & Technology

1997-09-01

Sepharose conjugated to three different lectins: ConA, wheat germ and lentil,. Each lectin bound many proteins in both the ECD-AP sup and the control 3T3 sup...control Lanes 13-14: Wheat germ ECD-AP Lanes 15-16: Wheat germ 3T3 control Odd lanes were eluted with a low sugar concentration; even lanes were...ECD-AP post incubation with lentil-Sepharose Lane 6: Protein remaining in pp ECD-AP post incubation with wheat germ -Sepharose Lane 8: Protein

A unique epidermal mucus lectin identified from catfish (Silurus asotus): first evidence of intelectin in fish skin slime.

PubMed

Tsutsui, Shigeyuki; Komatsu, Yukie; Sugiura, Takaya; Araki, Kyosuke; Nakamura, Osamu

2011-11-01

The present study reports a new type of skin mucus lectin found in catfish Silurus asotus. The lectin exhibited calcium-dependent mannose-binding activity. When mannose eluate from chromatography with mannose-conjugated agarose was analysed by SDS-PAGE, the lectin appeared as a single 35-kDa band. Gel filtration showed that the lectin forms monomers and dimers. A 1216-bp cDNA sequence obtained by RACE-PCR from the skin encoded a 308 amino acid secretory protein with homology to mammalian and fish intelectins. RT-PCR demonstrated that the lectin gene was expressed in the gill, kidney and skin. Subsequent sequencing revealed the presence of an isoform in the gills. Antiserum detected the intelectin protein in club cells in the skin and gill, renal tubules and blood plasma. Although intelectin gene expression was not induced by in vivo bacterial stimulation, the intelectin showed agglutination activity against the pathogenic bacterium Aeromonas salmonicida, suggesting that the lectin plays an important role in self-defence against bacteria in the skin surface of the catfish. These findings represent one of the few examples of characterization and functional analysis of a fish intelectin protein.

Lectins as probes for assessing the accessibility of N-linked glycans in relation to the conformational changes of fibronectin.

PubMed

Agniel, Rémy; Vendrely, Charlotte; Poulouin, Laurent; Bascetin, Rümeyza; Benachour, Hamanou; Gallet, Olivier; Leroy-Dudal, Johanne

2015-12-01

Fibronectin, a ≈ 450-kDa protein with 4-9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal-induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose-binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal-induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research. Copyright © 2015 John Wiley & Sons, Ltd.

Carbohydrate phenotyping of human and animal milk glycoproteins.

PubMed

Gustafsson, Anki; Kacskovics, Imre; Breimer, Michael E; Hammarström, Lennart; Holgersson, Jan

2005-03-01

Breast-milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. The plethora of carbohydrate epitopes in breast-milk is known to differ between species, with human milk expressing the most complex one. We have investigated the expression of protein-bound carbohydrate epitopes in milk from man, cow, goat, sheep, pig, horse, dromedary and rabbit. Proteins were separated by SDS-PAGE and the presence of carbohydrate epitopes on milk proteins were analysed by Western blotting using different lectins and carbohydrate-specific antibodies. We show that ABH, Lewis (Le)x, sialyl-Lex, Lea, sialyl-Lea and Leb carbohydrate epitopes are expressed mainly on man, pig and horse milk proteins. The blood group precursor structure H type 1 is expressed in all species investigated, while only pig, dromedary and rabbit milk proteins carry H type 2 epitopes. These epitopes are receptors for Helicobacter pylori (Leb and sialyl-Lex), enteropathogenic (H type 1, Lea and Lex) and enterotoxic Escherichia coli (heat-stable toxin; H type 1 and 2), and Campylobacter jejuni (H type 2). Thus, milk from these animals or their genetically modified descendants could have a therapeutic effect by inhibiting pathogen colonization and infection.

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.

The first trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae was found in Dendrobium pendulum: purification, characterization, and effects of stress factors.

PubMed

Siripipatthana, Patthraporn; Phaonakrop, Narumon; Roytrakul, Sittiruk; Senawong, Gulsiri; Mudalige-Jayawickrama, Rasika G; Sattayasai, Nison

2015-07-01

Trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae with two conformational forms was first studied in Dendrobium pendulum . It was highly expressed by stress factors. Using mannan-agarose column chromatography, a mannose-binding protein was purified from Dendrobium pendulum Roxb. pseudobulb. After heating in the presence of sodium dodecyl sulfate (SDS) with or without 2-mercaptoethanol, the protein showed one band with molecular mass of 14.0 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Without heating, three bands were found at positions of 14.0, 39.4, and 41.5 kDa, but a higher amount of 39.4 and 41.5 kDa protein bands were seen in the presence of 2-mercaptoethanol. Liquid chromatography-tandem mass spectrometry and database search indicated that the 14.0 kDa protein band contained three peptide fragments identical to parts of a lectin precursor from Dendrobiu m findleyanum Parish & Rchb.f. Native-PAGE and Ferguson plot showed that the purified protein had two native forms with molecular masses of 44.2 and 45.3 kDa, indicating three 14.0 kDa polypeptide subunits. The purified protein exhibited the agglutination activity with trypsinized chicken erythrocytes. It was then recognized as a Galanthus nivalis agglutinin-related lectin and named D. pendulum agglutinin (DPA). Using reverse transcription-polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of DPA precursor showed the highest homology (96.4%) with a lectin precursor of D. findleyanum and contained three mannose-binding sites. Greater amounts of DPA were found when the pseudobulbs were treated with stress factors including ultraviolet light, abscisic acid, hydrogen peroxide, and acetylene gas.

Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

PubMed

Böttger, Angelika; Doxey, Andrew C; Hess, Michael W; Pfaller, Kristian; Salvenmoser, Willi; Deutzmann, Rainer; Geissner, Andreas; Pauly, Barbara; Altstätter, Johannes; Münder, Sandra; Heim, Astrid; Gabius, Hans-Joachim; McConkey, Brendan J; David, Charles N

2012-01-01

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

Horizontal Gene Transfer Contributed to the Evolution of Extracellular Surface Structures: The Freshwater Polyp Hydra Is Covered by a Complex Fibrous Cuticle Containing Glycosaminoglycans and Proteins of the PPOD and SWT (Sweet Tooth) Families

PubMed Central

Böttger, Angelika; Doxey, Andrew C.; Hess, Michael W.; Pfaller, Kristian; Salvenmoser, Willi; Deutzmann, Rainer; Geissner, Andreas; Pauly, Barbara; Altstätter, Johannes; Münder, Sandra; Heim, Astrid; Gabius, Hans-Joachim; McConkey, Brendan J.; David, Charles N.

2012-01-01

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment. PMID:23300632

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

Lipopolysaccharide-specific binding C-type lectin with one CRD domain from Fenneropenaeus merguiensis (FmLC4) functions as a pattern recognition receptor in shrimp innate immunity.

PubMed

Utarabhand, Prapaporn; Thepnarong, Supattra; Runsaeng, Phanthipha

2017-10-01

In crustaceans, an innate immune system is solely required because they lack an adaptive immunity. One kind of pattern recognition receptors (PRRs) that plays a particular role in the innate immunity of aquatic shrimp is lectin. A new diverse C-type lectin (FmLC4) was cloned from the hepatopancreas of Fenneropenaeus merguiensis by using RT-PCR and 5' and 3' rapid amplification of cDNA ends approaches. A full-length FmLC4 cDNA comprises 706 bp with an open reading frame of 552 bp, encoding a peptide of 184 amino acids. The predicted primary sequence of FmLC4 consists of a signal peptide of 19 amino acids, a molecular mass of 20.4 kDa, an isoelectric point of 5.13, one carbohydrate recognition domain with a QPD motif and a Ca 2+ binding site as well as a double-loop characteristic supported by two conserved disulfide bonds. The FmLC4 mRNA expression was found only in the hepatopancreas of normal shrimp and significantly up-regulated upon challenge the shrimp with Vibrio harveyi or white spot syndrome virus (WSSV). Recombinant FmLC4 (rFmLC4) could agglutinate various bacterial strains with Ca 2+ -dependence. Lipopolysaccharide (LPS) could specifically inhibit the agglutinating activity and potently bind to rFmLC4, indicating that FmLC4 was LPS-specific binding C-type lectin. Moreover, rFmLC4 itself displayed the in vivo effective clearance of the pathogenic bacterium V. harveyi. Altogether, FmLC4 may serve as LPS-specific PRR to recognize opportunistic bacterial and viral pathogens, and thus to play a role in the immune defense of aquatic shrimp via the binding and agglutination. Copyright © 2017 Elsevier Ltd. All rights reserved.

Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights

PubMed Central

da Silva, Bruno Bezerra; Furtado, Gilvan Pessoa; Carneiro, Igor de Sa; Lobo, Marina Duarte Pinto; Guan, Yiwei; Guo, Jingxu; Coker, Alun R.; Lourenzoni, Marcos Roberto; Guedes, Maria Izabel Florindo; Owen, James S.; Abraham, David J.; Monteiro-Moreira, Ana Cristina de Oliveira; Moreira, Renato de Azevedo

2017-01-01

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin (FTP) and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. FTP is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically active recombinant FTP in Escherichia coli BL21, optimizing conditions with the best set yielding >40 mg/l culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/ml of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP–mannose and FTP–glucose crystals were obtained, and they diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å respectively. The best solution showed four monomers per asymmetric unit. Molecular dynamics (MD) simulation suggested that FTP displays higher affinity for mannose than glucose. Cell studies revealed that FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/ml and was also capable of stimulating cell migration at 50 µg/ml. In conclusion, our optimized expression system allowed high amounts of correctly folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example in wound healing and tissue regeneration. PMID:28684550

Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights.

PubMed

de Sousa, Felipe Domingos; da Silva, Bruno Bezerra; Furtado, Gilvan Pessoa; Carneiro, Igor de Sa; Lobo, Marina Duarte Pinto; Guan, Yiwei; Guo, Jingxu; Coker, Alun R; Lourenzoni, Marcos Roberto; Guedes, Maria Izabel Florindo; Owen, James S; Abraham, David J; Monteiro-Moreira, Ana Cristina de Oliveira; Moreira, Renato de Azevedo

2017-08-31

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin (FTP) and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. FTP is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically active recombinant FTP in Escherichia coli BL21, optimizing conditions with the best set yielding >40 mg/l culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/ml of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP-mannose and FTP-glucose crystals were obtained, and they diffracted X-rays to a resolution of 1.58 (P2 1 2 1 2 1 ), 1.70 (P3 1 21) and 1.60 (P3 1 21) Å respectively. The best solution showed four monomers per asymmetric unit. Molecular dynamics (MD) simulation suggested that FTP displays higher affinity for mannose than glucose. Cell studies revealed that FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/ml and was also capable of stimulating cell migration at 50 µg/ml. In conclusion, our optimized expression system allowed high amounts of correctly folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example in wound healing and tissue regeneration. © 2017 The Author(s).

Mechanisms of Mannose-Binding Lectin-Associated Serine Proteases-1/3 Activation of the Alternative Pathway of Complement

PubMed Central

Banda, Nirmal K.; Takahashi, Minoru; Takahashi, Kazue; Stahl, Gregory L.; Hyatt, Stephanie; Glogowska, Magdalena; Wiles, Timothy A.; Endo, Yuichi; Fujita, Teizo; Holers, V. Michael; Arend, William P.

2011-01-01

Mannose-binding lectin-associated serine proteases-1/3 (MASP-1/3) are essential in activating the alternative pathway (AP) of complement through cleaving pro-factor D (pro-Df) into mature Df. MASP are believed to require binding to mannose binding lectins (MBL) or ficolins (FCN) to carry out their biological activities. Murine sera have been reported to contain MBL-A, MBL-C, and FCN-A, but not FCN-B that exists endogenously in monocytes and is thought not to bind MASP-1. We examined some possible mechanisms whereby MASP-1/3 might activate the AP. Collagen antibody-induced arthritis, a murine model of inflammatory arthritis dependent on the AP, was unchanged in mice lacking MBL-A, MBL-C, and FCN-A (MBL−/−/FCN A−/− mice) in comparison to wild-type mice. The in vitro induction of the AP by adherent mAb to collagen II was intact using sera from MBL−/−/FCN A−/− mice. Furthermore, sera from MBL−/−/FCN A−/− mice lacked pro-Df and possessed only mature Df. Gel filtration of sera from MBL−/−/FCN A−/− mice showed the presence of MASP-1 protein in fractions containing proteins smaller than the migration of MBL-A and MBL-C in sera from C4−/− mice, suggesting possible binding of MASP-1 to an unknown protein. Lastly, we show that FCN-B was present in the sera of MBL−/−/FCN A−/−mice and that it was bound to MASP-1. We conclude that MASP-1 does not require binding to MBL-A, MBL-C, or FCN-A to activate the AP. MASP-1 may cleave pro-Df into mature Df through binding to FCN-B or to an unknown protein, or may function as an unbound soluble protein. PMID:21943708

Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

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

Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard

Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLAmore » complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an {alpha} helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.« less

Characterization of β-Glucan Recognition Site on C-Type Lectin, Dectin 1

PubMed Central

Adachi, Yoshiyuki; Ishii, Takashi; Ikeda, Yoshihiko; Hoshino, Akiyoshi; Tamura, Hiroshi; Aketagawa, Jun; Tanaka, Shigenori; Ohno, Naohito

2004-01-01

Dectin 1 is a mammalian cell surface receptor for (1→3)-β-d-glucans. Since (1→3)-β-d-glucans are commonly present on fungal cell walls, it has been suggested that dectin 1 is important for recognizing fungal invasion. In this study we tried to deduce the amino acid residues in dectin 1 responsible for β-glucan recognition. HEK293 cells transfected with mouse dectin 1 cDNA could bind to a gel-forming (1→3)-β-d-glucan, schizophyllan (SPG). The binding of SPG to a dectin 1 transfectant was inhibited by pretreatment with other β-glucans having a (1→3)-β-d-glucosyl linkage but not by pretreatment with α-glucans. Dectin 1 has a carbohydrate recognition domain (CRD) consisting of six cysteine residues that are highly conserved in C-type lectins. We prepared 32 point mutants with mutations in the CRD and analyzed their binding to SPG. Mutations at Trp221 and His223 resulted in decreased binding to β-glucan. Monoclonal antibody 4B2, a dectin- 1 monoclonal antibody which had a blocking effect on the β-glucan interaction, completely failed to bind the dectin-1 mutant W221A. A mutant with mutations in Trp221 and His223 did not have a collaborative effect on Toll-like receptor 2-mediated cellular activation in response to zymosan. These amino acid residues are distinct from residues in other sugar-recognizing peptide sequences of typical C-type lectins. These results suggest that the amino acid sequence W221-I222-H223 is critical for formation of a β-glucan binding site in the CRD of dectin 1. PMID:15213161

Effect of the lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) on the alpha-amylase secretion of rat pancreas in vitro and in vivo.

PubMed

Mikkat, U; Damm, I; Schröder, G; Schmidt, K; Wirth, C; Weber, H; Jonas, L

1998-05-01

Lectins are able to bind to cholecystokinin (CCK) receptors and other glycosylated membrane proteins. The lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) are used for affinity chromatography to isolate the highly glycosylated CCK-A receptor of pancreatic acinar cells. According to the working hypothesis that lectin binding to the CCK receptor should alter the ligand-receptor interaction, the effect of WGA and UEA-I on CCK-8-induced enzyme secretion was studied on isolated rat pancreatic acini in vitro. In vitro both lectins showed a dosage-dependent inhibition of CCK-8-induced alpha-amylase secretion of acini over 60 min. WGA showed a strong inhibitory effect on amylase secretion, approximately 40%, in vitro. UEA-I caused a smaller, but significant decrease, approximately 20%, in enzyme secretion of isolated acini. Additionally, both lectins inhibited cerulein/secretin- or cerulein-induced pancreatic secretion of rats in vivo, but not after secretin alone. The results are discussed with respect to a possible influence of both lectins on the interaction of CCK or cerulein with the CCK-A receptor.

Identification of structural and secretory lectin-binding glycoproteins of normal and cancerous human prostate.

PubMed

Lad, P M; Cooper, J F; Learn, D B; Olson, C V

1984-12-07

We have utilized the technique of lectin-loading of SDS gels with iodinated concanavalin A and wheat germ agglutinin to identify glycoproteins in prostatic and seminal fluids as well as in prostate tissue fractions. The following subunits which bound both lectins were detected: (a) 50, 43 and 38 kDa subunits common to prostatic and seminal fluids, and an additional 55 kDa subunit which predominates only in prostatic fluid; (b) 78, 55, 50 and 43 kDa subunits in prostatic tissue cytosol and (c) 195, 170, 135, 116 and 95 kDa subunits present in the particulate fractions of prostatic tissue. Immunoblotting using specific rabbit antibodies revealed the 50 kDa band to be prostatic acid phosphatase and the 38 kDa band to be prostate-specific antigen. Interestingly, antibodies directed toward prostatic acid phosphatase were found to cross-react with the 43 kDa band. Fractionation on sucrose gradients showed that several of these particulate glycoproteins were associated with a vesicle fraction enriched in adenylate cyclase activity, implying that they are plasma membrane glycoproteins. Comparison of soluble and particulate fractions of normal and cancerous tissue homogenates was made by densitometric scanning of autoradiograms of lectin-loaded gels. Similar relative intensities of lectin-binding were obtained for corresponding proteins in normal and cancerous tissue fractions. Also, immunoblotting showed no differences in prostatic acid phosphatase or prostate-specific antigen between normal and cancerous soluble homogenate fractions. Our results suggest that major lectin-binding proteins are conserved in the transition from normal to cancerous tissue. These results may be useful in developing a multiple-marker profile of metastatic prostate cancer and for the design of imaging agents, such as monoclonal antibodies, to prominent soluble and particulate prostate glycoproteins.

Carbohydrate–Aromatic Interactions in Proteins

PubMed Central

2015-01-01

Protein–carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C–H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C–H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C–H bonds engage more often in CH−π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate–aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C–H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein–carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein–carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites. PMID:26561965

Lectins for gastrointestinal targeting--15 years on.

PubMed

Woodley, J F

2000-01-01

In the mid-1980s, the concept of bioadhesion using synthetic polymers emerged, and brought with it the promise of improved efficiency for the delivery of drugs via mucosal surfaces. Studies in the author's laboratory concentrated on 'biological' bioadhesion using the naturally-occurring proteins, lectins, which recognise and bind sugars in glycoconjugates, such as those found on the surfaces of cells. Tomato Lectin (TL) was extensively studied as a putative non-toxic lectin with potential for drug targeting/delivery to the gastrointestinal (GI) tract. In vitro, the TL displayed impressive binding to the intestinal mucosa, but in vivo failed to significantly modify intestinal transit. A number of research groups have coupled the TL to microparticles, and significant systemic uptake of these has been observed in animal studies. Polymers with pendant sugars have also been shown to be bioadhesive, by interacting with endogenous lectins present on the cells of the GI tract. The use of lectins to target to Peyer's patches and diseased tissues in the colon is an interesting development, but much work remains to be done. Lectins also have potential in mucosal vaccines. Before advanced drug delivery systems using lectins can be realised, rigorous evaluation of their toxicity and immunogenicity will be required, but they clearly offer a number of possibilities for GI drug targeting systems in the future.

Purification and characterization of a glucosamine-binding antifungal lectin from Phaseolus vulgaris cv. Chinese pinto beans with antiproliferative activity towards nasopharyngeal carcinoma cells.

PubMed

Ang, Andrew Si Wo; Cheung, Randy Chi Fai; Dan, Xiuli; Chan, Yau Sang; Pan, Wenliang; Ng, Tzi Bun

2014-01-01

A lectin has successfully been isolated from Phaseolus vulgaris cv. Chinese pinto bean using affinity chromatography, ion exchange chromatography, and gel filtration in succession, with a 15.4-fold purification. Investigation of its characteristics revealed that Chinese pinto bean lectin (CPBL) was a 58-kDa dimeric glucosamine-binding protein. Its Mg(2+)-dependent hemagglutinating activity was stable at pH 7-8 and at or below 60 °C. When the purified lectin was tested against six fungal species including Phyllosticta citriasiana, Magnaporthe grisea, Bipolans maydis, Valsa mali, Mycosphaerella arachidicola, and Setosphaeria turcica, only the mycelial growth of V. mali was reduced by 30.6 % by the lectin at 30 μM. The lectin did not exert any discernible antiproliferative effects on breast cancer MCF-7 cells, but was able to suppress proliferation of nasopharyngeal carcinoma HONE-1 cells, with an IC50 of 17.3 μM, as revealed by the MTT assay. Since few plant lectins demonstrate antifungal activity against V. mali, and not many others have inhibitory effects on HONE-1 cells, CPBL is a distinctive lectin which may be exploited for development into an agent against V. mali and HONE-1 cells.

Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

PubMed

André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

1999-11-01

Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential of dendrimers for applications as lectin-targeting device, as attested by these observations.

Intravital lectin perfusion analysis of vascular permeability in human micro- and macro- blood vessels.

PubMed

Debbage, P L; Sölder, E; Seidl, S; Hutzler, P; Hugl, B; Ofner, D; Kreczy, A

2001-10-01

We previously applied intravital lectin perfusion in mouse models to elucidate mechanisms underlying vascular permeability. The present work transfers this technique to human models, analysing vascular permeability in macro- and microvessels. Human vascular endothelial surface carbohydrate biochemistry differs significantly from its murine counterpart, lacking alpha-galactosyl epitopes and expressing the L-fucose moiety in the glycocalyx; the poly-N-lactosamine glycan backbone is common to all mammals. We examined extensively lectin binding specificities in sections and in vivo, and then applied the poly-N-lactosamine-specific lectin LEA and the L-fucose-specific lectin UEA-I in human intravital perfusions. Transendothelial transport differed in macrovessels and microvessels. In microvessels of adult human fat tissue, rectal wall and rectal carcinomas, slow transendothelial transport by vesicles was followed by significant retention at the subendothelial basement membrane; paracellular passage was not observed. Passage time exceeded 1 h. Thus we found barrier mechanisms resembling those we described previously in murine tissues. In both adult and fetal macrovessels, the vena saphena magna and the umbilical vein, respectively, rapid passage across the endothelial lining was observed, the tracer localising completely in the subendothelial tissues within 15 min; vesicular transport was more rapid than in microvessels, and retention at the subendothelial basement membrane briefer.

Molecular dynamics simulations of pea (Pisum sativum) lectin structure with octyl glucoside detergents: the ligand interactions and dynamics.

PubMed

Konidala, Praveen; Niemeyer, Bernd

2007-07-01

The mitogenic pea (Pisum sativum) lectin is a legume protein of non-immunoglobulin nature capable of specific recognition of glucose derivatives without altering its structure. Molecular dynamics simulations were performed in a realistic environment to investigate the structure and interaction properties of pea lectin with various concentrations of n-octyl-beta-d-glucopyranoside (OG) detergent monomers distributed inside explicit solvent cell. In addition, the diffusion coefficients of the ligands (OG, Ca2+, Mn2+, and Cl-) and the water molecules were also reported. The structural flexibility of the lectin was conserved in all simulations. The self-assembly of OG monomers into a small micelle at the hydrophobic site of the lectin was noticed in the simulation with 20 OG monomers. The interaction energy analysis concludes that the lectin was appropriately termed an adaptive structure. One or rarely two binding sites were observed at an instant in each simulation that were electrostatically favoured for the OG to interact with the surface amino acid residues. Enhanced binding of OG to the pea lectin was quantified in the system containing only Ca2+ divalent ions. Interestingly, no binding was observed in the simulation without divalent ions. Furthermore, the lectin-ligand complex was stabilized by multiple hydrogen bonds and at least one water bridge. Finally, the work was also in accordance with the published work elsewhere that the simulations performed with different initial conditions and using higher nonbonded cutoffs for the van der Waals and electrostatic interactions provide more accurate information and clues than the single large simulation of the biomolecular system of interest.

Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

NASA Astrophysics Data System (ADS)

Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

2015-05-01

Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

Switching of bacterial adhesion to a glycosylated surface by reversible reorientation of the carbohydrate ligand.

PubMed

Weber, Theresa; Chandrasekaran, Vijayanand; Stamer, Insa; Thygesen, Mikkel B; Terfort, Andreas; Lindhorst, Thisbe K

2014-12-22

The surface recognition in many biological systems is guided by the interaction of carbohydrate-specific proteins (lectins) with carbohydrate epitopes (ligands) located within the unordered glycoconjugate layer (glycocalyx) of cells. Thus, for recognition, the respective ligand has to reorient for a successful matching event. Herein, we present for the first time a model system, in which only the orientation of the ligand is altered in a controlled manner without changing the recognition quality of the ligand itself. The key for this orientational control is the embedding into an interfacial system and the use of a photoswitchable mechanical joint, such as azobenzene. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

PubMed

Jančaříková, Gita; Houser, Josef; Dobeš, Pavel; Demo, Gabriel; Hyršl, Pavel; Wimmerová, Michaela

2017-08-01

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcβ1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed β-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

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

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

PubMed Central

Peiris, Diluka; Ossondo, Marlène; Fry, Simon; Loizidou, Marilena; Smith-Ravin, Juliette; Dwek, Miriam V.

2015-01-01

Background Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. Methodology In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. Results Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). Conclusion Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer. PMID:26495974

Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea).

PubMed

Ao, Jingqun; Ding, Yang; Chen, Yuanyuan; Mu, Yinnan; Chen, Xinhua

2015-12-10

The C-type lectin-like receptors (CTLRs) play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR) from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD), an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1-C6), a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp) and WYD (Trp-Tyr-Asp). Ca(2+) binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus) and guppy (Poecilia retitculata) CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR) protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca(2+)-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.

Two antibacterial C-type lectins from crustacean, Eriocheir sinensis, stimulated cellular encapsulation in vitro.

PubMed

Jin, Xing-Kun; Li, Shuang; Guo, Xiao-Nv; Cheng, Lin; Wu, Min-Hao; Tan, Shang-Jian; Zhu, You-Ting; Yu, Ai-Qing; Li, Wei-Wei; Wang, Qun

2013-12-01

The first step of host fighting against pathogens is that pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. In the present study, we investigated how invertebrate pattern recognition receptor (PRR) C-type lectins might be involved in the antimicrobial response in crustacean. Based on our previously obtained completed coding regions of EsLecA and EsLecG in Eriocheir sinensis, the recombinant EsLectin proteins were produced via prokaryotic expression system and affinity chromatography. Subsequently, both rEsLecA and rEsLecG were discovered to have wide spectrum binding activities towards microorganisms, and their microbial-binding was calcium-independent. Moreover, the binding activities of both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. Both microorganism growth inhibitory activities assays and antibacterial activities assays revealed their capabilities of suppressing microorganisms growth and directly killing microorganisms respectively. Furthermore, the encapsulation assays signified that both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. Collectively, data presented here demonstrated the successful expression and purification of two C-type lectins proteins in the Chinese mitten crab, and their critical role in the innate immune system of an invertebrate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Gene expression of galectin-9/ecalectin, a potent eosinophil chemoattractant, and/or the insertional isoform in human colorectal carcinoma cell lines and detection of frame-shift mutations for protein sequence truncations in the second functional lectin domain.

PubMed

Lahm, H; Hoeflich, A; Andre, S; Sordat, B; Kaltner, H; Wolf, E; Gabius, H J

2000-09-01

The family of Ca2+-independent galactoside-binding lectins with the beta-strand topology of the jelly-roll, referred to as galectins, is known to mediate and modulate a variety of cellular activities. Their functional versatility explains the current interest in monitoring their expression in cancer research, so far primarily focused on galectin-1 and -3. Tandem-repeat-type galectin-9 and its (most probably) allelic variant ecalectin, a potent eosinophil chemoattractant, are known to be human leukocyte products. We show by RT-PCR with primers specific for both that their mRNA is expressed in 17 of 21 human colorectal cancer lines. As also indicated by restriction analysis, in addition to the expected transcript of 571 bp an otherwise identical isoform coding for a 32-amino acid extension of the link peptide was detected. Positive cell lines differentially expressed either one (7 lines) or both transcripts (10 lines). Sequence analysis of RT-PCR products, performed in four cases, allowed to assign the standard transcript to ecalectin in the case of SW480 cells and detected two point mutations in the insert of the link peptide-coding sequence in WiDr and Colo205. Furthermore, this analysis identified the insertion of a single nucleotide into the coding sequence generating a frame-shift mutation, an event which has so far not been reported for any galectin. This alteration encountered in both transcripts of the WiDr line and the isoform transcript of Colo205 cells will most likely truncate the protein part within the second (C-terminal) carbohydrate recognition domain. Our results thus reveal the presence of mRNA for a galectin-9-isoform or a potent eosinophil chemoattractant (ecalectin) or a truncated version thereof with preserved N-terminal carbohydrate recognition domain in established human colon cancer cell lines.

Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation*

PubMed Central

Payne, Christina M.; Bomble, Yannick J.; Taylor, Courtney B.; McCabe, Clare; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2011-01-01

Proteins employ aromatic residues for carbohydrate binding in a wide range of biological functions. Glycoside hydrolases, which are ubiquitous in nature, typically exhibit tunnels, clefts, or pockets lined with aromatic residues for processing carbohydrates. Mutation of these aromatic residues often results in significant activity differences on insoluble and soluble substrates. However, the thermodynamic basis and molecular level role of these aromatic residues remain unknown. Here, we calculate the relative ligand binding free energy by mutating tryptophans in the Trichoderma reesei family 6 cellulase (Cel6A) to alanine. Removal of aromatic residues near the catalytic site has little impact on the ligand binding free energy, suggesting that aromatic residues immediately upstream of the active site are not directly involved in binding, but play a role in the glucopyranose ring distortion necessary for catalysis. Removal of aromatic residues at the entrance and exit of the Cel6A tunnel, however, dramatically impacts the binding affinity, suggesting that these residues play a role in chain acquisition and product stabilization, respectively. The roles suggested from differences in binding affinity are confirmed by molecular dynamics and normal mode analysis. Surprisingly, our results illustrate that aromatic-carbohydrate interactions vary dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, these results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering strategies for biomass utilization. Generally, these results suggest that nature employs aromatic-carbohydrate interactions with a wide range of binding affinities for diverse functions. PMID:21965672

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.

PubMed

Ding, Shi-You; Xu, Qi; Ali, Mursheda K; Baker, John O; Bayer, Edward A; Barak, Yoav; Lamed, Raphael; Sugiyama, Junji; Rumbles, Garry; Himmel, Michael E

2006-10-01

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.

A functional glycoprotein competitive recognition and signal amplification strategy for carbohydrate-protein interaction profiling and cell surface carbohydrate expression evaluation

NASA Astrophysics Data System (ADS)

Wang, Yangzhong; Chen, Zhuhai; Liu, Yang; Li, Jinghong

2013-07-01

A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes.A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes. Electronic supplementary information (ESI) available: Experimental details; characterization of probes; the influence of electrolyte pH; probe concentration and glucose concentration on the electrode ECL effect. See DOI: 10.1039/c3nr01598j

The Liverwort Contains a Lectin That Is Structurally and Evolutionary Related to the Monocot Mannose-Binding Lectins1

PubMed Central

Peumans, Willy J.; Barre, Annick; Bras, Julien; Rougé, Pierre; Proost, Paul; Van Damme, Els J.M.

2002-01-01

A mannose (Man)-binding lectin has been isolated and characterized from the thallus of the liverwort Marchantia polymorpha. N-terminal sequencing indicated that the M. polymorpha agglutinin (Marpola) shares sequence similarity with the superfamily of monocot Man-binding lectins. Searches in the databases yielded expressed sequence tags encoding Marpola. Sequence analysis, molecular modeling, and docking experiments revealed striking structural similarities between Marpola and the monocot Man-binding lectins. Activity and specificity studies further indicated that Marpola is a much stronger agglutinin than the Galanthus nivalis agglutinin and exhibits a preference for methylated Man and glucose, which is unprecedented within the family of monocot Man-binding lectins. The discovery of Marpola allows us, for the first time, to corroborate the evolutionary relationship between a lectin from a lower plant and a well-established lectin family from flowering plants. In addition, the identification of Marpola sheds a new light on the molecular evolution of the superfamily of monocot Man-binding lectins. Beside evolutionary considerations, the occurrence of a G. nivalis agglutinin homolog in a lower plant necessitates the rethinking of the physiological role of the whole family of monocot Man-binding lectins. PMID:12114560

Lectin conjugates as biospecific contrast agents for MRI. Coupling of Lycopersicon esculentum agglutinin to linear water-soluble DTPA-loaded oligomers.

PubMed

Pashkunova-Martic, Irena; Kremser, Christian; Galanski, Markus; Schluga, Petra; Arion, Vladimir; Debbage, Paul; Jaschke, Werner; Keppler, Bernhard

2011-06-01

Magnetic resonance imaging (MRI) requires synthesis of contrast media bearing targeting groups and numerous gadolinium chelating groups generating high relaxivity. This paper explores the results of linking the gadolinium chelates to the targeting group, a protein molecule, via various types of linkers. Polycondensates of diethylenetriaminepentaacetic acid (DTPA) with either diols or diamines were synthesised and coupled to the targeting group, a lectin (Lycopersicon esculentum agglutinin, tomato lectin) which binds with high affinity to specific oligosaccharide configurations in the endothelial glycocalyx. The polycondensates bear up to four carboxylic groups per constitutive unit. Gd-chelate bonds are created through dative interactions with the unshared pair of electrons on each oxygen and nitrogen atom on DTPA. This is mandatory for complexation of Gd(III) and avoidance of the severe toxicity of free gadolinium ions. The polymer-DTPA compounds were characterised by (1)H NMR and mass spectrometry. The final lectin-DTPA-polycondensate conjugates were purified by fast protein liquid chromatography (FPLC). The capacity for specific binding was assessed, and the MRI properties were examined in order to evaluate the use of these oligomers as components of selective perfusional contrast agents.

In vitro evaluation of biodegradable microspheres with surface-bound ligands.

PubMed

Keegan, Mark E; Royce, Sara M; Fahmy, Tarek; Saltzman, W Mark

2006-02-21

Protein ligands were conjugated to the surface of biodegradable microspheres. These microsphere-ligand conjugates were then used in two in vitro model systems to evaluate the effect of conjugated ligands on microsphere behavior. Microsphere retention in agarose columns was increased by ligands on the microsphere surface specific for receptors on the agarose matrix. In another experiment, conjugating the lectin Ulex europaeus agglutinin 1 to the microsphere surface increased microsphere adhesion to Caco-2 monolayers compared to control microspheres. This increase in microsphere adhesion was negated by co-administration of l-fucose, indicating that the increase in adhesion is due to specific interaction of the ligand with carbohydrate receptors on the cell surface. These results demonstrate that the ligands conjugated to the microspheres maintain their receptor binding activity and are present on the microsphere surface at a density sufficient to target the microspheres to both monolayers and three-dimensional matrices bearing complementary receptors.

Galectins in the Pathogenesis of Rheumatoid Arthritis

PubMed Central

Li, Song; Yu, Yangsheng; Koehn, Christopher D; Zhang, Zhixin; Su, Kaihong

2013-01-01

Rheumatoid arthritis (RA) is a complex and common systemic autoimmune disease characterized by synovial inflammation and hyperplasia. Multiple proteins, cells, and pathways have been identified to contribute to the pathogenesis of RA. Galectins are a group of lectins that bind to β-galactoside carbohydrates on the cell surface and in the extracellular matrix. They are expressed in a wide variety of tissues and organs with the highest expression in the immune system. Galectins are potent immune regulators and modulate a range of pathological processes, such as inflammation, autoimmunity, and cancer. Accumulated evidence shows that several family members of galectins play positive or negative roles in the disease development of RA, through their effects on T and B lymphocytes, myeloid lineage cells, and fibroblast-like synoviocytes. In this review, we will summarize the function of different galectins in immune modulation and their distinct roles in RA pathogenesis. PMID:24416634

Lectin Ulex europaeus agglutinin I specifically labels a subset of primary afferent fibers which project selectively to the superficial dorsal horn of the spinal cord.

PubMed

Mori, K

1986-02-19

To examine differential carbohydrate expression among different subsets of primary afferent fibers, several fluorescein-isothiocyanate conjugated lectins were used in a histochemical study of the dorsal root ganglion (DRG) and spinal cord of the rabbit. The lectin Ulex europaeus agglutinin I specifically labeled a subset of DRG cells and primary afferent fibers which projected to the superficial laminae of the dorsal horn. These results suggest that specific carbohydrates containing L-fucosyl residue is expressed selectively in small diameter primary afferent fibers which subserve nociception or thermoception.

Interfering with Gal-1–mediated angiogenesis contributes to the pathogenesis of preeclampsia

PubMed Central

Freitag, Nancy; Tirado-González, Irene; Barrientos, Gabriela; Herse, Florian; Thijssen, Victor L. J. L.; Weedon-Fekjær, Susanne M.; Schulz, Herbert; Wallukat, Gerd; Klapp, Burghard F.; Nevers, Tania; Sharma, Surendra; Staff, Anne Cathrine; Dechend, Ralf; Blois, Sandra M.

2013-01-01

Preeclampsia (PE) is a pregnancy-specific disorder characterized by sudden onset of hypertension and proteinuria in the second half of pregnancy (>20 wk). PE is strongly associated with abnormal placentation and an excessive maternal inflammatory response. Galectin-1 (Gal-1), a member of a family of carbohydrate-binding proteins, has been shown to modulate several processes associated with placentation and to promote maternal tolerance toward fetal antigens. Here, we show that Gal-1 exhibits proangiogenic functions during early stages of pregnancy, promoting decidual vascular expansion through VEGF receptor 2 signaling. Blocking Gal-1–mediated angiogenesis or lectin, galactoside-binding, soluble, 1 deficiency results in a spontaneous PE-like syndrome in mice, mainly by deregulating processes associated with good placentation and maternal spiral artery remodeling. Consistent with these findings, we observed a down-regulation of Gal-1 in patients suffering from early onset PE. Collectively, these results strengthen the notion that Gal-1 is required for healthy gestation and highlight Gal-1 as a valuable biomarker for early PE diagnosis. PMID:23798433

Isolation and molecular characterization of two lectins from dwarf elder (Sambucus ebulus L.) blossoms related to the Sam n1 allergen.

PubMed

Jimenez, Pilar; Cabrero, Patricia; Basterrechea, José E; Tejero, Jesús; Cordoba-Diaz, Damian; Girbes, Tomas

2013-10-14

Sambucus species contain a number of lectins with and without antiribosomal activity. Here, we show that dwarf elder (Sambucus ebulus L.) blossoms express two D-galactose-binding lectins that were isolated and purified by affinity chromatography and gel filtration. These proteins, which we named ebulin blo (A-B toxin) and SELblo (B-B lectin)--blo from blossoms--were subjected to molecular characterization and analysis by MALDI-TOF mass spectrometry and tryptic peptide fingerprinting. Both lectins share a high degree of amino acid sequence homology with Sambucus lectins related to the Sam n1 allergen. Ebulin blo, but not SELblo, was highly toxic by nasal instillation to mice. Overall, our results suggested that both lectins would belong to an allergen family exemplified by Sam n1 and could trigger allergy responses. Furthermore, they raise a concern about ebulin blo toxicity.

Isolation and Molecular Characterization of Two Lectins from Dwarf Elder (Sambucus ebulus L.) Blossoms Related to the Sam n1 Allergen

PubMed Central

Jimenez, Pilar; Cabrero, Patricia; Basterrechea, José E.; Tejero, Jesús; Cordoba-Diaz, Damian; Girbes, Tomas

2013-01-01

Sambucus species contain a number of lectins with and without antiribosomal activity. Here, we show that dwarf elder (Sambucus ebulus L.) blossoms express two d-galactose-binding lectins that were isolated and purified by affinity chromatography and gel filtration. These proteins, which we named ebulin blo (A-B toxin) and SELblo (B-B lectin)—blo from blossoms—were subjected to molecular characterization and analysis by MALDI-TOF mass spectrometry and tryptic peptide fingerprinting. Both lectins share a high degree of amino acid sequence homology with Sambucus lectins related to the Sam n1 allergen. Ebulin blo, but not SELblo, was highly toxic by nasal instillation to mice. Overall, our results suggested that both lectins would belong to an allergen family exemplified by Sam n1 and could trigger allergy responses. Furthermore, they raise a concern about ebulin blo toxicity. PMID:24129061

Effects of Hemagglutination Activity in the Serum of a Deep-Sea Vent Endemic Crab, Shinkaia Crosnieri, on Non-Symbiotic and Symbiotic Bacteria

PubMed Central

Fujiyoshi, So; Tateno, Hiroaki; Watsuji, Tomoo; Yamaguchi, Hideyuki; Fukushima, Daisuke; Mino, Sayaka; Sugimura, Makoto; Sawabe, Tomoo; Takai, Ken; Sawayama, Shigeki; Nakagawa, Satoshi

2015-01-01

In deep-sea hydrothermal environments, most invertebrates associate with dense populations of symbiotic microorganisms in order to obtain nutrition. The molecular interactions between deep-sea animals and environmental microbes, including their symbionts, have not yet been elucidated in detail. Hemagglutinins/lectins, which are carbohydrate-binding proteins, have recently been reported to play important roles in a wide array of biological processes, including the recognition and control of non-self materials. We herein assessed hemagglutination activity in the serum of a deep-sea vent endemic crab, Shinkaia crosnieri, which harbors chemosynthetic epibionts on its plumose setae. Horse and rabbit erythrocytes were agglutinated using this serum (opt. pH 7.5 and opt. temperature 15°C). Agglutinating activity was inhibited by eight kinds of sugars and several divalent cations, did not require any divalent metal ions, and remained detectable even after heating the serum at 100°C for 30 min. By using fluorescently labeled serum, we demonstrated that deep-sea crab serum components bound to the epibionts even in the presence of sugars. This study represents the first immunological assessment of a deep-sea vent endemic crab and demonstrated the possibility of a non-lectin-mediated symbiont-host interaction. PMID:26212518

Lectins of beneficial microbes: system organisation, functioning and functional superfamily.

PubMed

Lakhtin, M; Lakhtin, V; Alyoshkin, V; Afanasyev, S

2011-06-01

In this review our last results and proposals with respect to general aspects of lectin studies are summarised and compared. System presence, organisation and functioning of lectins are proposed, and accents on beneficial symbiotic microbial lectins studies are presented. The proposed general principles of lectin functioning allows for a comparison of lectins with other carbohydrate-recognition systems. A new structure-functional superfamily of symbiotic microbial lectins is proposed and its main properties are described. The proposed superfamily allows for extended searches of the biological activities of any microbial member. Prospects of lectins of beneficial symbiotic microorganisms are discussed.

Three novel B-type mannose-specific lectins of Cynoglossus semilaevis possess varied antibacterial activities against Gram-negative and Gram-positive bacteria.

PubMed

Sun, Yuan-yuan; Liu, Li; Li, Jun; Sun, Li

2016-02-01

Lectins are a group of sugar-binding proteins that are important factors of the innate immune system. In this study, we examined, in a comparative manner, the expression and function of three Bulb-type (B-type) mannose-specific lectins (named CsBML1, CsBML2, and CsBML3) from tongue sole. All three lectins possess three repeats of the conserved mannose binding motif QXDXNXVXY. Expression of CsBML1, CsBML2, and CsBML3 was most abundant in liver and upregulated by bacterial infection. Recombinant (r) CsBML1, CsBML2, and CsBML3 bound to a wide arrange of bacteria in a dose-dependent manner and with different affinities. All three lectins displayed mannose-specific and calcium-dependent agglutinating capacities but differed in agglutinating profiles. rCsBML1 and rCsBML2, but not rCsBML3, killed target bacteria in vitro and inhibited bacterial dissemination in fish tissues in vivo. These results indicate for the first time that in teleost, different members of B-type mannose-specific lectins likely play different roles in antibacterial immunity. Copyright © 2015 Elsevier Ltd. All rights reserved.

The zebrafish galectins Drgal1-L2 and Drgal3-L1 bind in vitro to the infectious hematopoietic necrosis virus (IHNV) glycoprotein and reduce viral adhesion to fish epithelial cells*

PubMed Central

Feng, Chiguang; González-Montalbán, Núria; Ravindran, Chinnarajan; Jackson, Shawn; de las Heras-Sánchez, Ana; Giomarelli, Barbara; Ahmed, Hafiz; Haslam, Stuart M.; Wu, Gang; Dell, Anne; Ammayappan, Arun; Vakharia, Vikram N.; Vasta, Gerardo R.

2015-01-01

The infectious hematopoietic necrosis virus (IHNV; Rhabdoviridae, Novirhabdovirus) infects teleost fish, such as salmon and trout, and is responsible for significant losses in the aquaculture industry and in wild fish populations. Although IHNV enters the host through the skin at the base of the fins, the viral adhesion and entry mechanisms are not fully understood. In recent years, evidence has accumulated in support of the key roles played by protein-carbohydrate interactions between host lectins secreted to the extracellular space and virion envelope glycoproteins in modulating viral adhesion and infectivity. In this study, we assessed in vitro the potential role(s) of zebrafish (Danio rerio) proto type galectin-1 (Drgal1-L2) and a chimera galectin-3 (Drgal3-L1) in IHNV adhesion to epithelial cells. Our results suggest that the extracellular Drgal1-L2 and Drgal3-L1 interact directly and in a carbohydrate-dependent manner with the IHNV glycosylated envelope and glycans on the epithelial cell surface, significantly reducing viral adhesion. PMID:26429411

Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph.

PubMed

Alpuche, Juan; Pereyra, Ali; Agundis, Concepción; Rosas, Carlos; Pascual, Cristina; Slomianny, Marie-Christine; Vázquez, Lorena; Zenteno, Edgar

2005-06-20

A 291-kDa lectin (LsL) was purified from the hemolymph of the white shrimp Litopenaeus setiferus by affinity chromatography on glutaraldehyde-fixed stroma from rabbit erythrocytes. LsL is a heterotetramer of two 80-kDa and two 52-kDa subunits, with no covalently-liked carbohydrate, and mainly composed by aspartic and glutamic acids, glycine and alanine, with relatively lower methionine and cysteine contents. Edman degradation indicated that the NH2-terminal of the 80-kDa subunit is composed DASNAQKQHDVNFLL, whereas the NH2-terminal of the 52-kDa subunit is blocked. The peptide mass fingerprint of LsL was predicted from tryptic peptides from each subunit by MALDI-TOF, and revealed that each subunit showed 23 and 22%, respectively, homology with the hemocyanin precursor from Litopenaeus vannamei. Circular dichroism analysis revealed beta sheet and alpha helix contents of 52.7 and 6.1%, respectively. LsL agglutinate at higher titers guinea pig, murine, and rabbit erythrocytes its activity is divalent cation-dependent. N-acetylated sugars, such as GlcNAc, GalNAc, and NeuAc, were the most effective inhibitors of the LsL hemagglutinating activity. Sialylated O-glycosylated proteins, such as bovine submaxillary gland mucin, human IgA, and fetuin, showed stronger inhibitory activity than sialylated N-glycosylated proteins, such as human orosomucoid, IgG, transferrin, and lactoferrin. Desialylation of erythrocytes or inhibitory glycoproteins abolished their capacity to bind LsL, confirming the relevance of sialic acid in LsL-ligand interactions.

Glycan Remodeling with Processing Inhibitors and Lectin-Resistant Eukaryotic Cells.

PubMed

Chang, Veronica T; Spooner, Robert A; Crispin, Max; Davis, Simon J

2015-01-01

Some of the most important and interesting molecules in metazoan biology are glycoproteins. The importance of the carbohydrate component of these structures is often revealed by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. On the other hand, the presence of large amounts of carbohydrate can often hinder the structural and functional analysis of glycoproteins. There are often good reasons, therefore, for wanting to engineer and predefine the N-glycans present on glycoproteins, e.g., in order to characterize the functions of the glycans or facilitate their subsequent removal. Here, we describe in detail two distinct ways in which to usefully interfere with oligosaccharide processing, one involving the use of specific processing inhibitors, and the other the selection of cell lines mutated at gene loci that control oligosaccharide processing, using cytotoxic lectins. Both approaches have the capacity for controlled, radical alteration of oligosaccharide processing in eukaryotic cells used for heterologous protein expression, and have great utility in the structural analysis of glycoproteins.

Adjuvant effects mediated by the carbohydrate recognition domain of Agrocybe aegerita lectin interacting with avian influenza H9N2 viral surface glycosylated proteins.

PubMed

Ma, Li-Bao; Xu, Bao-Yang; Huang, Min; Sun, Lv-Hui; Yang, Qing; Chen, Yi-Jie; Yin, Ya-Lin; He, Qi-Gai; Sun, Hui

To evaluate the potential adjuvant effect of Agrocybe aegerita lectin (AAL), which was isolated from mushroom, against a virulent H 9 N 2 strain in vivo and in vitro. In trial 1, 50 BALB/c male mice (8 weeks old) were divided into five groups (n=10 each group) which received a subcutaneous injection of inactivated H 9 N 2 (control), inactivated H 9 N 2 +0.2% (w/w) alum, inactivated H 9 N 2 +0.5 mg recombinant AAL/kg body weight (BW), inactivated H 9 N 2 +1.0 mg AAL/kg BW, and inactivated H 9 N 2 +2.5 mg AAL/kg BW, respectively, four times at 7-d intervals. In trial 2, 30 BALB/c male mice (8 weeks old) were divided into three groups (n=10 each group) which received a subcutaneous injection of inactivated H 9 N 2 (control), inactivated H 9 N 2 +2.5 mg recombinant wild-type AAL (AAL-wt)/kg BW, and inactivated H 9 N 2 +2.5 mg carbohydrate recognition domain (CRD) mutant AAL (AAL-mutR63H)/kg BW, respectively, four times at 7-d intervals. Seven days after the final immunization, serum samples were collected from each group for analysis. Hemagglutination assay, immunogold electron microscope, lectin blotting, and co-immunoprecipitation were used to study the interaction between AAL and H 9 N 2 in vitro. IgG, IgG1, and IgG2a antibody levels were significantly increased in the sera of mice co-immunized with inactivated H 9 N 2 and AAL when compared to mice immunized with inactivated H 9 N 2 alone. No significant increase of the IgG antibody level was detected in the sera of the mice co-immunized with inactivated H 9 N 2 and AAL-mutR63H. Moreover, AAL-wt, but not mutant AAL-mutR63H, adhered to the surface of H 9 N 2 virus. The interaction between AAL and the H 9 N 2 virus was further demonstrated to be associated with the CRD of AAL binding to the surface glycosylated proteins, hemagglutinin and neuraminidase. Our findings indicated that AAL could be a safe and effective adjuvant capable of boosting humoral immunity against H 9 N 2 viruses in mice through its interaction with the viral surface glycosylated proteins, hemagglutinin and neuraminidase.

Biophysical characterization and structural determination of the potent cytotoxic Psathyrella asperospora lectin.

PubMed

Ribeiro, João P; Ali Abol Hassan, Mohamed; Rouf, Razina; Tiralongo, Evelin; May, Tom W; Day, Christopher J; Imberty, Anne; Tiralongo, Joe; Varrot, Annabelle

2017-05-01

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N-acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X-ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969-975. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q-mediated complement system in the basal chordate.

PubMed

Gao, Zhan; Li, Mengyang; Ma, Jie; Zhang, Shicui

2014-12-01

The origin of the classical complement pathway remains open during chordate evolution. A C1q-like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan-binding lectin-associated serine protease-2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q-like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q-mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of highly water-soluble fluorescent conjugated glycopoly(p-phenylene)s for lectin and Escherichia coli.

PubMed

Xue, Cuihua; Jog, Sonali P; Murthy, Pushpalatha; Liu, Haiying

2006-09-01

Two facile, convenient, and versatile synthetic approaches are used to covalently attach carbohydrate residues to conjugated poly(p-phenylene)s (PPPs) for highly water-soluble PPPs bearing alpha-mannopyranosyl and beta-glucopyranosyl pendants (polymers A and B), which highly fluoresce in phosphate buffer (pH 7.0). The post-polymerization functionalization approach is to treat bromo-bearing PPP (polymer 1) with 1-thiolethyl-alpha-D-mannose tetraacetate or 1-thiol-beta-D-glucose tetraacetate in THF solution in the presence of K(2)CO(3) at room temperature through formation of thioether bridges, affording polymer 2a or 2b. The prepolymerization functionalization approach is to polymerize a well-defined sugar-carrying monomer, affording polymer 2a. Polymers 2a and 2b were deacetylated under Zemplén conditions in methanol and methylene chloride containing sodium methoxide, affording polymers A and B, respectively. The multivalent display of carbohydrates on the fluorescent conjugated glycopolymer overcomes the characteristic low binding affinity of the individual carbohydrates to their receptor proteins. Titration of concanavalin A (Con A) to alpha-mannose-bearing polymer A resulted in significant fluorescent quenching of the polymer with Stern-Volmer quenching constant of 4.5 x 10(7). Incubation of polymer A with Escherichia coli (E. coli) lead to formation of fluorescently stained bacterial clusters. Beta-glucose-bearing polymer B displayed no response to Con A and E. coli.

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

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

Preventing Pseudomonas aeruginosa and Chromobacterium violaceum infections by anti-adhesion-active components of edible seeds

PubMed Central

2012-01-01

Background Pseudomonas aeruginosa adhesion to animal/human cells for infection establishment involves adhesive proteins, including its galactose- and fucose-binding lectins PA-IL (LecA) and PA-IIL (LecB). The lectin binding to the target-cell receptors may be blocked by compatible glycans that compete with those of the receptors, functioning as anti-adhesion glycodecoys. The anti-adhesion treatment is of the utmost importance for abrogating devastating antibiotic-resistant P. aeruginosa infections in immunodeficient and cystic fibrosis (CF) patients. This strategy functions in nature in protecting embryos and neonates. We have shown that PA-IL, PA-IIL, and also CV-IIL (a PA-IIL homolog produced in the related pathogen Chromobacterium violaceum) are highly useful for revealing natural glycodecoys that surround embryos in diverse avian eggs and are supplied to neonates in milks and royal jelly. In the present study, these lectins were used as probes to search for seed embryo-protecting glycodecoys. Methods The lectin-blocking glycodecoy activities were shown by the hemagglutination-inhibition test. Lectin-binding glycoproteins were detected by Western blotting with peroxidase-labeled lectins. Results The present work reports the finding - by using PA-IL, PA-IIL, and CV-IIL - of rich glycodecoy activities of low (< 10 KDa) and high MW (> 10 kDa) compounds (including glycoproteins) in extracts of cashew, cocoa, coffee, pumpkin, and tomato seeds, resembling those of avian egg whites, mammal milks, and royal jelly. Conclusions Edible seed extracts possess lectin-blocking glycodecoys that might protect their embryos from infections and also might be useful for hampering human and animal infections. PMID:22336073

Transcriptome analyses to investigate symbiotic relationships between marine protists

PubMed Central

Balzano, Sergio; Corre, Erwan; Decelle, Johan; Sierra, Roberto; Wincker, Patrick; Da Silva, Corinne; Poulain, Julie; Pawlowski, Jan; Not, Fabrice

2015-01-01

Rhizaria are an important component of oceanic plankton communities worldwide. A number of species harbor eukaryotic microalgal symbionts, which are horizontally acquired in the environment at each generation. Although these photosymbioses are determinant for Rhizaria ability to thrive in oceanic ecosystems, the mechanisms for symbiotic interactions are unclear. Using high-throughput sequencing technology (i.e., 454), we generated large Expressed Sequence Tag (EST) datasets from four uncultured Rhizaria, an acantharian (Amphilonche elongata), two polycystines (Collozoum sp. and Spongosphaera streptacantha), and one phaeodarian (Aulacantha scolymantha). We assessed the main genetic features of the host/symbionts consortium (i.e., the holobiont) transcriptomes and found rRNA sequences affiliated to a wide range of bacteria and protists in all samples, suggesting that diverse microbial communities are associated with the holobionts. A particular focus was then carried out to search for genes potentially involved in symbiotic processes such as the presence of c-type lectins-coding genes, which are proteins that play a role in cell recognition among eukaryotes. Unigenes coding putative c-type lectin domains (CTLD) were found in the species bearing photosynthetic symbionts (A. elongata, Collozoum sp., and S. streptacantha) but not in the non-symbiotic one (A. scolymantha). More particularly, phylogenetic analyses group CTLDs from A. elongata and Collozoum sp. on a distinct branch from S. streptacantha CTLDs, which contained carbohydrate-binding motifs typically observed in other marine photosymbiosis. Our data suggest that similarly to other well-known marine photosymbiosis involving metazoans, the interactions of glycans with c-type lectins is likely involved in modulation of the host/symbiont specific recognition in Radiolaria. PMID:25852650

AMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding

PubMed Central

Bieri, Michael; Mobbs, Jesse I.; Koay, Ann; Louey, Gavin; Mok, Yee-Foong; Hatters, Danny M.; Park, Jong-Tae; Park, Kwan-Hwa; Neumann, Dietbert; Stapleton, David; Gooley, Paul R.

2012-01-01

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β1 and β2). Muscle-specific β2-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β1-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β2-CBM is concurrent with an increase in flexibility in β2-CBM, which may account for the affinity differences between the two isoforms. In contrast to β1-CBM, unbound β2-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β2-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β2-CBM mutant. Insertion of a threonine into the background of β1-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms. PMID:22339867

Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3.

PubMed

Diehl, Carl; Engström, Olof; Delaine, Tamara; Håkansson, Maria; Genheden, Samuel; Modig, Kristofer; Leffler, Hakon; Ryde, Ulf; Nilsson, Ulf J; Akke, Mikael

2010-10-20

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein-ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. (15)N and (2)H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein-carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

Changes in the levels of mannan-binding lectin and ficolins during head-down tilted bed rest.

PubMed

Kelsen, Jens; Sandahl, Thomas D; Storm, Line; Frings-Meuthen, Petra; Dahlerup, Jens F; Thiel, Steffen

2014-08-01

Spaceflight studies and ground-based analogues of microgravity indicate a weakening of human immunity. Mannan-binding lectin (MBL) and H-, L-, and M-ficolin together constitute the lectin pathway and mediate the clearance of pathogens through complement activation. We hypothesized that simulated microgravity may weaken human innate immune functions and studied the impact of 6° head-down tilted bed rest (HDT) for 21 d on MBL and ficolin levels. Within a 6-mo period, seven men underwent two periods of HDT. Blood samples were analyzed for MBL, H-, L-, and M-ficolin, mannose-binding lectin-associated protein of 44 kDa (MAp44), and collectin liver 1 (CL-L1) by time-resolved immunofluorometric assays (TRIFMA). We observed well-defined individual preintervention levels of MBL and ficolins. Remarkably similar intraindividual changes occurred for MBL and MBL levels decreased (mean 282 ng · ml⁻¹) in the recovery phase. Conversely, CL-L1, a protein with MBL-like properties, increased (mean 102 ng · ml⁻¹) during the recovery phase. M-ficolin increased (mean 79 ng · ml⁻¹) within the first 2 d of HDT, followed by a decrease (mean 112 ng · ml⁻¹) during the recovery phase. L-ficolin increased (mean 304 ng · ml⁻¹) during HDT, while H-ficolin was essentially unaffected. MAp44, a down-regulator of the lectin pathway, decreased initially (mean 78 ng · ml⁻¹) in the recovery phase followed by an increase (mean 131 ng · ml⁻¹). Alterations in MBL and ficolin levels were modest and with our current knowledge do not lead to overt immunodeficiency. Pronounced changes occurred when the subjects resumed the upright position. In selected individuals, these changes appear to be a conserved response to HDT.

AFM force spectroscopy reveals how subtle structural differences affect the interaction strength between Candida albicans and DC-SIGN.

PubMed

te Riet, Joost; Reinieren-Beeren, Inge; Figdor, Carl G; Cambi, Alessandra

2015-11-01

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C-type lectin dendritic cell-specific intracellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), because a detailed characterization at the structural level is lacking. DC-SIGN recognizes specific Candida-associated molecular patterns, that is, mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan-branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope-based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC-SIGN. We demonstrate that slight differences in the N-mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC-SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 kB T. The single-bond affinity of tetrameric DC-SIGN for wild-type C. albicans is ~10.7 kB T and a dissociation constant kD of 23 μM, which is relatively strong compared with other carbohydrate-protein interactions described in the literature. In conclusion, this study shows that DC-SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC-SIGN and its pathogenic ligands will lead to a better understanding of how fungal-associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti-fungal drugs. Copyright © 2015 John Wiley & Sons, Ltd.

Use of Protein A as the Primary Layer in Fluorescent Microsphere Technology.

DTIC Science & Technology

1992-08-01

mAb) to the galactose-binding adherence lectin of Entamoeba histolytica were assessed for their abilities to bind protein A, using BlAcore. Of the six...permission of the Commander, U.S. Army Chemical Research, Development and Engineering Center (CRDEC), ATTN: SMCCR- SPS -T, Aberdeen Proving Ground, MD...14 6 USE OF PROTEIN A AS THE PRIMARY LAYER IN FLUORESCENT MICROSPHERE TECHNOLOGY 1. INTRODUCTION Entamoeba histolytica causes amebic colitis worldwide

Evaluation of glycophenotype in breast cancer by quantum dot-lectin histochemistry

PubMed Central

Andrade, Camila G; Cabral Filho, Paulo E; Tenório, Denise PL; Santos, Beate S; Beltrão, Eduardo IC; Fontes, Adriana; Carvalho, Luiz B

2013-01-01

Cell surface glycoconjugates play an important role in differentiation/dedifferentiation processes and lectins are employed to evaluate them by several methodologies. Fluorescent probes are considered a valuable tool because of their ability to provide a particular view, and are more detailed and sensitive in terms of cell structure and molecular content. The aim of this study was to evaluate and compare the expression and distribution of glycoconjugates in normal human breast tissue, and benign (fibroadenoma), and malignantly transformed (invasive ductal carcinoma) breast tissues. For this, we used mercaptosuccinic acid-coated Cadmium Telluride (CdTe) quantum dots (QDs) conjugated with concanavalin A (Con A) or Ulex europaeus agglutinin I (UEA I) lectins to detect α-D-glucose/mannose and L-fucose residues, respectively. The QD-lectin conjugates were evaluated by hemagglutination activity tests and carbohydrate inhibition assays, and were found to remain functional, keeping their fluorescent properties and carbohydrate recognition ability. Fluorescence images showed that different regions of breast tissue expressed particular types of carbohydrates. While the stroma was preferentially and intensely stained by QD-Con A, ductal cells were preferentially labeled by QD-UEA I. These results indicate that QD-lectin conjugates can be used as molecular probes and can help to elucidate the glycoconjugate profile in biological processes. PMID:24324334

Development of Pulsating Tubules with Chiral Inversion

DTIC Science & Technology

2013-09-21

assembly of rod–coil block molecules. These supramolecular ligands agglutinated effectively specific bacterial cells through carbohydrate-mediated...transduction and cause Jurkat cells to release 100 to 300 times as much IL-2 as lectin-stimulated normal human peripheral blood lymphocytes. In our...were found to regulate T cell activation. The lengths as well as stability of the protein-coated supramolecular nanofibers could be manipulated by a

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance

PubMed Central

Simberg, Dmitri; Park, Ji-Ho; Karmali, Priya P.; Zhang, Wan-Ming; Merkulov, Sergei; McCrae, Keith; Bhatia, Sangeeta; Sailor, Michael; Ruoslahti, Erkki

2009-01-01

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles. PMID:19394687

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance.

PubMed

Simberg, Dmitri; Park, Ji-Ho; Karmali, Priya P; Zhang, Wan-Ming; Merkulov, Sergei; McCrae, Keith; Bhatia, Sangeeta N; Sailor, Michael; Ruoslahti, Erkki

2009-08-01

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles.

Amino Acid Change in the Carbohydrate Response Element Binding Protein is associated with lower triglycerides and myocardial infarction incidence depending on level of adherence to the Mediterranean diet in the PREDIMED trial

USDA-ARS?s Scientific Manuscript database

A variant (rs3812316, C771G, and Gln241His) in the MLXIPL (Max-like protein X interacting protein-like) gene encoding the carbohydrate response element binding protein has been associated with lower triglycerides. However, its association with cardiovascular diseases and gene-diet interactions modul...

Thermodynamics of alpha-Cyclodextrin-p-Nitrophenyl Glycoside Complexes. A Simple System To Understand the Energetics of Carbohydrate Interactions in Water.

PubMed

Junquera, Elena; Laynez, José; Menéndez, Margarita; Sharma, Sunil; Penadés, Soledad

1996-10-04

Thermodynamic studies of the binding of a series of p-nitrophenyl glycosides (PNPGly) of varying stereochemistry to alpha-cyclodextrin (alpha-CD) were performed at three different temperatures (25, 35, and 42 degrees C) using a microcalorimetric technique. The system p-nitrophenol (PNP) at pH = 3 and alpha-CD was also studied for the sake of comparison. All these complexes were found to be enthalpy driven with a favorable enthalpic term clearly dominant over an unfavorable entropic term. A clear enthalpy-entropy compensation effect was observed at all the temperatures, with a slope close to unity (alpha = 1.02) and an intercept TDeltaS degrees (o) = 2.91 kcal mol(-)(1). This thermodynamic pattern is in agreement with those usually found for lectin-carbohydrate associations and for the binding processes of several host-guest systems. This pattern is explained in terms of the contribution of primarily two driving forces: the van der Waals interactions between the host and the guest, and the solvation/desolvation processes which accompany the association reaction. The presence of the carbohydrate molecule in the PNP ring causes a slight destabilization of the complex at 25 degrees C with respect to the alpha-CD-PNP (pH = 3) complex, although a different behavior has been observed depending on the axial/equatorial configuration of the glycoside and the temperature. This behavior is modulated by the stereochemistry of the glycoside. Differences were observed between the deoxy-derivatives (LAra and LFuc) and those derivatives with a hydroxymethyl group (Glc, Gal, Man). DeltaC(p) degrees values were obtained from the dependency of DeltaH degrees on temperature (=( partial differentialDeltaH degrees / partial differentialT)(p)). These values are small and negative except for alphaMan complex. For the latter complex, discrepancy between the calorimetric and the calculated van't Hoff enthalpies was observed. Parallels are drawn between the thermodynamics of our model and those proposed for carbohydrate-protein associations.

Plant as a plenteous reserve of lectin

PubMed Central

Hivrale, AU; Ingale, AG

2013-01-01

Lectins are clusters of glycoproteins of nonimmune foundation that combine specifically and reversibly to carbohydrates, mainly the sugar moiety of glycoconjugates, resulting in cell agglutination and precipitation of glycoconjugates. They are universally distributed in nature, being established in plants, fungi, viruses, bacteria, crustacea, insects, and animals, but leguminacae plants are rich source of lectins. The present review reveals the structure, biological properties, and application of plant lectins. PMID:24084524

Characterization of the Grp94/OS-9 chaperone-lectin complex

PubMed Central

Seidler, Paul M.; Shinsky, Stephen A.; Hong, Feng; Li, Zihai; Cosgrove, Michael S.; Gewirth, Daniel T.

2014-01-01

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER associated degradation (ERAD) quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins (TMPs). To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal MRH lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains ‘mammalian-specific insets’ that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate, and additionally hints at a plausible biological role for the Grp94/OS-9 complex. PMID:25193139

Recognition and Binding of the PF2 Lectin to α-Amylase From Zabrotes subfasciatus (Coleoptera:Bruchidae) Larval Midgut

PubMed Central

Lagarda-Diaz, I.; Geiser, D.; Guzman-Partida, A.M.; Winzerling, J.; Vazquez-Moreno, L.

2014-01-01

Abstract Amylases are an important family of enzymes involved in insect carbohydrate metabolism that are required for the survival of insect larvae. For this reason, enzymes from starch-dependent insects are targets for insecticidal control. PF2 ( Olneya tesota ) is a lectin that is toxic to Zabrotes subfasciatus (Coleoptera: Bruchidae) larvae. In this study, we evaluated recognition of the PF2 lectin to α-amylases from Z. subfasciatus midgut and the effect of PF2 on α-amylase activity. PF2 caused a decrease of total amylase activity in vitro. Subsequently, several α-amylase isoforms were isolated from insect midgut tissues using ion exchange chromatography. Three enzyme isoforms were verified by an in-gel assay for amylase activity; however, only one isoform was recognized by antiamylase serum and PF2. The identity of this Z. subfasciatus α-amylase was confirmed by liquid chromatography−tandem mass spectrometry. The findings strongly suggest that a glycosylated α-amylase isoform from larval Z. subfasciatus midgut interacts with PF2, which interferes with starch digestion. PMID:25528751

A histochemical study of the distribution of lectin binding sites in the developing branchial area of the trout Salmo trutta.

PubMed Central

Rojo, M C; Blánquez, M J; González, M E

1996-01-01

A histochemical study of the branchial area of brown trout embryos from 35 to 71 d of incubation is reported. A battery of 6 different horseradish peroxidase-labelled lectins, the PAS reaction and Alcian blue staining were used to study the distribution of carbohydrate residues in glycoconjugates along the pharyngeal and branchial epithelia. Con A and WGA reacted at every site of the branchial region thus showing the ubiquitous presence of alpha-D-mannose and N-acetyl-D-glucosamine. WGA, DBA and SBA were good markers for the hatching gland cells (HGCs) and mucous cells. Other lectins, such as PNA and UEA I, reacted only for a short time at some sites during the considered period of incubation. From 35 d until posthatching stages, a manifest strong reaction was noted both in the dorsal epithelium of branchial arches and the HGCs as shown by SBA reactivity. This may be significant with regard to the controversial origin of HGCs, which is thought to be endodermal. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8982837

X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

PubMed Central

Chouquet, Anne; Païdassi, Helena; Ling, Wai Li; Frachet, Philippe; Houen, Gunnar; Arlaud, Gérard J.; Gaboriaud, Christine

2011-01-01

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca2+-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism. PMID:21423620

Biologically-Inspired Peptide Reagents for Enhancing IMS-MS Analysis of Carbohydrates

NASA Astrophysics Data System (ADS)

Bohrer, Brian C.; Clemmer, David E.

2011-09-01

The binding properties of a peptidoglycan recognition protein are translated via combinatorial chemistry into short peptides. Non-adjacent histidine, tyrosine, and arginine residues in the protein's binding cleft that associate specifically with the glycan moiety of a peptidoglycan substrate are incorporated into linear sequences creating a library of 27 candidate tripeptide reagents (three possible residues permutated across three positions). Upon electrospraying the peptide library and carbohydrate mixtures, some noncovalent complexes are observed. The binding efficiencies of the peptides vary according to their amino acid composition as well as the disaccharide linkage and carbohydrate ring-type. In addition to providing a charge-carrier for the carbohydrate, peptide reagents can also be used to differentiate carbohydrate isomers by ion mobility spectrometry. The utility of these peptide reagents as a means of enhancing ion mobility analysis of carbohydrates is illustrated by examining four glucose-containing disaccharide isomers, including a pair that is not resolved by ion mobility alone. The specificity and stoichiometry of the peptide-carbohydrate complexes are also investigated. Trihistidine demonstrates both suitable binding efficiency and successful resolution of disaccharides isomers, suggesting it may be a useful reagent in IMS analyses of carbohydrates.

Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin

USGS Publications Warehouse

Ottinger, C.A.; Johnson, S.C.; Ewart, K.V.; Brown, L.L.; Ross, N.W.

1999-01-01

We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonassalmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml−1 to virulent A. salmonicida failed to significantly reduce (P>0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (P≤0.05) dose-dependent increases in phagocytosis, and bactericidal activity. Significant increases (P≤0.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0–20.0 ng ml−1 but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml−1), a significant decrease (P≤0.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida.

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners

PubMed Central

Muramatsu, Takashi

2016-01-01

Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. PMID:26684586

Glycobiology, how to sugar-coat an undergraduate advanced biochemistry laboratory.

PubMed

McReynolds, Katherine D

2006-09-01

A second semester biochemistry laboratory has been implemented as an independent projects course at California State University, Sacramento since 1999. To incorporate aspects of carbohydrate biochemistry, or glycobiology, into our curriculum, projects in lectin isolation and purification were undertaken over the course of two semesters. Through this modification in course content, this class now offers a diverse, hands-on treatment of not only standard protein purification techniques but also carbohydrate techniques, specifically the study of carbohydrate-protein interactions through hemagglutination assays, a novel commercial assay known as the Instant™Chek assay, and the generation and use of appropriate affinity chromatography matrices. Throughout the semester, the students are in charge of all aspects of their projects, from planning to execution and completion. Specific examples of student projects are highlighted such that the breadth of protein-carbohydrate chemistry pursued in a 15-week semester can be appreciated. The feedback of the course was very favorable, indicating that the students came away with skills necessary for them to be successful in their future careers. Most importantly, however, aspects of glycobiology have now been incorporated effectively into a mainstream undergraduate biochemistry laboratory class. Copyright © 2006 International Union of Biochemistry and Molecular Biology, Inc.

Broad anti-HIV activity of the Oscillatoria agardhii agglutinin homologue lectin family.

PubMed

Férir, Geoffrey; Huskens, Dana; Noppen, Sam; Koharudin, Leonardus M I; Gronenborn, Angela M; Schols, Dominique

2014-10-01

Oscillatoria agardhii agglutinin homologue (OAAH) proteins belong to a recently discovered lectin family. The founding member OAA and a designed hybrid OAAH (OPA) recognize similar but unique carbohydrate structures of Man-9, compared with other antiviral carbohydrate-binding agents (CBAs). These two newly described CBAs were evaluated for their inactivating properties on HIV replication and transmission and for their potential as microbicides. Various cellular assays were used to determine antiviral activity against wild-type and certain CBA-resistant HIV-1 strains: (i) free HIV virion infection in human T lymphoma cell lines and PBMCs; (ii) syncytium formation assay using persistently HIV-infected T cells and non-infected CD4+ T cells; (iii) DC-SIGN-mediated viral capture; and (iv) transmission to uninfected CD4+ T cells. OAA and OPA were also evaluated for their mitogenic properties and potential synergistic effects using other CBAs. OAA and OPA inhibit HIV replication, syncytium formation between HIV-1-infected and uninfected T cells, DC-SIGN-mediated HIV-1 capture and transmission to CD4+ target T cells, thereby rendering a variety of HIV-1 and HIV-2 clinical isolates non-infectious, independent of their coreceptor use. Both CBAs competitively inhibit the binding of the Manα(1-2)Man-specific 2G12 monoclonal antibody (mAb) as shown by flow cytometry and surface plasmon resonance analysis. The HIV-1 NL4.3(2G12res), NL4.3(MVNres) and IIIB(GRFTres) strains were equally inhibited as the wild-type HIV-1 strains by these CBAs. Combination studies indicate that OAA and OPA act synergistically with Hippeastrum hybrid agglutinin, 2G12 mAb and griffithsin (GRFT), with the exception of OPA/GRFT. OAA and OPA are unique CBAs with broad-spectrum anti-HIV activity; however, further optimization will be necessary for microbicidal application. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy.

PubMed

Nestor, Gustav; Anderson, Taigh; Oscarson, Stefan; Gronenborn, Angela M

2017-05-03

NMR of a uniformly 13 C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the 13 C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the 13 C spectral dispersion of 13 C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.

Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

PubMed Central

Moreira, Gustavo M. S. G.; Conceição, Fabricio R.; McBride, Alan J. A.; Pinto, Luciano da S.

2013-01-01

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins. PMID:24260572

Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

PubMed

Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

2013-01-01

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

Maize root lectins mediate the interaction with Herbaspirillum seropedicae via N-acetyl glucosamine residues of lipopolysaccharides.

PubMed

Balsanelli, Eduardo; Tuleski, Thalita Regina; de Baura, Valter Antonio; Yates, Marshall Geoffrey; Chubatsu, Leda Satie; Pedrosa, Fabio de Oliveira; de Souza, Emanuel Maltempi; Monteiro, Rose Adele

2013-01-01

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.

Maize Root Lectins Mediate the Interaction with Herbaspirillum seropedicae via N-Acetyl Glucosamine Residues of Lipopolysaccharides

PubMed Central

Balsanelli, Eduardo; Tuleski, Thalita Regina; de Baura, Valter Antonio; Yates, Marshall Geoffrey; Chubatsu, Leda Satie; de Oliveira Pedrosa, Fabio; de Souza, Emanuel Maltempi; Monteiro, Rose Adele

2013-01-01

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization. PMID:24130823

Intracellular Mannose Binding Lectin Mediates Subcellular Trafficking of HIV-1 gp120 in Neurons

PubMed Central

Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, CL; Kaul, M; Singh, KK

2014-01-01

Human immunodeficiency virus -1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

PubMed

Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

2014-09-01

Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. Published by Elsevier Inc.

Purification of a PHA-like chitin-binding protein from Acacia farnesiana seeds: a time-dependent oligomerization protein.

PubMed

Santi-Gadelha, T; Rocha, B A M; Oliveira, C C; Aragão, K S; Marinho, E S; Gadelha, C A A; Toyama, M H; Pinto, V P T; Nagano, C S; Delatorre, P; Martins, J L; Galvani, F R; Sampaio, A H; Debray, H; Cavada, B S

2008-07-01

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 +/- 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

PubMed

Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

2010-01-01

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

Identification of Carbohydrate-Binding Domains in the Attachment Proteins of Type 1 and Type 3 Reoviruses

PubMed Central

Chappell, James D.; Duong, Joy L.; Wright, Benjamin W.; Dermody, Terence S.

2000-01-01

The reovirus attachment protein, ς1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The ς1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of ς1 that binds cell surface carbohydrate. Chimeric and truncated ς1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-ς1 antibodies, and oligomerization indicates that the chimeric and truncated ς1 proteins are properly folded. To assess carbohydrate binding, recombinant ς1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated ς1 proteins, the sialic acid-binding domain of type 3 ς1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted β-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of ς1 protein purified from virions. In contrast, the homologous region of T1L ς1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 ς1 tail. Furthermore, our findings indicate that T1L and T3D ς1 proteins contain different arrangements of receptor-binding domains. PMID:10954547

Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses.

PubMed

Chappell, J D; Duong, J L; Wright, B W; Dermody, T S

2000-09-01

The reovirus attachment protein, sigma1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The sigma1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of sigma1 that binds cell surface carbohydrate. Chimeric and truncated sigma1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-sigma1 antibodies, and oligomerization indicates that the chimeric and truncated sigma1 proteins are properly folded. To assess carbohydrate binding, recombinant sigma1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated sigma1 proteins, the sialic acid-binding domain of type 3 sigma1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted beta-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of sigma1 protein purified from virions. In contrast, the homologous region of T1L sigma1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 sigma1 tail. Furthermore, our findings indicate that T1L and T3D sigma1 proteins contain different arrangements of receptor-binding domains.

Integrative analysis workflow for the structural and functional classification of C-type lectins

PubMed Central

2011-01-01

Background It is important to understand the roles of C-type lectins in the immune system due to their ubiquity and diverse range of functions in animal cells. It has been observed that currently confirmed C-type lectins share a highly conserved domain known as the C-type carbohydrate recognition domain (CRD). Using the sequence profile of the CRD, an increasing number of putative C-type lectins have been identified. Hence, it is highly needed to develop a systematic framework that enables us to elucidate their carbohydrate (glycan) recognition function, and discover their physiological and pathological roles. Results Presented herein is an integrated workflow for characterizing the sequence and structural features of novel C-type lectins. Our workflow utilizes web-based queries and available software suites to annotate features that can be found on the C-type lectin, given its amino acid sequence. At the same time, it incorporates modeling and analysis of glycans - a major class of ligands that interact with C-type lectins. Thereafter, the results are analyzed together with context-specific knowledge to filter off unlikely predictions. This allows researchers to design their subsequent experiments to confirm the functions of the C-type lectins in a systematic manner. Conclusions The efficacy and usefulness of our proposed immunoinformatics workflow was demonstrated by applying our integrated workflow to a novel C-type lectin -CLEC17A - and we report some of its possible functions that warrants further validation through wet-lab experiments. PMID:22372988

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity

PubMed Central

Jančaříková, Gita; Demo, Gabriel; Hyršl, Pavel

2017-01-01

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcβ1–4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed β-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer—the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts. PMID:28806750

Histochemical study of lectin binding sites in fourth and fifth instar gypsy moth larval midgut epithelium

Treesearch

Algimantas P. Valaitis

2011-01-01

There is evidence that the gypsy moth, Lymantria dispar, midgut epithelial brush border membrane has membrane-bound glycoconjugates, such as BTR-270 and aminopeptidase N (APN), which function as high affinity binding sites (receptors) for the insecticidal proteins produced by Bacillus thuringiensis (Bt). As gypsy...

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

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

Zorn, Gilad, E-mail: zorn@ge.com; Castner, David G.; Tyagi, Anuradha

Perfluorophenylazide (PFPA) chemistry is a novel method for tailoring the surface properties of solid surfaces and nanoparticles. It is general and versatile, and has proven to be an efficient way to immobilize graphene, proteins, carbohydrates, and synthetic polymers. The main thrust of this work is to provide a detailed investigation on the chemical composition and surface density of the PFPA tailored surface. Specifically, gold surfaces were treated with PFPA-derivatized (11-mercaptoundecyl)tetra(ethylene glycol) (PFPA-MUTEG) mixed with 2-[2-(2-mercaptoethoxy)ethoxy]ethanol (MDEG) at varying solution mole ratios. Complementary analytical techniques were employed to characterize the resulting films including Fourier transform infrared spectroscopy to detect fingerprints ofmore » the PFPA group, x-ray photoelectron spectroscopy and ellipsometry to study the homogeneity and uniformity of the films, and near edge x-ray absorption fine structures to study the electronic and chemical structure of the PFPA groups. Results from these studies show that the films prepared from 90:10 and 80:20 PFPA-MUTEG/MDEG mixed solutions exhibited the highest surface density of PFPA and the most homogeneous coverage on the surface. A functional assay using surface plasmon resonance with carbohydrates covalently immobilized onto the PFPA-modified surfaces showed the highest binding affinity for lectin on the PFPA-MUTEG/MDEG film prepared from a 90:10 solution.« less

Expression of soybean lectin in transgenic tobacco results in enhanced resistance to pathogens and pests.

PubMed

Guo, Peipei; Wang, Yu; Zhou, Xiaohui; Xie, Yongli; Wu, Huijun; Gao, Xuewen

2013-10-01

Lectins are proteins of non-immune origin that specifically interact with carbohydrates, known to play important roles in the defense system of plants. In this study, in order to study the function of a new soybean lectin (SBL), the corresponding encoding gene lec-s was introduced into tobacco plants via Agrobacterium-mediated transformation. Southern blot analyses had revealed that the lec-s gene was stable integrated into the chromosome of the tobacco. The results of the reverse transcription polymerase chain reaction (RT-PCR) also indicated that the lec-s gene in the transgenic tobacco plants could be expressed under the control of the constitutive CaMV35S promoter. Evaluation agronomic of the performance had showed that the transgenic plants could resist to the infection of Phytophthora nicotianae. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed SBL significantly (P.0.05) reduced the weight gain of larvae of the beet armyworm (Spodoptera exigua). Further on, the lectins retarded the development of the larvae and their metamorphosis. These findings suggest that soybean lectins have potential as a protective agent against pathogens and insect pests through a transgenic approach. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.