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Sample records for c-type lectin surface

  1. Scavenger Receptor C-Type Lectin Binds to the Leukocyte Cell Surface Glycan Lewis By a Novel Mechanism

    SciTech Connect

    Feinberg, H.; Taylor, M.E.; Weis, W.I.; /Stanford U., Med. School /Imperial Coll., London

    2007-07-10

    The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewis(x) trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewis(x) and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewis(x) is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis.

  2. The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells*

    PubMed Central

    Manzo, Carlo; Torreno-Pina, Juan A.; Joosten, Ben; Reinieren-Beeren, Inge; Gualda, Emilio J.; Loza-Alvarez, Pablo; Figdor, Carl G.; Garcia-Parajo, Maria F.; Cambi, Alessandra

    2012-01-01

    The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection. PMID:23019323

  3. C-type lectins, fungi and Th17 responses

    PubMed Central

    Vautier, Simon; Sousa, Maria da Glória; Brown, Gordon D.

    2010-01-01

    Th17 cells are a recently discovered subset of T helper cells characterised by the release of IL-17, and are thought to be important for mobilization of immune responses against microbial pathogens, but which also contribute to the development of autoimmune diseases. The identification of C-type lectin receptors which are capable of regulating the balance between Th1 and Th17 responses has been of particular recent interest, which they control, in part, though the release of Th17 inducing cytokines. Many of these receptors recognise fungi, and other pathogens, and play key roles in driving the development of protective anti-microbial immunity. Here we will review the C-type lectins that have been linked to Th17 type responses and will briefly examine the role of Th17 responses in murine and human anti-fungal immunity. PMID:21075040

  4. Mosquito C-type lectins maintain gut microbiome homeostasis.

    PubMed

    Pang, Xiaojing; Xiao, Xiaoping; Liu, Yang; Zhang, Rudian; Liu, Jianying; Liu, Qiyong; Wang, Penghua; Cheng, Gong

    2016-01-01

    The long-term evolutionary interaction between the host immune system and symbiotic bacteria determines their cooperative rather than antagonistic relationship. It is known that commensal bacteria have evolved a number of mechanisms to manipulate the mammalian host immune system and maintain homeostasis. However, the strategies employed by the microbiome to overcome host immune responses in invertebrates still remain to be understood. Here, we report that the gut microbiome in mosquitoes utilizes C-type lectins (mosGCTLs) to evade the bactericidal capacity of antimicrobial peptides (AMPs). Aedes aegypti mosGCTLs facilitate colonization by multiple bacterial strains. Furthermore, maintenance of the gut microbial flora relies on the expression of mosGCTLs in A. aegypti. Silencing the orthologues of mosGCTL in another major mosquito vector (Culex pipiens pallens) also impairs the survival of gut commensal bacteria. The gut microbiome stimulates the expression of mosGCTLs, which coat the bacterial surface and counteract AMP activity. Our study describes a mechanism by which the insect symbiotic microbiome offsets gut immunity to achieve homeostasis. PMID:27572642

  5. Mosquito C-type lectins maintain gut microbiome homeostasis

    PubMed Central

    Pang, Xiaojing; Xiao, Xiaoping; Liu, Yang; Zhang, Rudian; Liu, Jianying; Liu, Qiyong; Wang, Penghua; Cheng, Gong

    2016-01-01

    The long-term evolutionary interaction between the host immune system and symbiotic bacteria determines their cooperative rather than antagonistic relationship. It is known that commensal bacteria have evolved a number of mechanisms to manipulate the mammalian host immune system and maintain homeostasis. However, the strategies employed by the microbiome to overcome host immune responses in invertebrates still remain to be understood. Here, we report that the gut microbiome in mosquitoes utilizes C-type lectins (mosGCTLs) to evade the bactericidal capacity of antimicrobial peptides (AMPs). Aedes aegypti mosGCTLs facilitate colonization by multiple bacterial strains. Furthermore, maintenance of the gut microbial flora relies on the expression of mosGCTLs in A. aegypti. Silencing the orthologues of mosGCTL in another major mosquito vector (Culex pipiens pallens) also impairs the survival of gut commensal bacteria. The gut microbiome stimulates the expression of mosGCTLs, which coat the bacterial surface and counteract AMP activity. Our study describes a mechanism by which the insect symbiotic microbiome offsets gut immunity to achieve homeostasis. PMID:27170846

  6. 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. PMID:24550728

  7. Inhibitory C-type lectin receptors in myeloid cells

    PubMed Central

    Redelinghuys, Pierre; Brown, Gordon D.

    2011-01-01

    C-type lectin receptors encoded by the natural killer gene complex play critical roles in enabling NK cell discrimination between self and non-self. In recent years, additional genes at this locus have been identified with patterns of expression that extend to cells of the myeloid lineage where many of the encoded inhibitory receptors have equally important functions as regulators of immune homeostasis. In the present review we highlight the roles of some of these receptors including recent insights gained with regard to the identification of exogenous and endogenous ligands, mechanisms of cellular inhibition and activation, regulated expression within different cellular and immune contexts, as well as functions that include the regulation of bone homeostasis and involvement in autoimmunity. PMID:20934454

  8. C-type lectins do not act as functional receptors for filovirus entry into cells

    SciTech Connect

    Matsuno, Keita; Nakayama, Eri; Noyori, Osamu; Marzi, Andrea; Ebihara, Hideki; Irimura, Tatsuro; Feldmann, Heinz; Takada, Ayato

    2010-12-03

    Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

  9. Identification and characterization of C-type lectin genes from the reniform nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    C-type lectins represent a large family of sugar-binding proteins which require calcium for their ligand-binding activity. C-type lectins play an important role in the innate immune response in all life forms when challenged by pathogens. Ligand binding occurs via conserved domain sequences which re...

  10. Targeting C-Type Lectin Receptors for Cancer Immunity

    PubMed Central

    Yan, Huimin; Kamiya, Tomomori; Suabjakyong, Papawee; Tsuji, Noriko M.

    2015-01-01

    C-type lectin receptors (CLRs) are a large family of soluble and trans-membrane pattern recognition receptors that are widely and primarily expressed on myeloid cells. CLRs are important for cell–cell communication and host defense against pathogens through the recognition of specific carbohydrate structures. Similar to a family of Toll-like receptors, CLRs signaling are involved in the various steps for initiation of innate immune responses and promote secretion of soluble factors such as cytokines and interferons. Moreover, CLRs contribute to endocytosis and antigen presentation, thereby fine-tune adaptive immune responses. In addition, there may also be a direct activation of acquired immunity. On the other hand, glycans, such as mannose structures, Lewis-type antigens, or GalNAc are components of tumor antigens and ligate CLRs, leading to immunoregulation. Therefore, agonists or antagonists of CLRs signaling are potential therapeutic reagents for cancer immunotherapy. We aim to overview the current knowledge of CLRs signaling and the application of their ligands on tumor-associating immune response. PMID:26379663

  11. C-type Lectin Receptors for Tumor Eradication: Future Directions

    PubMed Central

    Streng-Ouwehand, Ingeborg; Unger, Wendy W. J.; van Kooyk, Yvette

    2011-01-01

    Dendritic cells are key regulators in directing immune responses and therefore are under extensive research for the induction of anti-tumor responses. DCs express a large array of receptors by which they scan their surroundings for recognition and uptake of pathogens. One of the receptor-families is the C-type lectins (CLR), which bind carbohydrate structures and internalize antigens upon recognition. Intracellular routing of antigen through CLR enhances loading and presentation of antigen through MHC class I and II, inducing antigen-specific CD4+ and CD8+ T-cell proliferation and skewing T-helper cells. These characteristics make CLRs very interesting targets for DC-based immunotherapy. Profound research has been done on targeting specific tumor antigens to CLR using either antibodies or the natural ligands such as glycan structures. In this review we will focus on the current data showing the potency of CLR-targeting and discuss improvements that can be achieved to enhance anti-tumor activity in the near future. PMID:24212951

  12. Antibacterial membrane attack by a pore-forming intestinal C-type lectin

    PubMed Central

    Mukherjee, Sohini; Zheng, Hui; Derebe, Mehabaw; Callenberg, Keith; Partch, Carrie L.; Rollins, Darcy; Propheter, Daniel C.; Rizo, Josep; Grabe, Michael; Jiang, Qiu-Xing; Hooper, Lora V.

    2014-01-01

    Summary Human body surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota1,2. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate1,3 but the mechanism by which they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. Here we show that human RegIIIα (hRegIIIα, also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. We derive a three-dimensional model of the hRegIIIα pore by docking the hRegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and show that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits hRegIIIα pore-forming activity, explaining why hRegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota. PMID:24256734

  13. Isolation and cloning of a C-type lectin from the hexactinellid sponge Aphrocallistes vastus: a putative aggregation factor.

    PubMed

    Gundacker, D; Leys, S P; Schröder, H C; Müller, I M; Müller, W E

    2001-01-01

    Among the sponges (Porifera), the oldest group of metazoans in phylogenetic terms, the Hexactinellida is considered to have diverged earliest from the two other sponge classes, the Demospongiae and Calcarea. The Hexactinellida are unusual among all Metazoa in possessing mostly syncytial rather than cellular tissues. Here we describe the purification of a cell adhesion molecule with a size of 34 kDa (in its native form; 24 kDa after deglycosylation) from the hexactinellid sponge Aphrocallistes vastus. This adhesion molecule was previously found to agglutinate preserved cells and membranes in a non-species-specific manner (Müller, W. E. G., Zahn, R. K, Conrad, J., Kurelec, B., and Uhlenbruck, G. [1984] Cell adhesion molecules in the haxactinellid Aphrocallistes vastus: species-unspecific aggregationfactor. Differentiation, 26, 30--35). The fact that the aggregation process required Ca(2+) and was inhibited by bird's nest glycoprotein and D-galactose but not by D-mannose or N-acetyl-D-galactosamine suggests that this cell adhesion molecule is a C-type lectin. To test this assumption, two highly similar C-type lectins were cloned from A.vastus. The deduced polypeptides of the two cDNA species isolated classified these molecules as C-type lectins. The calculated M(r) of the 191 aa long sequences were 22,022 and 22,064, respectively. The C-type lectins showed highest similarity to C-type lectins (type-II membrane proteins) from higher metazoan phyla; these molecules are absent in non-Metazoa. The two sponge C-type lectins contain the conserved domains known from other C-type lectins (e.g., disulfide bonds, the amino acids known to be involved in Ca(2+)-binding, as well as the amino acids involved in the specificity of binding to D-galactose) and a hydrophobic N-terminal region. The N-terminal part of the purified C-type lectin was identical with the corresponding region of the deduced polypeptide from the cDNA. It is proposed that the A.vastus lectins might bind to the

  14. Cyanovirin-N inhibits mannose-dependent Mycobacterium-C-type lectin interactions but does not protect against murine tuberculosis

    PubMed Central

    Driessen, Nicole N.; Boshoff, Helena I.M.; Maaskant, Janneke J.; Gilissen, Sebastiaan A.C.; Vink, Simone; van der Sar, Astrid M.; Vandenbroucke-Grauls, Christina M.J.E.; Bewley, Carole A.; Appelmelk, Ben J.; Geurtsen, Jeroen

    2012-01-01

    Cyanovirin-N (CV-N) is a mannose-binding lectin that inhibits HIV-1 infection by blocking mannose-dependent target-cell entry via C-type lectins. Like HIV-1, Mycobacterium tuberculosis expresses mannosylated surface-structures and exploits C-type lectins to gain cell-access. Here we investigated whether CV-N, as for HIV-1, can inhibit M. tuberculosis infection. We found that CV-N specifically interacted with mycobacteria by binding to the mannose-capped lipoglycan lipoarabinomannan. Furthermore, CV-N competed with the C-type lectins DC-SIGN and mannose receptor for ligand binding and inhibited the binding of M. tuberculosis to dendritic cells but, unexpectedly, not to macrophages. Subsequent in vivo infection experiments in a mouse model demonstrated that CV-N, despite its activity, did not inhibit or delay M. tuberculosis infection. This outcome argues against a critical role for mannose-dependent C-type lectin interactions during initial stages of murine M. tuberculosis infection and suggests that, depending on the circumstances, M. tuberculosis can productively infect cells using different modes of entry. PMID:22942435

  15. The three-dimensional structure of codakine and related marine C-type lectins.

    PubMed

    Gourdine, Jean-Philippe; Markiv, Anatoly; Smith-Ravin, Juliette

    2007-10-01

    Codakine is a new Ca(2+)-dependent mannose-binding C-type lectin (MBL) isolated from the gill tissue of the tropical clam, Codakia orbicularis. Bioinformatic analyses with the BLAST program have revealed similarities with marine lectins involved in immunity whose three-dimensional (3D) structures were unknown up until recently. In this article, we present bioinformatic analyses of marine lectins that are homologous to codakine, in particular lectins from the sea worm Laxus oneistus, named mermaid. These lectins are involved in the symbiotic association with sulphur-oxidizing bacteria which are closely related to the C. orbicularis gill symbiont. Using homology modelling, folding that is characteristic of C-type lectins was observed in all the marine Ca(2+)-dependent lectins studied, with conservation of random coiled structures of the carbohydrate recognition domain (CRD) and Ca(2+)-binding sites. Like codakine, the marine lectins analysed contain a signal peptide commonly found in secreted and transmembrane proteins. The majority of the predictive 3D models established from the lectins exhibit a common feature, namely the involvement in invertebrate and vertebrate immunity (dendritic cell receptor, macrophage receptor, etc.). These bioinformatic analyses and the literature data support the hypothesis that codakine, like the L. oneistus mermaids, is probably involved in the cellular mediation of symbiosis and defence against pathogenic microorganisms. PMID:17493832

  16. A novel C-type lectin is involved in the innate immunity of Macrobrachium nipponense.

    PubMed

    Xiu, Yunji; Wang, Yinghui; Bi, Jingxiu; Liu, Yuhan; Ning, Mingxiao; Liu, Hui; Li, Shuang; Gu, Wei; Wang, Wen; Meng, Qingguo

    2016-03-01

    C-type lectins (CTLs) play important roles in invertebrate innate immunity by recognizing and eliminating pathogens. In the present study, a low-density lipoprotein receptor class A (LDLa) domain-containing CTL was identified from the oriental river prawn Macrobrachium nipponense, designated as MnCTLDcp1. The full-length cDNA of MnCTLDcp1 was composed of 1462 bp, with a 999-bp ORF encoding a 332-aa protein. An LDLa and a single C-type lectin-like domain (CTLD) were found. The mRNA transcripts of MnCTLDcp1 was expressed the highest in heart. After the prawns were challenged by Aeromonas hydrophila and Staphylococcus aureus, the expression level of MnCTLDcp1 in heart and hemocytes were all significantly up-regulated. Sugar binding assay revealed that the MnCTLDcp1 could bind to the glycoconjugates of bacteria surface, such as LPS, PGN and they can compete with bacterial as competitors. The recombinant MnCTLDcp1 agglutinates Gram-positive (S. aureus and Bacillus subtilis) and Gram-negative bacteria (A. hydrophila, Vibrio parahaemolyticus, Escherichia coli and Pseudomonas aeruginosa) in the presence of calcium and also could bind to these bacteria. These results clearly suggested that MnCTLDcp1 functions as a pattern-recognition receptor involved in the innate immunity of M. nipponense. PMID:26804648

  17. Antimicrobial properties of avian eggshell-specific C-type lectin-like proteins.

    PubMed

    Wellman-Labadie, Olivier; Lakshminarayanan, Rajamani; Hincke, Maxwell T

    2008-03-01

    C-type lectin-like proteins are major components of the calcified eggshell of multiple avian species. In this study, two representative avian C-type lectin-like proteins, ovocleidin-17 and ansocalcin, were purified from decalcified chicken and goose eggshell protein extracts and investigated for carbohydrate binding activity as well as antimicrobial activity. Purified ovocleidin-17 and ansocalcin were found to bind bacterial polysaccharides, and were bactericidal against Bacillus subtilis, Staphylococcus aureus and Pseudomona aeruginosa. Bactericidal activity was found to be enhanced in the presence of calcium but was not dependent on its presence. The results suggest that avian C-type lectin-like proteins may play an important antimicrobial role in defence of the avian embryo. PMID:18258195

  18. A C-Type Lectin from Bothrops jararacussu Venom Disrupts Staphylococcal Biofilms

    PubMed Central

    Klein, Raphael Contelli; Fabres-Klein, Mary Hellen; de Oliveira, Leandro Licursi; Feio, Renato Neves; Malouin, François; Ribon, Andréa de Oliveira Barros

    2015-01-01

    Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model. PMID:25811661

  19. A C-type lectin from Bothrops jararacussu venom disrupts Staphylococcal biofilms.

    PubMed

    Klein, Raphael Contelli; Fabres-Klein, Mary Hellen; de Oliveira, Leandro Licursi; Feio, Renato Neves; Malouin, François; Ribon, Andréa de Oliveira Barros

    2015-01-01

    Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model. PMID:25811661

  20. Characterization of beta-glucan recognition site on C-type lectin, dectin 1.

    PubMed

    Adachi, Yoshiyuki; Ishii, Takashi; Ikeda, Yoshihiko; Hoshino, Akiyoshi; Tamura, Hiroshi; Aketagawa, Jun; Tanaka, Shigenori; Ohno, Naohito

    2004-07-01

    Dectin 1 is a mammalian cell surface receptor for (1-->3)-beta-d-glucans. Since (1-->3)-beta-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 beta-glucan recognition. HEK293 cells transfected with mouse dectin 1 cDNA could bind to a gel-forming (1-->3)-beta-d-glucan, schizophyllan (SPG). The binding of SPG to a dectin 1 transfectant was inhibited by pretreatment with other beta-glucans having a (1-->3)-beta-d-glucosyl linkage but not by pretreatment with alpha-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 Trp(221) and His(223) resulted in decreased binding to beta-glucan. Monoclonal antibody 4B2, a dectin- 1 monoclonal antibody which had a blocking effect on the beta-glucan interaction, completely failed to bind the dectin-1 mutant W221A. A mutant with mutations in Trp(221) and His(223) 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 beta-glucan binding site in the CRD of dectin 1. PMID:15213161

  1. Computational and Experimental Prediction of Human C-Type Lectin Receptor Druggability

    PubMed Central

    Aretz, Jonas; Wamhoff, Eike-Christian; Hanske, Jonas; Heymann, Dario; Rademacher, Christoph

    2014-01-01

    Mammalian C-type lectin receptors (CTLRS) are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies, and lymphocyte homing. Despite a great interest in modulating CTLR recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 CTLRs using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable. To further explore these findings, we employed a fluorine-based nuclear magnetic resonance screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rate = 13.5%). A surface plasmon resonance-based follow-up assay confirmed 18 of these fragments (47%) and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7 and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular, with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of CTLRs can be developed. PMID:25071783

  2. Differential expression of two C-type lectins in grass carp Ctenopharyngodon idella and their response to grass carp reovirus.

    PubMed

    Ju, C S; He, L B; Pei, Y Y; Jiang, Y; Huang, R; Li, Y M; Liao, L J; Jang, S H; Wang, Y P

    2016-02-01

    The cDNAs of two C-type lectins in grass carp Ctenopharyngodon idella, galactose-binding lectin (galbl) and mannose-binding lectin (mbl), were cloned and analysed in this study. Both of them exhibited the highest expression level in liver, whereas their expression pattern differed in early phase of embryonic development. Following exposure to grass carp reovirus (GCRV), the mRNA expression level of galbl and mbl was significantly up-regulated in liver and intestine. PMID:26643267

  3. Sensing of cell death by myeloid C-type lectin receptors

    PubMed Central

    Sancho, David; Reis e Sousa, Caetano

    2015-01-01

    Molecules associated with dead or dying cells can be detected by receptors on macrophages and dendritic cells. Signals from these receptors impact myeloid cell function and play a role in determining whether death is silent or proinflammatory, tolerogenic or immunogenic. Prominent among myeloid receptors detecting dead cells are C-type lectin receptors (CLRs). Signals from these receptors variably induce endocytosis of cell corpses, corpse degradation, retrieval of dead cell-associated antigens and/or modulation of immune responses. The sensing of tissue damage by myeloid CLRs complements detection of pathogens in immunity and represents an ancient response aimed at restoring tissue homeostasis. PMID:23332826

  4. The evolution of HIV-1 interactions with coreceptors and mannose C-type lectin receptors.

    PubMed

    Borggren, Marie; Jansson, Marianne

    2015-01-01

    The phenotype of human immunodeficiency virus type 1 (HIV-1) commonly evolves between and within infected individuals, at virus transmission, and during disease progression. This evolution includes altered interactions between the virus and its coreceptors, i.e., chemokine receptors, as well as mannose C-type lectin receptors (CLRs). Transmitted/founder viruses are predominantly restricted to CCR5, whereas the subsequent intrapatient evolution of HIV-1 coreceptor use during progressive disease can be subdivided into two distinct pathways. Accordingly, the CCR5-restricted virus population is either gradually replaced by virus variants able to use CXCR4 or evolves toward an altered, more flexible use of CCR5. Despite a strong dependency on these coreceptors for host cell entry, HIV-1 also interacts with other cell surface molecules during target cell attachment, including the CLRs. The virus interaction with the CLRs may result either in the efficient transfer of virus to CD4(+) T cells or in the degradation of the virus in endosomal compartments. The determinants of the diverse outcomes depend on which CLR is engaged and also on the glycan makeup of the envelope glycoproteins, which may evolve with the strength of the immune pressure during the disease course. With the current clinical introduction of CCR5 antagonists and the development of additional entry inhibitors, knowledge on the evolution and baseline characteristics of HIV-1 interactions with coreceptor and CLR interactions may play important roles for individualized and optimized treatment strategies. This review summarizes our current understanding of the evolution of HIV-1 interactions with these receptors. PMID:25595802

  5. A C-type lectin is involved in the innate immune response of Chinese white shrimp.

    PubMed

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

    2009-10-01

    C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this work, a cDNA for a new C-type lectin, FcLec3, was obtained from Chinese white shrimp Fenneropenaeus chinensis using expressed sequence tag analysis and rapid amplification of the cDNA ends. FcLec3 contains an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that FcLec3 was mainly expressed in hepatopancreas and that the expression of FcLec3 was obviously up-regulated by Vibrio anguillarum or white spot syndrome virus (WSSV) challenge. Recombinant FcLec3 could agglutinate Gram-negative and -positive bacteria with the presence of calcium. A following agglutination inhibitory test indicated that FcLec3 could recognize muramic acid and peptidoglycan. Besides, pull-down assay showed that the recombinant protein could interact with VP28, one major envelope protein of WSSV. These results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp. PMID:19647083

  6. Genes encoding putative natural killer cell C-type lectin receptors in teleostean fishes

    PubMed Central

    Sato, Akie; Mayer, Werner E.; Overath, Peter; Klein, Jan

    2003-01-01

    Mammalian natural killer (NK) cells are cytotoxic lymphocytes that express receptors specific for MHC class I molecules. The NK cell receptors belong to two structurally unrelated families, the killer cell Ig-like receptors and the killer cell C-type lectin receptors. We describe a cDNA clone derived from the bony (cichlid) fish Paralabidochromis chilotes and show that it encodes a protein related to the CD94/NK cell group 2 (NKG2) subfamily of the killer cell C-type lectin receptors. The gene encoding this receptor in a related species, Oreochromis niloticus, has a similar structure to the human CD94/NKG2 genes and is a member of a multigene cluster that resembles the mammalian NK cell gene complex. Thus, the CD94/NKG2 subfamily of NK cell receptors must have arisen before the divergence of fish and tetrapods and may have retained its function (possibly monitoring the expression of MHC class I molecules) for >400 million years. PMID:12802013

  7. Structural and biological characterization of Nattectin, a new C-type lectin from the venomous fish Thalassophryne nattereri.

    PubMed

    Lopes-Ferreira, Mônica; Magalhães, Geraldo Santana; Fernandez, Jorge Hernandez; Junqueira-de-Azevedo, Inácio de Loiola M; Le Ho, Paulo; Lima, Carla; Valente, Richard H; Moura-da-Silva, Ana Maria

    2011-06-01

    Lectins are glycan-binding receptors that recognize glycan epitopes on foreign pathogens and in the host systems. They can be involved in functions that include innate immunity, development, immune regulation and homeostasis. Several lectins have been purified and characterized from fish species. In this work, using cation-exchange chromatography, a galactose-specific lectin belonging to the family of C-type lectins was isolated from the venom of the Brazilian venomous fish Thalassophryne nattereri. Nattectin is a basic, non-glycosilated, 15 kDa monomeric protein. It exhibits hemagglutination activity that is independent of Ca(2+). We also demonstrated a lectin activity for Nattectin in the innate immune system, especially in neutrophil mobilization in mice, indicating that marine organisms are source of immunomodulator agents. PMID:21396978

  8. MCL and Mincle: C-Type Lectin Receptors That Sense Damaged Self and Pathogen-Associated Molecular Patterns

    PubMed Central

    Richardson, Mark B.; Williams, Spencer J.

    2014-01-01

    Macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle) comprise part of an extensive repertoire of pattern recognition receptors with the ability to sense damage-associated and pathogen-associated molecular patterns. In this review, we cover the discovery and molecular characterization of these C-type lectin receptors, and highlight recent advances in the understanding of their roles in orchestrating the response of the immune system to bacterial and fungal infection, and damaged self. We also discuss the identification and structure–activity relationships of activating ligands, particularly trehalose dimycolate and related mycobacterial glycolipids, which have significant potential in the development of TH1/TH17 vaccination strategies. PMID:25002863

  9. Characterization of Multisugar-Binding C-Type Lectin (SpliLec) from a Bacterial-Challenged Cotton Leafworm, Spodoptera littoralis

    PubMed Central

    Seufi, AlaaEddeen M.; Galal, Fatma H.; Hafez, Elsayed E.

    2012-01-01

    Background Various proteins that display carbohydrate-binding activity in a Ca2+-dependent manner are classified into the C-type lectin family. They have one or two C-type carbohydrate-recognition domains (CRDs) composed of 110–130 amino acid residues in common. C-type lectins mediate cell adhesion, non-self recognition, and immuno-protection processes in immune responses and thus play significant roles in clearance of invaders, either as cell surface receptors for microbial carbohydrates or as soluble proteins existing in tissue fluids. The lectin of Spodoptera littoralis is still uncharacterized. Methodology A single orf encoding a deduced polypeptide consisting of an 18-residue signal peptide and a 291-residue mature peptide, termed SpliLec, was isolated from the haemolymph of the cotton leafworm, S. littoralis, after bacterial challenge using RACE-PCR. Sequence analyses of the data revealed that SpliLec consists of two CRDs. Short-form CRD1 and long-form CRD2 are stabilized by two and three highly conserved disulfide bonds, respectively. SpliLec shares homology with some dipteran lectins suggesting possible common ancestor. The purified SpliLec exhibited a 140-kDa molecular mass with a subunit molecular mass of 35 kDa. The hemagglutination assays of the SpliLec confirmed a thermally stable, multisugar-binding C-type lectin that binds different erythrocytes. The purified SpliLec agglutinated microorganisms and exhibited comparable antimicrobial activity against gram (+) and gram (−) bacteria too. Conclusions Our results suggested an important role of the SpliLec gene in cell adhesion and non-self recognition. It may cooperate with other AMPs in clearance of invaders of Spodoptera littoralis. PMID:22916161

  10. Critical roles of sea cucumber C-type lectin in non-self recognition and bacterial clearance.

    PubMed

    Wei, Xiumei; Liu, Xiangquan; Yang, Jianmin; Wang, Sheng; Sun, Guohua; Yang, Jialong

    2015-08-01

    C-type lectin is one important pattern recognition receptor (PRR) that plays crucial roles in multiple immune responses. A C-type lectin from sea cucumber Apostichopus japonicus (AjCTL-1) was characterized in the present study. The amino acid sequence of AjCTL-1 shared high similarities with other C-type lectins from invertebrates and vertebrates. The C-type lectin domain (CTLD) of AjCTL-1 contained a Ca(2+)-binding site 2 and four conserved cysteine residues. AjCTL-1 mRNA expression patterns in tissues and after bacterial challenge were then analysed. Quantitative PCR revealed that AjCTL-1 mRNA was widely expressed in the tested tissues of healthy sea cucumber. The highest expression level occurred in gonad followed by body wall, coelomocytes, tentacle, intestinum and longitudinal muscle, and the lowest expression level was in respiratory tree. AjCTL-1 mRNA expression in coelomocytes was significantly induced by gram-negative Listonella anguillarum and gram-positive Micrococcus luteus, with different up-regulation patterns post-challenge. Recombinant AjCTL-1 exhibited the ability to bind peptidoglycan directly, agglutinate M. luteus, Staphylococcus aureus and Escherichia coli, in a Ca(2+)-dependant manner, and enhance the phagocytosis of coelomocytes against E. coli in vitro. The results indicated that AjCTL-1 could act as a PRR in Apostichopus japonicus and had critical roles in non-self recognition and bacterial clearance against invading microbes. PMID:26052017

  11. Human CLEC18 Gene Cluster Contains C-type Lectins with Differential Glycan-binding Specificity.

    PubMed

    Huang, Ya-Lang; Pai, Feng-Shuo; Tsou, Yun-Ting; Mon, Hsien-Chen; Hsu, Tsui-Ling; Wu, Chung-Yi; Chou, Teh-Ying; Yang, Wen-Bin; Chen, Chung-Hsuan; Wong, Chi-Huey; Hsieh, Shie-Liang

    2015-08-28

    The human C-type lectin 18 (clec18) gene cluster, which contains three clec18a, clec18b, and clec18c loci, is located in human chromosome 16q22. Although the amino acid sequences of CLEC18A, CLEC18B, and CLEC18C are almost identical, several amino acid residues located in the C-type lectin-like domain (CTLD) and the sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain, also known as the cysteine-rich secretory proteins/antigen 5/pathogenesis-related 1 proteins (CAP) domain, are distinct from each other. Genotyping by real-time PCR and sequencing further shows the presence of multiple alleles in clec18a/b/c loci. Flow cytometry analysis demonstrates that CLEC18 (CLEC18A, -B, and -C) are expressed abundantly in human peripheral blood cells. Moreover, CLEC18 expression is further up-regulated when monocytes differentiate into macrophages and dendritic cells. Immunofluorescence staining reveals that CLEC18 are localized in the endoplasmic reticulum, Golgi apparatus, and endosome. Interestingly, CLEC18 are also detectable in human sera and culture supernatants from primary cells and 293T cells overexpressing CLEC18. Moreover, CLEC18 bind polysaccharide in Ca(2+)-independent manner, and amino acid residues Ser/Arg(339) and Asp/Asn(421) in CTLD domain contribute to their differential binding abilities to polysaccharides isolated from Ganoderma lucidum (GLPS-F3). The Ser(339) (CLEC18A) → Arg(339) (CLEC18A-1) mutation completely abolishes CLEC18A-1 binding to GLPS-F3, and a sugar competition assay shows that CLEC18 preferentially binds to fucoidan, β-glucans, and galactans. Because proteins with the SCP/TAPS/CAP domain are able to bind sterol and acidic glycolipid, and are involved in sterol transport and β-amyloid aggregation, it would be interesting to investigate whether CLEC18 modulates host immunity via binding to glycolipids, and are also involved in glycolipid transportation and protein aggregation in the future. PMID:26170455

  12. Fungal engagement of the C-type lectin mincle suppresses dectin-1-induced antifungal immunity.

    PubMed

    Wevers, Brigitte A; Kaptein, Tanja M; Zijlstra-Willems, Esther M; Theelen, Bart; Boekhout, Teun; Geijtenbeek, Teunis B H; Gringhuis, Sonja I

    2014-04-01

    Recognition of fungal pathogens by C-type lectin receptor (CLR) dectin-1 on human dendritic cells is essential for triggering protective antifungal TH1 and TH17 immune responses. We show that Fonsecaea monophora, a causative agent of chromoblastomycosis, a chronic fungal skin infection, evades these antifungal responses by engaging CLR mincle and suppressing IL-12, which drives TH1 differentiation. Dectin-1 triggering by F. monophora activates transcription factor IRF1, which is crucial for IL12A transcription via nucleosome remodeling. However, simultaneous F. monophora binding to mincle induces an E3 ubiquitin ligase Mdm2-dependent degradation pathway, via Syk-CARD9-mediated PKB signaling, that leads to loss of nuclear IRF1 activity, hence blocking IL12A transcription. The absence of IL-12 leads to impaired TH1 responses and promotes TH2 polarization. Notably, mincle is similarly exploited by other chromoblastomycosis-associated fungi to redirect TH responses. Thus, mincle is a fungal receptor that can suppress antifungal immunity and, as such, is a potential therapeutic target. PMID:24721577

  13. Signalling versatility following self and non-self sensing by myeloid C-type lectin receptors

    PubMed Central

    Iborra, Salvador; Sancho, David

    2015-01-01

    Among myeloid immune receptors, C-type lectin receptors (CLRs) have a remarkable capacity to sense a variety of self and non-self ligands. The coupling of CLRs to different signal transduction modules is influenced not only by the receptor, but also by the nature, density and architecture of the ligand, which can affect the rate of receptor internalization and trafficking to diverse intracellular compartments. Understanding how the variety of self and non-self ligands triggers differential CLR signalling and function presents a fascinating biological challenge. Non-self ligands usually promote inflammation and immunity, whereas self ligands are frequently involved in communication and tolerance. But pathogens can mimic self-inhibitory signals to escape immune surveillance, and endogenous ligands can contribute to the sensing of pathogens through CLRs. In this review, we survey the complexity and flexibility in functional outcome found in the myeloid CLRs, which is not only based on their differing intracellular motifs, but is also conditioned by the physical nature, affinity and avidity of the ligand. PMID:25269828

  14. Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle

    PubMed Central

    Ishikawa, Eri; Ishikawa, Tetsuaki; Morita, Yasu S.; Toyonaga, Kenji; Yamada, Hisakata; Takeuchi, Osamu; Kinoshita, Taroh; Akira, Shizuo; Yoshikai, Yasunobu

    2009-01-01

    Tuberculosis remains a fatal disease caused by Mycobacterium tuberculosis, which contains various unique components that affect the host immune system. Trehalose-6,6′-dimycolate (TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of M. tuberculosis. Despite five decades of research on TDM, its host receptor has not been clearly identified. Here, we demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed mycobacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of the bacteria; analysis of the lipid extracts identified TDM as a Mincle ligand. TDM activated macrophages to produce inflammatory cytokines and nitric oxide, which are completely suppressed in Mincle-deficient macrophages. In vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and characteristic lung inflammation, such as granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-deficient mice. Whole mycobacteria were able to activate macrophages even in MyD88-deficient background, but the activation was significantly diminished in Mincle/MyD88 double-deficient macrophages. These results demonstrate that Mincle is an essential receptor for the mycobacterial glycolipid, TDM. PMID:20008526

  15. Differential expression of skin mucus C-type lectin in two freshwater eel species, Anguilla marmorata and Anguilla japonica.

    PubMed

    Tsutsui, Shigeyuki; Yoshinaga, Tatsuki; Komiya, Kaoru; Yamashita, Hiroka; Nakamura, Osamu

    2016-08-01

    Two types of lactose-specific lectins, galectin (AJL-1) and C-type lectin (AJL-2), were previously identified in the mucus of adult Anguilla japonica. Here, we compared the expression profiles of these two homologous lectins at the adult and juvenile stages between the tropical eel Anguilla marmorata and the temperate eel A. japonica. Only one lectin, predicted to be an orthologue of AJL-1 by LC-MS/MS, was detected in the mucus of adult A. marmorata. We also found that an orthologous gene to AJL-2 was expressed at very low levels, or not at all, in the skin of adult A. marmorata. However, we detected the gene expression of an AJL-2-orthologue in the skin of juvenile A. marmorata, and a specific antibody also detected the lectin in the juvenile fish epidermis. These findings suggest that expression profiles of mucosal lectins vary during development as well as between species in the Anguilla genus. PMID:27026508

  16. Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

    PubMed Central

    Lear, Calli; Chen, Li; Yantosca, L. Michael; Scully, Corinne; Sarraju, Ashish; Sokolovska, Anna; Zariffard, M. Reza; Eisen, Damon P.; Mungall, Bruce A.; Kotton, Darrell N.; Omari, Amel; Huang, I-Chueh; Farzan, Michael; Takahashi, Kazue; Stuart, Lynda; Stahl, Gregory L.; Ezekowitz, Alan B.; Spear, Gregory T.; Olinger, Gene G.; Schmidt, Emmett V.; Michelow, Ian C.

    2013-01-01

    Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active

  17. Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors.

    PubMed

    Brudner, Matthew; Karpel, Marshall; Lear, Calli; Chen, Li; Yantosca, L Michael; Scully, Corinne; Sarraju, Ashish; Sokolovska, Anna; Zariffard, M Reza; Eisen, Damon P; Mungall, Bruce A; Kotton, Darrell N; Omari, Amel; Huang, I-Chueh; Farzan, Michael; Takahashi, Kazue; Stuart, Lynda; Stahl, Gregory L; Ezekowitz, Alan B; Spear, Gregory T; Olinger, Gene G; Schmidt, Emmett V; Michelow, Ian C

    2013-01-01

    Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active

  18. C-type Lectin Receptor Expression on Human Basophils and Effects of Allergen-Specific Immunotherapy.

    PubMed

    Lundberg, K; Rydnert, F; Broos, S; Andersson, M; Greiff, L; Lindstedt, M

    2016-09-01

    Basophils are emerging as immunoregulatory cells capable of interacting with their environment not only via their characteristic IgE-mediated activation, but also in an IgE-independent manner. Basophils are known to express and respond to stimulation via TLR2, TLR4, DC-SIGN and DCIR, but whether basophils also express other C-type lectin receptors (CLRs) is largely unknown. In this study, we investigate the CLR expression profile of human basophils using multicolour flow cytometry. As FcRs as well as some CLRs are associated with allergen recognition and shown to be involved in subsequent immune responses, the expression of CLRs and FcRs on peripheral blood basophils, as well as their frequency, was monitored for 1 year in subjects undergoing subcutaneous allergen-specific immunotherapy (AIT). Here, we show that human basophils express CLECSF14, DEC205, Dectin-1, Dectin-2 and MRC2. Furthermore, we demonstrate that the frequencies of basophils expressing the allergy-associated CLRs Dectin-1 and Dectin-2 were significantly reduced after 1 year and 8 weeks of AIT, respectively. In contrast, the frequency of basophils positive for FcγRII, as well as the fraction of total basophils, significantly increased after 1 year of AIT. The herein demonstrated expression of various CLRs on basophils, and their altered CLR and FcR expression profile upon AIT, suggest yet unexplored ways by which basophils can interact with antigens and may point to novel immunoregulatory functions targeted through AIT. PMID:27354239

  19. A Shrimp C-type Lectin Inhibits Proliferation of the Hemolymph Microbiota by Maintaining the Expression of Antimicrobial Peptides*

    PubMed Central

    Wang, Xian-Wei; Xu, Ji-Dong; Zhao, Xiao-Fan; Vasta, Gerardo Raul; Wang, Jin-Xing

    2014-01-01

    Some aquatic invertebrates such as shrimp contain low albeit stable numbers of bacteria in the circulating hemolymph. The proliferation of this hemolymph microbiota in such a nutrient-rich environment is tightly controlled in healthy animals, but the mechanisms responsible had remained elusive. In the present study, we report a C-type lectin (MjHeCL) from the kuruma shrimp (Marsupenaeus japonicus) that participates in restraining the hemolymph microbiota. Although the expression of MjHeCL did not seem to be modulated by bacterial challenge, the down-regulation of its expression by RNA interference led to proliferation of the hemolymph microbiota, ultimately resulting in shrimp death. This phenotype was rescued by the injection of recombinant MjHeCL, which restored the healthy status of the knockdown shrimp. A mechanistic analysis revealed that MjHeCL inhibited bacterial proliferation by modulating the expression of antimicrobial peptides. The key function of MjHeCL in the shrimp immune homeostasis might be related to its broader recognition spectrum of the hemolymph microbiota components than other lectins. Our study demonstrates the role of MjHeCL in maintaining the healthy status of shrimp and provides new insight into the biological significance of C-type lectins, a diversified and abundant lectin family in invertebrate species. PMID:24619414

  20. A four-CRD C-type lectin from Chlamys farreri mediating nonself-recognition with broader spectrum and opsonization.

    PubMed

    Huang, Mengmeng; Wang, Lingling; Yang, Jialong; Zhang, Huan; Wang, Leilei; Song, Linsheng

    2013-04-01

    C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins consisting of at least one carbohydrate-recognition domain (CRD), which participate in nonself-recognition and clearance of invaders. In invertebrate, some multidomain C-type lectins have been identified, but their relative functions and binding mechanism are still meager. In the present study, A C-type lectin (CfLec-4) with four CRDs from Chlamys farreri was selected to investigate its possible function in innate immunity. The mRNA expression of CfLec-4 in hemocytes was significantly up-regulated (P<0.01) after the stimulations of β-glucan, LPS or PGN, and reached the highest expression level at 3, 6, 12 h post-stimulation, which was 27.9-, 22.6- or 47.9-fold of that in blank group, respectively. Immunohistochemistry assay with polyclonal antibody specific for CfLec-4 revealed that the endogenous CfLec-4 was mainly located in the hepatopancreas, kidney and gonad of the scallops. The recombinant CfLec-4 (rCflec-4) could bind LPS, PGN, glucan and mannose in vitro, but could not bind LTA. Furthermore, rCflec-4 displayed a broader bacteria binding spectrum towards Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus as well as Gram-negative bacteria Escherichia coli, Vibrio anguillarum and fungi Pichia pastoris. Meanwhile, rCfLec-4 could significantly (P<0.01) enhance the phagocytosis of hemocytes in vitro. The results clearly suggested that four-CRD containing CfLec-4 not only served as PRR with wider recognition spectrum, but also functioned as an opsonin participating in the clearance of invaders in scallops. It could be inferred that the diversity and complexity of CRDs in C-type lectins endowed these receptors with comprehensive recognition spectrum and multiple immune functions against complex living environment. PMID:23276881

  1. Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea)

    PubMed Central

    Ao, Jingqun; Ding, Yang; Chen, Yuanyuan; Mu, Yinnan; Chen, Xinhua

    2015-01-01

    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). Ca2+ 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 Ca2+-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. PMID:26690423

  2. Collaboration between a soluble C-type lectin and calreticulin facilitates white spot syndrome virus infection in shrimp.

    PubMed

    Wang, Xian-Wei; Xu, Yi-Hui; Xu, Ji-Dong; Zhao, Xiao-Fan; Wang, Jin-Xing

    2014-09-01

    White spot syndrome virus (WSSV) mainly infects crustaceans through the digestive tract. Whether C-type lectins (CLs), which are important receptors for many viruses, participate in WSSV infection in the shrimp stomach remains unknown. In this study, we orally infected kuruma shrimp Marsupenaeus japonicus to model the natural transmission of WSSV and identified a CL (designated as M. japonicus stomach virus-associated CL [MjsvCL]) that was significantly induced by virus infection in the stomach. Knockdown of MjsvCL expression by RNA interference suppressed the virus replication, whereas exogenous MjsvCL enhanced it. Further analysis by GST pull-down and coimmunoprecipitation showed that MjsvCL could bind to viral protein 28, the most abundant and functionally relevant envelope protein of WSSV. Furthermore, cell-surface calreticulin was identified as a receptor of MjsvCL, and the interaction between these proteins was a determinant for the viral infection-promoting activity of MjsvCL. The MjsvCL-calreticulin pathway facilitated virus entry likely in a cholesterol-dependent manner. This study provides insights into a mechanism by which soluble CLs capture and present virions to the cell-surface receptor to facilitate viral infection. PMID:25070855

  3. A C-type lectin with an immunoglobulin-like domain promotes phagocytosis of hemocytes in crayfish Procambarus clarkii

    PubMed Central

    Zhang, Xiao-Wen; Wang, Yue; Wang, Xian-Wei; Wang, Lei; Mu, Yi; Wang, Jin-Xing

    2016-01-01

    C-type lectins are important immune molecules that participate in host defense response. The present work reports a novel C-type lectin (PcLec3) from the red swamp crayfish Procambarus clarkii. Sequence analysis found that PcLec3 encodes a polypeptide with252 amino acid residues, which contains an immunoglobulin-like domain (IG) and a C-type lectin domain (CTLD) arranged in tandem. Tissue distribution analysis indicated that PcLec3 is enriched expressed in hemocytes and hepatopancreas cells, in which PcLec3 was up-regulated following bacterial challenge by Vibrio anguillarum. Function analysis using recombinant full-length PcLec3, IG, and CTLD proteins revealed that these recombinant proteins had the capacity to bind carbohydrates and bacteria, while IG determined the cell binding activity. However, only full-length PcLec3 promotes the phagocytic activity of hemocytes and subsequent clearance of invasive bacteria. Taken together, these results manifest that PcLec3 acts as a hemocyte adhesion molecule to promote hemocyte phagocytosis against invasive V. anguillarum. PMID:27411341

  4. A C-type lectin with an immunoglobulin-like domain promotes phagocytosis of hemocytes in crayfish Procambarus clarkii.

    PubMed

    Zhang, Xiao-Wen; Wang, Yue; Wang, Xian-Wei; Wang, Lei; Mu, Yi; Wang, Jin-Xing

    2016-01-01

    C-type lectins are important immune molecules that participate in host defense response. The present work reports a novel C-type lectin (PcLec3) from the red swamp crayfish Procambarus clarkii. Sequence analysis found that PcLec3 encodes a polypeptide with252 amino acid residues, which contains an immunoglobulin-like domain (IG) and a C-type lectin domain (CTLD) arranged in tandem. Tissue distribution analysis indicated that PcLec3 is enriched expressed in hemocytes and hepatopancreas cells, in which PcLec3 was up-regulated following bacterial challenge by Vibrio anguillarum. Function analysis using recombinant full-length PcLec3, IG, and CTLD proteins revealed that these recombinant proteins had the capacity to bind carbohydrates and bacteria, while IG determined the cell binding activity. However, only full-length PcLec3 promotes the phagocytic activity of hemocytes and subsequent clearance of invasive bacteria. Taken together, these results manifest that PcLec3 acts as a hemocyte adhesion molecule to promote hemocyte phagocytosis against invasive V. anguillarum. PMID:27411341

  5. Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

    PubMed

    Léger, Psylvia; Tetard, Marilou; Youness, Berthe; Cordes, Nicole; Rouxel, Ronan N; Flamand, Marie; Lozach, Pierre-Yves

    2016-06-01

    Bunyaviruses represent a growing threat to humans and livestock globally. The receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely unidentified and poorly characterized. DC-SIGN is a C-type lectin highly expressed on dermal dendritic cells that has been found to act as an authentic entry receptor for many phleboviruses (Bunyaviridae), including Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Uukuniemi virus (UUKV). We found that these phleboviruses can exploit another C-type lectin, L-SIGN, for infection. L-SIGN shares 77% sequence homology with DC-SIGN and is expressed on liver sinusoidal endothelial cells. L-SIGN is required for UUKV binding but not for virus internalization. An endocytosis-defective mutant of L-SIGN was still able to mediate virus uptake and infection, indicating that L-SIGN acts as an attachment receptor for phleboviruses rather than an endocytic receptor. Our results point out a fundamental difference in the use of the C-type lectins L-SIGN and DC-SIGN by UUKV to enter cells, although both proteins are closely related in terms of molecular structure and biological function. This study sheds new light on the molecular mechanisms by which phleboviruses target the liver and also highlights the added complexity in virus-receptor interactions beyond attachment. PMID:26990254

  6. Mechanistic Insights into the Role of C-Type Lectin Receptor/CARD9 Signaling in Human Antifungal Immunity.

    PubMed

    Drummond, Rebecca A; Lionakis, Michail S

    2016-01-01

    Human CARD9 deficiency is an autosomal recessive primary immunodeficiency disorder caused by biallelic mutations in the gene CARD9, which encodes a signaling protein that is found downstream of many C-type lectin receptors (CLRs). CLRs encompass a large family of innate recognition receptors, expressed predominantly by myeloid and epithelial cells, which bind fungal carbohydrates and initiate antifungal immune responses. Accordingly, human CARD9 deficiency is associated with the spontaneous development of persistent and severe fungal infections that primarily localize to the skin and subcutaneous tissue, mucosal surfaces and/or central nervous system (CNS). In the last 3 years, more than 15 missense and nonsense CARD9 mutations have been reported which associate with the development of a wide spectrum of fungal infections caused by a variety of fungal organisms. The mechanisms by which CARD9 provides organ-specific protection against these fungal infections are now emerging. In this review, we summarize recent immunological and clinical advances that have provided significant mechanistic insights into the pathogenesis of human CARD9 deficiency. We also discuss how genetic mutations in CARD9-coupled receptors (Dectin-1, Dectin-2) and CARD9-binding partners (MALT1, BCL10) affect human antifungal immunity relative to CARD9 deficiency, and we highlight major understudied research questions which merit future investigation. PMID:27092298

  7. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    SciTech Connect

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.; Garboczi, David N.

    2010-11-03

    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

  8. Mechanistic Insights into the Role of C-Type Lectin Receptor/CARD9 Signaling in Human Antifungal Immunity

    PubMed Central

    Drummond, Rebecca A.; Lionakis, Michail S.

    2016-01-01

    Human CARD9 deficiency is an autosomal recessive primary immunodeficiency disorder caused by biallelic mutations in the gene CARD9, which encodes a signaling protein that is found downstream of many C-type lectin receptors (CLRs). CLRs encompass a large family of innate recognition receptors, expressed predominantly by myeloid and epithelial cells, which bind fungal carbohydrates and initiate antifungal immune responses. Accordingly, human CARD9 deficiency is associated with the spontaneous development of persistent and severe fungal infections that primarily localize to the skin and subcutaneous tissue, mucosal surfaces and/or central nervous system (CNS). In the last 3 years, more than 15 missense and nonsense CARD9 mutations have been reported which associate with the development of a wide spectrum of fungal infections caused by a variety of fungal organisms. The mechanisms by which CARD9 provides organ-specific protection against these fungal infections are now emerging. In this review, we summarize recent immunological and clinical advances that have provided significant mechanistic insights into the pathogenesis of human CARD9 deficiency. We also discuss how genetic mutations in CARD9-coupled receptors (Dectin-1, Dectin-2) and CARD9-binding partners (MALT1, BCL10) affect human antifungal immunity relative to CARD9 deficiency, and we highlight major understudied research questions which merit future investigation. PMID:27092298

  9. Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif.

    PubMed

    Hatakeyama, Tomomitsu; Kamiya, Takuro; Kusunoki, Masami; Nakamura-Tsuruta, Sachiko; Hirabayashi, Jun; Goda, Shuichiro; Unno, Hideaki

    2011-03-25

    CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity. PMID:21247895

  10. The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus

    PubMed Central

    Ng, Wy Ching; Londrigan, Sarah L.; Nasr, Najla; Cunningham, Anthony L.; Turville, Stuart; Brooks, Andrew G.

    2015-01-01

    ABSTRACT It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5+) but not late (Rab7+) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. IMPORTANCE On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of

  11. CfLec-3 from scallop: an entrance to non-self recognition mechanism of invertebrate C-type lectin

    PubMed Central

    Yang, Jialong; Huang, Mengmeng; Zhang, Huan; Wang, Lingling; Wang, Hao; Wang, Leilei; Qiu, Limei; Song, Linsheng

    2015-01-01

    A C-type lectin (CfLec-3) from Chlamys farreri with three carbohydrate-recognition domains (CRDs) was selected to dissect the possible mechanisms of PAMP binding and functional differentiation of invertebrate lectins. CfLec-3 distributed broadly, and its mRNA expression in hemocytes increased significantly after stimulations with LPS, PGN or β-glucan, but not poly(I:C). The recombinant CfLec-3 (rCfLec-3) could bind PAMPs and several microbes. rCfLec-3 mediated hemocytes phagocytosis against Escherichia coli and encapsulation towards agarose beads. Obvious functional differentiation occurred among the three CRDs, as CRD1 exhibited higher activity to bind PAMPs, while CRD2/3 were expert in promoting hemocyte mediated opsonisation. The tertiary structural differences were suspected to be associated with such functional differentiation. PAMP binding abilities of CfLec-3 were determined by Ca2+-binding site 2 motif. When Pro in this motif of each CRD was mutated into Ser, their PAMP binding abilities were deprived absolutely. rCRD2 acquired mannan binding capability when its EPD was replaced by EPN, but lost when EPN in rCRD3 was changed into EPD. The Pro in Ca2+-binding site 2 was indispensable for PAMPs binding, while Asn was determinant for specific binding to mannan. It shed new insight into PAMPs binding mechanism of invertebrate C-type lectins and their functional differentiation. PMID:25975813

  12. Identification and Characterization of a Spliced C-Type Lectin-Like Gene Encoded by Rat Cytomegalovirus

    PubMed Central

    Voigt, Sebastian; Sandford, Gordon R.; Ding, Lijun; Burns, William H.

    2001-01-01

    The English isolate of rat cytomegalovirus (RCMV) encodes a 20-kDa protein with a C-type lectin-like domain that is expressed in the delayed-early and late phases of the viral replication cycle. Genomic sequence analysis of the restriction fragment KpnR of RCMV revealed significant homology to several C-type lectin-containing molecules implicated in natural killer (NK) and T-cell interactions, as well as genes from four poxviruses and African swine fever virus. The gene is spliced into five exons and shows a splicing pattern with exon boundaries similar to those observed in the human differentiation antigen CD69. The cap site of the gene was mapped by RNase protection, 5′ rapid amplification of cDNA ends, and primer extension experiments. This analysis demonstrated that the core promoter of the RCMV lectin-like gene contains a GATA rather than a TATA box. Splicing patterns were confirmed with isolates from an infected-cell cDNA library. A unique aspect of the protein is that its translation is not initiated by the canonical methionine but rather by alanine. To study its role in virus replication and pathogenesis, a recombinant virus was constructed in which the gene is interrupted. Replication in tissue culture was similar to that of wild-type virus. PMID:11134273

  13. Ophioluxin, a convulxin-like C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI.

    PubMed

    Du, Xiao-Yan; Clemetson, Jeannine M; Navdaev, Alexei; Magnenat, Edith M; Wells, Timothy N C; Clemetson, Kenneth J

    2002-09-20

    Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family. PMID:12130642

  14. Isolation and characterization of two novel C-type lectins from the oriental river prawn, Macrobrachium nipponense.

    PubMed

    Xiu, Yunji; Hou, Libo; Liu, Xiaoqian; Wang, Yinghui; Gu, Wei; Meng, Qingguo; Wang, Wen

    2015-10-01

    C-type lectins are a family of calcium-dependent carbohydrate-binding proteins which are believed to play important roles in the innate immunity of invertebrates. This study identified two novel C-type lectins, designated as MnCTLDcp2 and MnCTLDcp3, from the oriental river prawn Macrobrachium nipponense. The full-length cDNA of MnCTLDcp2 was of 1582 bp with an open reading frame (ORF) of 972 bp encoding a polypeptide of 323 amino acids. The complete nucleotide sequence of MnCTLDcp3 cDNA was 583 bp, containing a 555 bp ORF encoding a putative protein of 184 deduced amino acids. The deduced MnCTLDcp2 and MnCTLDcp3 proteins both contained a single C-type lectin-like domain (CTLD). Besides, MnCTLDcp2 contains a signal peptide and an low-density lipoprotein receptor class A (LDLa) domain. Reverse transcription PCR showed that MnCTLDcp2 was expressed in the heart, gill, nerve hepatopancreas and intestine; MnCTLDcp3 was expressed in the hepatopancreas, heart, nerve, gill and muscle. Their expression in the heart tissue was regulated following challenge with bacteria. The microbial agglutination assay showed that both MnCTLDcp2 and MnCTLDcp3 could agglutinated bacteria in the presence of calcium. All these results suggested that MnCTLDcp2 and MnCTLDcp3 functioned as pattern recognition receptors in the immune system of M. nipponense. PMID:26208755

  15. A C-type lectin MGL1/CD301a plays an anti-inflammatory role in murine experimental colitis.

    PubMed

    Saba, Kengo; Denda-Nagai, Kaori; Irimura, Tatsuro

    2009-01-01

    Inflammatory bowel disease is caused by abnormal inflammatory and immune responses to harmless substances, such as commensal bacteria, in the large bowel. Such responses appear to be suppressed under healthy conditions, although the mechanism of such suppression is currently unclear. The present study aimed to reveal whether the recognition of bacterial surface carbohydrates by the macrophage galactose-type C-type lectin-1, MGL1/CD301a, induces both the production and secretion of interleukin (IL)-10. Dextran sulfate sodium salt (DSS) was orally administrated to mice that lacked MGL1/CD301a (Mgl1(-/-) mice) and their wild-type littermates. Mgl1(-/-) mice showed significantly more severe inflammation than wild-type mice after administration of DSS. MGL1-positive cells in the colonic lamina propria corresponded to macrophage-like cells with F4/80-high, CD11b-positive, and CD11c-intermediate expression. These cells in Mgl1(-/-) mice produced a lower level of IL-10 mRNA compared with wild-type mice after the administration of DSS for 2 days. Recombinant MGL1 was found to bind both Streptococcus sp. and Lactobacillus sp. among commensal bacteria isolated from mesenteric lymph nodes of DSS-treated mice. Heat-killed Streptococcus sp. induced an increase in IL-10 secretion by MGL1-positive colonic lamina propria macrophages, but not the macrophage population from Mgl1(-/-) mice. These results strongly suggest that MGL1/CD301a plays a protective role against colitis by effectively inducing IL-10 production by colonic lamina propria macrophages in response to invading commensal bacteria. PMID:19095961

  16. Abundant Expression of HIV Target Cells and C-Type Lectin Receptors in the Foreskin Tissue of Young Kenyan Men

    PubMed Central

    Hirbod, Taha; Bailey, Robert C.; Agot, Kawango; Moses, Stephen; Ndinya-Achola, Jeckoniah; Murugu, Ruth; Andersson, Jan; Nilsson, Jakob; Broliden, Kristina

    2010-01-01

    A biological explanation for the reduction in HIV-1 (HIV) acquisition after male circumcision may be that removal of the foreskin reduces the number of target cells for HIV. The expression of potential HIV target cells and C-type lectin receptors in foreskin tissue of men at risk of HIV infection were thus analyzed. Thirty-three foreskin tissue samples, stratified by Herpes simplex virus type 2 status, were obtained from a randomized, controlled trial conducted in Kenya. The samples were analyzed by confocal in situ imaging microscopy and mRNA quantification by quantitative RT-qPCR. The presence and location of T cells (CD3+CD4+), Langerhans cells (CD1a+Langerin/CD207+), macrophages (CD68+ or CD14+), and submucosal dendritic cells (CD123+BDCA-2+ or CD11c+DC-SIGN+) were defined. C-type lectin receptor expressing cells were detected in both the epithelium and submucosa, and distinct lymphoid aggregates densely populated with CD3+CD4+ T cells were identified in the submucosa. Although the presence of lymphoid aggregates and mRNA expression of selected markers varied between study subjects, Herpes simplex virus type 2 serostatus was not the major determinant for the detected differences. The detection of abundant and superficially present potential HIV target cells and submucosal lymphoid aggregates in foreskin mucosa from a highly relevant HIV risk group demonstrate a possible anatomical explanation that may contribute to the protective effect of male circumcision on HIV transmission. PMID:20395432

  17. C-type lectin receptors differentially induce th17 cells and vaccine immunity to the endemic mycosis of North America.

    PubMed

    Wang, Huafeng; LeBert, Vanessa; Hung, Chiung Yu; Galles, Kevin; Saijo, Shinobu; Lin, Xin; Cole, Garry T; Klein, Bruce S; Wüthrich, Marcel

    2014-02-01

    Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors exert divergent contributions to the development of antifungal Th17 cells and vaccine resistance against Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidioides posadasii. Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking Ag-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival, and migration. Whereas Card9 signaling is essential, C-type lectin receptors offer distinct and divergent contributions to vaccine immunity against these endemic fungal pathogens. Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells. PMID:24391211

  18. SHP-2 Mediates C-type Lectin Receptors-induced Syk Activation and Anti-fungal TH17 Responses

    PubMed Central

    Deng, Zihou; Ma, Shixin; Zhou, Hao; Zang, Aiping; Fang, Yiyuan; Li, Tiantian; Shi, Huanjing; Liu, Mei; Du, Min; Taylor, Patricia R.; Zhu, Helen H.; Chen, Jiangye; Meng, Guangxun; Li, Fubin; Chen, Changbin; Zhang, Yan; Jia, Xin-Ming; Lin, Xin; Zhang, Xiaoming; Pearlman, Eric; Li, Xiaoxia; Feng, Gen-Sheng; Xiao, Hui

    2015-01-01

    SUMMARY Fungal infection stimulates the canonical C-type lectin receptors (CLRs) signaling pathway via Syk activation. Here we show that SHP-2 plays a crucial role in mediating CLRs-induced Syk activation. Genetic ablation of Shp-2 (Ptpn11) in dendritic cells (DCs) and macrophages impaired Syk-mediated signaling and abrogated pro-inflammatory gene expression following fungal stimulation. Mechanistically, SHP-2 operates as a scaffold facilitating the recruitment of Syk to dectin-1 or FcRγ, through its N-SH2 domain and a previously unrecognized C-terminal ITAM motif. We demonstrate that DC-derived SHP-2 is crucial for the induction of IL-1β, IL-6 and IL-23, and anti-fungal TH17 cell responses to control Candida albicans infection. Together, these data reveal a mechanism by which SHP-2 mediates Syk activation in response to fungal infections PMID:25915733

  19. Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin

    PubMed Central

    Sancho, David; Mourão-Sá, Diego; Joffre, Olivier P.; Schulz, Oliver; Rogers, Neil C.; Pennington, Daniel J.; Carlyle, James R.; Reis e Sousa, Caetano

    2008-01-01

    The mouse CD8α+ DC subset excels at cross-presentation of antigen, which can elicit robust CTL responses. A receptor allowing specific antigen targeting to this subset and its equivalent in humans would therefore be useful for the induction of antitumor CTLs. Here, we have characterized a C-type lectin of the NK cell receptor group that we named DC, NK lectin group receptor-1 (DNGR-1). DNGR-1 was found to be expressed in mice at high levels by CD8+ DCs and at low levels by plasmacytoid DCs but not by other hematopoietic cells. Human DNGR-1 was also restricted in expression to a small subset of blood DCs that bear similarities to mouse CD8α+ DCs. The selective expression pattern and observed endocytic activity of DNGR-1 suggested that it could be used for antigen targeting to DCs. Consistent with this notion, antigen epitopes covalently coupled to an antibody specific for mouse DNGR-1 were selectively cross-presented by CD8α+ DCs in vivo and, when given with adjuvants, induced potent CTL responses. When the antigens corresponded to tumor-expressed peptides, treatment with the antibody conjugate and adjuvant could prevent development or mediate eradication of B16 melanoma lung pseudometastases. We conclude that DNGR-1 is a novel, highly specific marker of mouse and human DC subsets that can be exploited for CTL cross-priming and tumor therapy. PMID:18497879

  20. Molecular cloning and characterization of a C-type lectin from Ancylostoma ceylanicum: evidence for a role in hookworm reproductive physiology.

    PubMed Central

    Brown, Allison C.; Harrison, Lisa M.; Kapulkin, Wadim; Jones, Brian F.; Sinha, Anindita; Savage, Amy; Villalon, Nicholas; Cappello, Michael

    2007-01-01

    Lectins comprise a family of related proteins that mediate essential cell functions through binding to carbohydrates. Within this protein family, C-type lectins are defined by the requirement of calcium for optimal biologic activity. Using reverse transcription PCR, a cDNA corresponding to a putative C-type lectin has been amplified from the hookworm parasite Ancylostoma ceylanicum. The 550 nucleotide open reading frame of the Ancylostoma ceylanicum C-type Lectin-1 (AceCTL-1) cDNA corresponds to a 167 amino acid mature protein (18706 Da) preceded by a 17 amino acid secretory signal sequence. The recombinant protein (rAceCTL-1) was expressed in Drosophila S2 cells and purified using a combination of affinity chromatography and reverse phase HPLC. Using in vitro carbohydrate binding studies, it was determined that rAceCTL-1 binds N-acetyl-D-glucosamine, a common component of eukaryotic egg cell membranes. Using a polyclonal IgG raised against the recombinant protein, the native AceCTL-1 was identified in sperm and soluble protein extracts of adult male A. ceylanicum by immunoblot. Probing of adult hookworm sections with the polyclonal IgG demonstrated localization to the testes in males, as well as the spermatheca and developing embryos in females, consistent with its role as a sperm protein. Together, these data strongly suggest that AceCTL-1 is a male gender-specific C-type lectin with a function in hookworm reproductive physiology. PMID:17129620

  1. Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

    SciTech Connect

    Usami, Katsuaki; Matsuno, Keita; Igarashi, Manabu; Denda-Nagai, Kaori; Takada, Ayato; Irimura, Tatsuro

    2011-04-01

    Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.

  2. C-type lectin-like carbohydrate recognition of the hemolytic lectin CEL-III containing ricin-type -trefoil folds.

    PubMed

    Hatakeyama, Tomomitsu; Unno, Hideaki; Kouzuma, Yoshiaki; Uchida, Tatsuya; Eto, Seiichiro; Hidemura, Haruki; Kato, Norihisa; Yonekura, Masami; Kusunoki, Masami

    2007-12-28

    CEL-III is a Ca(2+)-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca(2+) located at the subdomains 1alpha, 1gamma, 2alpha, 2beta, and 2gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca(2+) ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2alpha and 2beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein. PMID:17977832

  3. Toll-Like Receptors and Dectin-1, a C-Type Lectin Receptor, Trigger Divergent Functions in CNS Macrophages

    PubMed Central

    Wang, Yan; Guan, Zhen; Beckwith, Kyle A.; Braun, Kaitlyn J.; Wei, Ping; McTigue, Dana M.

    2015-01-01

    Spinal cord injury (SCI) activates macrophages, endowing them with both reparative and pathological functions. The mechanisms responsible for these divergent functions are unknown but are likely controlled through stochastic activation of different macrophage receptor subtypes. Various danger-associated molecular patterns released from dying cells in the injured spinal cord likely activate distinct subtypes of macrophage pattern recognition receptors, including bacterial toll-like receptors (TLRs) and fungal C-type lectin receptors (e.g., dectin-1). To determine the in vivo consequences of activating these receptors, ligands specific for TLR2 or dectin-1 were microinjected, alone or in combination, into intact spinal cord. Both ligands elicit a florid macrophage reaction; however, only dectin-1 activation causes macrophage-mediated demyelination and axonal injury. Coactivating TLR2 reduced the injurious effects of dectin-1 activation. When injected into traumatically injured spinal cord, TLR2 agonists enhance the endogenous macrophage reaction while conferring neuroprotection. Indeed, dieback of axons was reduced, leading to smaller lesion volumes at the peak of the macrophage response. Moreover, the density of NG2+ cells expressing vimentin increased in and near lesions that were enriched with TLR2-activated macrophages. In dectin-1-null mutant (knock-out) mice, dieback of corticospinal tract axons also is reduced after SCI. Collectively, these data support the hypothesis that the ability of macrophages to create an axon growth-permissive microenvironment or cause neurotoxicity is receptor dependent and it may be possible to exploit this functional dichotomy to enhance CNS repair. SIGNIFICANCE STATEMENT There is a growing appreciation that macrophages exert diverse functions in the injured and diseased CNS. Indeed, both macrophage-mediated repair and macrophage-mediated injury occur, and often these effector functions are elicited simultaneously. Understanding the

  4. Super-Resolution Imaging of C-Type Lectin and Influenza Hemagglutinin Nanodomains on Plasma Membranes Using Blink Microscopy

    PubMed Central

    Itano, Michelle S.; Steinhauer, Christian; Schmied, Jürgen J.; Forthmann, Carsten; Liu, Ping; Neumann, Aaron K.; Thompson, Nancy L.; Tinnefeld, Philip; Jacobson, Ken

    2012-01-01

    Dendritic cells express DC-SIGN, a C-type lectin (CTL) that binds a variety of pathogens and facilitates their uptake for subsequent antigen presentation. DC-SIGN forms remarkably stable microdomains on the plasma membrane. However, inner leaflet lipid markers are able to diffuse through these microdomains suggesting that, rather than being densely packed with DC-SIGN proteins, an elemental substructure exists. Therefore, a super-resolution imaging technique, Blink Microscopy (Blink), was applied to further investigate the lateral distribution of DC-SIGN. Blink indicates that DC-SIGN, another CTL (CD206), and influenza hemagglutinin (HA) are all localized in small (∼80 nm in diameter) nanodomains. DC-SIGN and CD206 nanodomains are randomly distributed on the plasma membrane, whereas HA nanodomains cluster on length scales up to several microns. We estimate, as a lower limit, that DC-SIGN and HA nanodomains contain on average two tetramers or two trimers, respectively, whereas CD206 is often nonoligomerized. Two-color Blink determined that different CTLs rarely occupy the same nanodomain, although they appear colocalized using wide-field microscopy. What to our knowledge is a novel domain structure emerges in which elemental nanodomains, potentially capable of binding viruses, are organized in a random fashion; evidently, these nanodomains can be clustered into larger microdomains that act as receptor platforms for larger pathogens like yeasts. PMID:22500753

  5. Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

    SciTech Connect

    Le Coq, Johanne; Ghosh, Partho

    2012-06-19

    Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd ({approx}16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10{sup 20} potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.

  6. The C-Type Lectin Receptor MCL Mediates Vaccine-Induced Immunity against Infection with Blastomyces dermatitidis.

    PubMed

    Wang, Huafeng; Li, Mengyi; Lerksuthirat, Tassanee; Klein, Bruce; Wüthrich, Marcel

    2015-01-01

    C-type lectin receptors (CLRs) are essential in shaping the immune response to fungal pathogens. Vaccine-induced resistance requires Dectin-2 to promote differentiation of antifungal Th1 and Th17 cells. Since Dectin-2 and MCL heterodimerize and both CLRs use FcRγ as the signaling adaptor, we investigated the role of MCL in vaccine immunity to the fungal pathogen Blastomyces dermatitidis. MCL(-/-) mice showed impaired vaccine resistance against B. dermatitidis infection compared to that of wild-type animals. The lack of resistance correlated with the reduced recruitment of Th17 cells to the lung upon recall following experimental challenge and impaired interleukin-17 (IL-17) production by vaccine antigen-stimulated splenocytes in vitro. Soluble MCL fusion protein recognized and bound a water-soluble ligand from the cell wall of vaccine yeast, but the addition of soluble Dectin-2 fusion protein did not augment ligand recognition by MCL. Taken together, our data indicate that MCL regulates the development of vaccine-induced Th17 cells and protective immunity against lethal experimental infection with B. dermatitidis. PMID:26667836

  7. Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development.

    PubMed

    Yoshida, Shigeto; Shimada, Yohei; Kondoh, Daisuke; Kouzuma, Yoshiaki; Ghosh, Anil K; Jacobs-Lorena, Marcelo; Sinden, Robert E

    2007-12-01

    The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions. PMID:18159942

  8. The C-Type Lectin Receptor CLECSF8/CLEC4D Is a Key Component of Anti-Mycobacterial Immunity

    PubMed Central

    Wilson, Gillian J.; Marakalala, Mohlopheni J.; Hoving, Jennifer C.; van Laarhoven, Arjan; Drummond, Rebecca A.; Kerscher, Bernhard; Keeton, Roanne; van de Vosse, Esther; Ottenhoff, Tom H.M.; Plantinga, Theo S.; Alisjahbana, Bachti; Govender, Dhirendra; Besra, Gurdyal S.; Netea, Mihai G.; Reid, Delyth M.; Willment, Janet A.; Jacobs, Muazzam; Yamasaki, Sho; van Crevel, Reinout; Brown, Gordon D.

    2015-01-01

    Summary The interaction of microbes with pattern recognition receptors (PRRs) is essential for protective immunity. While many PRRs that recognize mycobacteria have been identified, none is essentially required for host defense in vivo. Here, we have identified the C-type lectin receptor CLECSF8 (CLEC4D, MCL) as a key molecule in anti-mycobacterial host defense. Clecsf8−/− mice exhibit higher bacterial burdens and increased mortality upon M. tuberculosis infection. Additionally, Clecsf8 deficiency is associated with exacerbated pulmonary inflammation, characterized by enhanced neutrophil recruitment. Clecsf8−/− mice show reduced mycobacterial uptake by pulmonary leukocytes, but infection with opsonized bacteria can restore this phagocytic defect as well as decrease bacterial burdens. Notably, a CLECSF8 polymorphism identified in humans is associated with an increased susceptibility to pulmonary tuberculosis. We conclude that CLECSF8 plays a non-redundant role in anti-mycobacterial immunity in mouse and in man. PMID:25674984

  9. Mitogenic activity of CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin, isolated from the marine invertebrate Cucumaria echinata (Holothuroidea).

    PubMed

    Jiang, Zedong; Kim, Daekyung; Yamasaki, Yasuhiro; Yamanishi, Tomohiro; Hatakeyama, Tomomitsu; Yamaguchi, Kenichi; Oda, Tatsuya

    2010-01-01

    An N-acetylgalactosamine (GalNAc)-specific Ca(2+)-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 microg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 microg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 microg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells. PMID:20699569

  10. Crystallization and preliminary crystallographic study of an invertebrate C-type lectin, CEL-I, from the marine invertebrate Cucumaria echinata.

    PubMed

    Hatakeyama, Tomomitsu; Matsuo, Noriaki; Aoyagi, Haruhiko; Sugawara, Hajime; Uchida, Tatsuya; Kurisu, Genji; Kusunoki, Masami

    2002-01-01

    CEL-I is a GalNAc-specific carbohydrate-binding protein (lectin) isolated from the sea cucumber Cucumaria echinata. This protein belongs to the widely distributed C-type lectin family of animal lectins, which require Ca(2+) for their carbohydrate-binding ability and play important roles in various molecular-recognition processes in organisms. CEL-I was crystallized with 2-methyl-2,4-pentanediol using the hanging-drop vapour-diffusion technique. The CEL-I crystals belong to the monoclinic space group C2, with unit-cell parameters a = 92.38 (3), b = 69.94 (3), c = 76.69 (3) A, beta = 136.46 (2) degrees. Diffraction data were collected to 2.0 A resolution using synchrotron radiation. The asymmetric unit contains one CEL-I molecule. PMID:11752793

  11. Novel sequences encoding venom C-type lectins are conserved in phylogenetically and geographically distinct Echis and Bitis viper species.

    PubMed

    Harrison, R A; Oliver, J; Hasson, S S; Bharati, K; Theakston, R D G

    2003-10-01

    Envenoming by Echis saw scaled vipers and Bitis arietans puff adders is the leading cause of death and morbidity in Africa due to snake bite. Despite their medical importance, the composition and constituent functionality of venoms from these vipers remains poorly understood. Here, we report the cloning of cDNA sequences encoding seven clusters or isoforms of the haemostasis-disruptive C-type lectin (CTL) proteins from the venom glands of Echis ocellatus, E. pyramidum leakeyi, E. carinatus sochureki and B. arietans. All these CTL sequences encoded the cysteine scaffold that defines the carbohydrate-recognition domain of mammalian CTLs. All but one of the Echis and Bitis CTL sequences showed greater sequence similarity to the beta than alpha CTL subunits in venoms of related Asian and American vipers. Four of the new CTL clusters showed marked inter-cluster sequence conservation across all four viper species which were significantly different from that of previously published viper CTLs. The other three Echis and Bitis CTL clusters showed varying degrees of sequence similarity to published viper venom CTLs. Because viper venom CTLs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis and Bitis CTLs on the basis of sequence alone. The extraordinary level of inter-specific and inter-generic sequence conservation exhibited by the Echis and Bitis CTLs leads us to speculate that antibodies to representative molecules should neutralise the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East and the Indian subcontinent. PMID:14557069

  12. Cytotoxicity of a GalNAc-specific C-type lectin CEL-I toward various cell lines.

    PubMed

    Kuramoto, Takuya; Uzuyama, Hitomi; Hatakeyama, Tomomitsu; Tamura, Tadashi; Nakashima, Takuji; Yamaguchi, Kenichi; Oda, Tatsuya

    2005-01-01

    We found that CEL-I was a potent cytotoxic lectin. MDCK, HeLa, and XC cells were highly sensitive to CEL-I cytotoxicity and killed in a dose-dependent manner, whereas CHO, L929, and RAW264.7 cells were relatively resistant to CEL-I, and no significant toxicity was observed up to 10 microg/ml. Among these cell lines, MDCK cells showed the highest susceptibility to CEL-I cytotoxicity. A binding study using FITC-labeled CEL-I (F-CEL-I) revealed that the amounts of bound F-CEL-I on the sensitive cell lines were evidently greater than those on the resistant cell lines, suggesting that the different susceptibility of the cell lines to CEL-I cytotoxicity is partly explained by different efficiencies of binding of CEL-I to these cell lines. Interestingly, the cytotoxicity of CEL-I toward MDCK cells was more potent than those of other lectins such as WGA, PHA-L, and Con A, even though these lectins were capable of binding to MDCK cells at comparable levels to CEL-I. Since the cytotoxicity of CEL-I was strongly inhibited by GalNAc, the binding to cell surface specific carbohydrates is essential for the CEL-I cytotoxicity. The trypan blue dye exclusion test indicated that CEL-I caused a disorder of plasma membrane integrity as a relatively early event. CEL-I failed to induce the release of carboxyfluorescein (CF) from CF-loaded MDCK cells as seen for pore-forming hemolytic isolectin CEL-III, suggesting that the primary cellular target of CEL-I may be the plasma membrane, but its action mechanism differs from that of CEL-III. Although CEL-I induced dramatic cellular morphological changes in MDCK cells, neither typical apoptotic nuclear morphological changes nor DNA fragmentation was observed in CEL-I-treated MDCK cells even after such cellular changes. Our results demonstrated that CEL-I showed a potent cytotoxic effect, especially on MDCK cells, by causing plasma membrane disorder without induction of apoptosis. PMID:15713882

  13. CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin, induces nitric oxide production in RAW264.7 mouse macrophage cell line.

    PubMed

    Yamanishi, Tomohiro; Hatakeyama, Tomomitsu; Yamaguchi, Kenichi; Oda, Tatsuya

    2009-08-01

    We found that CEL-I, a GalNAc-specific C-type lectin isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), induces inducible nitric oxide synthase (iNOS) expression and NO production in RAW264.7 cells. The NO production was inhibited by an iNOS inhibitor, L-NAME, but was not by a lipopolysaccharide (LPS) inhibitor, polymyxin B. In the presence of 0.1-M GalNAc, increased NO production by CEL-I-treated RAW264.7 cells was observed rather than the inhibition. Bovine serum albumin (BSA) significantly inhibited the CEL-I-induced NO production as well as the binding of FITC-labelled CEL-I on RAW264.7 cells. Three MAP kinase inhibitors (specific to extra-cellular regulated kinase, c-jun NH(2)-terminal kinase and p38 MAP kinase) inhibited CEL-I-induced NO production with different extents. Heat-treatment of CEL-I resulted in a decreased activity of CEL-I depending on the temperature. These results suggest that CEL-I induces NO production in RAW264.7 cells through the protein-cell interaction rather than the binding to the specific carbohydrate chains on the cell surface. PMID:19351706

  14. 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. PMID:10873458

  15. A novel C-type lectin with four CRDs is involved in the regulation of antimicrobial peptide gene expression in Hyriopsis cumingii.

    PubMed

    Zhao, Ling-Ling; Wang, Yu-Qing; Dai, Yun-Jia; Zhao, Li-Juan; Qin, Qiwei; Lin, Li; Ren, Qian; Lan, Jiang-Feng

    2016-08-01

    C-type lectins (CTLs) are found in a wide number of invertebrates, and have been reported to participate in immune responses, such as the activation of prophenoloxidase, cell adhesion, bacterial clearance and phagocytosis. Previous studies on CTLs focused on the function of their carbohydrate recognition domains (CRDs). Currently, studies on lectins with multi-CRDs are limited. In this study, a lectin with four CRDs was cloned from Hyriopsis cumingii, and called HcLec4. HcLec4 was widely distributed in several tissues and was significantly down-regulated at the early stage (2 h) of bacterial infection. We further analyzed the bacteria and carbohydrate binding activities of HcLec4. The results showed that HcLec4 could bind to several bacteria, lipopolysaccharide (LPS) and peptidoglycan (PGN). In HcLec4 knockdown mussels, the bacterial clearance rate was increased, and the expression level of antimicrobial peptides (AMPs) was up-regulated. This study reveals that HcLec4 exerts its antibacterial effect by regulating the expression of AMPs at the early stage of bacterial infection. PMID:27288254

  16. Genetic variability in the rat Aplec C-type lectin gene cluster regulates lymphocyte trafficking and motor neuron survival after traumatic nerve root injury

    PubMed Central

    2013-01-01

    Background C-type lectin (CLEC) receptors are important for initiating and shaping immune responses; however, their role in inflammatory reactions in the central nervous system after traumatic injuries is not known. The antigen-presenting lectin-like receptor gene complex (Aplec) contains a few CLEC genes, which differ genetically among inbred rat strains. It was originally thought to be a region that regulates susceptibility to autoimmune arthritis, autoimmune neuroinflammation and infection. Methods The inbred rat strains DA and PVG differ substantially in degree of spinal cord motor neuron death following ventral root avulsion (VRA), which is a reproducible model of localized nerve root injury. A large F2 (DAxPVG) intercross was bred and genotyped after which global expressional profiling was performed on spinal cords from F2 rats subjected to VRA. A congenic strain, Aplec, created by transferring a small PVG segment containing only seven genes, all C-type lectins, ontoDA background, was used for further experiments together with the parental strains. Results Global expressional profiling of F2 (DAxPVG) spinal cords after VRA and genome-wide eQTL mapping identified a strong cis-regulated difference in the expression of Clec4a3 (Dcir3), a C-type lectin gene that is a part of the Aplec cluster. Second, we demonstrate significantly improved motor neuron survival and also increased T-cell infiltration into the spinal cord of congenic rats carrying Aplec from PVG on DA background compared to the parental DA strain. In vitro studies demonstrate that the Aplec genes are expressed on microglia and upregulated upon inflammatory stimuli. However, there were no differences in expression of general microglial activation markers between Aplec and parental DA rats, suggesting that the Aplec genes are involved in the signaling events rather than the primary activation of microglia occurring upon nerve root injury. Conclusions In summary, we demonstrate that a genetic variation

  17. E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation.

    PubMed

    Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin

    2016-10-01

    C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response. PMID:27443248

  18. 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. PMID:23157284

  19. The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans.

    PubMed

    Ludington, Jacob G; Ward, Honorine D

    2016-05-01

    The apicomplexan parasite Cryptosporidium causes significant diarrheal disease worldwide. Effective anticryptosporidial agents are lacking, in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood. Previously, we identified and characterized a novel Cryptosporidium parvum C-type lectin domain-containing mucin-like glycoprotein, CpClec. In this study, we evaluated the mechanisms underlying interactions of CpClec with intestinal epithelial cells by using an Fc-tagged recombinant protein. CpClec-Fc displayed Ca(2+)-dependent, saturable binding to HCT-8 and Caco-2 cells and competitively inhibited C. parvum attachment to and infection of HCT-8 cells. Binding of CpClec-Fc was specifically inhibited by sulfated glycosaminoglycans, particularly heparin and heparan sulfate. Binding was reduced after the removal of heparan sulfate and following the inhibition of glycosaminoglycan synthesis or sulfation in HCT-8 cells. Like CpClec-Fc binding, C. parvum attachment to and infection of HCT-8 cells were inhibited by glycosaminoglycans and were reduced after heparan sulfate removal or inhibition of glycosaminoglycan synthesis or sulfation. Lastly, CpClec-Fc binding and C. parvum sporozoite attachment were significantly decreased in CHO cell mutants defective in glycosaminoglycan synthesis. Together, these results indicate that CpClec is a novel C-type lectin that mediates C. parvum attachment and infection via Ca(2+)-dependent binding to sulfated proteoglycans on intestinal epithelial cells. PMID:26975991

  20. Characteristic recognition of N-acetylgalactosamine by an invertebrate C-type Lectin, CEL-I, revealed by X-ray crystallographic analysis.

    PubMed

    Sugawara, Hajime; Kusunoki, Masami; Kurisu, Genji; Fujimoto, Tokiko; Aoyagi, Haruhiko; Hatakeyama, Tomomitsu

    2004-10-22

    CEL-I is a C-type lectin, purified from the sea cucumber Cucumaria echinata, that shows a high specificity for N-acetylgalactosamine (GalNAc). We determined the crystal structures of CEL-I and its complex with GalNAc at 2.0 and 1.7 A resolution, respectively. CEL-I forms a disulfide-linked homodimer and contains two intramolecular disulfide bonds, although it lacks one intramolecular disulfide bond that is widely conserved among various C-type carbohydrate recognition domains (CRDs). Although the sequence similarity of CEL-I with other C-type CRDs is low, the overall folding of CEL-I was quite similar to those of other C-type CRDs. The structure of the complex with GalNAc revealed that the basic recognition mode of GalNAc was very similar to that for the GalNAc-binding mutant of the mannose-binding protein. However, the acetamido group of GalNAc appeared to be recognized more strongly by the combination of hydrogen bonds to Arg115 and van der Waals interaction with Gln70. Mutational analyses, in which Gln70 and/or Arg115 were replaced by alanine, confirmed that these residues contributed to GalNAc recognition in a cooperative manner. PMID:15319425

  1. Fishing for lectins from diverse sequence libraries by yeast surface display - an exploratory study.

    PubMed

    Ryckaert, Stefan; Callewaert, Nico; Jacobs, Pieter P; Dewaele, Sylviane; Dewerte, Isabelle; Contreras, Roland

    2008-02-01

    The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein-carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or alpha-agglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates. PMID:18086821

  2. Lectin binding and surface glycoprotein pattern of human macrophage populations.

    PubMed

    Kreipe, H; Radzun, H J; Schumacher, U; Parwaresch, M R

    1986-01-01

    In the present study unstimulated and stimulated human blood monocytes, untreated and phorbol ester treated U-937 cells, as well as human peritoneal and alveolar macrophages were studied with respect to their surface membrane properties. Binding of different lectins and electrophoretic patterns of tritium labeled surface glycoproteins were compared. The analysis of surface glycoproteins could be interpreted as evidence for a common origin of the analysed cell populations. Furthermore, banding patterns of glycoproteins might be useful to define certain activation states within monocyte/macrophage differentiation. In contrast, lectin binding pattern did not clearly discriminate macrophage subpopulations. PMID:3102412

  3. Binding of sucrose octasulphate to the C-type lectin-like domain of the recombinant natural killer cell receptor NKR-P1A observed by NMR spectroscopy.

    PubMed

    Kogelberg, Heide; Frenkiel, Thomas A; Birdsall, Berry; Chai, Wengang; Muskett, Frederick W

    2002-11-01

    NKR-P1A is a C-type lectin-like receptor on natural killer cells believed to be involved in the cytotoxicity of these cells. Ligands for this protein are not known. Here, we describe the binding of a fully sulphated disaccharide, sucrose octasulphate, by the recombinant C-type lectin-like domain of NKR-P1A. The binding was observed by NMR spectroscopy methods that have recently been described for the screening of compound libraries for bioaffinities, namely the 2D NOESY and saturation transfer difference NMR experiments. (1)H titration studies indicate that the binding is specific. These findings raise the possibility that NKR-P1A recognises sulphated natural ligands in common with certain other members of the C-type lectin family. PMID:12404632

  4. Characterization of a recombinant C-type lectin, rCEL-IV, expressed in Escherichia coli cells using a synthetic gene.

    PubMed

    Hatakeyama, Tomomitsu; Hozawa, Takao; Hirotani, Iyo; Tsuda, Nobuaki; Kusunoki, Masami; Shiba, Kohei

    2006-03-01

    The body fluid of marine invertebrate Cucumaria echinata (Holothuroidea) contains four Ca2+-dependent galactose-specific lectins. One of these lectins, CEL-IV, is composed of a C-type carbohydrate-recognition domain homotetramer. CEL-IV exhibits higher specificity for alpha-galactosides than for beta-galactosides, while other C. echinata lectins show preferential binding of beta-galactosides. We constructed an artificial synthetic gene for recombinant CEL-IV (rCEL-IV) based on the amino acid sequence previously determined from the purified protein. rCEL-IV was expressed in Escherichia coli cells as inclusion bodies. After the refolding process, most of rCEL-IV spontaneously formed a homotetramer structure having interchain disulfide bonds. The secondary structure of rCEL-IV was similar to that of the native one, as judged by the comparison of the far UV-circular dichroism spectra of rCEL-IV and native CEL-IV (nCEL-IV). Carbohydrate-binding specificity of rCEL-IV was confirmed to be similar to that of nCEL-IV from the results of the binding-inhibition assay using liposomes composed of rabbit erythrocyte lipids. Crystals of rCEL-IV were obtained in a few days by the sitting drop vapor diffusion method. These results indicate that rCEL-IV achieved essentially correct three-dimensional structure, including the carbohydrate-binding sites, and it would be very useful for further study on the carbohydrate-recognition mechanism by mutational and X-ray crystallographic analyses. PMID:16503091

  5. Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata.

    PubMed

    Hatakeyama, Tomomitsu; Matsuo, Noriaki; Shiba, Kouhei; Nishinohara, Shoichi; Yamasaki, Nobuyuki; Sugawara, Hajime; Aoyagi, Haruhiko

    2002-01-01

    CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment. PMID:11866098

  6. Characterization of the Expression and Function of the C-Type Lectin Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration.

    PubMed

    Lo, Tsun-Ho; Silveira, Pablo A; Fromm, Phillip D; Verma, Nirupama D; Vu, Phi A; Kupresanin, Fiona; Adam, Rhonda; Kato, Masato; Cogger, Victoria C; Clark, Georgina J; Hart, Derek N J

    2016-08-01

    C-type lectin receptors play important roles in immune cell interactions with the environment. We described CD302 as the simplest, single domain, type I C-type lectin receptor and showed it was expressed mainly on the myeloid phagocytes in human blood. CD302 colocalized with podosomes and lamellopodia structures, so we hypothesized that it played a role in cell adhesion or migration. In this study, we used mouse models to obtain further insights into CD302 expression and its potential immunological function. Mouse CD302 transcripts were, as in humans, highest in the liver, followed by lungs, lymph nodes (LN), spleen, and bone marrow. In liver, CD302 was expressed by hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells. A detailed analysis of CD302 transcription in mouse immune cells revealed highest expression by myeloid cells, particularly macrophages, granulocytes, and myeloid dendritic cells (mDC). Interestingly, 2.5-fold more CD302 was found in migratory compared with resident mDC populations and higher CD302 expression in mouse M1 versus M2 macrophages was also noteworthy. CD302 knockout (CD302KO) mice were generated. Studies on the relevant immune cell populations revealed a decrease in the frequency and numbers of migratory mDC within CD302KO LN compared with wild-type LN. In vitro studies showed CD302KO and wild-type DC had an equivalent capacity to undergo maturation, prime T cells, uptake Ags, and migrate toward the CCL19/CCL21 chemokines. Nevertheless, CD302KO migratory DC exhibited reduced in vivo migration into LN, confirming a functional role for CD302 in mDC migration. PMID:27316686

  7. C-type lectin-like receptor LOX-1 promotes dendritic cell-mediated class-switched B cell responses.

    PubMed

    Joo, HyeMee; Li, Dapeng; Dullaers, Melissa; Kim, Tae-Whan; Duluc, Dorothee; Upchurch, Katherine; Xue, Yaming; Zurawski, Sandy; Le Grand, Roger; Liu, Yong-Jun; Kuroda, Marcelo; Zurawski, Gerard; Oh, SangKon

    2014-10-16

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses. PMID:25308333

  8. Critical Role for an Acidic Amino Acid Region in Platelet Signaling by the HemITAM (Hemi-immunoreceptor Tyrosine-based Activation Motif) Containing Receptor CLEC-2 (C-type Lectin Receptor-2)*

    PubMed Central

    Hughes, Craig E.; Sinha, Uma; Pandey, Anjali; Eble, Johannes A.; O'Callaghan, Christopher A.; Watson, Steve P.

    2013-01-01

    CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors. PMID:23264619

  9. Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene.

    PubMed

    Hatakeyama, Tomomitsu; Shiba, Kouhei; Matsuo, Noriaki; Fujimoto, Tokiko; Oda, Tatsuya; Sugawara, Hajime; Aoyagi, Haruhiko

    2004-01-01

    CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata. This lectin shows very high N-acetylgalactosamine-binding specificity. We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells. Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps. The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca(2+)-concentrations, whereas it was weaker at lower Ca(2+)-concentrations due to decreased Ca(2+)-binding affinity. rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GalNAc-binding specificity, as examined by hemagglutination inhibition assay. Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Ca(2+). Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures. Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity. These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I. Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I. PMID:14999015

  10. Molecular characterization of a transmembrane C-type lectin receptor gene from ayu (Plecoglossus altivelis) and its effect on the recognition of different bacteria by monocytes/macrophages.

    PubMed

    Zhang, Xue-Heng; Shi, Yu-Hong; Chen, Jiong

    2015-08-01

    C-type lectin receptors (CTLRs) play vital roles in immune responses as pattern-recognition receptors (PRRs). In this study, we identified a novel C-type lectin receptor (PaCTLRC) gene from ayu, Plecoglossus altivelis. Predicted PaCTLRC is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. Sequence comparison and phylogenetic tree analysis showed that PaCTLRC was most closely related to Atlantic salmon (Salmo salar) CLRC, but was significantly different from two other ayu CTLRs, aCLR and PaCD209L. PaCTLRC transcript was detected in all tested tissues and cells, with high levels in the liver; and its expression was significantly altered upon Vibrio anguillarum infection. Refolded recombinant PaCTLRC (rPaCTLRC) agglutinated three types of Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus and Streptococcus iniae) and four types of Gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, V. anguillarum and Vibrio parahaemolyticus) in a Ca(2+)-dependent manner in vitro, and Gram-positive bacteria were shown to be biologically relevant ligands for PaCTLRC. rPaCTLRC bound to d-mannose, d-galactose, l-fucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS) and peptidoglycan (PGN), exhibiting a relative binding strength to d-mannose and PGN. d-Mannose, l-fucose, GlcNAc, LPS and PGN could inhibit the agglutinating activity of rPaCTLRC, while d-galactose did not functioned. PaCTLRC neutralization using anti-PaCTLRC IgG resulted in the inhibition of phagocytosis by ayu monocytes/macrophages (MO/MΦ) of S. aureus but not of E. coli, and produced a consistently higher survival rate of S. aureus than that of E. coli. d-Mannose, LPS and PGN treatment had no significant influence on the phagocytosis of ayu MO/MΦ. These results suggest that PaCTLRC may serve as a Gram-positive bacteria-preferred PRR which is involved in pathogen recognition and signal transduction in ayu MO/MΦ. PMID:26010409

  11. Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells*

    PubMed Central

    Pollitt, Alice Y.; Poulter, Natalie S.; Gitz, Eelo; Navarro-Nuñez, Leyre; Wang, Ying-Jie; Hughes, Craig E.; Thomas, Steven G.; Nieswandt, Bernhard; Douglas, Michael R.; Owen, Dylan M.; Jackson, David G.; Dustin, Michael L.; Watson, Steve P.

    2014-01-01

    The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development. PMID:25368330

  12. C-type lectin Langerin is a β-glucan receptor on human Langerhans cells that recognizes opportunistic and pathogenic fungi

    PubMed Central

    de Jong, Marein A.W.P.; Vriend, Lianne E.M.; Theelen, Bart; Taylor, Maureen E.; Fluitsma, Donna; Boekhout, Teun; Geijtenbeek, Teunis B.H.

    2010-01-01

    Langerhans cells (LCs) lining the stratified epithelia and mucosal tissues are the first antigen presenting cells to encounter invading pathogens, such as viruses, bacteria and fungi. Fungal infections form a health threat especially in immuno-compromised individuals. LCs express C-type lectin Langerin that has specificity for mannose, fucose and GlcNAc structures. Little is known about the role of human Langerin in fungal infections. Our data show that Langerin interacts with both mannan and β-glucan structures, common cell-wall carbohydrate structures of fungi. We have screened a large panel of fungi for recognition by human Langerin and, strikingly, we observed strong binding of Langerin to a variety of Candida and Saccharomyces species and Malassezia furfur, but very weak binding was observed to Cryptococcus gattii and Cryptococcus neoformans. Notably, Langerin is the primary fungal receptor on LCs, since the interaction of LCs with the different fungi was blocked by antibodies against Langerin. Langerin recognizes both mannose and β-glucans present on fungal cell walls and our data demonstrate that Langerin is the major fungal pathogen receptor on human LCs that recognizes pathogenic and commensal fungi. Together these data may provide more insight in the role of LCs in fungal infections. PMID:20097424

  13. Antimicrobial functions of EsLecH, a C-type lectin, via JNK pathway in the Chinese mitten crab, Eriocheir sinensis.

    PubMed

    Zhu, You-Ting; Zhang, Xing; Wang, Shi-Chuang; Li, Wei-Wei; Wang, Qun

    2016-08-01

    C-type lectins (CTLs) are pattern recognition proteins that play significant roles in the innate immune system by identifying and eliminating pathogens. Here, we have reported a CTL (EsLecH) from the Chinese mitten crab that can bind to microorganisms and regulate antimicrobial peptide (AMP) expression via the c-Jun N-terminal kinase (JNK) pathway. EsLecH was found to have an N-terminal signal peptide and a single carbohydrate recognition domain. The EsLecH transcript was detected abundantly in various tissues, and it was significantly upregulated in hemocytes after challenging with lipopolysaccharides and bacteria. Recombinant (r)EsLecH could bind to microorganisms, but at different levels. Ca(2+) significantly increased rEsLecH binding affinity to microorganisms. Furthermore, growth inhibition by rEsLecH increased with increasing rEsLecH levels. Knockdown of EsLecH was accompanied by a significant reduction in AMP expression and JNK phosphorylation; AMP expression was reduced with JNK silencing and can not rescued by rEsLecH when absence of JNK. These results indicate that EsLecH could regulate AMPs via JNK signaling. PMID:27068761

  14. CLEC-38, a transmembrane protein with C-type lectin-like domains, negatively regulates UNC-40-mediated axon outgrowth and promotes presynaptic development in Caenorhabditis elegans.

    PubMed

    Kulkarni, Gauri; Li, Haichang; Wadsworth, William G

    2008-04-23

    In the developing nervous system, axons respond to various guidance cues to find their targets. The effects guidance cues have on an axon may change as an axon undergoes morphological changes, such as branching, turning, and synapse formation. The means by which these changes are regulated are not well understood. In Caenorhabditis elegans, the UNC-40/DCC (deleted in colorectal cancer) receptor mediates responses to the UNC-6/netrin guidance cue. Here, we show that CLEC-38, a protein with predicted transmembrane and C-type lectin-like domains, regulates UNC-40-mediated axon outgrowth as well as the organization of presynaptic terminals. We observe that, in genetic backgrounds sensitized for axon guidance defects, loss of clec-38 function can suppress defects in an UNC-40-dependent manner. Within migrating axons, clec-38 acts cell autonomously. Furthermore, loss of clec-38 function alters UNC-40::GFP (green fluorescent protein) expression. We also observe that loss of clec-38 function disrupts presynaptic patterning in animals with normal axon guidance and that there are genetic interactions between clec-38 and rpm-1, which encodes a protein implicated in regulating presynaptic assembly and axon morphology. We suggest CLEC-38 plays a role in promoting synapse assembly and refining axon outgrowth activity. PMID:18434533

  15. A single CRD C-type lectin from Eriocheir sinensis (EsLecB) with microbial-binding, antibacterial prophenoloxidase activation and hem-encapsulation activities.

    PubMed

    Fang, Zi-Yan; Li, Dan; Li, Xue-Jie; Zhang, Xing; Zhu, You-Ting; Li, Wei-Wei; Wang, Qun

    2016-03-01

    C-type lectins (CTLs) exist widely in crustaceans. To date, thirteen CTLs have been reported in crustaceans, and play significant roles in pathogen recognition, encapsulation of hemocytes and antimicrobial activity in the innate immune response. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, a novel CTL, designated as EsLecB, with a 470 bp open reading frame encodes a polypeptide of 156 amino acids, including a signal peptide of 19 amino acid residues and one carbohydrate-recognition domain of 131 aa residues, was cloned from the crustacean Eriocheir sinensis. By qRT-PCR analysis, EsLecB was detected in all tested tissues, and showed highest expression in hemocytes, hepatopancreas and heart. The expression of EsLecB was up-regulated following injections of PAMPs or bacteria. The recombinant protein (rEsLecB) expressed in Escherichia coli had a calcium-independent but carbohydrate-dependent microbial-binding and microbial-agglutinating, microorganism growth inhibitory and hem-encapsulation activities. Moreover, the rEsLecB could stimulate the activation of prophenoloxidase in vitro. These results indicated that EsLecB, as an antibacterial pattern recognition receptor is involved in innate immunity, and may act as an upstream detector of the prophenoloxidase activating system, which can detect pathogen invasion in E. sinensis. PMID:26826423

  16. C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity

    PubMed Central

    Wiersma, Valerie R; de Bruyn, Marco; Shi, Ce; Gooden, Marloes JM; Wouters, Maartje CA; Samplonius, Douwe F; Hendriks, Djoke; Nijman, Hans W; Wei, Yunwei; Zhou, Jin; Helfrich, Wijnand; Bremer, Edwin

    2015-01-01

    The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes. PMID:25760768

  17. 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. PMID:26724379

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

    SciTech Connect

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

    1987-05-01

    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 a 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.

  19. A single-CRD C-type lectin from oyster Crassostrea gigas mediates immune recognition and pathogen elimination with a potential role in the activation of complement system.

    PubMed

    Li, Hui; Zhang, Huan; Jiang, Shuai; Wang, Weilin; Xin, Lusheng; Wang, Hao; Wang, Lingling; Song, Linsheng

    2015-06-01

    C-type lectins (CTLs), serving as pattern recognition receptors (PRRs), are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins that participate in nonself-recognition and pathogen elimination. In the present study, a single carbohydrate-recognition domain (CRD) CTL was identified from oyster Crassostrea gigas (designated as CgCLec-2). There was only one CRD within the deduced amino acid sequence of CgCLec-2 consisting of 129 amino acid residues. A conserved EPN (Glu246-Pro247-Asn248) motif was found in Ca(2+)-binding site 2 of CgCLec-2. The CgCLec-2 mRNA could be detected in all the examined tissues at different expression levels in oysters. The mRNA expression of CgCLec-2 in hemocytes was up-regulated significantly at 6 h post Vibrio splendidus challenge. The recombinant CgCLec-2 (rCgCLec-2) could bind various Pathogen-Associated Molecular Patterns (PAMPs), including lipopolysaccharide, mannan and peptidoglycan, and displayed strong binding abilities to Vibrio anguillarum, V. splendidus and Yarrowiali polytica and week binding ability to Staphylococcus aureus. It could also enhance the phagocytic activity of oyster hemocytes to V. splendidus and exhibited growth suppression activity against gram-positive bacteria S. aureus but no effect on gram-negative bacteria V. splendidus. Furthermore, the interaction between rCgCLec-2 and rCgMASPL-1 was confirmed by GST Pull down. The results suggested that CgCLec-2 served as not only a PRR in immune recognition but also a regulatory factor in pathogen elimination, and played a potential role in the activation of complement system. PMID:25800112

  20. Preferential induction of CD4+ T cell responses through in vivo targeting of antigen to dendritic cell-associated C-type lectin-1.

    PubMed

    Carter, Robert W; Thompson, Clare; Reid, Delyth M; Wong, Simon Y C; Tough, David F

    2006-08-15

    Targeting of Ags and therapeutics to dendritic cells (DCs) has immense potential for immunotherapy and vaccination. Because DCs are heterogeneous, optimal targeting strategies will require knowledge about functional specialization among DC subpopulations and identification of molecules for targeting appropriate DCs. We characterized the expression of a fungal recognition receptor, DC-associated C-type lectin-1 (Dectin-1), on mouse DC subpopulations and investigated the ability of an anti-Dectin-1 Ab to deliver Ag for the stimulation of immune responses. Dectin-1 was shown to be expressed on CD8alpha-CD4-CD11b+ DCs found in spleen and lymph nodes and dermal DCs present in skin and s.c. lymph nodes. Injection of Ag-anti-Dectin-1 conjugates induced CD4+ and CD8+ T cell and Ab responses at low doses where free Ag failed to elicit a response. Notably, qualitatively different immune responses were generated by targeting Ag to Dectin-1 vs CD205, a molecule expressed on CD8alpha+CD4-CD11b- DCs, dermal DCs, and Langerhans cells. Unlike anti-Dectin-1, anti-CD205 conjugates failed to elicit an Ab response. Moreover, when conjugates were injected i.v., anti-Dectin-1 stimulated a much stronger CD4+ T cell response and a much weaker CD8+ T cell response than anti-CD205. The results reveal Dectin-1 as a potential targeting molecule for immunization and have implications for the specialization of DC subpopulations. PMID:16887988

  1. Functional evaluation of the role of C-type lectin domain family 16A at the chromosome 16p13 locus

    PubMed Central

    Zouk, H; D'Hennezel, E; Du, X; Ounissi-Benkalha, H; Piccirillo, C A; Polychronakos, C

    2014-01-01

    The type 1 diabetes-associated 16p13 locus contains the CLEC16A gene. Its preferential immune cell expression suggests involvement in autoimmunity. Given its elevated expression in dendritic and B cells – known professional antigen-presenting cells (APCs) – we hypothesize that C-type lectin domain family 16 member A (CLEC16A) may be involved in T cell co-stimulation and consequent activation and proliferation. We also sought to identify CLEC16A's subcellular localization. The effect of the CLEC16A knock-down (KD) on B cell co-stimulation and activation of T cells was tested in human lymphoblastoid cell lines (LCLs) by co-culture with CD4+ T cells. T cell activation and proliferation were determined by flow-cytometric analysis of CD69 and CD25 expression and carboxyfluorescein succinimidyl ester (CFSE) dilution, respectively. CLEC16A subcellular localization in K562 cells was examined by immunofluorescence. We show that the CLEC16A KD did not affect the tested indices of lymphoblastoid cell line (LCL) APC capacity. Additionally, the percentage of activated T cells following LCL co-culture was not affected significantly by the CLEC16A KD. T cells co-cultured with KD or control LCLs also exhibited similar cell division profiles. CLEC16A co-localized with an endoplasmic reticulum (ER) marker, suggesting that it may be an ER protein. In conclusion, CLEC16A may not be involved in T cell co-stimulation. Additional studies on CLEC16A, accounting for its ER localization, are needed to uncover its biological role. PMID:24237155

  2. Super-resolution imaging of C-type lectin spatial rearrangement within the dendritic cell plasma membrane at fungal microbe contact sites

    NASA Astrophysics Data System (ADS)

    Itano, Michelle; Graus, Matthew; Pehlke, Carolyn; Wester, Michael; Liu, Ping; Lidke, Keith; Thompson, Nancy; Jacobson, Ken; Neumann, Aaron

    2014-08-01

    Dendritic cells express DC-SIGN and CD206, C-type lectins (CTLs) that bind a variety of pathogens and may facilitate pathogen uptake for subsequent antigen presentation. Both proteins form punctate membrane nanodomains (~80 nm) on naïve cells. We analyzed the spatiotemporal distribution of CTLs following host-fungal particle contact using confocal microscopy and three distinct methods of cluster identification and measurement of receptor clusters in super-resolution datasets: DBSCAN, Pair Correlation and a custom implementation of the Getis spatial statistic. Quantitative analysis of confocal and super-resolution images demonstrated that CTL nanodomains become concentrated in the contact site relative to non-contact membrane after the first hour of exposure and established that this recruitment is sustained out to four hours. DC-SIGN nanodomains in fungal contact sites exhibit a 70% area increase and a 38% decrease in interdomain separation. Contact site CD206 nanodomains possess 90% greater area and 42% lower interdomain separation relative to non-contact regions. Contact site CTL clusters appear as disk-shaped domains of approximately 150-175 nm in diameter. The increase in length scale of CTL nanostructure in contact sites suggests that the smaller nanodomains on resting membranes may merge during fungal nanodomain structure, or that they become packed closely enough to achieve sub-resolution inter-domain edge separations of < 30 nm. This study provides evidence of local receptor spatial rearrangements on the nanoscale that occur in the plasma membrane upon pathogen binding and may direct important signaling interactions required to recognize and respond to the presence of a relatively large pathogen.

  3. Cell surface lectin array: parameters affecting cell glycan signature.

    PubMed

    Landemarre, Ludovic; Cancellieri, Perrine; Duverger, Eric

    2013-04-01

    Among the "omics", glycomics is one of the most complex fields and needs complementary strategies of analysis to decipher the "glycan dictionary". As an alternative method, which has developed since the beginning of the 21st century, lectin array technology could generate relevant information related to glycan motifs, accessibility and a number of other valuable insights from molecules (purified and non-purified) or cells. Based on a cell line model, this study deals with the key parameters that influence the whole cell surface glycan interaction with lectin arrays and the consequences on the interpretation and reliability of the results. The comparison between the adherent and suspension forms of Chinese Hamster Ovary (CHO) cells, showed respective glycan signatures, which could be inhibited specifically by neoglycoproteins. The modifications of the respective glycan signatures were also revealed according to the detachment modes and cell growth conditions. Finally the power of lectin array technology was highlighted by the possibility of selecting and characterizing a specific clone from the mother cell line, based on the slight difference determination in the respective glycan signatures. PMID:22899543

  4. A Lectin from Dioclea violacea Interacts with Midgut Surface of Lutzomyia migonei, Unlike Its Homologues, Cratylia floribunda Lectin and Canavalia gladiata Lectin

    PubMed Central

    Monteiro Tínel, Juliana Montezuma Barbosa; Benevides, Melina Fechine Costa; Frutuoso, Mércia Sindeaux; Rocha, Camila Farias; Arruda, Francisco Vassiliepe Sousa; Vasconcelos, Mayron Alves; Pereira-Junior, Francisco Nascimento; Cajazeiras, João Batista; do Nascimento, Kyria Santiago; Martins, Jorge Luiz; Teixeira, Edson Holanda; Cavada, Benildo Sousa; dos Santos, Ricardo Pires; Lima Pompeu, Margarida Maria

    2014-01-01

    Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand fly. Susceptibility and refractoriness to Leishmania depend on the outcome of multiple interactions that take place within the sand fly gut. Promastigote attachment to sand fly midgut epithelium is essential to avoid being excreted together with the digested blood meal. Promastigote and gut sand fly surface glycans are important ligands in this attachment. The purpose of the present study was to evaluate the interaction of three lectins isolated from leguminous seeds (Diocleinae subtribe), D-glucose and D-mannose-binding, with glycans on Lutzomyia migonei midgut. To study this interaction the lectins were labeled with FITC and a fluorescence assay was performed. The results showed that only Dioclea violacea lectin (DVL) was able to interact with midgut glycans, unlike Cratylia floribunda lectin (CFL) and Canavalia gladiata lectin (CGL). Furthermore, when DVL was blocked with D-mannose the interaction was inhibited. Differences of spatial arrangement of residues and volume of carbohydrate recognition domain (CRD) may be the cause of the fine specificity of DVL for glycans in the surface on Lu. migonei midgut. The findings in this study showed the presence of glycans in the midgut with glucose/mannose residues in its composition and these residues may be important in interaction between Lu. migonei midgut and Leishmania. PMID:25431778

  5. Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

    PubMed Central

    2014-01-01

    It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies. PMID:24629138

  6. Molecular characterization of the reniform nematode C-type lectin gene family reveals a likely role in mitigating environmental stresses during plant parasitism.

    PubMed

    Ganji, Satish; Jenkins, Johnie N; Wubben, Martin J

    2014-03-10

    The reniform nematode, Rotylenchulus reniformis, is a damaging semi-endoparasitic pathogen of more than 300 plant species. Transcriptome sequencing of R. reniformis parasitic females revealed an enrichment for sequences homologous to C-type lectins (CTLs), an evolutionarily ancient family of Ca(+2)-dependent carbohydrate-binding proteins that are involved in the innate immune response. To gain further insight as to the potential role of CTLs in facilitating plant parasitism by R. reniformis, we performed a comprehensive assessment of the CTL gene family. 5'- and 3'-RACE experiments identified a total of 11 R. reniformis CTL transcripts (Rr-ctl-1 through Rr-ctl-11) that ranged in length from 1083 to 1,194 bp and showed 93-99% identity with one another. An alignment of cDNA and genomic sequences revealed three introns with the first intron residing within the 5'-untranslated region. BLAST analyses showed the closest homologs belonging to the parasitic nematodes Heligmosomoides polygyrus and Heterodera glycines. Rr-ctl-1, -2, and -3 were expressed throughout the R. reniformis life cycle; whereas, the remaining Rr-ctl genes showed life stage-specific expression. Quantitative real time RT-PCR determined that Rr-ctl transcripts were 839-fold higher in sedentary female nematodes than the next most abundant life stage. Predicted Rr-CTL peptides ranged from 301 to 338 amino acids long, possessed an N-terminal signal peptide for secretion, and contained a conserved CLECT domain, including the mannose-binding motifs EPN and EPD and the conserved WND motif that is required for binding Ca(+2). In addition, Rr-CTL peptides harbored repeats of a novel 17-mer motif within their C-terminus that showed similarity to motifs associated with bacterial ice nucleation proteins. In situ hybridization of Rr-ctl transcripts within sedentary females showed specific accumulation within the hypodermis of the body regions exposed to the soil environment; those structures embedded within the

  7. A novel C-type lectin, Nattectin-like protein, with a wide range of bacterial agglutination activity in large yellow croaker Larimichthys crocea.

    PubMed

    Lv, Changhuan; Zhang, Dongling; Wang, Zhiyong

    2016-03-01

    C-type lectins (CTLs) are generally recognized as a superfamily of Ca(2+)-dependent carbohydrate-binding proteins, which serve as pattern recognition receptors (PRRs) in innate immunity of vertebrates. In this study, the molecular characterization and immune roles of a novel CTL from Larimichthys crocea (designated as LcNTC) were investigated. LcNTC is a novel protein that shared 33%-49% homology with other teleosts CTLs. The full-length cDNA of LcNTC was composed of 859 bp with a 465 bp open reading frame encoding a putative protein of 154 residues. LcNTC contained a single CRD with four conserved disulfide-bonded cysteine residues (Cys(57)-Cys(148), Cys(126)-Cys(140)) and EPN/AND motifs instead of invariant EPN/WND motifs required for carbohydrate-binding specificity and constructing Ca(2+)-binding sites. LcNTC mRNA was detected in all examined tissues with the most abundant in the gill. After challenged with poly I:C and Vibrio parahaemolyticus, the temporal expression of LcNTC was significantly up-regulated in the liver, spleen and head-kidney. LcNTC transcripts were also induced in the gill, skin, spleen and head-kidney post-infection with Cryptocaryon irritans. The recombinant LcNTC (rLcNTC) purified from Escherichia coli BL21 (DE3) exhibited strong agglutination activity against erythrocytes from human, rabbit and large yellow croaker in a Ca(2+)-dependent manner, and the agglutination could be inhibited by D-Mannose, D-Glucose, D-Fructose, α-Lactose, D-Maltose and LPS. Positive microbial agglutination activities of rLcNTC were observed against all tested bacteria in the presence of Ca(2+), including Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus and Micrococcus lysoleikticus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila). These findings collectively indicated that LcNTC might be involved in the innate immunity of L. crocea as a PRR. PMID:26828263

  8. U(VI) Reduction by Diverse Outer Surface c-Type Cytochromes of Geobacter sulfurreducens

    PubMed Central

    Leavitt, Janet J.; Comolli, Luis R.; Csencsits, Roseann; Janot, Noemie; Flanagan, Kelly A.; Gray, Arianna S.; Leang, Ching; Izallalen, Mounir; Mester, Tünde; Lovley, Derek R.

    2013-01-01

    Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors. PMID:23934497

  9. Lectins with anti-HIV activity: a review.

    PubMed

    Akkouh, Ouafae; Ng, Tzi Bun; Singh, Senjam Sunil; Yin, Cuiming; Dan, Xiuli; Chan, Yau Sang; Pan, Wenliang; Cheung, Randy Chi Fai

    2015-01-01

    Lectins including flowering plant lectins, algal lectins, cyanobacterial lectins, actinomycete lectin, worm lectins, and the nonpeptidic lectin mimics pradimicins and benanomicins, exhibit anti-HIV activity. The anti-HIV plant lectins include Artocarpus heterophyllus (jacalin) lectin, concanavalin A, Galanthus nivalis (snowdrop) agglutinin-related lectins, Musa acuminata (banana) lectin, Myrianthus holstii lectin, Narcissus pseudonarcissus lectin, and Urtica diocia agglutinin. The anti-HIV algal lectins comprise Boodlea coacta lectin, Griffithsin, Oscillatoria agardhii agglutinin. The anti-HIV cyanobacterial lectins are cyanovirin-N, scytovirin, Microcystis viridis lectin, and microvirin. Actinohivin is an anti-HIV actinomycete lectin. The anti-HIV worm lectins include Chaetopterus variopedatus polychaete marine worm lectin, Serpula vermicularis sea worm lectin, and C-type lectin Mermaid from nematode (Laxus oneistus). The anti-HIV nonpeptidic lectin mimics comprise pradimicins and benanomicins. Their anti-HIV mechanisms are discussed. PMID:25569520

  10. The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains.

    PubMed

    Koizumi, N; Imamura, M; Kadotani, T; Yaoi, K; Iwahana, H; Sato, R

    1999-01-25

    We recently isolated and characterized the lipopolysaccharide (LPS)-binding protein, BmLBP, from the larval hemolymph of the silkworm Bombyx mori. BmLBP is a pattern recognition molecule that recognizes the lipid A portion of LPS and participates in a cellular defense reaction. This paper describes the cDNA cloning of BmLBP. The deduced amino acid sequence of BmLBP revealed that BmLBP is a novel member of the C-type lectin superfamily with a unique structural feature that consists of two different carbohydrate-recognition domains in tandem, a short and a long form. PMID:9989592

  11. A New C-Type Lectin Similar to the Human Immunoreceptor DC-SIGN Mediates Symbiont Acquisition by a Marine Nematode†

    PubMed Central

    Bulgheresi, Silvia; Schabussova, Irma; Chen, Tie; Mullin, Nicholas P.; Maizels, Rick M.; Ott, Jörg A.

    2006-01-01

    Although thiotrophic symbioses have been intensively studied for the last three decades, nothing is known about the molecular mechanisms of symbiont acquisition. We used the symbiosis between the marine nematode Laxus oneistus and sulfur-oxidizing bacteria to study this process. In this association a monolayer of symbionts covers the whole cuticle of the nematode, except its anterior-most region. Here, we identify a novel Ca2+-dependent mannose-specific lectin that was exclusively secreted onto the posterior, bacterium-associated region of L. oneistus cuticle. A recombinant form of this lectin induced symbiont aggregation in seawater and was able to compete with the native lectin for symbiont binding in vivo. Surprisingly, the carbohydrate recognition domain of this mannose-binding protein was similar both structurally and functionally to a human dendritic cell-specific immunoreceptor. Our results provide a molecular link between bacterial symbionts and host-secreted mucus in a marine symbiosis and suggest conservation in the mechanisms of host-microbe interactions throughout the animal kingdom. PMID:16598002

  12. CEL-I, an invertebrate N-acetylgalactosamine-specific C-type lectin, induces TNF-alpha and G-CSF production by mouse macrophage cell line RAW264.7 cells.

    PubMed

    Yamanishi, Tomohiro; Yamamoto, Yoshiko; Hatakeyama, Tomomitsu; Yamaguchi, Kenichi; Oda, Tatsuya

    2007-11-01

    Our previous studies demonstrated that CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin purified from the marine invertebrate Cucumaria echinata (Holothuroidea) showed potent cytotoxicity to several cell lines such as HeLa, MDCK and XC cells. In this study, we found that CEL-I induced increased secretion of tumour necrosis factor-alpha (TNF-alpha) and granulocyte colony stimulation factor (G-CSF) by mouse macrophage cell line RAW264.7 cells in a dose-dependent manner, whereas this cell line was highly resistant to CEL-I cytotoxicity. The cytokine-inducing activity of CEL-I was stronger than that of phytohaemagglutinin (PHA-L). A binding study using FITC-labelled CEL-I (F-CEL-I) indicated that the amount of bound F-CEL-I on RAW264.7 cells was greater than that of F-PHA-L, suggesting that the greater activity of CEL-I to induce cytokine secretion by RAW264.7 cells is partly due to the higher binding ability. Since the cell binding and cytokine-inducing activity of CEL-I were partly but significantly inhibited by the specific sugar (GalNAc), it is considered that the binding of CEL-I to cell-surface-specific saccharide moieties, which may be recognized by CEL-I with higher affinity than GalNAc, is essential for the induction of cytokine secretion. The secretion of TNF-alpha and G-CSF from CEL-I-treated RAW264.7 cells were almost completely prevented by brefeldin A (BFA), whereas increase in mRNA levels of these cytokines were not affected by BFA. Bio-Plex beads assay suggested that temporal increase in phosphorylation of extracellular-regulated kinase (ERK), c-jun NH(2)-terminal kinase (JNK) and p38 MAP kinase occurred at relatively early time following CEL-I treatment. Furthermore, the secretion of TNF-alpha and G-CSF were inhibited by specific inhibitors for these MAP kinases. These results suggest that the intracellular signal transduction through the activation of MAP kinase system is involved in CEL-I-induced cytokine secretion. PMID:17846063

  13. Enhancement of solubility and yield of a β-glucan receptor Dectin-1 C-type lectin-like domain in Escherichia coli with a solubility-enhancement tag.

    PubMed

    Dulal, Hari Prasad; Nagae, Masamichi; Ikeda, Akemi; Morita-Matsumoto, Kana; Adachi, Yoshiyuki; Ohno, Naohito; Yamaguchi, Yoshiki

    2016-07-01

    Dectin-1 is a C-type lectin-like pattern recognition receptor for β(1-3)-glucans. It plays a crucial role in protecting against fungal invasion through binding to β-glucans which are commonly present on the fungal cell wall. To probe its ligand binding mechanism by NMR, we expressed the recombinant murine Dectin-1 C-type lectin-like domain (CTLD) in E. coli using pCold vector and purified it. However, the high concentration of Dectin-1 CTLD required for NMR analysis could not be attained due to its inherent low solubility and low bacterial expression. In this study, we tried to increase expression and solubility of Dectin-1 CTLD by codon optimization and fusion of a GB1 tag (B1 domain of streptococcal Protein G). GB1 was inserted on either the N-terminal (NT) or C-terminal end as well as both terminal ends of human and mouse Dectin-1 CTLDs. A pure monomeric sample was only obtained with NT-GB1 fused mouse Dectin-1. Expression of mouse Dectin-1 CTLD yielded 0.9 ± 0.2 mg/L culture, codon optimized mouse Dectin-1 CTLD produced 1.4 ± 0.2 mg/L, and the tag-fused domain 7.1 ± 0.3 mg/L. The tag also increased solubility from 0.1 mM to 1.4 mM. The recombinant protein was correctly folded, in a monomeric state, and specifically bound β-glucan laminarin. These results indicate that fusing GB1 to the N-terminus of mouse Dectin-1 domain advantageously increases yield and solubility, allows retention of native structure, and that the site of fusion is critical. PMID:27062941

  14. Lectin binding studies on a glycopolymer brush flow-through biosensor by localized surface plasmon resonance.

    PubMed

    Rosencrantz, Ruben R; Nguyen, Vu Hoa; Park, Hyunji; Schulte, Christine; Böker, Alexander; Schnakenberg, Uwe; Elling, Lothar

    2016-08-01

    A localized surface plasmon resonance biosensor in a flow-through configuration was applied for investigating kinetics of lectin binding to surface-grafted glycopolymer brushes. Polycarbonate filter membranes with pore sizes of 400 nm were coated with a 114-nm thick gold layer and used as substrate for surface-initiated atom-transfer radical polymerization of a glycomonomer. These grafted from glycopolymer brushes were further modified with two subsequent enzymatic reactions on the surface to yield an immobilized trisaccharide presenting brush. Specific binding of lectins including Clostridium difficile toxin A receptor domain to the glycopolymer brush surface could be investigated in a microfluidic setup with flow-through of the analytes and transmission surface plasmon resonance spectroscopy. Graphical abstract Glycopolymer brushes serve as high affinity ligands for lectin and toxin interactions in a sensitive, disposable flow-through LSPR biosensor. PMID:27277814

  15. Mannosylation Allows for Synergic (CD44/C-Type Lectin) Uptake of Hyaluronic Acid Nanoparticles in Dendritic Cells, but Only upon Correct Ligand Presentation.

    PubMed

    Gennari, Arianna; Pelliccia, Maria; Donno, Roberto; Kimber, Ian; Tirelli, Nicola

    2016-04-01

    The selective targeting of dendritic cells (DCs) can lead to more efficacious vaccines. Here, materials have been designed for a synergic DC targeting: interacting with CD44 through the use of hyaluronic acid (HA), and with mannose-binding lectins (typical DC pattern recognition receptors) through HA mannosylation. Negatively charged, HA-displaying nanoparticles are produced via polyelectrolyte complexation of (mannosylated) HA and high- or low- molecular-weight chitosan (CS, 36 and 656 kDa). Using CS36, HA is better exposed and the particles have a higher affinity for HA receptors; this means a higher number of receptors clustered around each particle and, due to the rather limited CD44 availability, an overall lower uptake per cell. Employing Langerhans-like XS106 cells, all particles show negligible toxicity or inflammatory activation. The cellular uptake kinetics are qualitatively similar to other leukocytic models and thus considered to be CD44-dominated; the uptake increases with increasing HA mannosylation and with the use of adjuvants (LPS, mannan) for CS36/HA but not for CS656//HA particles; this indicates that the interactions with mannose-binding receptors requires a correct ligand presentation, and only in that case can they be enhanced by appropriate adjuvants. In summary, mannose-binding receptors can be used to enhance the internalization of HA-based carriers, although this positive synergy depends on the mode of ligand presentation. PMID:26865006

  16. Hayabusa's follow-on mission for surface and sub-surface sample return from a C-type NEO

    NASA Astrophysics Data System (ADS)

    Yano, Hajime; Yoshikawa, M.; Yano, H.; Tsuda, Y.; Nakazawa, S.; Mimamino, H.; Terui, F.; Saiki, T.; Nishiyama, K.; Kubota, T.; Okada, T.; Morimoto, M. Y.; Ogawa, N.; Okamoto, C.; Takagi, Y.; Tachibana, S.; Nakamura, R.; Hirata, N.; Demura, H.

    n JAXA's Long-term Vision 2005-2025, acquiring the capability of deep space round trip be-yond the Earth-Moon system is one of key elements for the future space exploration and that has been Hayabusa's primary engineering goal. According to the solar system exploration sci-ence roadmap set by ISAS and JSPEC in 2007, a programmatic approach to small body sample returns from S-type, C-type and then P/D-type asteroids as well as dormant comets, i.e., 'the further, the smaller, the more primitive strategy', is recommended for strengthening Japan's unique position in the field of space exploration. In a more recent international context, NEOs and Martian satellites have been identified as critical targets for the future human space explo-ration en route to Mars; thus their robotic precursor missions with the round trip capability have become more important than ever. Thus, Hayabusa's immediate follow-on mission, nicknamed so far as 'Hayabusa-2', is to aim establishing round trip exploration capability with both technical and operational heritage and lessons leaerned from the original Haybusa mission. It will also conduct in-situ observation and surface and sub-surface sample returns of a C-type NEO after Hayabusa's investigation and sampling attempt at Itokawa, a sub-km, S-type NEO. Important to be reminded is that C-type asteroid exploration is not just matching with carbona-ceous chondrites and interplanetary dust but also enhancing chances to discover new extrater-restrial materials unknown to us today that may become clues to decode interactions among organic, inorganic compounds and "water" kept in various forms inside the object. These three groups of asteroidal materials are basic constituents of the planet Earth, its ocean and its life. Also physical probing inside solid planetary bodies has been recognized as an effective tool to open new scientific insights. By excavating sub-surface materials with artificial physical in-teractions such as an impactor

  17. Lectin binding to surface Ig variable regions provides a universal persistent activating signal for follicular lymphoma cells.

    PubMed

    Linley, Adam; Krysov, Sergey; Ponzoni, Maurilio; Johnson, Peter W; Packham, Graham; Stevenson, Freda K

    2015-10-15

    The vast majority of cases of follicular lymphoma (FL), but not normal B cells, acquire N-glycosylation sites in the immunoglobulin variable regions during somatic hypermutation. Glycans added to sites are unusual in terminating at high mannoses. We showed previously that the C-type lectins, dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor, bound to FL surface immunoglobulin (sIg), generating an intracellular Ca(2+) flux. We have now mapped further intracellular pathways activated by DC-SIGN in a range of primary FL cells with detection of phosphorylated ERK1/2, AKT, and PLCγ2. The SYK inhibitor (tamatinib) or the BTK inhibitor (ibrutinib) each blocked phosphorylation. Activation by DC-SIGN occurred in both IgM(+) and IgG(+) cases and led to upregulation of MYC expression, with detection in vivo observed in lymph nodes. Unlike cells of chronic lymphocytic leukemia, FL cells expressed relatively high levels of sIg, unchanged by long-term incubation in vitro, indicating no antigen-mediated downregulation in vivo. In contrast, expression of CXCR4 increased in vitro. Engagement of sIg in FL cells or normal B cells by anti-Ig led to endocytosis in vitro as expected, but DC-SIGN, even when cross-linked, did not lead to significant endocytosis of sIg. These findings indicate that lectin binding generates signals via sIg but does not mediate endocytosis, potentially maintaining a supportive antigen-independent signal in vivo. Location of DC-SIGN in FL tissue revealed high levels in sinusoidlike structures and in some colocalized mononuclear cells, suggesting a role for lectin-expressing cells at this site. PMID:26194765

  18. Surface array proteins of Campylobacter fetus block lectin-mediated binding to type A lipopolysaccharide.

    PubMed Central

    Fogg, G C; Yang, L Y; Wang, E; Blaser, M J

    1990-01-01

    Campylobacter fetus strains with type A lipopolysaccharide (LPS) and a surface array protein layer (S+) have been found to be pathogenic in humans and animals. Spontaneous laboratory mutants that lack surface array proteins (S-) are sensitive to the bactericidal activity of normal human serum. The ability of lectins to determine the presence of the S-layer and differentiate LPS type was assessed. We screened 14 lectins and found 3 (wheat germ agglutinin, Bandeiraea simplicifolia II, and Helix pomatia agglutinin) that agglutinated S- C. fetus strains with type A LPS but not S- strains with type B or type C LPS or S+ strains. However, the S+ type A strains were agglutinated after sequential water extraction, heat, or pronase treatment, all of which remove the S-layer, whereas there was no effect on the control strains. Specific carbohydrates for each lectin and purified LPS from a type A C. fetus strain specifically inhibited agglutination of an S- type A strain. In a direct enzyme-linked lectin assay, binding to the S- type A LPS strain was significantly greater than binding to the S+ strain (P = 0.01) or to a Campylobacter jejuni strain (P = 0.008). Consequently, these results indicate that the three lectins bind to the O side chains of C. fetus type A LPS but that the presence of the S-layer on intact cells blocks binding. Images PMID:2387622

  19. Vimentin and desmin possess GlcNAc-binding lectin-like properties on cell surfaces.

    PubMed

    Ise, Hirohiko; Kobayashi, Satoshi; Goto, Mitsuaki; Sato, Takao; Kawakubo, Masatomo; Takahashi, Masafumi; Ikeda, Uichi; Akaike, Toshihiro

    2010-07-01

    Vimentin and desmin are intermediate filament proteins found in various mesenchymal and skeletal muscle cells, respectively. These proteins play an important role in the stabilization of the cytoplasmic architecture. Here, we found, using artificial biomimicking glycopolymers, that vimentin and desmin possess N-acetylglucosamine (GlcNAc)-binding lectin-like properties on the cell surfaces of various vimentin- and desmin-expressing cells such as cardiomyocytes and vascular smooth muscle cells. The rod II domain of these proteins was demonstrated to be localized to the cell surface and to directly bind to the artificial biomimicking GlcNAc-bearing polymer, by confocal laser microscopy and surface plasmon resonance analysis. These glycopolymers strongly interact with lectins and are useful tools for the analysis of lectin-carbohydrate interactions, since glycopolymers binding to lectins can induce the clustering of lectins due to multivalent glycoside ligand binding. Moreover, immunocytochemistry and pull-down assay with His-tagged vimentin-rod II domain protein showed that the vimentin-rod II domain interacts with O-GlcNAc proteins. These results suggest that O-GlcNAc proteins might be one candidate for physiological GlcNAc-bearing ligands with which vimentin and desmin interact. These findings demonstrate a novel function of vimentin and desmin that does not involve stabilization of the cytoplasmic architecture by which these proteins interact with physiological GlcNAc-bearing ligands such as O-GlcNAc proteins on the cell surface through their GlcNAc-binding lectin-like properties. PMID:20332081

  20. 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.

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

    PubMed

    Gauthier, Julie D; Jenkins, Jill A; La Peyre, Jerome F

    2004-06-01

    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. PMID:15270084

  2. Fluorescein Isothiocyanate-Labeled Lectin Analysis of the Surface of the Nitrogen-Fixing Bacterium Azospirillum brasilense by Flow Cytometry

    PubMed Central

    Yagoda-Shagam, Janet; Barton, Larry L.; Reed, William P.; Chiovetti, Robert

    1988-01-01

    The cell surface of Azospirillum brasilense was probed by using fluorescein isothiocyanate (FITC)-labeled lectins, with binding determined by fluorescence-activated flow cytometry. Cells from nitrogen-fixing or ammonium-assimilating cultures reacted similarly to FITC-labeled lectins, with lectin binding in the following order: Griffonia simplicifolia II agglutinin > Griffonia simplicifolia I agglutinin > Triticum vulgaris agglutinin > Glycine max agglutinin > Canavalia ensiformis agglutinin > Limax flavus agglutinin > Lotus tetragonolobus agglutinin. The fluorescence intensity of cells labeled with FITC-labeled G. simplicifolia I, C. ensiformis, T. vulgaris, and G. max agglutinins was influenced by lectin concentration. Flow cytometry measurements of lectin binding to cells was consistent with measurements of agglutination resulting from lectin-cell interaction. Capsules surrounding nitrogen-fixing and ammonium-assimilating cells were readily demonstrated by light and transmission electron microscopies. Images PMID:16347693

  3. Destabilization of pea lectin by substitution of a single amino acid in a surface loop.

    PubMed

    Hoedemaeker, F J; van Eijsden, R R; Díaz, C L; de Pater, B S; Kijne, J W

    1993-09-01

    Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry. Inactivation of ANF is an important element in processing of food. In our study on the stability of Pisum sativum L. lectin (PSL), a conserved hydrophobic amino acid (Val103) in a surface loop was replaced with alanine. The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (Tm) by some 10 degrees C in comparison with wild-type (wt) PSL, and the denaturation energy (delta H) is only about 55% of that of wt PSL. Replacement of an adjacent amino acid (Phe104) with alanine did not result in a significant difference in stability in comparison with wt PSL. Both mutations did not change the sugar-binding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures. The double mutant, PSL V103A/F104A, was produced in Escherichia coli, but could not be isolated in an active (i.e. sugar-binding) form. Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 degrees C, as judged from haemagglutination assays. These results open the possibility of production of lectins that are active in planta at ambient temperatures, but are inactive and possibly non-toxic at 37 degrees C in the intestines of mammals. PMID:8400124

  4. An African swine fever virus ORF with similarity to C-type lectins is non-essential for growth in swine macrophages in vitro and for virus virulence in domestic swine.

    PubMed

    Neilan, J G; Borca, M V; Lu, Z; Kutish, G F; Kleiboeker, S B; Carrillo, C; Zsak, L; Rock, D L

    1999-10-01

    An African swine fever virus (ASFV) ORF, 8CR, with similarity to the C-type lectin family of adhesion proteins has been described in the pathogenic isolate Malawi Lil-20/1. The similarity of 8CR to cellular and poxvirus genes associated with cell adhesion, cell recognition and virus infectivity suggested that 8CR may be of significance to ASFV-host cell interactions. Sequence analysis of the 8CR ORF from additional pathogenic ASFV isolates demonstrated conservation among isolates from both pig and tick sources. Northern blot analysis demonstrated 8CR mRNA transcription late in the virus replication cycle. A Malawi Lil-20/1 8CR deletion mutant (delta8CR) was constructed to analyse 8CR function further. The growth characteristics in vitro of delta8CR in porcine macrophage cell cultures were identical to those observed for parental virus. In domestic swine, delta8CR exhibited an unaltered parental Malawi Lil-20/1 disease and virulence phenotype. Thus, although well conserved among pathogenic ASFV field isolates, 8CR is non-essential for growth in porcine macrophages in vitro and for virus virulence in domestic swine. PMID:10573162

  5. Enhanced cell adhesion on silk fibroin via lectin surface modification.

    PubMed

    Teuschl, Andreas H; Neutsch, Lukas; Monforte, Xavier; Rünzler, Dominik; van Griensven, Martijn; Gabor, Franz; Redl, Heinz

    2014-06-01

    Various tissue engineering (TE) approaches are based on silk fibroin (SF) as scaffold material because of its superior mechanical and biological properties compared to other materials. The translation of one-step TE approaches to clinical application has generally failed so far due to the requirement of a prolonged cell seeding step before implantation. Here, we propose that the plant lectin WGA (wheat germ agglutinin), covalently bound to SF, will mediate cell adhesion in a time frame acceptable to be part of a one-step surgical intervention. After the establishment of a modification protocol utilizing carbodiimide chemistry, we examined the attachment of cells, with a special focus on adipose-derived stromal cells (ASC), on WGA-SF compared to pure native SF. After a limited time frame of 20min the attachment of ASCs to WGA-SF showed an increase of about 17-fold, as compared to pure native SF. The lectin-mediated cell adhesion further showed an enhanced resistance to trypsin (as a protease model) and to applied fluid shear stress (mechanical stability). Moreover, we could demonstrate that the adhesion of ASCs on the WGA-SF does not negatively influence proliferation or differentiation potential into the osteogenic lineage. To test for in vitro immune response, the proliferation of peripheral blood mononuclear cells in contact with the WGA-SF was determined, showing no alterations compared to plain SF. All these findings suggest that the WGA modification of SF offers important benefits for translation of SF scaffolds into clinical applications. PMID:24530561

  6. C-type lectin receptor dectin-3 mediates trehalose 6,6'-dimycolate (TDM)-induced Mincle expression through CARD9/Bcl10/MALT1-dependent nuclear factor (NF)-κB activation.

    PubMed

    Zhao, Xue-Qiang; Zhu, Le-Le; Chang, Qing; Jiang, Changying; You, Yun; Luo, Tianming; Jia, Xin-Ming; Lin, Xin

    2014-10-24

    Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6'-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection. PMID:25202022

  7. A new C-type lectin (RVsnaclec) purified from venom of Daboia russelii russelii shows anticoagulant activity via inhibition of FXa and concentration-dependent differential response to platelets in a Ca²⁺-independent manner.

    PubMed

    Mukherjee, Ashis K; Dutta, Sumita; Mackessy, Stephen P

    2014-11-01

    This is the first report on the characterization of a snaclec (RVsnaclec) purified from Daboia russelii russelii venom. The RVsnaclec is a heterodimer of two subunits, α (15.1 kDa) and β (9 kDa). These subunits are covalently linked to form multimeric (αβ)₂ and (αβ)₄ structures. Peptide mass fingerprinting analysis of RVsnaclec via LC-MS/MS demonstrated its similarity to snaclecs purified from other viperid snake venoms. Two tryptic peptide sequences of RVsnaclec revealed the putative conserved domains of C-type lectin (CTL). RVsnaclec dose-dependently increased the Ca-clotting time and prothrombin time of platelet-poor plasma (PPP); however, it did not affect the partial thromboplastin time (APTT) or thrombin time of PPP. The in vitro and in vivo anticoagulant activity of RVsnaclec is correlated to its binding and subsequent uncompetitive inhibition of FXa (Ki = 0.52 μmole) in a Ca(2+)-independent manner; however, supplementation with 0.25 mM Ca(2+) enhanced the Xa binding potency of RVsnaclec. Monovalent or polyvalent antivenom failed to neutralize its anticoagulant potency, and RVsnaclec did not inhibit trypsin, chymotrypsin, thrombin or plasmin. RVsnaclec was devoid of hemolytic activity or cytotoxicity against several human cancer cell lines, demonstrated concentration-dependent aggregation and deaggregation of human platelets, and inhibited the ADP-induced aggregation of platelet. RVsnaclec (5.0 mg/kg body weight) was non-lethal to mice and showed no adverse pharmacological effects, suggesting that it has potential as a lead compound for future therapeutic applications in cardiovascular disorders. PMID:25281435

  8. Lectin-functionalized poly(glycidyl methacrylate)-block-poly(vinyldimethyl azlactone) surface supports for high avidity microbial capture

    SciTech Connect

    Hansen, Ryan R; Hinestrosa Salazar, Juan P; Shubert, Katherine R; Morrell, Jennifer L.; Pelletier, Dale A; Messman, Jamie M; Kilbey, II, S Michael; Lokitz, Bradley S; Retterer, Scott T

    2013-01-01

    Microbial exopolysaccharides (EPS) play a critical and dynamic role in shaping the interactions between microbial community members and their local environment. The capture of targeted microbes using surface immobilized lectins that recognize specific extracellular oligosaccharide moieties offers a non-destructive method for functional characterization based on EPS content. In this report, we evaluate the use of the block co-polymer, poly(glycidyl methacrylate)-block-4,4-dimethyl-2-vinylazlactone (PGMA-b-PVDMA), as a surface support for lectin-specific microbial capture. Arrays of circular polymer supports ten micron in diameter were generated on silicon substrates to provide discrete, covalent coupling sites for Triticum vulgare and Lens culinaris lectins. These supports promoted microbe adhesion and colony formation in a lectin-specific manner. Silicon posts with similar topography containing only physisorbed lectins showed significantly less activity. These results demonstrate that micropatterned PGMA-b-PVDMA supports provide a unique platform for microbial capture and screening based on EPS content by combining high avidity lectin surfaces with three-dimensional topography.

  9. Analysis of unconventional approaches for the rapid detection of surface lectin binding ligands on human cell lines.

    PubMed

    Welty, Lily Anne Y; Heinrich, Eileen L; Garcia, Karina; Banner, Lisa R; Summers, Michael L; Baresi, Larry; Metzenberg, Stan; Coyle-Thompson, Cathy; Oppenheimer, Steven B

    2006-01-01

    For over a decade our laboratory has developed and used a novel histochemical assay using derivatized agarose beads to examine the surface properties of various cell types. Most recently, we have used this assay to examine lectin binding ligands on two human cell types, CCL-220, a colon cancer cell line, and CRL-1459, a non-cancer colon cell line. We found that CCL-220 cells bound specific lectins better than CRL-1459, and this information was used to test for possible differential toxicity of these lectins in culture, as a possible approach in the design of more specific anti-cancer drugs. Although we have examined the validity of the bead-binding assay in sea urchin cell systems, we have not previously validated this technique for mammalian cells. Here the binding results of the bead assay are compared with conventional fluorescence assays, using lectins from three species (Triticum vulgaris, Phaseolus vulgaris, and Lens culinaris) on the two colon cell lines. These lectins were chosen because they seemed to interact with the two cell lines differently. Binding results obtained using both assays were compared for frozen, thawed and fixed; cultured and fixed; and live cells. Both qualitative and quantitative fluorescence results generally correlated with those using the bead assay. Similar results were also obtained with all of the three different cell preparation protocols. The fluorescence assay was able to detect lower lectin binding ligand levels than the bead assay, while the bead assay, because it can so rapidly detect cells with large numbers of lectin binding ligands, is ideal for initial screening studies that seek to identify cells that are rich in surface binders for specific molecules. The direct use of frozen, thawed and fixed cells allows rapid mass screening for surface molecules, without the requirement for costly and time consuming cell culture. PMID:16414103

  10. A New Surface Plasmon Resonance Assay for In Vitro Screening of Mannose-Binding Lectin Inhibitors.

    PubMed

    Stravalaci, Matteo; De Blasio, Daiana; Orsini, Franca; Perego, Carlo; Palmioli, Alessandro; Goti, Giulio; Bernardi, Anna; De Simoni, Maria-Grazia; Gobbi, Marco

    2016-08-01

    Mannose-binding lectin (MBL) is a circulating protein that acts as a soluble pattern recognition molecule of the innate immunity. It binds to carbohydrate patterns on the surface of pathogens or of altered self-cells, with activation of the lectin pathway of the complement system. Recent evidence indicates that MBL contributes to the pathophysiology of ischemia-reperfusion injury and other conditions. Thus, MBL inhibitors offer promising therapeutic strategies, since they prevent the interaction of MBL with its target sugar arrays. We developed and characterized a novel assay based on surface plasmon resonance for in vitro screening of these compounds, which may be useful before the more expensive and time-consuming in vivo studies. The assay measures the inhibitor's ability to interfere with the binding of murine MBL-A or MBL-C, or of human recombinant MBL, to mannose residues immobilized on the sensor chip surface. We have applied the assay to measure the IC50 of synthetic glycodendrimers, two of them with neuroprotective properties in animal models of MBL-mediated injuries. PMID:26969323

  11. Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria

    PubMed Central

    Carlson, Hans K.; Iavarone, Anthony T.; Gorur, Amita; Yeo, Boon Siang; Tran, Rosalie; Melnyk, Ryan A.; Mathies, Richard A.; Auer, Manfred; Coates, John D.

    2012-01-01

    Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium. PMID:22307634

  12. Molecular recognition of surface-immobilized carbohydrates by a synthetic lectin

    PubMed Central

    Rauschenberg, Melanie; Fritz, Eva-Corrina; Schulz, Christian; Kaufmann, Tobias

    2014-01-01

    Summary The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of synthetic carbohydrate receptors (“synthetic lectins”) constitutes a key advance in biomedical technology. In this article we report a synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontaneously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA), β-D-galactose, β-D-glucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobilized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluorescence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins and that the recognition of synthetic and natural lectins is strictly orthogonal. PMID:24991289

  13. Kinetics and thermodynamics of glycans and glycoproteins binding to Holothuria scabra lectin: a fluorescence and surface plasmon resonance spectroscopic study.

    PubMed

    Gowda, Nagaraj M; Gaikwad, Sushama M; Khan, M Islam

    2013-11-01

    Holothuria scabra produces a monomeric lectin (HSL) of 182 kDa. HSL showed strong antibacterial activity and induced bacterial agglutination under in vitro conditions, indicating its role in animals' innate immune responses. Very few lectins have been reported from echinoderms and none of these lectins have been explored in detail for their sugar-binding kinetics. Affinity, kinetics and thermodynamic analysis of glycans and glycoproteins binding to HSL were studied by fluorescence and surface plasmon resonance spectroscopy. Lectin binds with higher affinity to O-linked than N-linked asialo glycans, and the affinities were relatively higher than that for sialated glycans and glycoproteins. T-antigen α-methyl glycoside was the most potent ligand having the highest affinity (Ka 8.32 ×10(7) M(-1)). Thermodynamic and kinetic analysis indicated that the binding of galactosyl Tn-antigen and asialo glycans is accompanied by an enthalpic contribution in addition to higher association rate coupled by low activation energy for the association process. Presence of sialic acid or protein matrix inhibits binding. Higher affinity of HSL for O-glycans than N-glycans had biological implications; since HSL specifically recognizes bacteria, which have mucin or O-glycan cognate on their cell surfaces and play a major role in animal innate immunity. Since, HSL had higher affinity to T-antigen, makes it a useful tool for cancer diagnostic purpose. PMID:23736907

  14. Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics

    PubMed Central

    Soga, Keisuke; Abo, Hirohito; Qin, Sheng-Ying; Kyoutou, Takuya; Hiemori, Keiko; Tateno, Hiroaki; Matsumoto, Naoki; Hirabayashi, Jun; Yamamoto, Kazuo

    2015-01-01

    Leguminous lectins have a conserved carbohydrate recognition site comprising four loops (A–D). Here, we randomly mutated the sequence and length of loops C and D of peanut agglutinin (PNA) and expressed the proteins on the surface of mouse green fluorescent protein (GFP)-reporter cells. Flow cytometry, limiting dilution, and cDNA cloning were used to screen for several mutated PNAs with distinct properties. The mutated PNA clones obtained using NeuAcα2-6(Galβ1-3)GalNAc as a ligand showed preference for NeuAcα2-6(Galβ1-3)GalNAc rather than non-sialylated Galβ1-3GlcNAc, whereas wild-type PNA binds to Galβ1-3GlcNAc but not sialylated Galβ1-3GalNAc. Sequence analyses revealed that for all of the glycan-reactive mutated PNA clones, (i) loop C was eight amino acids in length, (ii) loop D was identical to that of wild-type PNA, (iii) residue 127 was asparagine, (iv) residue 125 was tryptophan, and (v) residue 130 was hydrophobic tyrosine, phenylalanine, or histidine. The sugar-binding ability of wild-type PNA was increased nine-fold when Tyr125 was mutated to tryptophan, and that of mutated clone C was increased more than 30-fold after His130 was changed to tyrosine. These results provide an insight into the relationship between the amino acid sequences of the carbohydrate recognition site and sugar-binding abilities of leguminous lectins. PMID:26287256

  15. Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics.

    PubMed

    Soga, Keisuke; Abo, Hirohito; Qin, Sheng-Ying; Kyoutou, Takuya; Hiemori, Keiko; Tateno, Hiroaki; Matsumoto, Naoki; Hirabayashi, Jun; Yamamoto, Kazuo

    2015-01-01

    Leguminous lectins have a conserved carbohydrate recognition site comprising four loops (A-D). Here, we randomly mutated the sequence and length of loops C and D of peanut agglutinin (PNA) and expressed the proteins on the surface of mouse green fluorescent protein (GFP)-reporter cells. Flow cytometry, limiting dilution, and cDNA cloning were used to screen for several mutated PNAs with distinct properties. The mutated PNA clones obtained using NeuAcα2-6(Galβ1-3)GalNAc as a ligand showed preference for NeuAcα2-6(Galβ1-3)GalNAc rather than non-sialylated Galβ1-3GlcNAc, whereas wild-type PNA binds to Galβ1-3GlcNAc but not sialylated Galβ1-3GalNAc. Sequence analyses revealed that for all of the glycan-reactive mutated PNA clones, (i) loop C was eight amino acids in length, (ii) loop D was identical to that of wild-type PNA, (iii) residue 127 was asparagine, (iv) residue 125 was tryptophan, and (v) residue 130 was hydrophobic tyrosine, phenylalanine, or histidine. The sugar-binding ability of wild-type PNA was increased nine-fold when Tyr125 was mutated to tryptophan, and that of mutated clone C was increased more than 30-fold after His130 was changed to tyrosine. These results provide an insight into the relationship between the amino acid sequences of the carbohydrate recognition site and sugar-binding abilities of leguminous lectins. PMID:26287256

  16. The presence of multiple c-type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper.

    PubMed

    Karlsen, O A; Lillehaug, J R; Jensen, H B

    2008-10-01

    Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure. PMID:18681943

  17. Assembly, organization and regulation of cell-surface receptors by lectin-glycan complexes.

    PubMed

    Elola, María T; Blidner, Ada G; Ferragut, Fátima; Bracalente, Candelaria; Rabinovich, Gabriel A

    2015-07-01

    Galectins are a family of β-galactoside-binding lectins carrying at least one consensus sequence in the carbohydrate-recognition domain. Properties of glycosylated ligands, such as N- and O-glycan branching, LacNAc (N-acetyl-lactosamine) content and the balance of α2,3- and α2,6-linked sialic acid dramatically influence galectin binding to a preferential set of counter-receptors. The presentation of specific glycans in galectin-binding partners is also critical, as proper orientation and clustering of oligosaccharide ligands on multiple carbohydrate side chains increase the binding avidity of galectins for particular glycosylated receptors. When galectins are released from the cells, they typically concentrate on the cell surface and the local matrix, raising their local concentration. Thus galectins can form their own multimers in the extracellular milieu, which in turn cross-link glycoconjugates on the cell surface generating galectin-glycan complexes that modulate intracellular signalling pathways, thus regulating cellular processes such as apoptosis, proliferation, migration and angiogenesis. Subtle changes in receptor expression, rates of protein synthesis, activities of Golgi enzymes, metabolite concentrations supporting glycan biosynthesis, density of glycans, strength of protein-protein interactions at the plasma membrane and stoichiometry may modify galectin-glycan complexes. Although galectins are key contributors to the formation of these extended glycan complexes leading to promotion of receptor segregation/clustering, and inhibition of receptor internalization by surface retention, when these complexes are disrupted, some galectins, particularly galectin-3 and -4, showed the ability to drive clathrin-independent mechanisms of endocytosis. In the present review, we summarize the data available on the assembly, hierarchical organization and regulation of conspicuous galectin-glycan complexes, and their implications in health and disease. PMID:26173257

  18. Reverse micellar extraction of lectin from black turtle bean (Phaseolus vulgaris): optimisation of extraction conditions by response surface methodology.

    PubMed

    He, Shudong; Shi, John; Walid, Elfalleh; Zhang, Hongwei; Ma, Ying; Xue, Sophia Jun

    2015-01-01

    Lectin from black turtle bean (Phaseolus vulgaris) was extracted and purified by reverse micellar extraction (RME) method. Response surface methodology (RSM) was used to optimise the processing parameters for both forward and backward extraction. Hemagglutinating activity analysis, SDS-PAGE, RP-HPLC and FTIR techniques were used to characterise the lectin. The optimum extraction conditions were determined as 77.59 mM NaCl, pH 5.65, AOT 127.44 mM sodium bis (2-ethylhexyl) sulfosuccinate (AOT) for the forward extraction; and 592.97 mM KCl, pH 8.01 for the backward extraction. The yield was 63.21 ± 2.35 mg protein/g bean meal with a purification factor of 8.81 ± 0.17. The efficiency of RME was confirmed by SDS-PAGE and RP-HPLC, respectively. FTIR analysis indicated there were no significant changes in the secondary protein structure. Comparison with conventional chromatographic method confirmed that the RME method could be used for the purification of lectin from the crude extract. PMID:25053033

  19. A quantitative method to discriminate between non-specific and specific lectin-glycan interactions on silicon-modified surfaces.

    PubMed

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

    2016-02-15

    Essential to the success of any surface-based carbohydrate biochip technology is that interactions of the particular interface with the target protein be reliable and reproducible and not susceptible to unwanted nonspecific adsorption events. This condition is particularly important when the technology is intended for the evaluation of low-affinity interactions such as those typically encountered between lectins and their monomeric glycan ligands. In this paper, we describe the fabrication of glycan (mannoside and lactoside) monolayers immobilized on hydrogenated crystalline silicon (111) surfaces. An efficient conjugation protocol featuring a key "click"-based coupling step has been developed which ensures the obtention of interfaces with controlled glycan density. The adsorption behavior of these newly developed interfaces with the lectins, Lens culinaris and Peanut agglutinin, has been probed using quantitative IR-ATR and the data interpreted using various isothermal models. The analysis reveals that protein physisorption to the interface is more prevalent than specific chemisorption for the majority of washing protocols investigated. Physisorption can be greatly suppressed through application of a strong surfactinated rinse. The coexistence of chemisorption and physisorption processes is further demonstrated by quantification of the amounts of adsorbed proteins distributed on the surface, in correlation with the results obtained by atomic force microscopy (AFM). Taken together, the data demonstrates that the nonspecific adsorption of proteins to these glycan-terminated surfaces can be effectively eliminated through the proper control of the chemical structure of the surface monolayer combined with the implementation of an appropriate surface-rinse protocol. PMID:26619130

  20. Lectins in the investigation of receptors

    NASA Astrophysics Data System (ADS)

    Lakhtin, V. M.; Yamskov, Igor A.

    1991-08-01

    Problems of the purification and characterisation are considered for approximately 270 receptors (including cell surface and organelle enzymes), which are glycoconjugates (mainly glycoproteins) from animals, plants and microorganisms, using various lectins (mainly lectin sorbents). An analysis has been carried out of the stages of lectin affinity chromatography of receptors (choice of detergent, use of organic solvents, elution with carbohydrates, etc.). Examples are given of procedures for the purification of receptors, including the use of paired columns and combination chromatography on lectins. The possibility of separating sub-populations of receptors using lectins has been demonstrated. Examples are given of the use of lectins in the analysis of the oligosaccharide structure of receptors. Cases are recorded of the interaction of receptors with endogenous lectins and of receptor lectins with endogenous glycoconjugates. It has been shown that lectins, in combination with glycosidases and antibodies, may be useful in the investigation of receptors. The bibliography contains 406 references.

  1. Cell-surface changes in cadmium-resistant Euglena: Studies using lectin-binding techniques and flow cytometry

    SciTech Connect

    Bonaly, J.; Brochiero, E.

    1994-01-01

    Most in vitro studies on contaminants focus on the short-term effects of pollutants on cells, without regard to long-term effects and the ability of cells or microorganisms to develop a specific resistance to a pollutant. Cadmium is ubiquitous environmental contaminant. This heavy metal enters the aquatic environment mainly through vapor emissions and fallout during smelting operations. Diverse mechanisms of algal resistance to toxic metals are known. Among these, the most general mechanism is the development of metal-binding proteins. In cadmium-resistant unicellular Euglena gracilis Z algae cells, the metal did not appear to be sequestered on soluble metal-binding ligands. Previous experiments have shown that resistance development is related to a diminution of cadmium penetration into cells, implicating cell surface or membrane alteration. This research investigates the mechanisms of development of cadmium resistance in Euglena cells at the cell-surface level. Sugar chains of glycoproteins and glycolipids are a predominant feature of the surface of cells. Moreover, the cell-response to environmental changes is often orchestrated through surface macromolecules such as glycoproteins. In this study, we applied this lectin method to investigate surface carbohydrate expression during and after resistance development. Our interest was twofold: (1) to learn more about the carbohydrate composition of the cell-surface of Euglena; and (2) to determine whether transition from wild cells to Cd-resistant cells changes the expression of cell-surface carbohydrates. 13 refs., 2 figs., 1 tab.

  2. Electrochemical synthesis of nanostructured gold film for the study of carbohydrate–lectin interactions using localized surface plasmon resonance spectroscopy

    PubMed Central

    Bhattarai, Jay K.; Sharma, Abeera; Fujikawa, Kohki; Demchenko, Alexei V.; Stine, Keith J.

    2014-01-01

    Localized surface plasmon resonance (LSPR) spectroscopy is a label-free chemical and biological molecular sensing technique whose sensitivity depends upon development of nanostructured transducers. Herein, we report an electrodeposition method for fabricating nanostructured gold films (NGFs) that can be used as transducers in LSPR spectroscopy. The NGF was prepared by electrodepositing gold from potassium dicyanoaurate solution onto a flat gold surface using two sequential controlled potential steps. Imaging by scanning electron microscopy reveals a morphology consisting of randomly configured block-like nanostructures. The bulk refractive index sensitivity of the prepared NGF is 100 ± 2 nm RIU−1 and the initial peak in the reflectance spectrum is at 518 ± 1 nm under N2(g). The figure of merit is 1.7. In addition, we have studied the interaction between carbohydrate (mannose) and lectin (Concanavalin A) on the NGF surface using LSPR spectroscopy by measuring the interaction of 8-mercaptooctyl-α-D-mannopyranoside (αMan-C8-SH) with Concanavalin A by first immobilizing αMan-C8-SH in mixed SAMs with 3,6-dioxa-8-mercaptooctanol (TEG-SH) on the NGF surface. The interaction of Con A with the mixed SAMs is confirmed using electrochemical impedance spectroscopy. Finally, the NGF surface was regenerated to its original sensitivity by removing the SAM and the bound biomolecules. The results from these experiments contribute toward the development of inexpensive LSPR based sensors that could be useful for studying glycan–protein interactions and other bioanalytical purposes. PMID:25442712

  3. Characterization of the Decaheme c-Type Cytochrome OmcA in Solution and on Hematite Surfaces by Small Angle X-Ray Scattering and Neutron Reflectometry

    PubMed Central

    Johs, A.; Shi, L.; Droubay, T.; Ankner, J.F.; Liang, L.

    2010-01-01

    Abstract The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate shuttling of electrons to extracellular acceptors that include solid metal oxides such as hematite (α-Fe2O3). No information is yet available concerning OmcA structure in physiologically relevant conditions such as aqueous environments. We purified OmcA and characterized its solution structure by small angle x-ray scattering (SAXS), and its interaction at the hematite-water interface by neutron reflectometry. SAXS showed that OmcA is a monomer that adopts a flat ellipsoidal shape with an overall dimension of 34 × 90 × 65 Å3. To our knowledge, we obtained the first direct evidence that OmcA undergoes a redox state-dependent conformational change in solution whereby reduction decreases the overall length of OmcA by ∼7 Å (the maximum dimension was 96 Å for oxidized OmcA, and 89 Å for NADH and dithionite-reduced OmcA). OmcA was also found to physically interact with electron shuttle molecules such as flavin mononucleotide, resulting in the formation of high-molecular-weight assemblies. Neutron reflectometry showed that OmcA forms a well-defined monomolecular layer on hematite surfaces, where it assumes an orientation that maximizes its contact area with the mineral surface. These novel insights into the molecular structure of OmcA in solution, and its interaction with insoluble hematite and small organic ligands, demonstrate the fundamental structural bases underlying OmcA's role in mediating redox processes. PMID:20550916

  4. Characterization of the Decaheme c-Type Cytochrome OmcA in Solution and on Hematite Surfaces by Small Angle X-Ray Scattering and Neutron Reflectometry

    SciTech Connect

    Johs, Alexander; Shi, Liang; Droubay, Timothy C.; Ankner, John F.; Liang, L.

    2010-06-01

    The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate shuttling of electrons to extracellular acceptors that include solid metal oxides such as hematite (a-Fe2O3). No information is yet available concerning OmcA structure in physiologically relevant conditions such as aqueous environments. We purified OmcA and characterized its solution structure by small angle x-ray scattering (SAXS), and its interaction at the hematite-water interface by neutron reflectometry. SAXS showed that OmcA is a monomer that adopts a flat ellipsoidal shape with an overall dimension of 34 x 90 x 65A˚ 3. To our knowledge, we obtained the first direct evidence that OmcA undergoes a redox state-dependent conformational change in solution whereby reduction decreases the overall length of OmcA by ~7 A˚ (the maximum dimension was 96 A˚ for oxidized OmcA, and 89 A˚ for NADH and dithionite-reduced OmcA). OmcA was also found to physically interact with electron shuttle molecules such as flavin mononucleotide, resulting in the formation of high-molecular-weight assemblies. Neutron reflectometry showed that OmcA forms a well-defined monomolecular layer on hematite surfaces, where it assumes an orientation that maximizes its contact area with the mineral surface. These novel insights into the molecular structure of OmcA in solution, and its interaction with insoluble hematite and small organic ligands, demonstrate the fundamental structural bases underlying OmcA’s role in mediating redox processes.

  5. MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

    PubMed

    Hasan, Imtiaj; Sugawara, Shigeki; Fujii, Yuki; Koide, Yasuhiro; Terada, Daiki; Iimura, Naoya; Fujiwara, Toshiyuki; Takahashi, Keisuke G; Kojima, Nobuhiko; Rajia, Sultana; Kawsar, Sarkar M A; Kanaly, Robert A; Uchiyama, Hideho; Hosono, Masahiro; Ogawa, Yukiko; Fujita, Hideaki; Hamako, Jiharu; Matsui, Taei; Ozeki, Yasuhiro

    2015-12-01

    MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface. PMID:26694420

  6. MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

    PubMed Central

    Hasan, Imtiaj; Sugawara, Shigeki; Fujii, Yuki; Koide, Yasuhiro; Terada, Daiki; Iimura, Naoya; Fujiwara, Toshiyuki; Takahashi, Keisuke G.; Kojima, Nobuhiko; Rajia, Sultana; Kawsar, Sarkar M. A.; Kanaly, Robert A.; Uchiyama, Hideho; Hosono, Masahiro; Ogawa, Yukiko; Fujita, Hideaki; Hamako, Jiharu; Matsui, Taei; Ozeki, Yasuhiro

    2015-01-01

    MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface. PMID:26694420

  7. Electrochemical synthesis of nanostructured gold film for the study of carbohydrate-lectin interactions using localized surface plasmon resonance spectroscopy.

    PubMed

    Bhattarai, Jay K; Sharma, Abeera; Fujikawa, Kohki; Demchenko, Alexei V; Stine, Keith J

    2015-03-20

    Localized surface plasmon resonance (LSPR) spectroscopy is a label-free chemical and biological molecular sensing technique whose sensitivity depends upon development of nanostructured transducers. Herein, we report an electrodeposition method for fabricating nanostructured gold films (NGFs) that can be used as transducers in LSPR spectroscopy. The NGF was prepared by electrodepositing gold from potassium dicyanoaurate solution onto a flat gold surface using two sequential controlled potential steps. Imaging by scanning electron microscopy reveals a morphology consisting of randomly configured block-like nanostructures. The bulk refractive index sensitivity of the prepared NGF is 100±2 nmRIU(-1) and the initial peak in the reflectance spectrum is at 518±1 nm under N2(g). The figure of merit is 1.7. In addition, we have studied the interaction between carbohydrate (mannose) and lectin (Concanavalin A) on the NGF surface using LSPR spectroscopy by measuring the interaction of 8-mercaptooctyl-α-d-mannopyranoside (αMan-C8-SH) with Concanavalin A by first immobilizing αMan-C8-SH in mixed SAMs with 3,6-dioxa-8-mercaptooctanol (TEG-SH) on the NGF surface. The interaction of Con A with the mixed SAMs is confirmed using electrochemical impedance spectroscopy. Finally, the NGF surface was regenerated to its original sensitivity by removing the SAM and the bound biomolecules. The results from these experiments contribute toward the development of inexpensive LSPR based sensors that could be useful for studying glycan-protein interactions and other bioanalytical purposes. PMID:25442712

  8. Use of lectins in immunohematology

    PubMed Central

    Gorakshakar, Ajit C.; Ghosh, Kanjaksha

    2016-01-01

    Lectins are carbohydrate binding proteins present in seeds of many plants, especially corals and beans, in fungi and bacteria, and in animals. Apart from their hemagglutinating property, a wide range of functions have been attributed to them. Their importance in the area of immunohematology is immense. They are used to detect specific red cell antigens, to activate different types of lymphocytes, in order to resolve problems related to polyagglutination and so on. The introduction of advanced biotechnological tools generates new opportunities to exploit the properties of lectins, which were not used earlier. Stem cell research is a very important area in transplant medicine. Certain lectins detect surface markers of stem cell. Hence, they are used to understand the developmental biology of stem cells. The role of various lectins in the areas of transfusion and transplant medicine is discussed in detail in this review. PMID:27011665

  9. 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

  10. Discrimination between lectins with similar specificities by ratiometric profiling of binding to glycosylated surfaces; a chemical ‘tongue’ approach† †Electronic supplementary information (ESI) is available: This includes protein preparation, surface functionalisation and LDA analysis. See DOI: 10.1039/c5ra08857g Click here for additional data file.

    PubMed Central

    Otten, L.

    2015-01-01

    Carbohydrate–lectin interactions dictate a range of signalling and recognition processes in biological systems. The exploitation of these, particularly for diagnostic applications, is complicated by the inherent promiscuity of lectins along with their low affinity for individual glycans which themselves are challenging to access (bio)synthetically. Inspired by how a ‘tongue’ can discriminate between hundreds of flavours using a minimal set of multiplexed sensors and a training algorithm, here individual lectins are ‘profiled’ based on their unique binding profile (barcode) to a range of monosaccharides. By comparing the relative binding of a panel of 5 lectins to 3 monosaccharide-coated surfaces, it was possible to generate a training algorithm that enables correct identification of lectins, even those with similar glycan preferences. This is demonstrated to be useful for discrimination between the cholera and ricin toxin lectins showing the potential of this minimalist approach for exploiting glycan complexity. PMID:27019703

  11. Specific adhesion of glycophorin liposomes to a lectin surface in shear flow.

    PubMed Central

    Wattenbarger, M R; Graves, D J; Lauffenburger, D A

    1990-01-01

    The adhesion of cells to other cells or to surfaces by receptor-ligand binding in a shear field is an important aspect of many different biological processes and various cell separation techniques. The purpose of this study was to observe the adhesion of model cells with receptor molecules embedded in their surfaces to a ligand-coated surface under well-defined flow conditions in a parallel plate flow chamber. Liposomes containing glycophorin were used as the model cells to permit a variation in the adhesion parameters and then to observe the effect on adhesion. A mathematical model for cell sedimentation was created to predict the deposition time and the velocity preceding adhesion for the selection of experimental operating conditions and the methods useful for data analysis. The likelihood of cell attachment was represented by a quantity called the sticking probability which was defined as the inverse of the number of times a liposome made contact with the surface before attachment occurred. The sticking probability decreased as the cell receptor concentration was lowered from approximately 10(4) to 10(2) receptors per 4-microns diam liposome and as the shear rate increased from 5 to 22 s-1. The effect of the wall shear rate and particle diameter on detachment of liposomes from a surface was also observed. PMID:2344463

  12. SEM visualization of glycosylated surface molecules using lectin-coated microspheres

    NASA Technical Reports Server (NTRS)

    Duke, J.; Janer, L.; Campbell, M.

    1985-01-01

    There are several techniques currently used to localize glycosylated surface molecules by scanning electron microscopy (Grinnell, 1980; Molday, 1976; Linthicum and Sell, 1975; Nicolson, 1974; Lo Buglio, et al, 1972). A simple and rapid method, using a modification of Grinnell's technique is reported here. Essentially, microspheres coated with Concavalin A are used to bind to glycosylated regions of the palatal shelf epithelium and are visualized in the scanning electron microscope (SEM).

  13. Lectins from opportunistic bacteria interact with acquired variable-region glycans of surface immunoglobulin in follicular lymphoma

    PubMed Central

    Schneider, Dunja; Dühren-von Minden, Marcus; Alkhatib, Alabbas; Setz, Corinna; van Bergen, Cornelis A. M.; Benkißer-Petersen, Marco; Wilhelm, Isabel; Villringer, Sarah; Krysov, Sergey; Packham, Graham; Zirlik, Katja; Römer, Winfried; Buske, Christian; Stevenson, Freda K.; Veelken, Hendrik

    2015-01-01

    B-cell antigen receptor (BCR) expression is a key feature of most B-cell lymphomas, but the mechanisms of BCR signal induction and the involvement of autoantigen recognition remain unclear. In follicular lymphoma (FL) B cells, BCR expression is retained despite a chromosomal translocation that links the antiapoptotic gene BCL2 to the regulatory elements of immunoglobulin genes, thereby disrupting 1 heavy-chain allele. A remarkable feature of FL-BCRs is the acquisition of potential N-glycosylation sites during somatic hypermutation. The introduced glycans carry mannose termini, which create potential novel binding sites for mannose-specific lectins. Here, we investigated the effect of N-linked variable-region glycosylation for BCR interaction with cognate antigen and with lectins of different origins. N-glycans were found to severely impair BCR specificity and affinity to the initial cognate antigen. In addition, we found that lectins from Pseudomonas aeruginosa and Burkholderia cenocepacia bind and stimulate FL cells. Human exposure to these bacteria can occur by contact with soil and water. In addition, they represent opportunistic pathogens in susceptible hosts. Understanding the role of bacterial lectins might elucidate the pathogenesis of FL and establish novel therapeutic approaches. PMID:25784678

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

    SciTech Connect

    Bradshaw, William J.; Kirby, Jonathan M.; Thiyagarajan, Nethaji; Chambers, Christopher J.; Davies, Abigail H.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

    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) 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.

  15. Role of the lectin complement pathway in kidney transplantation.

    PubMed

    Farrar, Conrad A; Zhou, Wuding; Sacks, Steven H

    2016-10-01

    In the last 15 years two major advances in the role of complement in the kidney transplant have come about. The first is that ischaemia reperfusion injury and its profound effect on transplant outcome is dependent on the terminal product of complement activation, C5b-9. The second key observation relates to the function of the small biologically active fragments C3a and C5a released by complement activation in increasing antigen presentation and priming the T cell response that results in transplant rejection. In both cases local synthesis of C3 principally by the renal tubule cells plays an essential role that overshadows the role of the circulating pool of C3 generated largely by hepatocyte synthesis. More recent efforts have investigated the molecules expressed by renal tissue that can trigger complement activation. These have revealed a prominent effect of collectin-11 (CL-11), a soluble C-type lectin that is expressed in renal tissue and aligns with its major ligand L-fucose at sites of complement activation following ischaemic stress. Biochemical studies have shown that interaction between CL-11 and L-fucose results in complement activation by the lectin complement pathway, precisely targeting the innate immune response to the ischaemic tubule surface. Therapeutic approaches to reduce inflammatory and immune stimulation in ischaemic kidney have so far targeted C3 or its activation products and several are in clinical trials. The finding that lectin-fucose interaction is an important trigger of lectin pathway complement activation within the donor organ opens up further therapeutic targets where intervention could protect the donor kidney against complement. PMID:27286717

  16. Lectin staining and flow cytometry reveals female-induced sperm acrosome reaction and surface carbohydrate reorganization

    PubMed Central

    Kekäläinen, Jukka; Larma, Irma; Linden, Matthew; Evans, Jonathan P.

    2015-01-01

    All cells are covered by glycans, an individually unique layer of oligo- and polysaccharides that are critical moderators of self-recognition and other cellular-level interactions (e.g. fertilization). The functional similarity between these processes suggests that gamete surface glycans may also have an important, but currently overlooked, role in sexual selection. Here we develop a user-friendly methodological approach designed to facilitate future tests of this possibility. Our proposed method is based on flow cytometric quantification of female-induced sperm acrosome reaction and sperm surface glycan modifications in the Mediterranean mussel Mytilus galloprovincialis. In this species, as with many other taxa, eggs release water-soluble factors that attract conspecific sperm (chemoattraction) and promote potentially measurable changes in sperm behavior and physiology. We demonstrate that flow cytometry is able to identify sperm from other seawater particles as well as accurately measure both acrosome reaction and structural modifications in sperm glycans. This methodological approach can increase our understanding of chemically-moderated gamete-level interactions and individual-specific gamete recognition in Mytilus sp. and other taxa with similar, easily identifiable acrosome structure. Our approach is also likely to be applicable to several other species, since carbohydrate-mediated cellular-level interactions between gametes are universal among externally and internally fertilizing species. PMID:26470849

  17. Purification and Characterization of OmcZ, an Outer-Surface, Octaheme c-Type Cytochrome Essential for Optimal Current Production by Geobacter sulfurreducens▿ †

    PubMed Central

    Inoue, Kengo; Qian, Xinlei; Morgado, Leonor; Kim, Byoung-Chan; Mester, Tünde; Izallalen, Mounir; Salgueiro, Carlos A.; Lovley, Derek R.

    2010-01-01

    Previous studies have demonstrated that Geobacter sulfurreducens requires the c-type cytochrome OmcZ, which is present in large (OmcZL; 50-kDa) and small (OmcZS; 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZS was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZS and molecular weight measurements indicated that OmcZS is a cleaved product of OmcZL retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX14CH. The purified OmcZS was remarkably thermally stable (thermal-denaturing temperature, 94.2°C). Redox titration analysis revealed that the midpoint reduction potential of OmcZS is approximately −220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (−420 to −60 mV). OmcZS transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of G. sulfurreducens. PMID:20400562

  18. Oviduct fluid and heparin induce similar surface changes in bovine sperm during capacitation: a flow cytometric study using lectins.

    PubMed

    Mahmoud, A I; Parrish, J J

    1996-04-01

    Eight different lectins conjugated to fluorescein isothiocyanate (FITC) were used to screen for sperm plasma membrane changes during in vitro capacitation of bovine sperm. Analysis of lectin binding to sperm was done using flow cytometry. Of the eight lectins, only Triticum vulgaris (wheat germ agglutinin, WGA) binding to sperm was altered with capacitation. Capacitation of bovine sperm by heparin was found to decrease WGA binding to sperm by 78% (P < 0.05). The effect of capacitation by oviduct fluid was next compared with capacitation by heparin for changes in WGA binding to sperm. The effect of inhibiting capacitation with glucose on WGA binding was also determined. WGA-bound sperm were detected by flow cytometry as being present in two fluorescence peaks defined as low fluorescence (A) or high fluorescence (B) intensity. The percentage of sperm in peak A was greater for heparin and oviduct fluid-treated sperm compared to sperm incubated under noncapacitating conditions in only culture medium (P < 0.001). Capacitation with either heparin or oviduct fluid was inhibited by glucose as assessed by the ability of lysophosphatidylcholine (100 micrograms/ml) to induce acrosome reactions. Glucose also reduced the percentage of sperm in peak A for both heparin- and oviduct fluid-treated sperm (P < 0.01). We conclude that heparin or oviduct fluid induced changes on the sperm plasma membrane during capacitation. Binding sites for WGA on sperm were either structurally altered or lost during capacitation. PMID:9052948

  19. Diversified Carbohydrate-Binding Lectins from Marine Resources

    PubMed Central

    Ogawa, Tomohisa; Watanabe, Mizuki; Naganuma, Takako; Muramoto, Koji

    2011-01-01

    Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families. PMID:22312473

  20. Functional Specialty of CD40 and Dendritic Cell Surface Lectins for Exogenous Antigen Presentation to CD8(+) and CD4(+) T Cells.

    PubMed

    Yin, Wenjie; Gorvel, Laurent; Zurawski, Sandra; Li, Dapeng; Ni, Ling; Duluc, Dorothée; Upchurch, Katherine; Kim, JongRok; Gu, Chao; Ouedraogo, Richard; Wang, Zhiqing; Xue, Yaming; Joo, HyeMee; Gorvel, Jean-Pierre; Zurawski, Gerard; Oh, SangKon

    2016-03-01

    Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific T cells. Delivering antigen to DCs via surface receptors is thus an appealing strategy to evoke cellular immunity. Nonetheless, which DC surface receptor to target to yield the optimal CD8(+) and CD4(+) T cell responses remains elusive. Herein, we report the superiority of CD40 over 9 different lectins and scavenger receptors at evoking antigen-specific CD8(+) T cell responses. However, lectins (e.g., LOX-1 and Dectin-1) were more efficient than CD40 at eliciting CD4(+) T cell responses. Common and distinct patterns of subcellular and intracellular localization of receptor-bound αCD40, αLOX-1 and αDectin-1 further support their functional specialization at enhancing antigen presentation to either CD8(+) or CD4(+) T cells. Lastly, we demonstrate that antigen targeting to CD40 can evoke potent antigen-specific CD8(+) T cell responses in human CD40 transgenic mice. This study provides fundamental information for the rational design of vaccines against cancers and viral infections. PMID:27077111

  1. Functional Specialty of CD40 and Dendritic Cell Surface Lectins for Exogenous Antigen Presentation to CD8+ and CD4+ T Cells

    PubMed Central

    Yin, Wenjie; Gorvel, Laurent; Zurawski, Sandra; Li, Dapeng; Ni, Ling; Duluc, Dorothée; Upchurch, Katherine; Kim, JongRok; Gu, Chao; Ouedraogo, Richard; Wang, Zhiqing; Xue, Yaming; Joo, HyeMee; Gorvel, Jean-Pierre; Zurawski, Gerard; Oh, SangKon

    2016-01-01

    Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific T cells. Delivering antigen to DCs via surface receptors is thus an appealing strategy to evoke cellular immunity. Nonetheless, which DC surface receptor to target to yield the optimal CD8+ and CD4+ T cell responses remains elusive. Herein, we report the superiority of CD40 over 9 different lectins and scavenger receptors at evoking antigen-specific CD8+ T cell responses. However, lectins (e.g., LOX-1 and Dectin-1) were more efficient than CD40 at eliciting CD4+ T cell responses. Common and distinct patterns of subcellular and intracellular localization of receptor-bound αCD40, αLOX-1 and αDectin-1 further support their functional specialization at enhancing antigen presentation to either CD8+ or CD4+ T cells. Lastly, we demonstrate that antigen targeting to CD40 can evoke potent antigen-specific CD8+ T cell responses in human CD40 transgenic mice. This study provides fundamental information for the rational design of vaccines against cancers and viral infections. PMID:27077111

  2. Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: A receptor for lectin-mediated interbacterial adherence

    SciTech Connect

    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 of 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.

  3. 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

  4. Application of fluorescence resonance energy transfer techniques to the study of lectin-binding site distribution on Paramecium primaurelia (Protista, Ciliophora) cell surface.

    PubMed

    Locatelli, D; Delmonte Corrado, M U; Politi, H; Bottiroli, G

    1998-01-01

    Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon occurring between the molecules of two fluorochromes with suitable spectral characteristics (donor-acceptor dye pair), and consisting in an excitation energy migration through a non-radiative process. Since the efficiency of the process is strictly dependent on the distance and reciprocal orientation of the donor and acceptor molecules, FRET-based techniques can be successfully applied to the study of biomolecules and cell component organisation and distribution. These techniques have been employed in studying Paramecium primaurelia surface membrane for the reciprocal distribution of N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc) glycosidic residues, which were found to be involved in mating cell pairing. NeuAc and GlcNAc were detected by their specific binding lectins, Limulus polyphemus agglutinin (LPA) and wheat germ agglutinin (WGA), respectively. Microspectrofluorometric analysis afforded the choice of fluorescein isothiocyanate and Texas red conjugated with LPA and WGA, respectively, as a suitable donor-acceptor couple efficiently activating FRET processes. Studies performed both in solution and in cells allowed to define the experimental conditions favourable for a FRET analysis. The comparative study carried out both on the conjugating-region and the non conjugating region of the surface membrane, indicates that FRET distribution appears quite homogeneous in mating-competent mating type (mt) I, whereas, in mating-competent mt II cells, FRET distribution seems to be preferentially localised on the conjugating-region functionally involved in mating cell pairing. This difference in the distribution of lectin-binding sites is suggested to be related to mating-competence acquisition. PMID:9857246

  5. ON VASCULAR STENOSIS, RESTENOSIS AND MANNOSE BINDING LECTIN.

    PubMed

    Kahlow, Barbara Stadler; Nery, Rodrigo Araldi; Skare, Thelma L; Ribas, Carmen Australia Paredes Marcondes; Ramos, Gabriela Piovezani; Petisco, Roberta Dombroski

    2016-03-01

    Mannose binding lectin is a lectin instrumental in the innate immunity. It recognizes carbohydrate patterns found on the surface of a large number of pathogenic micro-organisms, activating the complement system. However, this protein seems to increase the tissue damage after ischemia. In this paper is reviewed some aspects of harmful role of the mannose binding lectin in ischemia/reperfusion injury. PMID:27120743

  6. ON VASCULAR STENOSIS, RESTENOSIS AND MANNOSE BINDING LECTIN

    PubMed Central

    KAHLOW, Barbara Stadler; NERY, Rodrigo Araldi; SKARE, Thelma L; RIBAS, Carmen Australia Paredes Marcondes; RAMOS, Gabriela Piovezani; PETISCO, Roberta Dombroski

    2016-01-01

    Mannose binding lectin is a lectin instrumental in the innate immunity. It recognizes carbohydrate patterns found on the surface of a large number of pathogenic micro-organisms, activating the complement system. However, this protein seems to increase the tissue damage after ischemia. In this paper is reviewed some aspects of harmful role of the mannose binding lectin in ischemia/reperfusion injury. PMID:27120743

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

    PubMed Central

    Bradshaw, William J.; Kirby, Jonathan M.; Thiyagarajan, Nethaji; Chambers, Christopher J.; Davies, Abigail H.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

    2014-01-01

    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) 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. PMID:25004975

  8. Lectins of marine hydrobionts.

    PubMed

    Chernikov, O V; Molchanova, V I; Chikalovets, I V; Kondrashina, A S; Li, W; Lukyanov, P A

    2013-07-01

    Data from the literature and results of our research on lectins isolated from some kinds of marine hydrobionts such as clams, ascidians, sea worms, sponges, and algae are presented in this review. Results of comparative analysis of the basic physicochemical properties and biological activity of lectins isolated from various sources are discussed. PMID:24010839

  9. Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

    SciTech Connect

    de Miranda Santos, I.K.; Pereira, M.E.

    1984-09-01

    Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various /sup 125/I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeus and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.

  10. Coloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques.

    PubMed

    Roth, J; Binder, M

    1978-03-01

    Three markers, colloidal gold, ferritin and peroxidase, were checked for usefulness in double labeling of lectin-binding sites. The amount of various lectins for the stabilization of good sols of a different particle size was evaluated. Several lectin-gold complexes were prepared for electron microscopic labeling purposes, and the optimal amount of various lectins needed for stabilization of gold solutions of a different particle size was determined. The following combinations were investigated for their usefulness in labeling two different lectin-binding sites: lectin-gold and lectin-gold (different particle size), lectin-gold and lectin-ferritin, as well as lectin-ferritin and lectin-peroxidase. Of these combinations the latter did not give satisfactory results for double labeling. In all single and double labeling techniques with the above mentioned markers the quantitative evaluation of the number of lectin-binding sites is not feasible, but these techniques will be of considerable value for the investigation of the dynamics of different lectin-binding sites on the cell surface. PMID:632554

  11. Lectin-binding properties of Aeromonas caviae strains

    PubMed Central

    Rocha-de-Souza, Cláudio M.; Hirata-Jr, Raphael; Mattos-Guaraldi, Ana L.; Freitas-Almeida, Angela C.; Andrade, Arnaldo F. B.

    2008-01-01

    The cell surface carbohydrates of four strains of Aeromonas caviae were analyzed by agglutination and lectin-binding assays employing twenty highly purified lectins encompassing all sugar specificities. With the exception of L-fucose and sialic acid, the sugar residues were detected in A. caviae strains. A marked difference, however, in the pattern of cell surface carbohydrates in different A. caviae isolates was observed. Specific receptors for Tritricum vulgaris (WGA), Lycopersicon esculentum (LEL) and Solanum tuberosum (STA) (D-GlcNAc-binding lectins) were found only in ATCC 15468 strain, whereas Euonymus europaeus (EEL, D-Gal-binding lectin) sites were present exclusively in AeQ32 strain, those for Helix pomatia (HPA, D-GalNAc-binding lectin) in AeC398 and AeV11 strains, and for Canavalia ensiformes (Con A, D-Man-binding lectin) in ATCC 15468, AeC398, AeQ32 and AeV11 strains, after bacterial growing at 37°C. On the other hand, specific receptors for WGA and EEL were completely abrogated growing the bacteria at 22°C. Binding studies with 125I- labeled lectins from WGA, EEL and Con A were performed. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the A. caviae strains. PMID:24031204

  12. Cytokine production by human epithelial and endothelial cells following exposure to oral viridans streptococci involves lectin interactions between bacteria and cell surface receptors.

    PubMed Central

    Vernier, A; Diab, M; Soell, M; Haan-Archipoff, G; Beretz, A; Wachsmann, D; Klein, J P

    1996-01-01

    In order to examine the possible implication of human epithelial and endothelial cells in the pathogenesis of various diseases associated with oral viridans streptococci, we tested the immunomodulatory effects of 11 representative strains of oral viridans streptococci on human epithelial KB cells and endothelial cells. We then examined the possible role of two major adhesins from oral viridans streptococci, protein I/II and rhamnose-glucose polymers (RGPs), in this process. In this study we demonstrate that oral viridans streptococci are potent stimulators of interleukin-8 (IL-8) production from KB cells and of IL-6 and IL-8 production from endothelial cells. The ability of protein I/II and RGPs to contribute to these effects was then examined. Using biotinylated protein I/IIf and RGPs from Streptococcus mutans OMZ 175, we showed that these adhesins bind to KB and endothelial cells through specific interactions and that the binding of these molecules initiates the release of IL-8 from KB cells and of IL-6 and IL-8 from endothelial cells. These results suggest that protein I/IIf and RGPs play an important role in the interactions between bacteria and KB and endothelial cells in that similar cytokine profiles are obtained when cells are stimulated with bacteria or surface components. We also provide evidence that protein I/IIf binds to and stimulates KB and endothelial cells through lectin interactions and that N-acetyl neuraminic acid (NANA) and fucose present on cell surface glycoproteins may form the recognition site since binding and cytokine release can be inhibited by dispase and periodate treatment of cells and by NANA and fucose. These results demonstrate that oral viridans streptococci, probably by engaging two cell surface adhesins, exert immunomodulatory effects on human KB and endothelial cells. PMID:8757828

  13. Determination of hyperglycosylated human chorionic gonadotropin produced by malignant gestational trophoblastic neoplasias and male germ cell tumors using a lectin-based immunoassay and surface plasmon resonance

    PubMed Central

    Kelly, Lisa S.; Birken, Steven; Puett, David

    2007-01-01

    The ability to reliably detect aberrant glycosylation of human chorionic gonadotropin (hCG) may have profound implications for the diagnosis and monitoring of malignant gestational trophoblastic neoplasia, germ cell tumors, other malignancies, and pregnancy complications. To become a clinically useful assay, however, this discrimination of glycoforms should be possible on minimally treated biological specimens. Towards this end, we have developed a lectin-based sandwich-type immunoassay to compare the glycosylation patterns of hCG among urine specimens from patients presenting with a normal pregnancy, invasive mole, choriocarcinoma, and male germ cell tumors using carbohydrate-free antibody fragments as capture reagents and a panel of eight lectins, five recognizing neutral sugars and three recognizing sialic acid. There was no significant difference in the binding of any of the lectins to hCG in the urine of women over the gestational range of 6 – 38 weeks. Three lectins, however, exhibited differential binding to urinary hCG derived from these normal pregnant controls and that from patients with malignant forms of gestational trophoblastic disease and male germ cell tumors. Galanthus nivalis agglutinin and Maackia amurensis lectin, which bind terminal mannose and α(2–3)sialic acid, respectively, preferentially bound pregnancy-derived hCG, whereas the lectin, wheat germ agglutinin, which binds sialic acid and β(1–4)N-acetylglucosamine, exhibited decreased binding to pregnancy-derived hCG compared to that from patients with male germ cell tumors and malignant gestational trophoblastic neoplasia. The differential binding observed with these three promising lectins is most encouraging and warrants further examination. The experimental paradigm also holds promise for the development of comparable assays for other glycosylated tumor markers. PMID:17081681

  14. Use of amaranthus leucocarpus lectin to differentiate cervical dysplasia (CIN).

    PubMed

    Santaella-Verdejo, Arturo; Gallegos, Belem; Pérez-Campos, Eduardo; Hernández, Pedro; Zenteno, Edgar

    2007-01-01

    Alterations in O-glycosylation of proteins in cell surfaces can originate disorder in cellular function, as well as in cell transformation and tumoral differentiation. In this work, we investigate changes in O-glycosylation in cervical intraepithelial dysplasia (CIN) at different stages of differentiation (CIN I, CIN II, and CIN III) using lectins specific for O-glycosidically linked glycans. Twenty cases with CIN I, CIN II, and CIN III dysplasias each, and 20 normal cases were studied by lectin histochemistry and evaluated under optical microscopy. The lectins from Glycine max and Griffonia simplicifolia showed no differences in their recognition pattern among the different CIN stages and normal tissue. Dolichos Biflorus lectin recognized CIN I dysplasia. Lectin from Amaranthus leucocarpus showed increased reactivity in the presence of CIN II dysplasia, compared with CIN I and CIN III. These results suggest that subtle modifications in the O-glycosylation pattern could be considered in diagnosis or prognosis of cervical precancerous stages. PMID:17516251

  15. Structure-function relationship of monocot mannose-binding lectins.

    PubMed Central

    Barre, A; Van Damme, E J; Peumans, W J; Rougé, P

    1996-01-01

    The monocot mannose-binding lectins are an extended superfamily of structurally and evolutionarily related proteins, which until now have been isolated from species of the Amaryllidaceae, Alliaceae, Araceae, Orchidaceae, and Liliaceae. To explain the obvious differences in biological activities, the structure-function relationships of the monocot mannose-binding lectins were studied by a combination of glycan-binding studies and molecular modeling using the deduced amino acid sequences of the currently known lectins. Molecular modeling indicated that the number of active mannose-binding sites per monomer varies between three and zero. Since the number of binding sites is fairly well correlated with the binding activity measured by surface plasmon resonance, and is also in good agreement with the results of previous studies of the biological activities of the mannose-binding lectins, molecular modeling is of great value for predicting which lectins are best suited for a particular application. PMID:8972598

  16. 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

  17. Histological and lectin histochemical studies on the olfactory and respiratory mucosae of the sheep.

    PubMed

    Ibrahim, Dalia; Nakamuta, Nobuaki; Taniguchi, Kazumi; Yamamoto, Yoshio; Taniguchi, Kazuyuki

    2014-03-01

    The olfactory and respiratory mucosae of the Corriedale sheep were examined using lectin histochemistry in order to clarify the histochemical and glycohistochemical differences between these two tissues. The olfactory epithelium was stained with 13 lectins out of 21 lectins examined, while the respiratory epithelium was positive to 16 lectins. The free border of both of the olfactory and respiratory epithelia was stained with 12 lectins: Wheat germ agglutinin (WGA), succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL), Datura stramonium lectin (DSL), Soybean agglutinin (SBA), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-120), Erythrina cristagalli lectin (ECL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L). The associated glands of the olfactory mucosa, Bowman's glands, were stained with 13 lectins. While both the goblet cells and mucous nasal glands were stained with 8 lectins; five of them (WGA, s-WGA, STL, Vicia villosa agglutinin (VVA) and ECL) were mutually positive among the Bowman's glands, mucous nasal glands and the goblet cells. These findings indicate that the glycohistochemical characteristics of the free borders of both olfactory and respiratory epithelia are similar to each other, suggesting that secretions from the Bowman's glands and those of the goblet cells and mucous nasal glands are partially exchanged between the surface of two epithelia to contribute the functions of the respiratory epithelium and the olfactory receptor cells, respectively. PMID:24200894

  18. Glycan profiling of endometrial cancers using lectin microarray.

    PubMed

    Nishijima, Yoshihiro; Toyoda, Masashi; Yamazaki-Inoue, Mayu; Sugiyama, Taro; Miyazawa, Masaki; Muramatsu, Toshinari; Nakamura, Kyoko; Narimatsu, Hisashi; Umezawa, Akihiro; Mikami, Mikio

    2012-10-01

    Cell surface glycans change during the process of malignant transformation. To characterize and distinguish endometrial cancer and endometrium, we performed glycan profiling using an emerging modern technology, lectin microarray analysis. The three cell lines, two from endometrial cancers [well-differentiated type (G1) and poorly differentiated type (G3)] and one from normal endometrium, were successfully categorized into three independent groups by 45 lectins. Furthermore, in cancer cells, a clear difference between G1 and G3 type was observed for the glycans recognized with six lectins, Ulex europaeus agglutinin I (UEA-I), Sambucus sieboldiana agglutinin (SSA), Sambucus nigra agglutinin (SNA), Trichosanthes japonica agglutinin I (TJA-I), Amaranthus caudatus agglutinin (ACA), and Bauhinia purpurea lectin (BPL). The lectin microarray analysis using G3 type tissues demonstrated that stage I and stage III or IV were distinguished depending on signal pattern of three lectins, Dolichos biflorus agglutinin (DBA), BPL, and ACA. In addition, the analysis of the glycans on the ovarian cancer cells showed that only anticancer drug-sensitive cell lines had almost no activities to specific three lectins. Glycan profiling by the lectin microarray may be used to assess the characteristics of tumors and potentially to predict the success of chemotherapy treatment. PMID:22957961

  19. Carbohydrate-lectin interactions assayed by SPR.

    PubMed

    Duverger, Eric; Lamerant-Fayel, Nathalie; Frison, Natacha; Monsigny, Michel

    2010-01-01

    Surface plasmon resonance is a valuable tool to determine the affinity between glycoconjugates and sugar-binding proteins such as plant and animal lectins. The main interest of using such an approach is that neither the lectins - which are proteins - nor their ligands - natural compounds such as glycoproteins, oligosaccharides, polysaccharides, or synthetic glycoconjugates such as glycoclusters or neoglycoproteins - require any tag. Because lectins bear several binding sites, they behave like immunoglobulin eliciting avidity phenomena. This peculiarity may lead to erroneous results if special conditions are not applied. We obtained best and reproducible results when the lectin was immobilized and its ligands were used as soluble analytes. With heterogeneous glycoconjugates such as neoglycoproteins (which are heterogeneous in terms of nature, number, and position of sugar residues) or a mixture of oligosaccharides, the data may be more accurately gathered by using the Sips approach, which has been used to determine mean binding constants of polyclonal antibodies. With small analytes such as oligosaccharides, we found it convenient to determine binding constants by using an inhibitory approach: a neoglycoprotein (M (r) = approximately 80,000) was allowed to bind to the immobilized lectin and small oligosaccharides were used as inhibitors. With larger glycoconjugates such as peptides substituted with glycoclusters, direct binding measurements gave accurate results. Because of the availability of low-cost simple sugars (mono- or disaccharides) it is very convenient to use large concentrations of such carbohydrates to clean the sensor chips instead of more drastic cleaning solutions such as acids or alkali, in such a way that the immobilized lectin is stable for many experiments. PMID:20217620

  20. Enhancement of Localized Surface Plasmon Resonance polymer based biosensor chips using well-defined glycopolymers for lectin detection.

    PubMed

    Jin, Yan; Wong, Kok Hou; Granville, Anthony Michael

    2016-01-15

    Poly(methyl methacrylate) polymer based Localized Surface Plasmon Resonance biosensor chips were successfully fabricated using glycopolymer brushes carrying glucose moieties for the detection of concanavalin A. Poly(pentafluorostyrene), with pre-determined polymer chain lengths, were synthesized via a reversible addition-fragmentation chain transfer polymerization technique. The synthesized poly(pentafluorostyrene), was subsequently converted into glycopolymers via a para-fluoro-thiol "click" reaction and grafted onto the surface of sensor chips. The "glycocluster effect" induced by pendent carbohydrate moieties enabled a stronger affinity for concanavalin A binding, which resulted in a dramatic expansion of the sensors' response range. It was discovered that the longer polymer brushes did not guarantee additional enhancements for the sensor chips. Instead, they could lead to higher detection limits. In this study, the limit of detection for the sensor chips was discovered to be 1.3nmolL(-1) with a saturated response at 1054.2nmolL(-1). In addition to the superior performance, the capabilities of the reported sensor chips can be easily manipulated to detect a diverse range of analytes by "clicking" various sensing elements onto the polymer brushes. PMID:26433474

  1. Fucose-binding Lotus tetragonolobus lectin binds to human polymorphonuclear leukocytes and induces a chemotactic response.

    PubMed

    VanEpps, D E; Tung, K S

    1977-09-01

    Fucose-binding L. tetragonolobus lectin to the surface of human polymorphonuclear leukocytes (PMN) and induces a chemotactic response. Both surface binding and chemotaxis are inhibited by free fucose but not by fructose, mannose, or galactose. The lectin-binding sites on PMN are unrelated to the A, B, or O blood group antigen. Utilization of this lectin should be a useful tool in isolating PMN membrane components and in analyzing the mechanism of neutrophil chemotaxis. PMID:330752

  2. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides

    PubMed Central

    2015-01-01

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs. PMID:26407233

  3. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides.

    PubMed

    Mitchell, Daniel E; Gibson, Matthew I

    2015-10-12

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs. PMID:26407233

  4. Lectin reactivities as intermediate biomarkers in premalignant colorectal epithelium.

    PubMed

    Boland, C R; Martin, M A; Goldstein, I J

    1992-01-01

    Normal colonic epithelial cells undergo maturation as they traverse the crypt to the lumenal surface. The binding of lectins to goblet cell mucins and other glycoconjugates changes as the cells migrate and differentiate. Additional stepwise modifications in glycoconjugate expression occur in premalignant and malignant neoplasms that may be detected by lectin binding studies. The lectins Dolichos biflorus agglutinin (DBA) and soybean agglutinin (SBA) have been developed as markers of differentiation in normal-appearing colonic epithelium. Using a quantitative biometric system to score tissues, reduced levels of lectin binding have been found in rectal tissue from patients with familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer. The lectin Amaranthus caudatus agglutinin (ACA) binds to a cytoplasmic glycoconjugate expressed at the base of the colonic crypt and serves as a possible proliferation marker in the distal, but not proximal, colon. ACA binding increases in tandem with increased levels of proliferation (using BrdU incorporation) in neoplastic tissues. Binding by the peanut lectin (PNA) occurs late in the adenoma-to-carcinoma sequence--in larger adenomas and in cancers--and serves as a marker of advancing neoplasia. Lectins identify the stepwise changes that occur during normal differentiation, proliferation and in advancing neoplasia. By selecting the appropriate probe, biomarkers may be developed for early, intermediate, and late events in colorectal cancer. PMID:1469891

  5. Lectin domains at the frontiers of plant defense

    PubMed Central

    Lannoo, Nausicaä; Van Damme, Els J. M.

    2014-01-01

    Plants are under constant attack from pathogens and herbivorous insects. To protect and defend themselves, plants evolved a multi-layered surveillance system, known as the innate immune system. Plants sense their encounters upon perception of conserved microbial structures and damage-associated patterns using cell-surface and intracellular immune receptors. Plant lectins and proteins with one or more lectin domains represent a major part of these receptors. The whole group of plant lectins comprises an elaborate collection of proteins capable of recognizing and interacting with specific carbohydrate structures, either originating from the invading organisms or from damaged plant cell wall structures. Due to the vast diversity in protein structures, carbohydrate recognition domains and glycan binding specificities, plant lectins constitute a very diverse protein superfamily. In the last decade, new types of nucleocytoplasmic plant lectins have been identified and characterized, in particular lectins expressed inside the nucleus and the cytoplasm of plant cells often as part of a specific plant response upon exposure to different stress factors or changing environmental conditions. In this review, we provide an overview on plant lectin motifs used in the constant battle against pathogens and predators during plant defenses. PMID:25165467

  6. Interaction of Helicobacter pylori with C-Type Lectin Dendritic Cell-Specific ICAM Grabbing Nonintegrin

    PubMed Central

    Miszczyk, Eliza; Rudnicka, Karolina; Moran, Anthony P.; Fol, Marek; Kowalewicz-Kulbat, Magdalena; Druszczyńska, Magdalena; Matusiak, Agnieszka; Walencka, Maria; Rudnicka, Wiesława; Chmiela, Magdalena

    2012-01-01

    In this study we asked whether Helicobacter pylori whole cells and lipopolysaccharide (LPS) utilize sugar moieties of Lewis (Le) antigenic determinants to interact with DC-SIGN (dendritic cell specific ICAM grabbing nonintegrin) receptor on dendritic cells (DCs). For this purpose the soluble DC-SIGN/Fc adhesion assay and the THP-1 leukemia cells with induced expression of DC-SIGN were used. We showed that the binding specificity of DC-SIGN with H. pylori LeX/Y positive whole cells and H. pylori LPS of LeX/Y type was fucose dependent, whereas in LeXY negative H. pylori strains and LPS preparations without Lewis determinants, this binding was galactose dependent. The binding of soluble synthetic LeX and LeY to the DC-SIGN-like receptor on THP-1 cells was also observed. In conclusion, the LeXY dependent as well as independent binding of H. pylori whole cells and H. pylori LPS to DC-SIGN was described. Moreover, we demonstrated that THP-1 cells may serve as an in vitro model for the assessment of H. pylori-DC-SIGN interactions mediated by LeX and LeY determinants. PMID:22550396

  7. Effects of lectin ingestion on animal growth and internal organs.

    PubMed

    Pusztai, A

    1998-01-01

    Lectins are essential and omnipresent plant constituents. As many foods are of plant origin, the daily ingestion of lectins by both humans and animals is appreciable. For example, in an ad hoc survey, 53 edible plants were shown to contain lectins and approx 30% of fresh and processed food regularly consumed by humans had significant hemagglutinating activity (1). The situation is potentially even more acute in animal nutrition because animal diet is less diverse than that of humans, and in most instances foodstuffs are not thoroughly heat-treated. This is particularly significant in the light of our finding a correlation between lectin activity and antinutritional effects (2). As in evolution, the mammalian gut has been regularly exposed to lectins, they must have played an important part in the development of the digestive system. Although based on experience, most overtly toxic plants have been eliminated from the diet, many plants with appreciable lectin content are still consumed because it has not been easy to relate growth retardation and antinutritional, mild allergic or other subclinical symptoms to the food consumed or a particular component of it. As some lectins are at least partially heat stable and most survive the passage through the gut in functionally and immunologically intact form, their interaction with the gut surface epithelium (3) can damage the gut at high dietary intakes and this may lead to digestive disorders/diseases in some instances. However, it is not generally appreciated that not all lectins are antinutrients and indeed some may have beneficial effects and be of potential value in nutritional practice. Accordingly, it is of considerable importance to establish whether a lectin has deleterious or potentially beneficial effects for mammals. Unfortunately at present there are no adequate in vitro methods to do this reliably and it is usually necessary to carry out in vivo animal feeding studies, despite their relatively cumbersome

  8. 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). PMID:24270074

  9. Glycan and lectin biosensors

    PubMed Central

    Belický, Štefan; Katrlík, Jaroslav

    2016-01-01

    A short description about the importance of glycan biorecognition in physiological (blood cell type) and pathological processes (infections by human and avian influenza viruses) is provided in this review. Glycans are described as much better information storage media, compared to proteins or DNA, due to the extensive variability of glycan structures. Techniques able to detect an exact glycan structure are briefly discussed with the main focus on the application of lectins (glycan-recognising proteins) in the specific analysis of glycans still attached to proteins or cells/viruses. Optical, electrochemical, piezoelectric and micromechanical biosensors with immobilised lectins or glycans able to detect a wide range of analytes including whole cells/viruses are also discussed. PMID:27365034

  10. Glycan and lectin biosensors.

    PubMed

    Belický, Štefan; Katrlík, Jaroslav; Tkáč, Ján

    2016-06-30

    A short description about the importance of glycan biorecognition in physiological (blood cell type) and pathological processes (infections by human and avian influenza viruses) is provided in this review. Glycans are described as much better information storage media, compared to proteins or DNA, due to the extensive variability of glycan structures. Techniques able to detect an exact glycan structure are briefly discussed with the main focus on the application of lectins (glycan-recognising proteins) in the specific analysis of glycans still attached to proteins or cells/viruses. Optical, electrochemical, piezoelectric and micromechanical biosensors with immobilised lectins or glycans able to detect a wide range of analytes including whole cells/viruses are also discussed. PMID:27365034

  11. 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

  12. Lectins stain cells differentially in the coral, Montipora capitata.

    PubMed

    Work, Thierry M; Farah, Yael

    2014-03-01

    A limitation in our understanding of coral disease pathology and cellular pathogenesis is a lack of reagents to characterize coral cells. We evaluated the utility of plant lectins to stain tissues of a dominant coral, Montipora capitata, from Hawaii. Of 22 lectins evaluated, nine of these stained structures in the upper or basal body wall of corals. Specific structures revealed by lectins that were not considered distinct or evident on routine hematoxylin and eosin sections of coral tissues included apical and basal granules in gastrodermis and epidermis, cnidoglandular tract and actinopharynx cell surface membranes, capsules of mature holotrichous isorhizas, and perivitelline and periseminal cells. Plant lectins could prove useful to further our understanding of coral physiology, anatomy, cell biology, and disease pathogenesis. PMID:24518620

  13. Lectins stain cells differentially in the coral, Montipora capitata

    USGS Publications Warehouse

    Work, Thierry M.; Farah, Yael

    2014-01-01

    A limitation in our understanding of coral disease pathology and cellular pathogenesis is a lack of reagents to characterize coral cells. We evaluated the utility of plant lectins to stain tissues of a dominant coral, Montipora capitata, from Hawaii. Of 22 lectins evaluated, nine of these stained structures in the upper or basal body wall of corals. Specific structures revealed by lectins that were not considered distinct or evident on routine hematoxylin and eosin sections of coral tissues included apical and basal granules in gastrodermis and epidermis, cnidoglandular tract and actinopharynx cell surface membranes, capsules of mature holotrichous isorhizas, and perivitelline and periseminal cells. Plant lectins could prove useful to further our understanding of coral physiology, anatomy, cell biology, and disease pathogenesis.

  14. C-type transitions in methyl formate

    NASA Technical Reports Server (NTRS)

    Plummer, Grant M.; Herbst, Eric; De Lucia, Frank C.

    1987-01-01

    Based on previously determined spectral constants for methyl formate in its ground torsional degenerate substate, the frequencies and intensities of forbidden c-type transitions in this molecule, which is represented by a large number of lines in OMC-1, are calculated along with other 'forbidden' transitions labeled x-type. The stronger c-type transitions below 300 GHz with angular momentum quantum number of 30 or less and with upper state rotational energy of 350/cm or less are included in a list of spectral frequencies presented in this paper. Because the c-type transitions borrow intensity from the b-type transitions, the intensities of strongly affected b-type spectra are recalculated and presented.

  15. MMBL proteins: from lectin to bacteriocin.

    PubMed

    Ghequire, Maarten G K; Loris, Remy; De Mot, René

    2012-12-01

    Arguably, bacteriocins deployed in warfare among related bacteria are among the most diverse proteinacous compounds with respect to structure and mode of action. Identification of the first prokaryotic member of the so-called MMBLs (monocot mannose-binding lectins) or GNA (Galanthus nivalis agglutinin) lectin family and discovery of its genus-specific killer activity in the Gram-negative bacteria Pseudomonas and Xanthomonas has added yet another kind of toxin to this group of allelopathic molecules. This novel feature is reminiscent of the protective function, on the basis of antifungal, insecticidal, nematicidal or antiviral activity, assigned to or proposed for several of the eukaryotic MMBL proteins that are ubiquitously distributed among monocot plants, but also occur in some other plants, fish, sponges, amoebae and fungi. Direct bactericidal activity can also be effected by a C-type lectin, but this is a mammalian protein that limits mucosal colonization by Gram-positive bacteria. The presence of two divergent MMBL domains in the novel bacteriocins raises questions about task distribution between modules and the possible role of carbohydrate binding in the specificity of target strain recognition and killing. Notably, bacteriocin activity was also demonstrated for a hybrid MMBL protein with an accessory protease-like domain. This association with one or more additional modules, often with predicted peptide-hydrolysing or -binding activity, suggests that additional bacteriotoxic proteins may be found among the diverse chimaeric MMBL proteins encoded in prokaryotic genomes. A phylogenetic survey of the bacterial MMBL modules reveals a mosaic pattern of strongly diverged sequences, mainly occurring in soil-dwelling and rhizosphere bacteria, which may reflect a trans-kingdom acquisition of the ancestral genes. PMID:23176516

  16. Fine carbohydrate recognition of Euphorbia milii lectin.

    PubMed

    Irazoqui, Fernando J; Vozari-Hampe, Magdolna M; Lardone, Ricardo D; Villarreal, Marcos A; Sendra, Victor G; Montich, Guillermo G; Trindade, Vera M; Clausen, Henrik; Nores, Gustavo A

    2005-10-14

    Glycans are key structures involved in biological processes such as cell attachment, migration, and invasion. Information coded on cell-surface glycans is frequently deciphered by proteins, as lectins, that recognize specific carbohydrate topology. Here, we describe the fine carbohydrate specificity of Euphorbia milii lectin (EML). Competitive assays using various sugars showed that GalNAc was the strongest inhibitor, and that the hydroxyl axial position of C4 and acetamido on C2 of GalNAc are critical points of EML recognition. A hydrophobic locus adjacent to GalNAc is also an important region for EML binding. Direct binding assays of EML revealed a stereochemical requirement for a structure adjacent to terminal GalNAc, showing that GalNAc residue is a necessary but not sufficient condition for EML interaction. The capacity of EML to bind epithelial tumor cells makes it a potentially useful tool for study of some over-expressed GalNAc glycoconjugates. PMID:16122701

  17. Weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume Dolichos biflorus.

    PubMed

    Buts, L; Dao-Thi, M H; Loris, R; Wyns, L; Etzler, M; Hamelryck, T

    2001-05-25

    The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation, which becomes important when they interact with multivalent glycoconjugates, for instance those on cell surfaces. Recently, it has become clear that certain lectins form weakly associated oligomers. This phenomenon may play a role in the regulation of receptor crosslinking and subsequent signal transduction. The crystal structure of DB58, a dimeric lectin from the legume Dolichos biflorus reveals a separate dimer of a previously unobserved type, in addition to a tetramer consisting of two such dimers. This tetramer resembles that formed by DBL, the seed lectin from the same plant. A single amino acid substitution in DB58 affects the conformation and flexibility of a loop in the canonical dimer interface. This disrupts the formation of a stable DBL-like tetramer in solution, but does not prohibit its formation in suitable conditions, which greatly increases the possibilities for the cross-linking of multivalent ligands. The non-canonical DB58 dimer has a buried symmetrical alpha helix, which can be present in the crystal in either of two antiparallel orientations. Two existing structures and datasets for lectins with similar quaternary structures were reconsidered. A central alpha helix could be observed in the soybean lectin, but not in the leucoagglutinating lectin from Phaseolus vulgaris. The relative position and orientation of the carbohydrate-binding sites in the DB58 dimer may affect its ability to crosslink mulitivalent ligands, compared to the other legume lectin dimers. PMID:11491289

  18. Microencapsulation of lectin anti-cancer agent and controlled release by alginate beads, biosafety approach.

    PubMed

    El-Aassar, M R; Hafez, Elsayed E; El-Deeb, Nehal M; Fouda, Moustafa M G

    2014-08-01

    Hepatocellular carcinoma (HCC) is considered as one of the most aggressive cancer worldwide. In Egypt, the prevalence of HCC is increasing during last years. Recently, drug-loaded microparticles were used to improve the efficiency of various medical treatments. This study is designed to evaluate the anticancer potentialities of lectins against HCC while hinting to its safety usage. The aim is also extended to encapsulate lectins in alginate microbeads for oral drug delivery purposes. The extracted lectins showed anti-proliferative effect against HCC with a percentage of 60.76% by using its nontoxic dose with an up-regulation of P53 gene expression. Concerning the handling of lectin alginate microbeads for oral drug delivery, the prepared lectin alginate beads were ∼100μm in diameter. The efficiency of the microcapsules was checked by scanning electron microscopy, the SEM showed the change on the alginate beads surface revealing the successful lectin encapsulation. The release of lectins from the microbeads depended on a variety of factors as the microbeads forming carriers and the amount-encapsulated lectins. The Pisum sativum extracted lectins may be considered as a promising agent in controlling HCC and this solid dosage form could be suitable for oral administration complemented with/or without the standard HCC drugs. PMID:24857870

  19. Bjcul, a snake venom lectin, modulates monocyte-derived macrophages to a pro-inflammatory profile in vitro.

    PubMed

    Dias-Netipanyj, M F; Boldrini-Leite, L M; Trindade, E S; Moreno-Amaral, A N; Elifio-Esposito, S

    2016-06-01

    Macrophages are cells of high plasticity and can act in different ways to ensure that the appropriate immune response remains controlled. This study shows the effects of the C-type Bothrops jararacussu venom lectin (BJcuL) on the activation of human macrophages derived from the U937 cell line. BJcuL binds on the cell surface, and this event is inhibited by its specific carbohydrate. It induced phagocytosis and production of H2O2, and expression of antigen presentation molecules. It also enhanced the production of TNF-α, GM-CSF and IL-6 by macrophages and indirectly induced T cells to an increased production of TNF-α, IFN-γ and IL-6 in the presence of LPS. Our results suggest that BJcuL can modulate macrophage functional activation towards an M1 state. PMID:26944802

  20. A review of fish lectins.

    PubMed

    Ng, Tzi Bun; Fai Cheung, Randy Chi; Wing Ng, Charlene Cheuk; Fang, Evandro Fei; Wong, Jack Ho

    2015-01-01

    Lectins have been reported from various tissues of a diversity of fish species including Japanese eel, conger eel, electric eel, bighead carp, gibel carp, grass carp, Arabian Gulf catfish, channel catfish, blue catfish, catfish, pike perch, perch, powan, zebrafish, toxic moray, cobia fish, steelhead trout, Japanese trout, Atlantic salmon, chinook salmon, olive rainbow smelt, rainbow smelt, white-spotted charr, tilapia, blue gourami, ayu, Potca fish, Spanish mackerel, gilt head bream, tench, roach, rudd, common skate, and sea lamprey. The tissues from which the lectins were isolated comprise gills, eggs, electric organ, stomach, intestine, and liver. Lectins have also been isolated from skin, mucus serum, and plasma. The lectins differ in molecular weight, number of subunits, glycosylation, sugar binding specificity and amino acid sequence. Their activities include antimicrobial, antitumor, immunoregulatory and a role in development. PMID:25929869

  1. 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. PMID:12777210

  2. Complexity of lectin-mediated reactions in bacteria-induced histamine release.

    PubMed

    Jensen, C; Stahl Skov, P; Norn, S; Espersen, F; Bøg-Hansen, T C; Lihme, A

    1984-08-01

    We have earlier suggested that bacteria-induced histamine release is caused by different mechanisms, including allergic and non-immunological mechanisms, and that the latter probably depends on lectin-mediated reactions. Two possibilities of lectin-mediated reactions were examined in this study, bacterial surface lectins bind to sugars on the basophil cell membrane leading to histamine release, and the reverse reaction where bacterial aminosugars react with lectins on the basophil cell surface. In the bacterial histamine release caused by the Staph. aureus strain Wood 46 it was possible to demonstrate a reverse reaction, but not a bacterial lectin-mediated reaction. The reaction seems to be complex, as lower concentrations of sugars might potentiate the release of histamine by binding to the target cell or bacteria, while the release is inhibited by higher concentrations. PMID:6208803

  3. Use of labeled tomato lectin for imaging vasculature structures.

    PubMed

    Robertson, Richard T; Levine, Samantha T; Haynes, Sherry M; Gutierrez, Paula; Baratta, Janie L; Tan, Zhiqun; Longmuir, Kenneth J

    2015-02-01

    Intravascular injections of fluorescent or biotinylated tomato lectin were tested to study labeling of vascular elements in laboratory mice. Injections of Lycopersicon esculentum agglutinin (tomato lectin) (50-100 µg/100 µl) were made intravascularly, through the tail vein, through a cannula implanted in the jugular vein, or directly into the left ventricle of the heart. Tissues cut for thin 10- to 12-µm cryostat sections, or thick 50- to 100-µm vibratome sections, were examined using fluorescence microscopy. Tissue labeled by biotinylated lectin was examined by bright field microscopy or electron microscopy after tissue processing for biotin. Intravascular injections of tomato lectin led to labeling of vascular structures in a variety of tissues, including brain, kidney, liver, intestine, spleen, skin, skeletal and cardiac muscle, and experimental tumors. Analyses of fluorescence in serum indicated the lectin was cleared from circulating blood within 2 min. Capillary labeling was apparent in tissues collected from animals within 1 min of intravascular injections, remained robust for about 1 h, and then declined markedly until difficult to detect 12 h after injection. Light microscopic images suggest the lectin bound to the endothelial cells that form capillaries and endothelial cells that line some larger vessels. Electron microscopic studies confirmed the labeling of luminal surfaces of endothelial cells. Vascular labeling by tomato lectin is compatible with a variety of other morphological labeling techniques, including histochemistry and immunocytochemistry, and thus appears to be a sensitive and useful method to reveal vascular patterns in relationship to other aspects of parenchymal development, structure, and function. PMID:25534591

  4. Bi- to tetravalent glycoclusters presenting GlcNAc/GalNAc as inhibitors: from plant agglutinins to human macrophage galactose-type lectin (CD301) and galectins.

    PubMed

    André, Sabine; O'Sullivan, Shane; Koller, Christiane; Murphy, Paul V; Gabius, Hans-Joachim

    2015-04-14

    Emerging insights into the functional spectrum of tissue lectins leads to identification of new targets for the custom-made design of potent inhibitors, providing a challenge for synthetic chemistry. The affinity and selectivity of a carbohydrate ligand for a lectin may immensely be increased by a number of approaches, which includes varying geometrical or topological features. This perspective leads to the design and synthesis of glycoclusters and their testing using assays of physiological relevance. Herein, hydroquinone, resorcinol, benzene-1,3,5-triol and tetra(4-hydroxyphenyl)ethene have been employed as scaffolds and propargyl derivatives obtained. The triazole-containing linker to the α/β-O/S-glycosides of GlcNAc/GalNAc presented on these scaffolds was generated by copper-catalysed azide-alkyne cycloaddition. This strategy was used to give a panel of nine glycoclusters with bi-, tri- and tetravalency. Maintained activity for lectin binding after conjugation was ascertained for both sugars in solid-phase assays with the plant agglutinins WGA (GlcNAc) and DBA (GalNAc). Absence of cross-reactivity excluded any carbohydrate-independent reactivity of the bivalent compounds, allowing us to proceed to further testing with a biomedically relevant lectin specific for GalNAc. Macrophage galactose(-binding C)-type lectin, involved in immune defence by dendritic cells and in virus uptake, was produced as a soluble protein without/with its α-helical coiled-coil stalk region. Binding to ligands presented on a matrix and on cell surfaces was highly susceptible to the presence of the tetravalent inhibitor derived from the tetraphenylethene-containing scaffold, and presentation of GalNAc with an α-thioglycosidic linkage proved favorable. Cross-reactivity of this glycocluster to human galectins-3 and -4, which interact with Tn-antigen-presenting mucins, was rather small. Evidently, the valency and spatial display of α-GalNAc residues is a key factor to design potent and

  5. Probing the cons and pros of lectin-induced immunomodulation: case studies for the mistletoe lectin and galectin-1.

    PubMed

    Gabius, H J

    2001-07-01

    When imagining to monitor animal cells through a microscope with resolution at the molecular level, a salient attribute of their surfaces will be the abundance of glycan chains. They present galactosides at their termini widely extending like tentacles into the extracellular space. Their spatial accessibility and their potential for structural variability endow especially these glycan parts with capacity to act as docking points for molecular sensors (sugar receptors such as lectins). Binding and ligand clustering account for transmission of post-binding signals into the cell interior. The range of triggered activities has turned plant lectins into popular tools in cell biology and immunology. Potential for clinical application has been investigated rigorously only in recent years. As documented in vitro and in vivo for the galactoside-specific mistletoe lectin, its apparent immunomodulatory capacity reflected in upregulation of production of proinflammatory cytokines will not necessarily be clinically favorable but a double-edged sword. In fact, lectin application has been shown to stimulate tumor growth in cell lines, histocultures of human tumors and in two animal models using chemical carcinogenesis or tumor transplantation. When testing immunological effects of the endogenous lectin galectin-1, protection against disorders mediated by activated T cells came up for consideration. Elimination of these cells via CD7-dependent induction of apoptosis, and a shift to the Th2 response by the galectin, are factors to ameliorate disease states. This result encourages further efforts with other galectins. Functional redundancy, synergism, diversity or antagonism among galectins are being explored to understand the actual role of this class of endogenous lectins in inflammation. Regardless of the results of further preclinical testing for galectin-1, these two case studies break new ground in our understanding how glycans as ligands for lectins convey reactivity to

  6. Characterization of IgE-binding epitopes of peanut (Arachis hypogaea) PNA lectin allergen cross-reacting with other structurally related legume lectins.

    PubMed

    Rougé, Pierre; Culerrier, Raphaël; Granier, Claude; Rancé, Fabienne; Barre, Annick

    2010-08-01

    Sera from peanut allergic patients contain IgE that specifically interact with the peanut lectin PNA and other closely related legume lectins like LcA from lentil, PsA from pea and PHA from kidney bean. The IgE-binding activity of PNA and legume lectins was assessed by immunoblotting, surface plasmon resonance (SPR) and ELISA measurements, using sera from peanut allergic patients as a IgE source. This IgE-binding cross-reactivity most probably depends on the occurrence of structurally related epitopes that have been identified on the molecular surface of PNA and other legume lectins. These epitopes definitely differ from those responsible for the allergenicity of the major allergens Ara h 1, Ara h 2 and Ara h 3, also recognized by the IgE-containing sera of peanut allergic patients. Peanut lectin PNA and other legume lectins have been characterized as potential allergens for patients allergic to edible legume seeds. However, the clinical significance of the lectin-IgE interaction has to be addressed. PMID:20541807

  7. Inhibitory potential of chemical substitutions at bioinspired sites of β-D-galactopyranose on neoglycoprotein/cell surface binding of two classes of medically relevant lectins.

    PubMed

    Giguère, Denis; André, Sabine; Bonin, Marc-André; Bellefleur, Marc-André; Provencal, Alexandre; Cloutier, Philipe; Pucci, Bernard; Roy, René; Gabius, Hans-Joachim

    2011-05-15

    Galactose is the key contact site for plant AB-toxins and the human adhesion/growth-regulatory galectins. Natural anomeric extensions and 3'-substitutions enhance its reactivity, thus prompting us to test the potential of respective chemical substitutions of galactose in the quest to develop potent inhibitors. Biochemical screening of a respective glycoside library with 60 substances in a solid-phase assay was followed by examining the compounds' activity to protect cells from lectin binding. By testing 32 anomeric extensions, 18 compounds with additional 3'-substitution, three lactosides and two Lewis-type trisaccharides rather mild effects compared to the common haptenic inhibitor lactose were detected in both assays. When using trivalent glycoclusters marked enhancements with 6- to 8-fold increases were revealed for the toxin and three of four tested galectins. Since the most potent compound and also 3'-substituted thiogalactosides reduced cell growth of a human tumor line at millimolar concentrations, biocompatible substitutions and scaffolds will be required for further developments. The synthesis of suitable glycoclusters, presenting headgroups which exploit differences in ligand selection in interlectin comparison to reduce cross-reactivity, and the documented strategic combination of initial biochemical screening with cell assays are considered instrumental to advance inhibitor design. PMID:21524586

  8. Hayabusa2, C-type Asteroid Sample Return Mission

    NASA Astrophysics Data System (ADS)

    Takagi, Y.; Yoshikawa, M.; Abe, M.; Tachibana, S.; Okada, T.; Kitazato, K.; Nakamura, R.; Hirata, N.; Yano, H.; Demura, H.; Nakazawa, S.; Iijima, Y.; Shirai, K.; Hayakawa, M.; Hayabusa 2 Project Team

    2011-12-01

    be created by the device is expected to be a small one with a few meters in diameter, but still, by acquiring samples from the surface that is exposed by a collision, we can get fresh samples that are less weathered by the space environment or heat. Hayabusa2 is scheduled for launch in 2014 (or 2015 as a backup.) It should arrive at the C-type asteroid in mid 2018, staying around there for one and half years before leaving the asteroid at the end of 2019 and returning to Earth around the end of 2020.

  9. Signalling through MyD88 drives surface expression of the mycobacterial receptors MCL (Clecsf8, Clec4d) and Mincle (Clec4e) following microbial stimulation.

    PubMed

    Kerscher, Bernhard; Dambuza, Ivy M; Christofi, Maria; Reid, Delyth M; Yamasaki, Sho; Willment, Janet A; Brown, Gordon D

    2016-01-01

    The heterodimeric mycobacterial receptors, macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle), are upregulated at the cell surface following microbial challenge, but the mechanisms underlying this response are unclear. Here we report that microbial stimulation triggers Mincle expression through the myeloid differentiation primary response gene 88 (MyD88) pathway; a process that does not require MCL. Conversely, we show that MCL is constitutively expressed but retained intracellularly until Mincle is induced, whereupon the receptors form heterodimers which are translocated to the cell surface. Thus this "two-step" model for induction of these key receptors provides new insights into the underlying mechanisms of anti-mycobacterial immunity. PMID:27005451

  10. Molecular cloning, characterization and expression analysis of F-type lectin from pearl oyster Pinctada fucata.

    PubMed

    Anju, A; Jeswin, J; Thomas, P C; Vijayan, K K

    2013-07-01

    F-type lectin is an important type of pattern recognition receptor that can recognize and bind carbohydrate moieties on the surface of potential pathogens through its carbohydrate recognition domains (CRDs). This paper reports the cloning of an F-type lectin (designated as pfF-type lectin) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this pfF-type lectin contains an open reading frame (ORF) of 588 bp coding for196 amino acids. A signal peptide at the N-terminus of the deduced polypeptide was predicted by the signal P program and the cleavage site is located between the positions of Gly(19)and Tyr(20). Conserved domain search at NCBI revealed the pfF-type lectin domain extends from Lys(55)to Val(192). Semi-quantitative analysis in adult tissues showed that the pfF-type lectin mRNA was abundantly expressed in haemocytes and gill and rarely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfF-type lectin mRNA in haemocytes was increased, reaching the highest level at 4 h, then dropping to basal levels at 36 h. These results suggest that F-type lectin play a critical role in the innate immune system of the pearl oyster P. fucata. PMID:23624143

  11. Antinutritional properties of plant lectins.

    PubMed

    Vasconcelos, Ilka M; Oliveira, José Tadeu A

    2004-09-15

    Lectins are carbohydrate binding (glyco)proteins which are ubiquitous in nature. In plants, they are distributed in various families and hence ingested daily in appreciable amounts by both humans and animals. One of the most nutritionally important features of plant lectins is their ability to survive digestion by the gastrointestinal tract of consumers. This allows the lectins to bind to membrane glycosyl groups of the cells lining the digestive tract. As a result of this interaction a series of harmful local and systemic reactions are triggered placing this class of molecules as antinutritive and/or toxic substances. Locally, they can affect the turnover and loss of gut epithelial cells, damage the luminal membranes of the epithelium, interfere with nutrient digestion and absorption, stimulate shifts in the bacterial flora and modulate the immune state of the digestive tract. Systemically, they can disrupt lipid, carbohydrate and protein metabolism, promote enlargement and/or atrophy of key internal organs and tissues and alter the hormonal and immunological status. At high intakes, lectins can seriously threaten the growth and health of consuming animals. They are also detrimental to numerous insect pests of crop plants although less is presently known about their insecticidal mechanisms of action. This current review surveys the recent knowledge on the antinutritional/toxic effects of plant lectins on higher animals and insects. PMID:15302522

  12. 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. PMID:23828036

  13. Investigation of lectinized liposomes as M-cell targeted carrier-adjuvant for mucosal immunization.

    PubMed

    Gupta, Prem N; Vyas, Suresh P

    2011-01-01

    In the present investigation hepatitis B surface antigen (HBsAg) encapsulated liposomes were developed and coupled with Ulex europaeus agglutinin 1 (UEA-1) to increase transmucosal uptake by M-cells of the Peyer's patches. The liposomes were characterized for shape, size, polydispersity and encapsulation efficiency. Bovine submaxillary mucin (BSM) was used as a biological model for the in vitro determination of lectin activity and specificity. Dual staining technique was used to investigate targeting of lectinized liposomes to the M-cells. Anti-HBsAg IgG response in serum and anti-HBsAg sIgA level in various mucosal fluids was estimated by using ELISA, following oral immunization with lectinized and non-lectinized liposomes in Balb/c mice. Additionally, interleukin-2 (IL-2) and interferon-γ (IFN-γ) level in the spleen homogenates was determined. The results suggest that lectinized liposomes were successfully developed, exhibited increased activity with BSM as compared to non-lectinized liposomes and α-l-fucose specificity of the lectinized liposomes was also maintained. The lectinized liposomes were predominantly targeted to the M-cells. The serum anti-HBsAg IgG titre obtained after 3 consecutive days oral immunizations with HBsAg encapsulated lectinized liposomes and boosting after third week was comparable with the titre recorded after single intramuscular prime and third week boosting with alum-HBsAg. Moreover, lectinized liposomes induced higher sIgA level in mucosal secretions and cytokines level in the spleen homogenates. The results showed that the developed surface modified liposomes could be a potential module for the development of effective mucosal vaccines. PMID:20843665

  14. A novel bifunctional hybrid with marine bacterium alkaline phosphatase and Far Eastern holothurian mannan-binding lectin activities.

    PubMed

    Balabanova, Larissa; Golotin, Vasily; Kovalchuk, Svetlana; Bulgakov, Alexander; Likhatskaya, Galina; Son, Oksana; Rasskazov, Valery

    2014-01-01

    A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens. PMID:25397876

  15. A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

    PubMed Central

    Balabanova, Larissa; Golotin, Vasily; Kovalchuk, Svetlana; Bulgakov, Alexander; Likhatskaya, Galina; Son, Oksana; Rasskazov, Valery

    2014-01-01

    A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25±5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens. PMID:25397876

  16. Antibacterial and Antifungal Activities of Lectin Extracted from Fruiting Bodies of the Korean Cauliflower Medicinal Mushroom, Sparassis latifolia (Agaricomycetes).

    PubMed

    Chandrasekaran, Gayathri; Lee, Young-Chul; Park, Hyun; Wu, Yuanzheng; Shin, Hyun-Jae

    2016-01-01

    In this article we describe the isolation and characterization of a novel lectin from fruiting bodies of the mushroom Sparassis latifolia. The antibacterial activity of the purified lectin against Escherichia coli and resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa as well as the antifungal activity against Candida and Fusarium species were determined. Circular dichroism spectroscopy and the tryptophan blue shift assay indicated that the lectin interacts with microbial surfaces. This suggests the potential of the lectin isolated from S. latifolia, a valuable source of bioactive constituents, as a therapeutic in pharmaceutical agent. PMID:27481295

  17. Crystal structure of extracellular domain of human lectin-like transcript 1 (LLT1), the ligand for natural killer receptor-P1A.

    PubMed

    Kita, Shunsuke; Matsubara, Haruki; Kasai, Yoshiyuki; Tamaoki, Takaharu; Okabe, Yuki; Fukuhara, Hideo; Kamishikiryo, Jun; Krayukhina, Elena; Uchiyama, Susumu; Ose, Toyoyuki; Kuroki, Kimiko; Maenaka, Katsumi

    2015-06-01

    Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended β-sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function. PMID:25826155

  18. Binding of FITC-labelled lectins to the gastrointestinal epithelium of the rat.

    PubMed

    Baintner, K; Jakab, G; Gyôri, Z; Kiss, P

    2000-01-01

    Biotechnology uses lectin genes to transfect into crop plants for protection against insects and nematodes. On the other hand, the information is limited on lectin-binding properties of cells in the gastrointestinal tract. Therefore, binding of a panel of FITC-labelled plant lectins to gastrointestinal cells of the rat was studied. In the stomach, cytoplasmic staining of parietal cells by PHA appeared to be due to glycoproteins attached to the tubulovesicles. PNA also stained the parietal cells, but only in the isthmus and neck regions, reacting with desialylated glycoproteins. WGA bound to the mucous neck cells with higher affinity than to the surface and foveolar mucous cells. The mucous cells were also stained by SNA-I, UEA-I and, less intensively, by LCA. Chief cells did not show detectable reaction with any of the applied lectins. Binding of PHA to gastric cells showed differences when compared with the results of in vivostudies. Small intestinal brush border was stained with UEA-I and SNA-I, the latter lectin also strongly stained the surface of small intestinal crypts. Both lectins reacted with the mucus of goblet cells. In the large intestine UEA-I and SNA-I stained the goblet cells at the base and upper part of the crypts, respectively. Accordingly, we provided evidences for the unique lectin-binding phenotype of the various segments of the gastrointestinal tract. PMID:11033457

  19. Lectin binding of human sperm associates with DEFB126 mutation and serves as a potential biomarker for subfertility

    PubMed Central

    Xin, Aijie; Cheng, Li; Diao, Hua; Wu, Yancheng; Zhou, Shumin; Shi, Changgen; Sun, Yangyang; Wang, Peng; Duan, Shiwei; Zheng, Jufen; Wu, Bin; Yuan, Yao; Gu, Yihua; Chen, Guowu; Sun, Xiaoxi; Shi, Huijuan; Tao, Shengce; Zhang, Yonglian

    2016-01-01

    Coating on the sperm surface, glycocalyx, plays a key role in sperm motility, maturation and fertilization. A comprehensive profile of sperm surface glycans will greatly facilitate both basic researches and clinical studies. Because of the capability of recognizing different glycan moieties, lectins are widely used in glycobiology. However, lacking high-throughput technology, limited lectins have been reported for analyzing the glycan of human sperm. In this study, we employed a lectin microarray for profiling the surface glycans of human sperm, on which 54 out of 91 lectins showed positive binding. Based on this technique, we compared lectin binding profiling of sperm with homozygous DEFB126 mutation (del/del) with that of wild type (wt/wt). DEFB126 was reported to contribute to the sialylation on sperm surface and its homozygous mutation was related to male subfertility. Six lectins (Jacalin/AIA, GHA, ACL, MPL, VVL and ABA) were found to develop lower binding affinity to sperm with del/del. Further validation showed that these lectins, especially ABA and MPL, can be potential biomarkers for clinical diagnosis of subfertility due to the mutation of DEFB126. Our research provides insight into the detection of some unexplained male subfertility, and the lectin microarray is generally applicable for infertility/subfertility sperm biomarker discovery. PMID:26832966

  20. Lectin binding of human sperm associates with DEFB126 mutation and serves as a potential biomarker for subfertility.

    PubMed

    Xin, Aijie; Cheng, Li; Diao, Hua; Wu, Yancheng; Zhou, Shumin; Shi, Changgen; Sun, Yangyang; Wang, Peng; Duan, Shiwei; Zheng, Jufen; Wu, Bin; Yuan, Yao; Gu, Yihua; Chen, Guowu; Sun, Xiaoxi; Shi, Huijuan; Tao, Shengce; Zhang, Yonglian

    2016-01-01

    Coating on the sperm surface, glycocalyx, plays a key role in sperm motility, maturation and fertilization. A comprehensive profile of sperm surface glycans will greatly facilitate both basic researches and clinical studies. Because of the capability of recognizing different glycan moieties, lectins are widely used in glycobiology. However, lacking high-throughput technology, limited lectins have been reported for analyzing the glycan of human sperm. In this study, we employed a lectin microarray for profiling the surface glycans of human sperm, on which 54 out of 91 lectins showed positive binding. Based on this technique, we compared lectin binding profiling of sperm with homozygous DEFB126 mutation (del/del) with that of wild type (wt/wt). DEFB126 was reported to contribute to the sialylation on sperm surface and its homozygous mutation was related to male subfertility. Six lectins (Jacalin/AIA, GHA, ACL, MPL, VVL and ABA) were found to develop lower binding affinity to sperm with del/del. Further validation showed that these lectins, especially ABA and MPL, can be potential biomarkers for clinical diagnosis of subfertility due to the mutation of DEFB126. Our research provides insight into the detection of some unexplained male subfertility, and the lectin microarray is generally applicable for infertility/subfertility sperm biomarker discovery. PMID:26832966

  1. Molecular cloning and expression analysis of mannose receptor C type 1 in grass carp (Ctenopharyngodon idella).

    PubMed

    Wang, Li; Liu, Lichun; Zhou, Yang; Zhao, Xiaoheng; Xi, Mingjun; Wei, Shun; Fang, Rui; Ji, Wei; Chen, Nan; Gu, Zemao; Liu, Xueqin; Wang, Weimin; Asim, Muhammad; Liu, Xiaoling; Lin, Li

    2014-03-01

    Mannose receptor C type 1 (MRC1) is a pattern-recognition receptor (PRR) which plays a significant role in immune responses. Much work on MRC1 has been done in mammals and birds while little in fish. In this study, we cloned and characterized MRC1 in grass carp (gcMR). The full-length gcMR contained 5291bp encoding a putative protein of 1432 amino acids. The predicted amino acid sequences showed that gcMR contained a signal peptide, a cysteine-rich (CR) domain, a fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. gcMR were constitutively expressed in different organs with the higher expression in spleen and head kidney. During embryonic development, gcMR transcript levels were highest at cleavage stage. The up-regulation expression of gcMR, IL-1β and TNF-α in liver, spleen, head kidney and intestine after Aeromonas hydrophila infection indicating it involved in innate immune regulation during bacterial infections. PMID:24184700

  2. Electron microscopic demonstration of lectin binding sites in the taste buds of the European catfish Silurus glanis (Teleostei).

    PubMed

    Witt, M; Reutter, K

    1990-01-01

    Taste buds in the European catfish Silurus glanis were examined with electron microscopic lectin histochemistry. For detection of carbohydrate residues in sensory cells and adjacent epithelial cells, gold-, ferritin- and biotin-labeled lectins were used. A post-embedding procedure carried out on tissue sections embedded in LR-White was applied to differentiate between the sensory cells: The lectins from Helix pomatia (HPA) and Triticum vulgare (WGA) bound to N-acetyl-galactosamine and to N-acetylglucosamine residues occurring especially in vesicles of dark sensory cells. This indicates a secretory function of these cells. Most light sensory cells--with some exceptions, probably immature cells--, are HPA-negative. The mucus of the receptor field and at the top of the adjacent epithelial cells was strongly HPA-positive. Pre-embedding studies were performed in order to obtain information about the reaction of the mucus with lectins under supravital conditions. The mucus of the taste bud receptor field exhibited intensive binding to WGA, but not to the other lectins tested. Most lectins bound predominantly to the surface mucus of the nonsensory epithelium and to the marginal cells close to the receptor field. The strong lectin binding to mucins and the relatively weak lectin binding to cell surface membranes in pre-embedding studies suggest that the mucus possibly serves as a barrier which is passed selectively only by a small amount of lectins or lectin-carbohydrate complexes. Lectin-carbohydrate interactions may play a role in recognition phenomena on the plasmalemmata of the taste bud sensory cells. Recognition processes directed to bacteria or viruses should be considered as well. PMID:2279957

  3. Lectin staining patterns in human gastric mucosae with and without exposure to Helicobacter pylori

    PubMed Central

    Melo-Junior, Mario R.; Cavalcanti, Carmelita L.B.; Pontes-Filho, Nicodemos T.; Carvalho Jr, Luiz B.; Beltrão, Eduardo I. C.

    2008-01-01

    The aim of the present study was to evaluate qualitative changes in the glycoconjugate expression in human gastric tissue of positive and negative patients for Helicobacter pylori, through lectins: Wheat Germ Agglutinin (WGA) and Concanavalin A (Con A). The lectins recognized differently the glycoconjugates in the superficial mucous layer at the gastric tissues. The results suggest a significant change in the carbohydrate moieties present on the surface of the gastric cells during infection. PMID:24031208

  4. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  5. Putative glycoprotein and glycolipid polymorphonuclear leukocyte receptors for the Actinomyces naeslundii WVU45 fimbrial lectin.

    PubMed Central

    Sandberg, A L; Ruhl, S; Joralmon, R A; Brennan, M J; Sutphin, M J; Cisar, J O

    1995-01-01

    Recognition of receptors on sialidase-treated polymorphonuclear leukocytes (PMNs) by the Gal/GalNAc lectin associated with the type 2 fimbriae of certain strains of actinomyces results in activation of the PMNs, phagocytosis, and destruction of the bacteria. In the present study, plant lectins were utilized as probes to identify putative PMN receptors for the actinomyces lectin. The Gal-reactive lectin from Ricinus communis (RCAI), the Gal/GalNAc-reactive lectins from R. communis (RCAII) and Bauhinia purpurea (BPA), as well as the Gal beta 1-3GalNAc-specific lectins from Arachis hypogaea (PNA) and Agaricus bisporus (ABA) inhibited killing of Actinomyces naeslundii WVU45 by sialidase-treated PMNs. These five lectins detected a 130-kDa surface-labeled glycoprotein on nitrocellulose transfers of PMN extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This glycoprotein was revealed only after treatment of the transfers with sialidase, a condition analogous to the sialidase dependence of the lectin-mediated biological responses of the PMNs to the actinomyces. The mannose-reactive lectin concanavalin A did not inhibit killing of the actinomyces and failed to detect the 130-kDa glycoprotein but did block PMN-dependent killing of Escherichia coli B, a bacterium that possesses mannose-sensitive fimbriae. Therefore, the PMN glycoprotein receptor for A. naeslundii is clearly distinct from those recognized by E. coli. Two major putative glycolipid receptors were also identified by actinomyces and RCAI overlays on sialidase-treated thin-layer chromatograms of PMN gangliosides. Thus, both a 130-kDa glycoprotein and certain gangliosides are implicated in the attachment of the actinomyces to PMNs. PMID:7790078

  6. Sizes and Albedos of Young C-type Asteroids

    NASA Astrophysics Data System (ADS)

    Tamblyn, Peter; Chapman, Clark; Durda, Dan; Merline, William; Nesvorny, David

    2005-06-01

    We propose to measure the sizes and albedos of 8 very young C-type asteroids with IRAC 8um and near-simultaneous ground-based visible photometry. Asteroid families are created from major collisions between asteroids and are identified from clustering of orbital elements. Co-I Nesvorny has recently identified an exceptionally-young family (Veritas) and precisely-dated it at only 8.3+/-0.5 Myr (just 0.2% of the age of the solar system). We will compare our results for this family with those obtained by our similar Spitzer GO-1 program where we study an even younger S-type family, Karin. C-type asteroids are composed of primitive material (as opposed to the more processed silicate-rich S-types) and comprise the majority of asteorids in the Main Belt, yet their compositions and properties remain elusive. These recent breakup events provide unparalleled opportunities to study compositions, dynamics, and collisions of asteroids. They allow tests of the rates of physical processes that happen on time scales comparable with the family age. Space weathering, for example, appears to affect C- and S-type asteroids very differently. We will test directly whether the Veritas fragments have similar albedos; we will also test if their albedos differ from those of similar asteroids with much older surfaces by study of a second C-type family, Themis. We will compare our observations with those made of larger asteroids of both families, from a companion ground-based program. We will quantify any correlation of size with albedo, a dominant uncertainty in standard size estimates. The size distribution will be used to calibrate hydrocode models of asteroid collisions. To do this will require observations at the smallest practical sizes. In addition, the measured sizes will be immediately applicable to a novel measurement of the Yarkovsky Effect. We have already demonstrated in our GO-1 program that we can make similar Spitzer observations and provide the ground-based visible support.

  7. 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

  8. Lectin Binding to the Root and Root Hair Tips of the Tropical Legume Macroptilium atropurpureum Urb

    PubMed Central

    Ridge, R. W.; Rolfe, B. G.

    1986-01-01

    Ten fluorescein isothiocyanate-labeled lectins were tested on the roots of the tropical legume Macroptilium atropurpureum Urb. Four of these (concanavalin A, peanut agglutinin, Ricinis communis agglutinin I [RCA-I], wheat germ agglutinin) were found to bind to the exterior of root cap cells, the root cap slime, and the channels between epidermal cells in the root elongation zone. One of these lectins, RCA-I, bound to the root hair tips in the mature and emerging hair zones and also to sites at which root hairs were only just emerging. There was no RCA-I binding to immature trichoblasts. Preincubation of these lectins with their hapten sugars eliminated all types of root cell binding. By using a microinoculation technique, preincubation of the root surface with RCA-I lectin was found to inhibit infection and nodulation by Rhizobium spp. Preincubation of the root surface with the RCA-I hapten β-d-galactose or a mixture of RCA-I lectin and its hapten failed to inhibit nodulation. Application of RCA-I lectin to the root surface caused no apparent detrimental effects to the root hair cells and did not prevent the growth of root hairs. The lectin did not prevent Rhizobium sp. motility or viability even after 24 h of incubation. It was concluded that the RCA-I lectin-specific sugar β-d-galactose may be involved in the recognition or early infection stages, or both, in the Rhizobium sp. infection of M. atropurpureum. Images PMID:16346989

  9. Mitogenic activity of edible mushroom lectins.

    PubMed

    Ho, J C K; Sze, S C W; Shen, W Z; Liu, W K

    2004-03-17

    A special group of lectins were isolated from three popular Asian edible mushrooms: Volvariella volvacea, Pleurotus flabellatus and Hericium erinacium, and their mitogenic activities towards mouse T cells were compared to the extensively investigated Agaricus bisporus lectin (ABL) and the Jack bean lectin, Concanavalin A (Con A). Among the four mushroom lectins tested, V. volvacea lectin (VVL) exhibited strong mitogenic activity as demonstrated by 3H-thymidine incorporation, which was at least 10-fold more effective than that of Con A, and the other mushroom lectins did not exhibit any proliferative activity. Treatment with VVL and ABL resulted in activation of the protein tyrosine kinase, p56lck, and expression of early activation markers, CD69 and CD25, but only VVL induced intracellular calcium influx while ABL triggered cell death. The calcium influx was sensitive to calcium channel antagonists such as nifedipine and verapamil. The P. flabellatus lectin (PFL) and H. erinacium lectin (HEL) did not stimulate p56lck expression and cell proliferation. Neither of these lectins interfered with Con A-mediated lymphocyte proliferation, which further indicated that both PFL and HEL were non-mitogenic. Taken all results together, VVL induced mitogenesis through T cell receptors and the subsequent calcium signaling pathway. PMID:15026140

  10. Agglutination of Sindbis Virus and of Cells Infected with Sindbis Virus by Plant Lectins

    PubMed Central

    Birdwell, Charles R.; Strauss, James H.

    1973-01-01

    We have examined the agglutination of Sindbis virus and of chick and hamster cells infected with Sindbis virus by two of the plant lectins, concanavalin A and Ricinus communis agglutinin. Both lectins agglutinate the virus by binding to the polysaccharide chains of the envelope glycoproteins. Both chick and hamster cells exhibit increased agglutination by the lectins after infection by Sindbis virus. In the case of chick cells infected with Sindbis virus, this increase in agglutinability occurs between 3 and 5 h after infection. Infected and mock-infected cells bind the same amount of 3H-labeled concanavalin A, which suggests that the increase in agglutination after infection is due to rearrangements at the cell surface rather than to insertion of new lectin binding sites per se. PMID:4735591

  11. Lectin-mediated microfluidic capture and release of leukemic lymphocytes from whole blood.

    PubMed

    Vickers, Dwayne A L; Hincapie, Marina; Hancock, William S; Murthy, Shashi K

    2011-06-01

    Lectins are a group of proteins that bind specifically and reversibly to mono- and oligosaccharide carbohydrate structures that are present on the surfaces of mammalian cells. The use of lectins as capture agents in microfluidic channels was examined with a focus on cells associated with T and B lymphocytic leukemia. In addition to examining the adhesion of Jurkat T and Raji B lymphocytes to a broad panel of lectins, this work also examined the capture of these cells from whole blood. Captured T and B lymphocytes were eluted from the microfluidic devices with a solution of the lectin's inhibiting sugar. The capture and release steps were accomplished in under 1 h. The significance of this work lies within the realm of low-cost capture of abundant target cells with non-stimulatory elution capability. PMID:21455756

  12. Biophysical characterization of lectin-glycan interactions for therapeutics, vaccines and targeted drug-delivery.

    PubMed

    Christie, Michelle P; Toth, Istvan; Simerská, Pavla

    2014-01-01

    Lectin-glycan interactions play a role in biological processes, host-pathogen interactions and in disease. A more detailed understanding of these interactions is not only useful for the elucidation of their biological function but can also be applied in immunology, drug development and delivery and diagnostics. We review some commonly used biophysical techniques for studying lectin-glycan interactions; namely: frontal affinity chromatography, glycan/lectin microarray, surface plasmon resonance, electrochemical impedance spectroscopy, isothermal titration calorimetry, fluorescent assays, enzyme linked lectin sorbent assay and saturation transfer difference nuclear magnetic resonance spectroscopy. Each method is evaluated on efficiency, cost and throughput. We also consider the advantages and limitations of each technique and provide examples of their application in biology, drug discovery and delivery, immunology, glycoprofiling and biosensing. PMID:25531972

  13. Bishydrazide glycoconjugates for lectin recognition and capture of bacterial pathogens.

    PubMed

    Adak, Avijit Kumar; Leonov, Alexei P; Ding, Ning; Thundimadathil, Jyothi; Kularatne, Sumith; Low, Philip S; Wei, Alexander

    2010-11-17

    Bishydrazides are versatile linkers for attaching glycans to substrates for lectin binding and pathogen detection schemes. The α,ω-bishydrazides of carboxymethylated hexa(ethylene glycol) (4) can be conjugated at one end to unprotected oligosaccharides, then attached onto carrier proteins, tethered onto activated carboxyl-terminated surfaces, or functionalized with a photoactive cross-linking agent for lithographic patterning. Glycoconjugates of bishydrazide 4 can also be converted into dithiocarbamates (DTCs) by treatment with CS(2) under mild conditions, for attachment onto gold substrates. The immobilized glycans serve as recognition elements for cell-surface lectins and enable the detection and capture of bacterial pathogens such as Pseudomonas aeruginosa by their adsorption onto micropatterned substrates. A detection limit of 10³ cfu/mL is demonstrated, using a recently introduced method based on optical pattern recognition. PMID:20925370

  14. Bishydrazide Glycoconjugates for Lectin Recognition and Capture of Bacterial Pathogens

    PubMed Central

    Adak, Avijit Kumar; Leonov, Alexei P.; Ding, Ning; Thundimadathil, Jyothi; Kularatne, Sumith; Low, Philip S.; Wei, Alexander

    2010-01-01

    Bishydrazides are versatile linkers for attaching glycans to substrates for lectin binding and pathogen detection schemes. The α,ω-bishydrazides of carboxymethylated hexaethylene glycol (4) can be conjugated at one end to unprotected oligosaccharides, then attached onto carrier proteins, tethered onto activated carboxyl-terminated surfaces, or functionalized with a photoactive crosslinking agent for lithographic patterning. Glycoconjugates of bishydrazide 4 can also be converted into dithiocarbamates (DTCs) by treatment with CS2 under mild conditions, for attachment onto gold substrates. The immobilized glycans serve as recognition elements for cell-surface lectins and enable the detection and capture of bacterial pathogens such as Psuedomonas aeruginosa by their adsorption onto micropatterned substrates. A detection limit of 103 cfu/mL is demonstrated, using a recently introduced method based on optical pattern recognition. PMID:20925370

  15. Molecular and biological characterization of a mannan-binding lectin from the holothurian Apostichopus japonicus.

    PubMed

    Bulgakov, Aleksandr A; Eliseikina, Marina G; Petrova, Irina Yu; Nazarenko, Evgeny L; Kovalchuk, Svetlana N; Kozhemyako, Valery B; Rasskazov, Valery A

    2007-12-01

    To elucidate the origin and evolution of mannan-binding lectins (MBL), a new C-type lectin (CTL) specific for high-mannose glycans (MBL-AJ) was isolated from the coelomic plasma of the holothurian Apostichopus japonicus. MBL-AJ has oligomeric forms with identical 17-kDa subunits on SDS-PAGE. Among natural ligands, lectin hemagglutination activity was competitively inhibited by extracellular low-branched, but not high-branched, alpha-D-mannans isolated from marine halophilic bacteria and composed of alpha-1,2 and alpha-1,6 linked D-mannose residues. This suggests that the lectin interacts with backbone or inner side chain mannose residues, but not with terminal ones. The activity of the lectin was Ca(2+)-, pH-, and temperature-dependent. MBL-AJ cDNA was cloned from a holothurian coelomocyte cDNA library. The subunit of the mature protein has 159 amino acids and a single carbohydrate-recognition domain (CRD) of CTL. CRD contains a Glu-Pro-Asp amino acid sequence (EPN-motif) conserved for all known MBLs. A monospecific polyclonal antibody against MBL-AJ was obtained using the 34-kDa lectin dimer as an immunogen. The MBL-AJ has demonstrated immunochemical identity to the earlier isolated mannan-binding CTL from another holothurian, Cucumaria japonica. But a more interesting finding was cross-reactivity of MBL-AJ and human serum MBL detected by the antibody against MBL-AJ. Taking into consideration such MBL-AJ peculiarities as its carbohydrate specificity, the presence of a conserved region forming the mannose-binding site, common antigenic determinants with human MBL, and participation in defense reactions, it is possible that MBL-AJ belongs to the family of evolutionary conserved mannan-binding proteins. PMID:17890508

  16. Disruption of the C. elegans Intestinal Brush Border by the Fungal Lectin CCL2 Phenocopies Dietary Lectin Toxicity in Mammals.

    PubMed

    Stutz, Katrin; Kaech, Andres; Aebi, Markus; Künzler, Markus; Hengartner, Michael O

    2015-01-01

    Lectins are non-immunoglobulin carbohydrate-binding proteins without enzymatic activity towards the bound carbohydrates. Many lectins of e.g. plants or fungi have been suggested to act as toxins to defend the host against predators and parasites. We have previously shown that the Coprinopsis cinerea lectin 2 (CCL2), which binds to α1,3-fucosylated N-glycan cores, is toxic to Caenorhabditis elegans and results in developmental delay and premature death. In this study, we investigated the underlying toxicity phenotype at the cellular level by electron and confocal microscopy. We found that CCL2 directly binds to the intestinal apical surface and leads to a highly damaged brush border with loss of microvilli, actin filament depolymerization, and invaginations of the intestinal apical plasma membrane through gaps in the terminal web. We excluded several possible toxicity mechanisms such as internalization and pore-formation, suggesting that CCL2 acts directly on intestinal apical plasma membrane or glycocalyx proteins. A genetic screen for C. elegans mutants resistant to CCL2 generated over a dozen new alleles in bre 1, ger 1, and fut 1, three genes required for the synthesis of the sugar moiety recognized by CCL2. CCL2-induced intestinal brush border defects in C. elegans are similar to the damage observed previously in rats after feeding the dietary lectins wheat germ agglutinin or concanavalin A. The evolutionary conserved reaction of the brush border between mammals and nematodes might allow C. elegans to be exploited as model organism for the study of dietary lectin-induced intestinal pathology in mammals. PMID:26057124

  17. Disruption of the C. elegans Intestinal Brush Border by the Fungal Lectin CCL2 Phenocopies Dietary Lectin Toxicity in Mammals

    PubMed Central

    Stutz, Katrin; Kaech, Andres; Aebi, Markus; Künzler, Markus; Hengartner, Michael O.

    2015-01-01

    Lectins are non-immunoglobulin carbohydrate-binding proteins without enzymatic activity towards the bound carbohydrates. Many lectins of e.g. plants or fungi have been suggested to act as toxins to defend the host against predators and parasites. We have previously shown that the Coprinopsis cinerea lectin 2 (CCL2), which binds to α1,3-fucosylated N-glycan cores, is toxic to Caenorhabditis elegans and results in developmental delay and premature death. In this study, we investigated the underlying toxicity phenotype at the cellular level by electron and confocal microscopy. We found that CCL2 directly binds to the intestinal apical surface and leads to a highly damaged brush border with loss of microvilli, actin filament depolymerization, and invaginations of the intestinal apical plasma membrane through gaps in the terminal web. We excluded several possible toxicity mechanisms such as internalization and pore-formation, suggesting that CCL2 acts directly on intestinal apical plasma membrane or glycocalyx proteins. A genetic screen for C. elegans mutants resistant to CCL2 generated over a dozen new alleles in bre 1, ger 1, and fut 1, three genes required for the synthesis of the sugar moiety recognized by CCL2. CCL2-induced intestinal brush border defects in C. elegans are similar to the damage observed previously in rats after feeding the dietary lectins wheat germ agglutinin or concanavalin A. The evolutionary conserved reaction of the brush border between mammals and nematodes might allow C. elegans to be exploited as model organism for the study of dietary lectin-induced intestinal pathology in mammals. PMID:26057124

  18. The first crystal structure of a Mimosoideae lectin reveals a novel quaternary arrangement of a widespread domain.

    PubMed

    Gallego del Sol, Francisca; Nagano, Celso; Cavada, Benildo S; Calvete, Juan J

    2005-10-28

    The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of beta-prism domains, and highlight the adaptability of the beta-prism fold as a building block in the evolution of plant lectins. The P.platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroid-shaped structure of the P.platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P.platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms. PMID:16185708

  19. A C-type lectin receptor pathway is responsible for the pathogenesis of acute cyclophosphamide-induced cystitis in mice.

    PubMed

    Dejima, Takashi; Shibata, Kensuke; Yamada, Hisakata; Takeuchi, Ario; Hara, Hiromitsu; Eto, Masatoshi; Naito, Seiji; Yoshikai, Yasunobu

    2013-12-01

    Hemorrhagic cystitis often arises after cyclophosphamide (CYP) administration. As yet, however, the mechanism involved in its pathogenesis is unknown. In this study, it was found that the Fc receptor γ chain (FcRγ)- caspase recruitment domain-containing protein 9 (CARD9)-dependent pathway rather than the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway is involved in the pathogenesis of acute CYP-induced cystitis in mice. Rapid and transient production of interleukin (IL)-6 and IL-1β was detected in the bladder at 4 hr, preceding IL-23 and IL-17A production and an influx of neutrophils, which reached a peak at 24 hr after injection. As assessed by weight, edema and neutrophil infiltration, cystitis was significantly attenuated in CARD9 knockout (KO) and FcRγKO mice, this attenuation being accompanied by impaired production of IL-1β, IL-6, IL-23 and IL-17A. The major source of IL-17A is the vesical γδ T cell population: IL-17AKO, CδKO and Tyk2KO mice showed little IL-17A production and reduced neutrophil infiltration in the bladder after CYP injection. These results suggest that FcRγ-CARD9-dependent production of proinflammatory cytokines such as IL-1β, IL-6, and IL-23 and the subsequent activation of IL-17A-producing γδ T cells are at least partly involved in the pathogenesis of acute CYP-induced cystitis in mice. PMID:24102807

  20. Allergy-Protective Arabinogalactan Modulates Human Dendritic Cells via C-Type Lectins and Inhibition of NF-κB.

    PubMed

    Peters, Marcus; Guidato, Patrick M; Peters, Karin; Megger, Dominik A; Sitek, Barbara; Classen, Birgit; Heise, Esther M; Bufe, Albrecht

    2016-02-15

    Arabinogalactan (AG) isolated from dust of a traditional farm prevents disease in murine models of allergy. However, it is unclear whether this polysaccharide has immune regulatory properties in humans. The aim of this study was to test the influence of AG on the immune-stimulating properties of human dendritic cells (DCs). Moreover, we sought to identify the receptor to which AG binds. AG was produced from plant callus tissue under sterile conditions to avoid the influence of pathogen-associated molecular patterns in subsequent experiments. The influence of AG on the human immune system was investigated by analyzing its impact on monocyte-derived DCs. To analyze whether the T cell stimulatory capacity of AG-stimulated DCs is altered, an MLR with naive Th cells was performed. We revealed that AG reduced T cell proliferation in a human MLR. In the search for a molecular mechanism, we found that AG binds to the immune modulatory receptors DC-specific ICAM-3 -: grabbing non integrin (DC-SIGN) and macrophage mannose receptor 1 (MMR-1). Stimulation of these receptors with AG simultaneously with TLR4 stimulation with LPS increased the expression of the E3 ubiquitin-protein ligase tripartite motif -: containing protein 21 and decreased the phosphorylation of NF-κB p65 in DCs. This led to a reduced activation profile with reduced costimulatory molecules and proinflammatory cytokine production. Blocking of MMR-1 or DC-SIGN with neutralizing Abs partially inhibits this effect. We conclude that AG dampens the activation of human DCs by LPS via binding to DC-SIGN and MMR-1, leading to attenuated TLR signaling. This results in a reduced T cell activation capacity of DCs. PMID:26746190

  1. Plasmon waveguide resonance for sensing glycan-lectin interactions.

    PubMed

    Alves, Isabel; Kurylo, Ievgen; Coffinier, Yannick; Siriwardena, Aloysius; Zaitsev, Vladimir; Harté, Etienne; Boukherroub, Rabah; Szunerits, Sabine

    2015-05-11

    Carbohydrate-modified interfaces have been shown to be valuable tools for the study of protein-glycan recognition events. Label-free approache such as plasmonic based techniques are particularly attractive. This paper describes a new analytical platform for the sensitive and selective screening of carbohydrate-lectin interactions using plasmon waveguide resonance. Planar optical waveguides (POW), consisting of glass prisms coated with silver (50 nm) and silica (460 nm) layers were derivatized with mannose or lactose moieties. The specific association of the resulting interface with selected lectins was assessed by following the changes in its plasmonic response. The immobilization strategy investigated in this work is based on the formation of a covalent bond between propargyl-functionalized glycans and surface-linked azide groups via a Cu(I) "click" chemistry. Optimization of the surface architecture through the introduction of an oligo(ethylene glycol) spacer between the plasmonic surface and the glycan ligands provided an interface which allowed screening of glycan-lectin interactions in a highly selective manner. The limit of detection (LOD) of this method for this particular application was found to be in the subnanomolar range (0.5 nM), showing it to constitute a promising analytical platform for future development and use in a pharmaceutical or biomedical setting. PMID:25911432

  2. Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection

    PubMed Central

    Vázquez, Alicia; de Dios Ruiz-Rosado, Juan; Terrazas, Luis I.; Juárez, Imelda; Gomez-Garcia, Lorena; Calleja, Elsa; Camacho, Griselda; Chávez, Ana; Romero, Miriam; Rodriguez, Tonathiu; Espinoza, Bertha; Rodriguez-Sosa, Miriam

    2014-01-01

    The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 104 T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection. PMID:25170304

  3. Lectin binding patterns to plasmalemmal glycoconjugates of goblet cells undergoing differentiation in vitro.

    PubMed

    Frisch, E B; Phillips, T E

    1990-09-01

    The plasmalemmal glycoconjugates of the HT29-18N2 (N2) cell line were characterized on cells grown as 1) undifferentiated multilayers in glucose-containing culture media and 2) monolayers of columnar cells acquiring the goblet cell phenotype in glucose-free media. Lectins were unable to bind sheets of detached N2 cells in the absence of fixation. Following fixation with aldehydes, a dramatic unmasking of lectin binding sites was seen. When fixed monolayers were stained prior to embedding, biotinylated lectins, visualized by the avidin-biotin-complexed peroxidase technique, were more efficient than collodial gold-coupled lectins. Lectin binding sites could also be detected by using collodial gold-coupled lectins to stain monolayers embedded in LR White, Lowicryl K4M, and Lowicryl HM20. The binding of 5 lectins (wheat germ, Dolichos bifluros, peanut, soybean, and Ulex europeus) was found to be independent of the stage of differentiation; "pre-differentiated" columnar cells which had prominent microvilli and no or few mucous secretory granules had identical staining patterns as well-differentiated goblet cells with large numbers of secretory granules. Ricinus communis I was the only lectin whose binding was influenced by the stage of differentiation; it intensely labeled undifferentiated multilayers of N2 cells but only weakly labeled basolateral membranes of differentiated monolayers. Canavalia ensiformas (ConA) caused a moderate and even labeling of both apical and basolateral membranes of fixed monolayers stained prior to embedding, but post-embedding labeling revealed heavy labeling along the lateral margins of all columnar cells and weak to moderate binding along the apical and basal cell surface. PMID:2213229

  4. Density variant glycan microarray for evaluating cross-linking of mucin-like glycoconjugates by lectins.

    PubMed

    Godula, Kamil; Bertozzi, Carolyn R

    2012-09-26

    Interactions of mucin glycoproteins with cognate receptors are dictated by the structures and spatial organization of glycans that decorate the mucin polypeptide backbone. The glycan-binding proteins, or lectins, that interact with mucins are often oligomeric receptors with multiple ligand binding domains. In this work, we employed a microarray platform comprising synthetic glycopolymers that emulate natural mucins arrayed at different surface densities to evaluate how glycan valency and spatial separation affect the preferential binding mode of a particular lectin. We evaluated a panel of four lectins (Soybean agglutinin (SBA), Wisteria floribunda lectin (WFL), Vicia villosa-B-4 agglutinin (VVA), and Helix pomatia agglutin (HPA)) with specificity for α-N-acetylgalactosamine (α-GalNAc), an epitope displayed on mucins overexpressed in many adenocarcinomas. While these lectins possess the ability to agglutinate A(1)-blood cells carrying the α-GalNAc epitope and cross-link low valency glycoconjugates, only SBA showed a tendency to form intermolecular cross-links among the arrayed polyvalent mucin mimetics. These results suggest that glycopolymer microarrays can reveal discrete higher-order binding preferences beyond the recognition of individual glycan epitopes. Our findings indicate that glycan valency can set thresholds for cross-linking by lectins. More broadly, well-defined synthetic glycopolymers enable the integration of glycoconjugate structural and spatial diversity in a single microarray screening platform. PMID:22967056

  5. Multivalent Carbohydrate-Lectin Interactions: How Synthetic Chemistry Enables Insights into Nanometric Recognition.

    PubMed

    Roy, René; Murphy, Paul V; Gabius, Hans-Joachim

    2016-01-01

    Glycan recognition by sugar receptors (lectins) is intimately involved in many aspects of cell physiology. However, the factors explaining the exquisite selectivity of their functional pairing are not yet fully understood. Studies toward this aim will also help appraise the potential for lectin-directed drug design. With the network of adhesion/growth-regulatory galectins as therapeutic targets, the strategy to recruit synthetic chemistry to systematically elucidate structure-activity relationships is outlined, from monovalent compounds to glyco-clusters and glycodendrimers to biomimetic surfaces. The versatility of the synthetic procedures enables to take examining structural and spatial parameters, alone and in combination, to its limits, for example with the aim to produce inhibitors for distinct galectin(s) that exhibit minimal reactivity to other members of this group. Shaping spatial architectures similar to glycoconjugate aggregates, microdomains or vesicles provides attractive tools to disclose the often still hidden significance of nanometric aspects of the different modes of lectin design (sequence divergence at the lectin site, differences of spatial type of lectin-site presentation). Of note, testing the effectors alone or in combination simulating (patho)physiological conditions, is sure to bring about new insights into the cooperation between lectins and the regulation of their activity. PMID:27187342

  6. Crystal structure of a symbiosis-related lectin from octocoral.

    PubMed

    Kita, Akiko; Jimbo, Mitsuru; Sakai, Ryuichi; Morimoto, Yukio; Miki, Kunio

    2015-09-01

    D-Galactose-binding lectin from the octocoral, Sinularia lochmodes (SLL-2), distributes densely on the cell surface of microalgae, Symbiodinium sp., an endosymbiotic dinoflagellate of the coral, and is also shown to be a chemical cue that transforms dinoflagellate into a non-motile (coccoid) symbiotic state. SLL-2 binds with high affinity to the Forssman antigen (N-acetylgalactosamine(GalNAc)α1-3GalNAcβ1-3Galα1-4Galβ1-4Glc-ceramide), and the presence of Forssman antigen-like sugar on the surface of Symbiodinium CS-156 cells was previously confirmed. Here we report the crystal structures of SLL-2 and its GalNAc complex as the first crystal structures of a lectin involved in the symbiosis between coral and dinoflagellate. N-Linked sugar chains and a galactose derivative binding site common to H-type lectins were observed in each monomer of the hexameric SLL-2 crystal structure. In addition, unique sugar-binding site-like regions were identified at the top and bottom of the hexameric SLL-2 structure. These structural features suggest a possible binding mode between SLL-2 and Forssman antigen-like pentasaccharide. PMID:26022515

  7. 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.

  8. Using lectins to harvest the plasma/serum glycoproteome.

    PubMed

    Fanayan, Susan; Hincapie, Marina; Hancock, William S

    2012-07-01

    Aberrant protein glycosylation has been shown to be associated with disease processes and identification of disease-specific glycoproteins and glycosylation changes may serve as potential diagnostic and therapeutic biomarkers. However despite recent advances in proteomic-based biomarker discovery, this knowledge has not yet translated into an extensive mining of the glycoproteome for potential biomarkers. The major challenge for a comprehensive glycoproteomics analysis arises primarily from the enormous complexity and the large dynamic range in protein constituent in biological samples. Methods that specifically target glycoproteins are therefore necessary to facilitate their selective enrichment prior to their identification by MS-based analysis. The use of lectins, with selective affinities for specific carbohydrate epitopes, to enrich glycoprotein fractions coupled with modern MS, have greatly enhanced the identification of the glycoproteome. On account of their ability to specifically bind cell surface carbohydrates lectins have, during the recent past, found extensive applications in elucidation of the architecture and dynamics of cell surface carbohydrates, glycoconjugate purification, and structural characterization. Combined with complementary depletion and MS technologies, lectin affinity chromatography is becoming the most widely employed method of choice for biomarker discovery in cancer and other diseases. PMID:22740463

  9. 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.

  10. Comparison of the 2D and 3D Nanostructured Lectin-Based Biosensors for In Situ Detection of Sialic Acid on Glycoproteins

    PubMed Central

    Bertok, Tomas; Sediva, Alena; Vikartovska, Alica; Tkac, Jan

    2016-01-01

    We present here comparison of a build-up of two ultrasensitive lectin biosensors based on 2D or 3D architecture. A 2D lectin biosensor was prepared by a covalent immobilisation of lectin Sambucus nigra agglutinin (SNA) recognising sialic acid directly on a mixed self-assembled monolayer (SAM) on planar gold surfaces. A 3D biosensor was prepared by covalent immobilisation of SNA lectin on a mixed SAM layer formed on gold nanoparticles. Surface plasmon resonance technique allowed to follow kinetics of a mixed SAM (1:1 mixture of 11-mercaptoundecanoic acid and 6-mercaptohexanol) formation on a bare gold electrode and on an electrode modified by 5 nm and 20 nm gold nanoparticles (AuNPs). Results from the study revealed that a mixed SAM formation is slower on surfaces with increased curvature, the process of SAM formation on all surfaces is completed within 6 min, but a density of thiols on such surfaces differs significantly. Quartz crystal microbalance experiments showed that a surface density of immobilised lectin of (2.53 ± 0.01) pmol cm-2 was higher on planar gold surface compared to the surface modified by 20 nm AuNPs with a surface density of (0.94 ± 0.01) pmol cm-2. Even though a larger amount of SNA lectin was immobilised on a surface of the 2D biosensor compared to the 3D biosensor, lectin molecules immobilised on AuNPs were more accessible for its analytes – glycoproteins fetuin and asialofetuin, containing different amount of sialic acid on the protein surface. Most likely a better accessibility of lectin for its analytes on a 3D surface and proper interfacial properties of a 3D surface are behind unprecedented detection limit down to aM level for the lectin biosensor based on such a nanoscale tuned interface.