Molecular Cloning and Characteristics of a Lectin from the Bivalve Glycymeris yessoensis.

C-type lectins (CTLs) are a family of carbohydrate-binding proteins that mediate multiple biological events, including adhesion between cells, the turnover of serum glycoproteins, and innate immune system reactions to prospective invaders. Here, we describe the cDNA cloning of lectin from the bivalve Glycymeris yessoensis (GYL), which encodes 161 amino acids and the C-type carbohydrate recognition domain (CRD) with EPN and WND motifs. The deduced amino acid sequence showed similarity to other CTLs. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans are made up of xylose, mannose, D-glucosamine, 3-O-methylated galactose, D-quinovoses, and 3-O-methylated 6-deoxy-D-glucose. The potential CRD tertiary structure of the GYL adopted CTL-typical long-form double-loop structure and included three disulfide bridges at the bases of the loops. Additionally, when confirming the GYL sequence, eight isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using the glycan microarray approach, natural carbohydrate ligands were established, and the glycotope for GYL was reconstructed as "Galß1-4GlcNAcß obligatory containing an additional fragment", like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues.

New l-Rhamnose-Binding Lectin from the Bivalve Glycymeris yessoensis: Purification, Partial Structural Characterization and Antibacterial Activity.

In this study, a new l-rhamnose-binding lectin (GYL-R) from the hemolymph of bivalve Glycymeris yessoensis was purified using affinity and ion-exchange chromatography and functionally characterized. Lectin antimicrobial activity was examined in different ways. The lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as l-rhamnose, d-galactose, lactose, l-arabinose and raffinose. Using the glycan microarray approach, natural carbohydrate ligands were established for GYL-R as l-Rha and glycans containing the α-Gal residue in the terminal position. The GYL-R molecular mass determined by MALDI-TOF mass spectrometry was 30,415 Da. The hemagglutination activity of the lectin was not affected by metal ions. The lectin was stable up to 75 °C and between pH 4.0 and 12.0. The amino acid sequence of the five GYL-R segments was obtained with nano-ESI MS/MS and contained both YGR and DPC-peptide motifs which are conserved in most of the l-rhamnose-binding lectin carbohydrate recognition domains. Circular dichroism confirmed that GYL is a α/ß-protein with a predominance of the random coil. Furthermore, GYL-R was able to bind and suppress the growth of the Gram-negative bacteria E. coli by recognizing lipopolysaccharides. Together, these results suggest that GYL-R is a new member of the RBL family which participates in the self-defense mechanism against bacteria and pathogens with a distinct carbohydrate-binding specificity.

Spatial Structure of Lectin from the Mussel Mytilus trossulus: In-Sights from Molecular Modelling and Practical Proof.

Most proteins have the ability to self-associate into homooligomeric protein complexes, which consist of two or more identical subunits. Today, modern methods of molecular modeling are an integral part of the study of many biologically active molecules. In silico methods are widely used in structure establishing and function and activity prediction of lectins - carbohydrate-binding proteins. Here, we described by computer simulation the spatial organization of lectin isolated from the mantle of the mussel Mytilus trossulus (MTL). It was shown that the dimerization of MTL gives a total of six ligand binding sites that may be important for the manifestation its biological properties. The ability of MTL to form a dimeric and oligomeric structure was confirmed by dynamic light scattering and SDS-PAGE methods.

Identification and Characterization of a Novel Lectin from the Clam Glycymeris yessoensis and Its Functional Characterization under Microbial Stimulation and Environmental Stress.

Lectin from the bivalve Glycymeris yessoensis (GYL) was purified by affinity chromatography on porcine stomach mucin-Sepharose. GYL is a dimeric protein with a molecular mass of 36 kDa, as established by SDS-PAGE and MALDI-TOF analysis, consisting of 18 kDa subunits linked by a disulfide bridge. According to circular dichroism data, GYL is a ß/α-protein with the predominance of ß-structure. GYL preferentially agglutinates enzyme-treated rabbit erythrocytes and recognizes glycoproteins containing O-glycosidically linked glycans, such as porcine stomach mucin (PSM), fetuin, thyroglobulin, and ovalbumin. The amino acid sequences of five segments of GYL were acquired via mass spectrometry. The sequences have no homology with other known lectins. GYL is Ca2+-dependent and stable over a range above a pH of 8 and temperatures up to 20 °C for 30 min. GYL is a pattern recognition receptor, as it binds common pathogen-associated molecular patterns, such as peptidoglycan, LPS, ß-1,3-glucan and mannan. GYL possesses a broad microbial-binding spectrum, including Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Vibrio proteolyticus), but not the fungus Candida albicans. Expression levels of GYL in the hemolymph were significantly upregulated after bacterial challenge by V. proteolyticus plus environmental stress (diesel fuel). Results indicate that GYL is probably a new member of the C-type lectin family, and may be involved in the immune response of G. yessoensis to bacterial attack.