Two Lectins From Marine Bivalve Mollusks And Their Antibacterial Mechanism

Marine bivalve mollusks belonging to invertebrate without the vertebrate immune system, had the unique innate immune systems. Their lectins were one of significant humoral immune molecules. In the complex ocean environment, bivalve mollusks produced a variety of different carbohydrate specificity of lectins with specific biological activities. Some bivalve mollusks lectins had the functions of inhibiting the growth of bacteria. This suggested that these lectins could be the potential and important sources of the antimicrobial agents.A novel yeast-binding Ca2+-dependent lectin, Cyclina sinensis (Gmelin) lectin (CSL), was isolated from hemolymph of C. sinensis by ion-exchange on Cellulose DE52and purified by gel filtration on Sephadex G-100and HPLC on TSK-gel G4000PWXL.SDS-PAGE showed that the CSL protein had a molecular weight of72kDa, containing40and18kDa subunits. The native lectin behaved as a non-aggregation molecule in the native conformation. The N-terminal sequence of its18kDa subunit was AKPYDDAKYD. CSL digested by trypsin following by nano-ESI-MS/MS assay, five small peptides were obtained, which were YVAEEADR, LAITEVDLER, ENALDKAEQLEQK, SIADDDRIDQLEK and EVDRLEDELLAEK, respectively. N-terminal sequence and the five peptide sequences indicated that CSL and other marine bivalve mollusc lectins were heterogenous lectins. CSL contained abundant acidic amino acid Asx and Glx, but less Met, Phe and Tyr. Its total carbohydrate content was found to be16.2%. The linkage between the oligosaccharide moiety and peptide moiety of CSL belonged to O-glucosidic linkage. CSL had higher hemagglutination activity at0-40℃, and its hemagglutination activity was stable at pH6-7. D-Man and N-acetyl-D-galactosamine (GalNAc) could inhibit its hemagglutination. CSL could agglutinate E. coli and S. cerevisiae, especially the binding to yeast cells could be inhibited by D-Man.A lectin, designed as MCL-T, molecular mass148kDa, containing62kDa and36kDa subunits, was purified from Manila clam Ruditapes philippinarum using a combination of ion-exchange on Cellulose DE52and gel filtration on Sephadex G-100and HPLC on TSK-gel G3000PWXL.GalNAc and mucin from porcine stomach (PSM) inhibited its hemagglutination. MCL-T was digested by trypsin followed by nano-ESI-MS/MS assay, and five small peptides were obtained, which were VQRVTMD, MAPGFVNNK, NAIFYVDVDK, ERVHPPRPGLQR, AKIDMASPFIVMR and IDMASPFIVMRDPVLYR, respectively. According to molecular mass and the6peptide sequences, MCL-T was different to other reported lectins from Manila clam. MCL-T had higher hemagglutination activity at0-40℃, and its hemagglutination activity was stable at pH5-8. MCL-T could agglutinate E. coli, S. aureus, C. perfringens and Shewanella sp. treated with HCl, but could not agglutinate native E. coli, S. aureus, C. perfringens, B. subtilis and Shewanella sp.Antibacterial activities of CSL, MCL-T and CGL were studied. The results showed that CSL inhibited the growth of E. coli, C. perfringens and B. subtilis. Inhibition of respiration indicated that CSL inhibited Embden-Meyerhof Pathway (EMP) of E. coli and C. perfringens as well as Hexose Monophophate Pathway (HMP) of B. subtilis. When E. coli, C. perfringens and B. subtilis were incubated with CSL, DNA, RNA and soluble proteins decreased with increasing concentrations of CSL, but the NO contents increased at first and then decreased. MCL-T was found to show the antibacterial activities against S. aureus、B. subtilis and Shewanella sp.. Inhibition of respiration indicated that MCL-T inhibited HMP of S. aureus and B. subtilis as well as tricarboxylic acid (TCA) cycle of Shewanella sp.. When B. subtilis, S. aureus and Shewanella sp. were incubated with MCL-T, DNA, RNA and soluble proteins of B. subtilis and S. aureus decreased with increasing concentrations of MCL-T, but the NO contents increased at first and then decreased. CGL inhibited the growth of E. coli, B. subtilis and Shewanella sp.. Inhibition of respiration indicated that CGL inhibitied TCA cycle of E. coli, HMP of Shewanella sp. and EMP of B. subtilis. When E. coli, Shewanella sp. and B. subtilis were incubated with CGL, DNA contents of Shewanella sp. and B. subtilis, RNA and soluble proteins contents of the three bacteria decreased with increasing concentrations of CGL, but the NO contents of them increased at first and then decreased.Interactions between MCL-T and OMP of S. aureus were studied, and MCL-T binding to OMP of S. aureus was found. In addition, the binding is concentration-dependent and inhibited by PSM and GalNAc. CSL, MCL-T and CGL had RNase activities. ATP contents of bacteria assay showed that the three lectins did not lead to ATP leakage when they showed antibacterial activities. SEM studies showed the death of bacteria was not because of the disruption of the cells. MCL-T had domains of binding to OMP of S. aureus and RNase activities, CSL and CGL had domains of RNase activities.Two novel lectins from marine bivalve mollusks, CSL and MCL-T, were found in this study. Their structures, physicochemical characteristics, and carbohydrate specificity were studied. In addition, antibacterial mechanism of CSL, MCL-T and CGL were investigated, which provided a solid foundation for their application.