PURIFICATION AND CHARACTERIZATION OF 2 CA2-DEPENDENT LECTINS FROM CELOMIC PLASMA OF SEA-CUCUMBER, STICHOPUS-JAPONICUS()

Two structurally distinct lectins were purified from the coelomic plas ma of holothurian, Stichopus japonicus, by affinity chromatography on a porcine stomach mucin-conjugated agarose column, gel filtration on a Superose 6 column, and ion-exchange chromatography on a HiTrap Q-FPLC . The two lectins showed apparent molecular masses of about 400 kDa (S PL-1) and 60 kDa (SPL-2) on gel filtration, but about 17 kDa on SDS-PA GE under reducing conditions. Both lectins showed hemagglutination act ivity toward rabbit erythrocytes in the presence of Ca2+ ions. The N-t erminal amino acid sequences were highly homologous to but distinct fr om those of a Ca2+-dependent (C-type) lectin named SJL-1 purified from the same species. In addition to porcine stomach mucin, the hemagglut ination activity of SPL-1 was strongly inhibited by uronic acids such as galacturonic acid, and glucuronic acid, while the activity of SPL-2 was inhibited by GalNAc and galactosides. Both lectins were adsorbed on clotted coelomocytes in the presence of Ca2+ but not in the presenc e of inhibitory sugars or EGTA, suggesting the presence of an endogeno us carbohydrate ligand(s) for plasma C-type lectins in the clot. Howev er, coelomocyte clotting occurred normally even in the presence of inh ibitory sugars, but was strongly inhibited by synthetic GRGDSP peptide or EGTA, suggesting the participation of integrin but not the lectin- carbohydrate interaction in the clotting events.