Purification and characterization of two Ca(2+)-dependent lectins from coelomic plasma of sea cucumber, Stichopus japonicus.
Two structurally distinct lectins were purified from the coelomic plasma 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 (SPL-1) and 60 kDa (SPL-2) on gel filtration, but about 17 kDa on SDS-PAGE under reducing conditions. Both lectins showed hemagglutination activity toward rabbit erythrocytes in the presence of Ca2+ ions. The N-terminal amino acid sequences were highly homologous to but distinct from those of a Ca(2+)-dependent (C-type) lectin named SJL-I purified from the same species. In addition to porcine stomach mucin, the hemagglutination 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 presence of inhibitory sugars or EGTA, suggesting the presence of an endogenous carbohydrate ligand(s) for plasma C-type lectins in the clot. However, coelomocyte clotting occurred normally even in the presence of inhibitory 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.
Matsui
T
T
Division of Biomedical Polymer Science, Fujita Health University, Aichi.
Ozeki
Y
Y
Suzuki
M
M
Hino
A
A
Titani
K
K
D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion; D004912:Erythrocytes / Q000187:drug effects; D016023:Integrins / Q000378:metabolism; D037102:Lectins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009842:Oligopeptides; D012616:Sea Cucumbers / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D016023:Integrins; D037102:Lectins; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid