Study On An Opsonin-like Molecule In Coelomic Fluid Of Sea Cucumber Apostichopus Japonicus (Selenka)

Apostichopus japonicus, an important sea cucumber in some province of China, such as Liaoning, Shandong and Hebei province. Phagocytosis is known to play an important role in echinoderm innate immune system. Opsonins are collective term of phagocytosis-enhancing serum proteins. They can bind to the surface of many kinds of pathogens and tag them as targets for phagocytosis in vertebrates. Opsonization to augment phagocytosis (or encapsulation) is the general molecular response of echinoderms to foreign pathogens. In order to determine if opsonin-like protein exists in the coelomic fluid of sea cucumber Apostichopus japonicus and identify the properties of this opsonin-like protein, immunochemical methods were performed including phagocytosis assay, SDS-PAGE, gel filtration and mass spectrometric analysis. The phagocytosis of phagocytes from the sea cucumber Apostichopus japonicus was analyzed in a quantitative in vitro assay using heat-killed yeast cells as target cells. Phagocytes of the sea cucumber were capable of phagocytosis in isotonic buffer, cell-free coelomic fluid and yeast cell-adsorbed cell-free coelomic fluid, but the phagocytosis level was enhanced when the incubation medium was cell-free coelomic fluid. SDS-PAGE analysis indicated that the about 18 kDa molecule can bind to the yeast cells. By using gel filtration we isolated the opsonin-like molecule of 18 kDa from coelomic fluid of the sea cucumber Apostichopus japonicus. Functional test of the opsonin-like molecule suggested that the about 18 kDa molecule is a phagocytosis-enhancing opsonin-like molecule. But the phagocytosis-enhancing function of the opsonin-like molecule is dependent on some other factors in the cell-free coelomic fluid. The properties of this opsonin-like molecule were analyzed by mass spectrometric analysis and peptide sequencing. We suggest that this is a new member of opsonin-like molecule found in invertebtrates.Changes in the amounts of opsonin-like molecule in coelomic fluid are followed over time by Western blots and ELISA. In the animals receiving lipopolysaccharids (LPS), initial increases in opsonin-like molecule are observed within 1 h post-injection (P<0.05). From 1 h to day 2, the level of opsonin-like molecule is significantly higher than 15 min prior to the first injection (P<0.05). Overall, these 9 animals shows a maximum amount of opsonin-like molecule in coelomic fluid between days 3 and 6, and the level is significantly higher than day 2 (P<0.05). After day 6, opsonin-like molecule levels are decreased.While the earliest response in the animals receiving sterile sea water (SSW) was day 2. From day 2 until day 8, the level of opsonin-like molecule is significantly higher than day 15 prior to the first injection (P<0.05).Although all animals responded to injury with increased levels of opsonin-like molecule in the coelomic fluid, those challenged with LPS had greater amounts of opsonin-like molecule in coelomic fluid than those receving SSW. This indicates that whether challenge with LPS or in response to injury, animals shows increases amounts of opsonin-like molecule in coelomic fluid. But those responses to a combination of LPS plus injury resulted in greater increases.The opsonin-like molecule probably is a type of lectin. It is nonspecific in agglutination for any type of human erythrocytes. The agglutination for chicken erythrocytes is the highest, and then the rabbits, humans (A, B, O, and AB), mouse and golden carp. The opsonin-like molecule shows strong broad spectrum antibacterial activity against both grampositiveand gram-negative bacteria. The agglutination for Vibrio anguillarum is the highest. The lectin still shows aggllutinating activity after being treated with 70℃for 30 minutes. There are no aggllutinating activities after being treated with 80℃for 30 minutes, indicating that it had a middle heat resistance. This lectin has the maximum hemagglutinating activity at pH 6.0-8.0, with activity at pH 5.0-9.0. The hemagglutinating activity for rabbit could not be inhibited by D-Glueose, D-Galaetose,L-Arabinose,D-Xylose,D-Mannose,L-Rhamnose,D-Frutose and LPS, but inhibited by GlcNAc,GalNAc,NeuAc,BSM,Fetuin,Asialo-BSM,Asialo- Fetuin. The minimum inhibitory concentration are 20.15 mmol/L,12.50 mmol/L,0.25 mmol/L,0.39 mg/mL,1.56 mg/mL,3.12 mg/mL and 6.26 mg/mL. The hemagglutinating activity is not inhibited by treatment with EDTA and Ca²⁺,Mg²⁺,Mn²⁺,K⁺.