Abstract

Objective: In this study, we hypothesized that endotoxin-induced protein coagulation in the snail is mediated by lectins and serine proteases.
Methods: Experiments were carried out to determine the lectin activities of the hemolymph fractions and those of their coagulates and supernatants. The plasma was also exposed to 5000 EU/ml endotoxin, incubated for 1 h at 37 °C and the supernatant’s protein profile was compared on SDS-PAGE to a non-treated plasma. In addition, LPS-agarose was used as an affinity column to isolate lipopolysaccharide binding protein(s). Eluted proteins were subjected to SDS-PAGE, mass spectrometry and proteomic analysis. Finally, endotoxin-induced coagulation was tested for Ca2+ ion- and serine protease-dependence.
Results: Results from this study indicated that lectins were involved in the coagulation process induced by endotoxin. There was a loss in total lectin actvity from the plasma fraction on exposure to endotoxin. Several mono- and dissaccharides inhibited the agglutination of the reconstituted lyophilized plasma by rabbit erythrocytes, indicating the possible presence of several types of lectins in the plasma. Endotoxin-absorbed plasma resulted in a reduction of proteins in the >250 kDa range, ~66 kDa and a 37 kDa protein based on SDS-PAGE analysis. Excision of those bands and subsequent proteomic analyses predicted the presence of hemocyanin (389-394 kDa), an allergen (69 kDa) and a Gram-negative binding protein (50 kDa). Endotoxin-induced protein coagulation was inhibited by a trypsin protease inhbitor. No coagulaton was detected in the absence of Ca2+ ion.
Conclusions: From these results, we conclude that endotoxin-induced protein coagulation in Archachatina marginata hemolymph is lectin and serine protease-mediated and Ca2+ ion dependent.

Key words: Agglutinins (lectins); Archachatina marginata; hemolymph coagulation; endotoxin; innate immunity; hemocyanin; serine proteases.