Role of serine proteases and agglutinins in the endotoxininduced protein coagulation in Archachatina marginata hemolymph

Abstract

Musa Oyewole Salawu, Timothy Yoshino, Hussein Oyelola Bukoye Oloyede,Adenike Kuku, Mikail Olugbemiro Nafiu, Laura Gonzalez, Xiao Jun Wu

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, endotoxininduced 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.