The role of amoebocytes in endotoxin-mediated coagulation in the innate immunity of achatina fulica snails

Abstract

Achatina amoebocyte lysate (AAL) derived from amoebocytes of Achatina fulica was activated by Gram-negative bacterial endotoxins in a time-dependent manner resulting in gel formation/coagulation. The activation and maximum proliferation of amoebocytes was observed 40 min after intramuscular injection (20 µg/snail) of endotoxin. Endotoxin-mediated proteolytic activity of AAL towards a serine-protease-specific chromogenic substrate was maximum at pH 8.0, 37°C and within 15 min in a divalent-cation-dependent manner. The AAL activity induced by the endotoxin was directly dependent on the endotoxin concentration, showed a high specificity and saturated at higher endotoxin concentrations. An endotoxin-sensitive factor (ESF) was purified from AAL to apparent homogeneity by single-step affinity chromatography on a heparin-Sepharose 4B column. Native ESF of molecular weight 140 000 was composed of two identical subunits of molecular weight 70 000 attached through non-covalent association. A strong binding to endotoxin (Escherichia coli 055:B5) was exhibited by ESF with a 40-fold higher biological activity than AAL. The ESF was shown to have a unique Phe-Ile active site with regard to its alternate activation by α -chymotrypsin instead of endotoxin. The ESF was characterized as a serine protease type as evidenced by potent inhibition with specific inhibitors.