Characterization of a 14 kDa plant-related serine protease inhibitor and regulation of cytotoxic activity in earthworm coelomic fluid

Abstract

We have purified and characterized the serine protease inhibitor activity contained in the coelomic fluid of the earthworms, Eisenia. Serine protease inhibitor activity was stable between pH 3 and 9.5, not flocculable by pH 3.0 and resistant to 100°C for 15 min. or to 4°C for 24h. Ten μL of coelomic fluid was sufficient to inhibit in vitro the protease activity of 0.12 μg of trypsin. Injection of living bacteria into earthworms resulted in increased serine protease activity 1–2 days post-injection, and increased serine protease inhibitor activity on day 4, suggesting that serine protease inhibitor is responsible for serine protease neutralization. Purified to homogeneity by affinity chromatography on trypsin, the serine protease inhibitor of Eisenia is a monomer of 14 kDa. Its partial NH 2 amino acid sequence revealed a basic hydrophobic fragment which shared 68–75% homologies and 47–60% identities with several plant serine protease inhibitors. Eisenia cytotoxic activity due to the two fetidins of 40 and 45 kDa was stimulable in vitro by several serine proteases. Incubation with soybean trypsin inhibitor variant a (STIa) resulted in less cytotoxicity. The inhibitory effect occurred only when STIa was added before cell disruption. Interpretative cytotoxic scheme involving the release of intracellular cytotoxic proteins, intracellular trypsin-like activator and extracellular serine protease inhibitor suggests regulatory mechanisms for cellular/humoral immune system of earthworms.