Characterization of inflamin, the first member of a new family of snake venom proteins that induces inflammation

Unlike other sea snakes, Aipysurus eydouxii feeds exclusively on fish eggs. This unusual feeding habit prompted us to search for unique transcripts in their venom glands. In the present study we expressed a novel cysteine-rich secretory protein containing 94 amino acid residues that was identified in its cDNA library. As it induced inflammation and writhing in animals, this protein was named inflamin. It induced two waves of prostanoid production. The first wave peaked at 10 min and 6-oxo PGF_1α (prostaglandin F_1α) (6-keto PGF_1α) was the major product. The second wave, specifically of 6-oxo PGF_1α and PGE₂ (prostanglandin E₂), started after 2 h. In RAW 264.7 cells, COX-1 (cyclo-oxygenase-1) activity showed a transient increase at 10 min and is responsible for the first wave, but its expression was unaffected. COX-2 was induced after 3 h and is responsible for the second wave. Using specific inhibitors, we showed that cPLA₂ (calcium-dependent phospholipase A₂), and not sPLA₂ (secretory phospholipase A₂), iPLA₂ (calcium-independent phospholipase A₂) or DAG (diacylglycerol) lipase, plays a key role in arachidonate release. The cPLA₂ activity showed a transient increase of 62% at 10 min; this increase was due to its phosphorylation and not due to an increase in its expression. Thus inflamin, the first member of a new family of snake venom proteins, leads to an increase in the cPLA₂ and COX-1 activity resulting in inflammation and pain.