Isolation, characterization and molecular cloning of AnMIP, a new α-type phospholipase A 2 myotoxin inhibitor from the plasma of the snake Atropoides nummifer (Viperidae: Crotalinae)

Abstract

A new phospholipase A 2 (PLA 2)-inhibitory protein was isolated from the plasma of Atropoides nummifer, a crotaline snake from Central America. This inhibitor was named AnMIP, given its ability to neutralize the activity of basic PLA 2 myotoxins of its own and related venoms. The cDNA of AnMIP was cloned and sequenced, showing that it belongs to the α group of phospholipase A 2 inhibitors (PLIs). AnMIP appears as a homotrimer in the native state, held together by non-covalent forces, with a subunit molecular mass of 22,247–22,301 and an isoelectric point of 4.1–4.7. This trimeric structure is the first observed in a PLIα from American crotaline snakes, previously reported only in Asian species. Sequencing, mass spectrometry, and analytical isoelectrofocusing indicated the existence of isoforms, as reported for other PLIαs isolated from snake plasma. The inhibitory profile of AnMIP showed specificity towards group II PLA 2s, either belonging to the catalytically-active (D49) or -inactive (K49) subtypes, exemplified in this study by Bothrops asper myotoxin I and A. nummifer myotoxin II, respectively. By phylogenetic analysis it was shown that AnMIP is closely related to CgMIP-II, previously isolated from the plasma of Cerrophidion godmani, showing 93% amino acid sequence identity.