A phospholipase A2 inhibitor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Protein structure, genomic structure, and mechanism of action.

ABSTRACT

The lethal toxicity of the South American rattlesnake (Crotalus durissus terrificus) venom can be attributed mainly to the presence of a pre-synaptic neurotoxin, crotoxin, with phospholipase A2 activity. Crotoxin is a heterodimer of an acidic protein (CA) and a basic phospholipase A2 (CB). An anti-toxic protein of subunit molecular mass 23.6 kDa that neutralizes both lethal and PLA2 activity of crotalid venom and crotoxin has been previously purified from the plasma of this snake (Fortes-Dias, C., Fonseca, B. C. B., Kochva, E., and Diniz, C. R. (1991) Toxicon 29, 997-1008). The protein has been named CNF for Crotalus neutralizing factor. In the present study, we have shown that CNF exists as an oligomeric aggregate of (CNF)n, where n = 6-8, and when it interacts with crotoxin, it replaces the acidic protein CA of crotoxin to form a stable near stoichiometric complex of CNF.CB. The CNF.CB complex no longer exhibits PLA2 activity and is inert in vivo. Thus, the exchange reaction between CA.CB of crotoxin and CNF to form CNF.CB and free CA is reminiscent of the interaction of crotoxin with its target receptor at the neuromuscular transmission site in the presynaptic cells. A cDNA encoding CNF has been isolated from a liver cDNA library using an appropriate nucleotide probe. The nucleotide sequence codes for a 19-residue signal peptide, followed by a 181-residue protein of which 16 are half-cystines. Calculated molecular mass is 20.06 kDa, and there is a putative N-linked carbohydrate site at Asn157.