Protein And cDNA Structures Of Neurotoxic Phospholipase A2 From Crotalus durissus collilineatus.

Fagundes, F.H.R., Ponce-Soto, L.A., Bonfim, V.L., Damico, D.C.S. and Marangoni. S.

Department of Biochemistry Institute of Biology - UNICAMP – BRAZIL. 

A phospholipase A2 from the Crotalus durissus collilineatus (Cdcoll) (PLA2 F6) was isolated by Ponce-Soto, L.A., et al.,(2002). Both, protein sequencing and cDNA sequencing solved the primary structures. We have determined the amino acid and cDNA sequences of PLA2 F6 of Cdcoll. The protein primary structure was solved by sequencing the peptides generated by enzymatic fragmentation with SV-8 and clostripain. The cDNA synthesized from venom gland mRNA was amplified by the polymerase-chain reaction using a pair of synthetic oligonucleotide primers corresponding to the N- and C-terminal flanking regions of the enzymes. The nucleotide sequencing by dideoxynucleotide chain termination method using products of the polymerase chain reaction directly in automated DNA sequencer. The deduced amino acid sequences of PLA₂ F6 were 99% identical to those of the sequenced protein. PLA2 F6 consists of a signal peptides of 16 amino acid residues followed by a polypeptide chain of 122 amino acid residues and with a primary structure of: mralwivavl lvgveghllq fnkmikfetr rnaipfyafy gcycgwggrg rpkdatdrcc ivhdccygkl akcntkwdfy ryslrsgyft cgkgtwceqq icecdrvaae clrrslstyr ygymiypdsr crgpsetc. its molecular mass and isoeletric point were 14276.90 Da and 8.65, both estimated by two dimensional electrophoresis and mass spectrometry (MALD-TOF). PLA2 F6 purified from venom of the Cdcoll showed neurotoxic activity in a chick biventer cervicis preparation. Alignments and amino acid analyzes was maked using program DNAstar. The amino acid sequence of the PLA2 F6 revealed high sequence homology with others neurotoxics PLA2s especially the D49 PLA2s, its revealed highly conserved amino acid sequences principally for calcium binding loop and active site helix. This is the first report of cDNA sequence of Cdcoll venom PLA2.

Financial Support: CAPESand FAPESP

Correspondence to: fabiofagundes@hotmail.com