6 ISOFORMS OF CARDIOTOXIN IN MALAYAN SPITTING COBRA (NAJA-NAJA-SPUTATRIX) VENOM - CLONING AND CHARACTERIZATION OF CDNAS

Cardiotoxins are the most abundant toxin components of cobra venom. Al though many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra(Naja atm) and two more of uncertain ori gin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein anal ysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CT X 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardio toxin genes from a single species of a spitting cobra. The cDNAs encod ing these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. T he native and recombinant cardiotoxins were first characterized by Wes tern blotting and N-terminal protein sequencing. These proteins were a lso found to have different levels of cytolytic activity on cultured b aby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 ( 20% constitution) is highly homologous to the cardiotoxins of N. n. at m and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic i soform (CTX 5) could have evolved first followed by the hydrophobic is oforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras coul d be the modern descendants of the African and Egyptian counterparts. (C) 1998 Elsevier Science B.V.