CLONING AND CHARACTERIZATION OF GENES ENCODING SNAKE CARDIOTOXIN AND PHOSPHOLIPASE A2

Abstract

SDS-PAGE of the total venom of Naja naja sputatrix showed that it consisted largely of low as well as high molecular weight proteins and enzymes. ?-neurotoxins (two species; m.wt. 7 kDa each; 6.5% of venom protein) cause neuromuscular blockade by binding to the nicotinic acetylcholine receptors at the post synaptic neuromuscular junction. In contrast, the ?-neurotoxins (three species; m.wt. 14 kDa each; 14% of venom protein) possess phospholipase A2 enzymatic activity and they bring about neuromuscular blockade by acting presynaptically. Cardiotoxins (three species; m.wt. 7 kDa each; 60% of venom protein) are basic proteins and they cause systemic cardiac arrest in vivo. They also depolarize cardiac and other muscle and nerve cells. At high concentrations, they are haemolytic, and they also lyse normal and tumour cells in vitro. Fifty-two types of cardiotoxins have been sequenced so far and they all share a high degree of conserved residues. As cardiotoxins form the major component of venom protein, it bears extension that these proteins, together with the neurotoxins, are responsible for the major symptoms of envenomation by the N. n. sputatrix. The last decade of work on snake cardiotoxins have focused on structural and functional studies at the protein level. Very little information was available on the cardiotoxin gene. For this reason, the project focused on the study of the gene of the cardiotoxin and attempts were made to produce the recombinant cardiotoxin in bacteria in the laboratory. The nucleotide sequence of a cDNA encoding the cardiotoxin has been determined. It is 372 bp long; the sequence has been deposited at the Los Alamos National Laboratory under the accession number L04640. The open reading frame of this cDNA contains a 21 -residue signal peptide followed by the 60-amino acid cardiotoxin protein. Polyclonal antibodies against the total venom have been produced and the titres have been determined. They were mainly used for Western blotting experiments and immunoscreening. The novel method of RNA-PCR has been employed to isolate the structural genes encoding the cardiotoxin and PLA2. The sequences of these structural genes have been verified by dideoxy sequencing. Their amino acid sequences, which have been translated from the structural gene sequences, were found to have some degree of homology to reported amino acid sequences. Hence the method of RNA-PCR has been established and can potentially be applied to isolate any other toxin genes from snake total RNA, provided that a good source of RNA was available and suitable primers were present. The structural gene of cardiotoxin was subcloned into a phage expression vector, ?gt11, and a plasmid expression vector, pXa3. In both cases, ?-galactosidase fusion proteins were produced. ?gt11 was used initially as it provided an easy and convenient means of identifying clones expressing the cardiotoxin by immunoscreening. The pXa3 vector containing the cardiotoxin structural gene produced a tripartite protein, consisting of ?-galactosidase/collagen and the recombinant cardiotoxin. This fusion protein was purified using an immunoaffinity column, and the recombinant cardiotoxin was cleaved off from rest of the fusion protein using endoprotease factor Xa. Animal toxicity and cytotoxicity experiments carried out using the recombinant cardiotoxin protein showed that it was ten times less toxic than the total venom. A more meaningful analysis could be made using cardiotoxin purified from the snake venom, but this was not available at the time of the experiment.