CLONING AND CHARACTERIZATION OF GENES ENCODING CARDIOTOXINS IN NAJA NAJA SPUTATRIX

Abstract

Cardiotoxins (CTX) are the most abundant toxin components of cobra venom. The cloning of the cardiotoxin structural gene from Malayan spitting cobra, Naja naja sputatrix, was carried out using cDNA fragments generated by reverse transcription- polymerase chain reaction (RT-PCR). Putative clones were sequenced on both strands and sequence data analysis revealed the presence of nine cDNA sequences encoding six cardiotoxins isoforms (Ctx-1, 2a, 3, 4a, 4b and 5) in N. n. sputatrix. The cDNAs encoding these isoforms were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N- terminal sequencing. These proteins were also found to have different level of cytolytic activity on cultured baby 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 present study suggests that the most hydrophilic isoform (Ctx-5) could have evolved first followed by the hydrophobic isoforms (Ctx-1, 2, 3 and 4). It is speculated that Asiatic cobras could be the modern descendents of the African and Egyptian counterparts. Using the combination of "genomic PCR" and "genome walking" procedures, the N. n. sputatrix cardiotoxin gene was cloned, sequenced and analyzed. The present study reports on the genomic structure, organization and polymorphic nature of the cardiotoxin gene encoding cardiotoxins of N.n.sputatrix. The polymorphic gene consists of six CTX isoforms, each (2.2 kb) having 3 exons and 2 introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6 / C/EBP, NF-kB and PuF have been identified in the 5'-promoter region of the gene. The CDX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3 which, correspond to the functional domains in the three-finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process. Using N. n. sputatrix as a model, the present study reports on the mechanism of toxin gene expression by studying the mRNA turnover and protein synthesis rates for cardiotoxins, neurotoxins (NTX) and phospholipase A2 (PLA2). The study was carried out for over a period of 8 days after the stimulation of venom synthesis by manual "milking" of the venom gland. Immunofluorescence detection and in situ hybridization were used to localize the toxins and their mRNAs in venom gland sections. Elevation of mRNA levels were observed within 2 hours indicating that transcriptional induction of toxin genes is an early event following milking of the venom gland, which correlates to the accelerated transcription of amylase gene in stimulated rat parotid gland. The rate of mRNA turnover is highest for CTX followed by PLA2 and NTX. The mRNA levels in the milked glands remained significantly higher than that of unmilked (control) glands for up to 192 hours (8 days) post- milking. The rate of protein synthesis as determined by immunofluorescence and liquid chromatography-mass spectrometry (LC-MS) techniques, increased in parallel with the increase in the toxin mRNA content in the secretory epithelial cells, suggesting that transcriptional regulation of the toxin genes may be involved. The gradual decrease in toxin synthesis with the concomitant increase in the secretion and accumulation of venom in the tubular lumina indicates that the synthesis of toxins is coordinated with its secretion in a feed back mechanism. The higher rates of synthesis, secretion and accumulation of venom in spitting cobras as compared to in vipers could be the result of an evolutionary adaptation. This makes perfect biological sense as the N. n. sputatrix expends huge quantity of its venom for spitting, believed to be a unique defensive reaction.