MOLECULAR CLONING, CHARACTERISATION AND EXPRESSION OF SHORT ?-NEUROTOXIN GENES IN NAJA NAJA SPUTATRIX

Abstract

The venom of spitting cobra, Naja naja sputatrix contains highly potent ?-neurotoxins (NTXs) in addition to phospholipase A2 and cardiotoxin. In this study, two isoforms (nNTX1 and nNTX4) of short ?-neurotoxins have been identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), followed by capillary electrophoresis and mass spectrometry. These neurotoxins have also been characterised by N-terminal amino acid sequencing and receptor binding. Competitive binding using [125I]-?-BTX showed that the neurotoxins have high affinities to both neuronal (rat brain synaptosomes) and peripheral (Torpedo electric organ) nAChRs. Four cDNAs (NTX1, NTX2, NTX3, NTX4) encoding four short ?-neurotoxin isoforms were cloned and characterised using reverse trancription-polymerase chain reaction (RT-PCR) of the mRNAs obtained from venom glands. The cDNAs, which were 285 bp in size, contained a sequence encoding a signal peptide of 21 amino acid residues. The leader sequence has been found to be identical in all of the clones analysed. Based on the variable amino acid residues present in the protein, the neurotoxins in N. n. sputatrix could be assigned to two major groups, 10E11T, 10Q11A, which can be further subdivided into 10E11T28S, 10E11T28G and 10Q11A28S, 10Q11A28G. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. The coding regions of the mature NTX toxins were cloned and the proteins were expressed as glutathione-S-transferase (GST) fusion proteins in E. coli. After affinity purification and digestion with thrombin, the recombinant toxins were purified by RP-HPLC. N-terminal amino acid sequencing showed that they are identical to the native NTXs. Three genes (NTX1 , NTX2 and NTX4) that are responsible for the synthesis of three isoforms of ?-neurotoxins in the venom of single spitting cobra have been completely characterised. DNA amplification by long distance-polymerase chain reaction (LD-PCR) and genome walking have provided information on NTX gene structure including their promoters and 5' and 3' untranslated regions (UTRs). Each NTX isoform is ~4 kb in size and contains three exons and two introns. Two possible transcription-initiation sites were identified by primer extension analysis and they corresponded to the adenine (A) nucleotide at positions +1 and -45. The promoter region contains two TATA boxes and a CCAAT box. Putative binding sites for transcriptional factors AP-2 and GATA are also shown. The NTX gene isoforms show nucleotide variation in exon 2, which corresponds to the functional domain in the three-fingered loop structure of the neurotoxin molecule. These observations together with the Southern blot hybridisation results suggest that the neurotoxin genes could be found in multiple copies in the genome of the snake. The high similarity observed among the NTX gene isoforms of N. n. sputatrix as well as with ?-NTX and ?-NTX genes from other snakes suggests that the NTX genes have probably evolved through a positive Darwinian selection process from a common ancestral gene. Phylogenetic analysis based on the molecular properties or these toxins at both the protein and gene level is consistent with the speculation that evolutionary-related but functionally distinct toxins have developed from an ancestral gene. Further studies on the short neurotoxins and their functions will provide valuable information on their role(s) in physiology and biochemistry of neurological abnormalities as well as in the design and development of pharmaceutical products related to the treatment of diseases. The venom of spitting cobra, Naja naja sputatrix contains highly potent ?-neurotoxins (NTXs) in addition to phospholipase A2 and cardiotoxin. In this study, two isoforms (nNTX1 and nNTX4) of short ?-neurotoxins have been identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), followed by capillary electrophoresis and mass spectrometry. These neurotoxins have also been characterised by N-terminal amino acid sequencing and receptor binding. Competitive binding using [125I]-?-BTX showed that the neurotoxins have high affinities to both neuronal (rat brain synaptosomes) and peripheral (Torpedo electric organ) nAChRs. Four cDNAs (NTX1, NTX2, NTX3, NTX4) encoding four short ?-neurotoxin isoforms were cloned and characterised using reverse trancription-polymerase chain reaction (RT-PCR) of the mRNAs obtained from venom glands. The cDNAs, which were 285 bp in size, contained a sequence encoding a signal peptide of 21 amino acid residues. The leader sequence has been found to be identical in all of the clones analysed. Based on the variable amino acid residues present in the protein, the neurotoxins in N. n. sputatrix could be assigned to two major groups, 10E11T, 10Q11A, which can be further subdivided into 10E11T28S, 10E11T28G and 10Q11A28S, 10Q11A28G