Molecular cloning and evolution of the genes encoding the precursors of taiwan cobra cardiotoxin and cardiotoxin-like basic protein.

Genomic DNAs encoding the precursors of eight cardiotoxins and two cardiotoxin-like basic proteins (CLBP) were isolated from the liver of Naja naja atra (Taiwan cobra). The cardiotoxin and CLBP genes have three exons like alpha-neurotoxin precursors. The promoter regions of these genes are highly conserved and contain the consensus transcriptional factor-binding sites for TBP, NF-1, CACCC-binding site, Spl and EFII, suggesting that these genes are regulated using similar transcriptional mechanisms. The introns and flanking regions of these genes share a high degree of nucleotide sequence identity, but except for the signal peptide domain the protein-coding regions are much more diversified than introns. The ratio of nonsynonymous to synonymous substitution is higher than one, reflecting that adaptive selection occurred during the evolution of cardiotoxin and CLBP proteins. Phylogenetic trees separate CLBPs and cardiotoxins into two clusters, suggesting that the CLBP gene and the cardiotoxin gene diverged earlier before the appearance of numerous cardiotoxins and CLBP.

Novel neurotoxins from Taiwan banded krait (Bungarus multicinctus) venom: purification, characterization and gene organization.

Two novel neurotoxins BM10-1 and BM10-2 were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combinations of chromatography on a SP-Sephadex C-25 column and a reverse phase HPLC column. BM10-1 contained 66 amino acid residues including 10 Cys residues, while BM10-2 consisted of 65 amino acid residues with 8 Cys residues. The secondary structure of both BM10-1 and BM10-2 was dominated with beta-sheet, but their gross conformation differed as evidenced by CD spectra and acrylamide quenching studies. BM10-1 inhibited carbachol-induced muscle contraction in a reversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. BM10-2 exhibited an irreversible but weak inhibition on carbachol-induced muscle contraction. Sequence alignment of neurotoxins with BM10-1 and BM10-2 suggested that the manner in the manifestation of their activity could be partly elucidated by the residues at toxin second loop. The genomic DNAs encoding BM10-1 and BM10-1-like protein (BM10-1L) were amplified by PCR. The two genes shared virtually identical structural organization and high degree of sequence identity with B. multicinctus neurotoxin genes. Compared to intron sequences of these genes, the protein-coding regions were highly variable. The difference between BM10-1 gene and BM10-1L gene notably arose from the third exon. These results suggest the evolution of B. multicinctus neurotoxins via the path of gene duplication.

Muscarinic toxin-like proteins from Taiwan banded krait (Bungarus multicinctus) venom: purification, characterization and gene organization.

Two novel proteins, BM8 and BM14, were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase HPLC column. Both proteins contained 82 amino acid residues including 10 cysteine residues, but there were two amino acid substitutions at positions 37 and 38 (Glu37-Ala38 in BM8; Lys37-Lys38 in BM14). CD spectra and acrylamide quenching studies revealed that the gross conformation of BM8 and BM14 differed. In contrast to BM8, BM14 inhibited the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine (mAchR) receptor subtype. Trinitrophenylation of Lys residues abolished the mAchR-binding activity of BM14, indicating that the Lys substitutions at positions 37 and 38 played a crucial role in the activity of BM14. The genomic DNA encoding the precursor of BM14 was amplified by PCR. The gene shared virtually identical structural organization with alpha-neurotoxin and cardiotoxin genes. The intron sequences of these genes shared a sequence identity up to 84%, but the protein-coding regions were highly variable. These results suggest that BM8, BM14, neurotoxins and cardiotoxins may have originated from a common ancestor, and the evolution of snake venom proteins shows a tendency to diversify their functions.

Organization and phylogenetic analysis of kappa-bungarotoxin genes from Bungarus multicinctus (Taiwan banded krait).

Two genomic DNAs were isolated from the liver of Bungarus multicinctus (Taiwan banded krait) encoded kappa1-bungarotoxin and kappa3-bungarotoxin precursors, respectively. They shared virtually identical overall organization with three exons separated by two introns and a high degree of nucleotide-sequence identity with alpha-neurotoxin genes, including similar intron insertions. This suggests that kappa-neurotoxins and alpha-neurotoxins might have originated from a common ancestor. The consensus transcriptional factor binding sites within the promoter regions of these genes indicate that their transcriptions are, at least in part, regulated under the same mechanism. Comparative analyses on kappa-bungarotoxin and alpha-neurotoxin genes revealed that the protein-coding regions of exons were much more diversified than introns except for the signal peptide domain. Phylogenetic analyses on the exon and intron regions of kappa-bungarotoxin and alpha-neurotoxin genes showed that the evolution of exon regions were not in consensus with that of intron regions. The ratio of nonsynonymous to synonymous substitution is higher than 1, reflecting the occurrence of an adaptive selection during the evolution of kappa-bungarotoxins. In contrast to a conserved size of the second intron, segmental insertions and/or deletions within the first intron accelerate the evolutionary divergence of kappa- and alpha-neurotoxin genes.

Characterization and gene organization of Taiwan banded krait (Bungarus multicinctus) gamma-bungarotoxin.

gamma-Bungarotoxin was isolated from Bungarus multicinctus (Taiwan banded krait) venom using a combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase high-performance liquid chromatography column. Circular dichroism (CD) measurement revealed that its secondary structure was dominant with beta-sheet structure as is that of snake venom alpha-neurotoxins and cardiotoxins. gamma-Bungarotoxin exhibits activity on inhibiting the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine receptor subtype, and competes weakly with radioiodinated alpha-bungarotoxin for binding to the Torpedo nicotinic acetylcholine receptor. Moreover, the toxin inhibits collagen-induced platelet aggregation, with an IC50 of approximately 200 nM. The genomic DNA encoding the gamma-bungarotoxin precursor is amplified by polymerase chain reaction (PCR). The gene is organized with three exons separated by two introns, and shares virtually identical overall organization with those reported for alpha-neurotoxin and cardiotoxin genes, including similar intron insertions. The intron sequences of these genes share sequence identity up to 85%, but the exon sequences are highly variable. These observations suggest that gamma-bungarotoxin, alpha-neurotoxins, and cardiotoxins originate from a common ancestor, and the evolution of these genes shows a tendency to diversify the functions of snake venom proteins.