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Novel neurotoxins from Bungarus multicinctus: Purification, Characterization, and Gene Organization.Ca-Ling, Chung 17 May 2001 (has links)
Three three-finger proteins (BM8, BM11, BM14) were isolated from Bungarus multicinctus (Taiwan banded krait) venom using successive chromatography on a ion-exchanger column and reverse phase HPLC column. BM8 and BM14 contain 82 amino acid residues including 10 cysteine residues, and BM11 contain 68 amino acid residues including 10 cysteine residues. Unlike snake venom cardiotoxins and a-neurotoxins, the three proteins contain two additional cysteine residues in their N-terminal region. Noticeably, only two amino acid substitutions occurred at positions 37 and 38 were observed between BM8 and BM14. CD measurement revealed that their secondary structures were dominant with b-sheet structure as noted with cardiotoxins and a-neurotoxins. However, the gross conformations of BM8, BM11 and BM14 were not the same as evidenced by CD spectra and acrylamide quenching studies. In contract to BM8, BM11 and BM14 exhibit an activity on blocking [3H]QNB binding to muscarinic acetylcholine receptor (mAChR). Modification of Lys residues in BM14 resulted in a decreased binding activity, indicating that the Lys residues may be involved in the mAChR binding. The genomic DNA with the size of approximate 2.3 kb and 2.4 kb, respectively, encoded the precursors of BM14 and BM11 was amplified by PCR. The genes shared virtually identical overall organization with snake venom cardiotoxin and£\-neurotoxin genes, composed of three exons and two introns. Comparison of BM11 and BM14 genes with cardiotoxins and a-neurotoxins genes showed that the exon regions were more diversified than intron regions. This implicated that the accelerated evolution may be involved in the evolution of these genes in order to acquire newly arising functions. Nevertheless, several transcriptional factor binding sites including SP-1, CACCC-binding factor and NF-KB were highly conserved in the promoter regions. It reflected a common regulation mechanism in controlling the transcription of these genes. These observations indicate that the three proteins are evolutionarily related to cardiotoxins and a-neurotoxins.
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Site-direct mutagenesis on alpha-bungarotoxin: structural dependence on the integrity of disulfide bondsCheng, Ching-Wen 16 May 2001 (has links)
£\-Bungarotoxin (£\-Bgt), a £\-neurotoxin from Taiwan banded krait (Bungarus multicinctus), consists of a single polypeptide chain of 74 amino acid residues cross-linking with five disulfide bonds. In order to explore the structural dependence of the disulfide bonds in the toxin molecule, the mutants with deleting one out of the five disulfide bonds were prepared by site-directed mutagenesis. The mutated and wild-type cDNAs were subcloned into the expression vectors pET14b as well as pET32a(+), and then transformed into Escherichia coli, BL-21(DE3). The recombinant proteins derived from pET14b expression system were isolated from inclusion bodies of E. coli, and refolding into their folded structure in vitro. Alternatively, the proteins derived from pET32a(+) system were expressed as a fusion protein and purified on a His-Bind resin column. Moreover, the recombinant proteins were further purified using a reverse phase column, and the homogeneity of recombinant proteins was determined by SDS-PAGE analyses. Noticeably, the refolding reaction was beneficially achieved by deleting the disulfide bond Cys29-Cys33. Although SDS-PAGE analyses showed that the recombinant proteins were homogeneous in molecular weight, several disulfide isomers appeared in each protein preparation as revealed by native gel analyses. These observations suggest that the formation of disulfide bonds and folding of£\-Bgt may pass through multiple pathways.
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Taiwan Banded Krait beta-Bungarotoxins: Novel Isotoxins, Targeting and Gene OrganizationChu, Yuan-Ping 11 June 2002 (has links)
beta-Bungarotoxin (beta-Bgt), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, two novel beta-Bgt isotoxins were purified from Bungarus multicinctus venom by the combinations of ion-exchange chromatography and reverse phase HPLC. Amino acid sequencing, peptide mapping and mass analyses revealed that they probably contained the same A chain, but their B chain differed. Consequently, the discrepancies in their biological activity and fine structure reflected the role of B chain in intact of beta-Bgt. In Yeast-Two-Hybrid system, a potassium channel binding protein was identified to interact with the B chain of beta-Bgt. Although the recombinant potassium channel binding protein functionally bound with Ca2+, but it could not prove to bind with BM12 and BM13 as revealed by in vitro cross-linking assay.
The A chain genes including A1 chain, A2 chain and A8 chain genes were amplified by PCR reaction. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with A chain cDNAs revealed that the A1 chain gene was organized with four exon and three intron, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponds to the first exon of A1 chain gene is skipped instead of inclusion of intronic sequence adjacent to the second intron. The resulting A2 chain mRNA encoded a 25 residues signal peptide, which different from A1 chain mRNA with a 27 residues signal peptide. Comparative analyses on phospholipase A2 genes and cDNAs suggest that this is the first report on skipping of exon changes the signal peptide sequence of snake venom proteins.
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