cDNA sequence analysis and mutagenesis studies on the A chain of beta-bungarotoxin from Taiwan banded krait.
The cDNA encoding the A chain of beta-bungarotoxin (beta-Bgt) was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait). The deduced amino acid sequence encoding the A chain revealed that the determined chain was different from the known A chains (A1, A2, A3, A4, and A5). Nevertheless, the amino acid sequence and the cDNA sequence of the novel A chain were highly homologous with those of other A chains. The gene encoding the A chain of beta-Bgt was subjected to mutagenesis, and the Tyr-11, Cys-15, and Leu-72 of the A chain were substituted by Cys-11, Ser-15, and Cys-72, respectively. Instead of the six disulfide bonds observed with the A chain, the resulting mutant contained seven disulfide linkages in its molecular structure which simulated those of presynaptic PLA2 neurotoxins and PLA2 enzymes. However, the mutant did not exhibit a higher phospholipase activity than that noted with the recombinant A chain. These results seem to suggest that, in the absence of the B chain, the six pairs of disulfide bonds in the recombinant A-chain molecule are enough to maintain its active conformation for exerting the phospholipase activity.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan, ROC.
Wu
P F
PF
Chang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002038:Bungarotoxins / Q000737:chemistry*; D017838:Bungarus; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017421:Sequence Analysis; D012910:Snake Venoms / Q000737:chemistry*
D002038:Bungarotoxins; D018076:DNA, Complementary; D004220:Disulfides; D011994:Recombinant Proteins; D012910:Snake Venoms; D010741:Phospholipases A; D054467:Phospholipases A2