cDNA cloning of brevinase, a heterogeneous two-chain fibrinolytic enzyme from Agkistrodon blomhoffii brevicaudus snake venom, by serial hybridization-polymerase chain reaction.
Brevinase is a heterogeneous two-chain fibrinolytic enzyme, different from all of the known single-chain enzymes. A cDNA encoding brevinase was cloned from the venom gland of Agkistrodon blomhoffii brevicaudus by serial hybridization-PCR. Serial hybridization-PCR effectively amplified the complete cDNA of brevinase from the mixture of closely related transcripts. The cDNA sequence of 744 nucleotides was determined. The cDNA sequence included an open reading frame of 233 amino acids composed of an A chain (77 residues) and a B chain (156 residues). The deduced amino acid sequence included a potential N-glycosylation site (N54-X-S56), O-glycosylation site (Ser179), and RGD sequence. Brevinase included a unique Arg77 residue at the C-terminus of the A chain, distinguishing it from all of the compared homologous single-chain proteases. It could be assumed that a posttranslational cleavage site is located between Arg77 and Asn78. Based on the sequence similarity to those of the venom proteases, we could deduce that the critical catalytic residues are His40, Asn78, Asp85, and Ser179 and that the six potential disulfide bonds are Cys7-Cys138, Cys26-Cys41, Cys73-Cys231, Cys117-Cys185, Cys149-Cys164, and Cys175-Cys200. Despite the conservation of critical sequences, the phylogenetic tree showed that two-chain brevinase might be evolved separately from the homologous single-chain proteases.
Lee
J W
JW
Department of Microbiology, College of Natural Sciences, Kyungpook National University, Taegu, Korea.
Park
W
W
D017836:Agkistrodon / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003435:Crotalid Venoms / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D019143:Evolution, Molecular; D015723:Gene Library; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*
D003435:Crotalid Venoms; D018076:DNA, Complementary; D012697:Serine Endopeptidases; C120502:brevinase