Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis.
The major coagulating fibrinogenase of Deinagkistrdon acutus venom, designated acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Extensive screening of the venom gland cDNA species after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X. The complete amino acid sequences of these enzymes were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosylation sites. The purified acutobin (40 kDa) contains approx. 30% carbohydrate by weight, which could be partly removed by N-glycanase. The phylogenetic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolution of the three major venom enzyme subtypes: coagulating enzymes, kininogenases and plasminogen activators. The possible structural elements responsible for the functional specificity of each subtype are discussed.
Wang
Y M
YM
Institute of Biological Chemistry, Academia Sinica, P.O. Box 23-106, Taipei, Taiwan.
Wang
S R
SR
Tsai
I H
IH
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000201:enzymology*; D018076:DNA, Complementary; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D003435:Crotalid Venoms; D018076:DNA, Complementary; D007527:Isoenzymes; D012697:Serine Endopeptidases