Isolation and cloning of a metalloproteinase from king cobra snake venom.
A 50 kDa fibrinogenolytic protease, ohagin, from the venom of Ophiophagus hannah was isolated by a combination of gel filtration, ion-exchange and heparin affinity chromatography. Ohagin specifically degraded the alpha-chain of human fibrinogen and the proteolytic activity was completely abolished by EDTA, but not by PMSF, suggesting it is a metalloproteinase. It dose-dependently inhibited platelet aggregation induced by ADP, TMVA and stejnulxin. The full sequence of ohagin was deduced by cDNA cloning and confirmed by protein sequencing and peptide mass fingerprinting. The full-length cDNA sequence of ohagin encodes an open reading frame of 611 amino acids that includes signal peptide, proprotein and mature protein comprising metalloproteinase, disintegrin-like and cysteine-rich domains, suggesting it belongs to P-III class metalloproteinase. In addition, P-III class metalloproteinases from the venom glands of Naja atra, Bungarus multicinctus and Bungarus fasciatus were also cloned in this study. Sequence analysis and phylogenetic analysis indicated that metalloproteinases from elapid snake venoms form a new subgroup of P-III SVMPs.
Guo
Xiao-Xi
XX
Biotoxin Units, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan 650223, China.
Zeng
Lin
L
Lee
Wen-Hui
WH
Zhang
Yun
Y
Jin
Yang
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004546:Elapid Venoms / Q000737:chemistry / Q000201:enzymology*; D019143:Evolution, Molecular; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D004546:Elapid Venoms; D010975:Platelet Aggregation Inhibitors; D005340:Fibrinogen; D045726:Metalloproteases