Purification and characterization of kaouthiagin, a von Willebrand factor-binding and -cleaving metalloproteinase from Naha kaouthia cobra venom.
A von Willebrand factor (vWF)-binding and -cleaving metalloproteinase, termed "kaouthiagin", was purified from the venom of cobra snake Naja kaouthia. Kaouthiagin is a monomer with a molecular mass of about 46 kDa and 51 kDa under non-reducing and reducing conditions, respectively, and the N-terminal amino acid sequence is homologous to high molecular mass snake venom metalloproteinases. Kaouthiagin bound to vWF in a divalent ion-independent manner, but the reduced kaouthiagin failed to interact with vWF, suggesting that the protein conformation maintained by intrachain-disulfide linkages of the molecule is essential for the binding to vWF. Neither botrocetin nor bitiscetin, vWF-binding modulators from another snake venom, interfered with the binding between kaouthiagin and vWF, but a monoclonal antibody VW92-3 specific to the N-terminal region of vWF (residues 1-910) inhibited the binding. Without affecting platelet GPIb/IX and GPIIb/IIIa, kaouthiagin specifically cleaved vWF between residues Pro-708 and Asp-709 in a divalent ion-dependent manner to diminish the multimeric structure of vWF in plasma, resulting in the loss of ristocetin-induced platelet aggregability and the collagen-binding activity of vWF. These results indicate that kaouthiagin is a unique metalloproteinase which specifically binds to and cleaves vWF at a specific site and that it will be a useful tool for functional dissection of vWF.
Department of Medical Information Technology, Fujita Health University College, Toyoake, Japan.
J
J
Hamako
T
T
Matsui
S
S
Nishida
S
S
Nomura
Y
Y
Fujimura
M
M
Ito
Y
Y
Ozeki
K
K
Titani
D000595:Amino Acid Sequence; D000818:Animals; D004546:Elapid Venoms / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D014841:von Willebrand Factor / Q000378:metabolism*
D004546:Elapid Venoms; D014841:von Willebrand Factor; D008666:Metalloendopeptidases