TY - JOUR
T1 - Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation
AU - Takatsuka, Hideo
AU - Sakurai, Yoshihiko
AU - Yoshioka, Akira
AU - Kokubo, Tetsuro
AU - Usami, Yoshiko
AU - Suzuki, Masami
AU - Matsui, Taei
AU - Titani, Koiti
AU - Yagi, Hideo
AU - Matsumoto, Masanori
AU - Fujimura, Yoshihiro
N1 - Funding Information:
This work was supported in part by Research Grants from the Japanese Ministry of Health and Welfare for Cardiovascular Diseases (Y.F.) and from the Japanese Ministry of Education Culture and Science (Y.F.), and by a Grant-in-Aid (K.T.) from the Fujita Health University.
PY - 2001/1/12
Y1 - 2001/1/12
N2 - L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 μM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of l-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin αIIb/β3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.
AB - L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 μM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of l-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin αIIb/β3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.
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U2 - 10.1016/S0167-4838(00)00229-6
DO - 10.1016/S0167-4838(00)00229-6
M3 - Article
C2 - 11341935
AN - SCOPUS:0035847123
SN - 1570-9639
VL - 1544
SP - 267
EP - 277
JO - BBA - Protein Structure
JF - BBA - Protein Structure
IS - 1-2
ER -