Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation.
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 microM, 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 alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.
Takatsuka
H
H
Department of Pediatrics, Nara Medical University, Kashihara, Japan.
Sakurai
Y
Y
Yoshioka
A
A
Kokubo
T
T
Usami
Y
Y
Suzuki
M
M
Matsui
T
T
Titani
K
K
Yagi
H
H
Matsumoto
M
M
Fujimura
Y
Y
D017836:Agkistrodon; D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D018076:DNA, Complementary; D006801:Humans; D050830:L-Amino Acid Oxidase; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010975:Platelet Aggregation Inhibitors; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase