Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain.
Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell-matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5'-RACE, the 2040bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3mg/L of culture medium. The molecular weights were 11kDa in reduced and 22kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC(50) of 976nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.
Singhamatr
Pon
P
Faculty of Medicine, Department of Medicine, Chulalongkorn University, Rama IV Road, Patumwan, Bangkok 10330, Thailand.
Rojnuckarin
Ponlapat
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019483:Disintegrins / Q000737:chemistry*; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017841:Trimeresurus
D003435:Crotalid Venoms; D019483:Disintegrins; D011994:Recombinant Proteins; D045726:Metalloproteases; C527483:albolatin, Trimeresurus albolabris