title: Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation.
abstract: Molecular cloning and functional characterization of P-III snake venom metalloproteinases (SVMPs) will give us deeper insights in the pathogenesis of viper bites. This may lead to novel therapy for venom-induced local tissue damages, the complication refractory to current antivenom. The aim of this study was to elucidate the in vitro activities of a new SVMP from the green pit viper (GPV) using recombinant DNA technology. We report, here, a new cDNA clone from GPV (Cryptelytrops albolabris) venom glands encoding 614 amino acid residues P-III SVMP, termed albocollagenase. The conceptually translated protein comprised a signal peptide and prodomain, followed by a metalloproteinase domain containing a zinc-binding motifs, HEXGHXXGXXH-CIM and 9 cysteine residues. The disintegrin-like and cysteine-rich domains possessed 24 cysteines and a DCD (Asp-Cys-Asp) motif. The albocollagenase deduced amino acid sequence alignments showed approximately 70% identity with other P-III SVMPs. Notably, the prodomain was highly conserved, while the metalloproteinase, disintegrin-like and cysteine-rich domains contained several differences. Albocollagenase without the signal peptide and prodomain was expressed in Pichia pastoris with an N-terminal six-histidine tag. After affinity purification from the supernatant of methanol-induced media, SDS-PAGE and Western blot analysis in both reducing and non-reducing conditions showed a protein band of approximately 62 kDa. The recombinant albocollagenase could digest human type IV collagen from human placenta basement membrane within 1 min. After 10-min incubation, it also inhibited collagen-induced platelet aggregation with 50% inhibitory concentration (IC₅₀) of 70 nM. This is the first report of the active recombinant SVMP enzymes expressed in P. pastoris. The results suggest the significant roles of P-III SVMP in local and systemic pathology of envenomated patients. Inhibitors of this SVMP will be investigated in further studies to find a better treatment for viper bites.
authors.affiliation: Division of Hematology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Anuwat
authors.initials: A
authors.lastname: Pinyachat
authors.forename: Ponlapat
authors.initials: P
authors.lastname: Rojnuckarin
authors.forename: Chuanchom
authors.initials: C
authors.lastname: Muanpasitporn
authors.forename: Pon
authors.initials: P
authors.lastname: Singhamatr
authors.forename: Surang
authors.initials: S
authors.lastname: Nuchprayoon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D045726:Metalloproteases / Q000235:genetics / Q000633:toxicity*; D008969:Molecular Sequence Data; D010843:Pichia; D010974:Platelet Aggregation / Q000187:drug effects*; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000633:toxicity*; D017422:Sequence Analysis, DNA; D014757:Viper Venoms / Q000737:chemistry*; D017819:Viperidae
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014757:Viper Venoms; D003094:Collagen; D045726:Metalloproteases