title: Molecular cloning, expression and characterization of albolamin: a type P-IIa snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris).
abstract: Snake venom metalloproteinases (SVMPs) can damage vessel wall, degrade clotting factors, inhibit integrins and block platelet functions. Studying them not only gives us deeper insights in pathogenesis of snakebites, but also potentially yields novel therapeutic agents. Here, we discovered a clone of an RGD-containing SVMP from the green pit viper (Cryptelytrops albolabris) venom gland cDNA library. Sequence analysis revealed that it belonged to the P-IIa subclass of SVMP comprising signal peptide, prodomain, metalloproteinase and disintegrin. Compared with other P-II SVMPs, it contained 2 additional conserved cysteines that were predicted to prevent the release of disintegrin from the metalloproteinase domain in the mature protein. The N-terminal histidine-tagged construct of metalloproteinase and disintegrin domains of albolamin was inserted into the pPICZαA vector and expressed in Pichia pastoris. The recombinant protein molecular weight was approximately 35 kDa on Western blot probed with anti-polyhistidine antibody. The recombinant albolamin could digest human type IV collagen starting within 15 min after incubation. In addition, it dose-dependently inhibited collagen-induced platelet aggregation with the IC50 of 1.8 μM. However, there was no effect on ADP-induced platelet aggregation. Therefore, the inhibition mechanism is probably through blocking collagen receptor(s). Albolamin activities probably contributed to pathology of green pit viper bites. Its disintegrin domain deserves further studies for the potential to be a useful agent affecting platelet functions.
authors.affiliation: Department of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok 10330, Thailand.
authors.forename: Panchalee
authors.initials: P
authors.lastname: Jangprasert
authors.affiliation: Department of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok 10330, Thailand. Electronic address: rojnuckarinp@gmail.com.
authors.forename: Ponlapat
authors.initials: P
authors.lastname: Rojnuckarin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D024141:Collagen Type IV / Q000378:metabolism; D005786:Gene Expression Regulation; D015723:Gene Library; D020128:Inhibitory Concentration 50; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012910:Snake Venoms / Q000737:chemistry*; D017841:Trimeresurus / Q000378:metabolism*
chemical_list: D024141:Collagen Type IV; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012910:Snake Venoms; D045726:Metalloproteases
keywords: Albolamin; Collagen degradation; Cryptelytrops albolabris; Disintegrin; Platelet aggregation; Snake venom metalloproteinase