Expression and characterization of a recombinant fibrinogenolytic serine protease from green pit viper (Trimeresurus albolabris) venom.
Viper venom serine proteases (SPs) display several effects on hemostatic system. Molecular cloning showed that Trimeresurus albolabris venom comprised a mixture of five SPs with thrombin-like (2), fibrinogenase (2) and plasminogen-activating (1) activities. Because only few fibrinogenolytic SP sequences were reported, we decided to express albofibrase, a novel fibrinogenase from T. albolabris using Pichia pastoris system. The recombinant active form of enzyme was 30 kDa including 2.2 kDa of glycosylation. Albofibrase showed an alpha fibrinogenase activity. In addition, a plasminogen activating and clotting effect were detectable. Albofibrase prolonged APTT and PT in a time-dependent manner. The effect was neutralized by pre-incubation with equine antivenom to T. albolabris. Therefore, the protein is potentially useful as a new anticoagulant as the antidote is clinically available. Sequence analysis compared with other snake venom fibrinogenases and SPs could not find any unique residues responsible for their various effects. Structure-function relationship should be further studied using mutagenesis in order to explore the mechanisms of venom protease functional diversity.
Muanpasitporn
Chuanchom
C
Department of Medicine, Faculty of Medicine, Chulalongkorn University, Rama IV Road, Patumwan, Bangkok 10330, Thailand.
Rojnuckarin
Ponlapat
P
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D003435:Crotalid Venoms / Q000201:enzymology*; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia; D011994:Recombinant Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017841:Trimeresurus
D000925:Anticoagulants; D003435:Crotalid Venoms; D017931:DNA Primers; D011994:Recombinant Proteins; C029748:Trimeresurus venoms; D005340:Fibrinogen; D012697:Serine Endopeptidases