Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity.
The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.
Filippovich
Igor
I
The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Herston, Australia.
Sorokina
Natasha
N
St Pierre
Liam
L
Flight
Simone
S
de Jersey
John
J
Perry
Naomi
N
Masci
Paul P
PP
Lavin
Martin F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation; D001779:Blood Coagulation Factors / Q000235:genetics* / Q000378:metabolism; D001780:Blood Coagulation Tests; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004546:Elapid Venoms / Q000235:genetics* / Q000378:metabolism; D017815:Elapidae; D004789:Enzyme Activation; D005170:Factor X / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D011516:Prothrombin; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000235:genetics*; D012756:Sheep
D001779:Blood Coagulation Factors; D004546:Elapid Venoms; D011993:Recombinant Fusion Proteins; D011516:Prothrombin; D005170:Factor X; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C505354:prothrombin activator protease, Pseudonaja textilis