Expression and functional characterization of chimeras between human and bovine vitamin-K-dependent protein-S-defining modules important for the species specificity of the activated protein C cofactor activity.
Vitamin-K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C (APC) in the degradation of factors Va and VIIIa. The APC-cofactor function of protein S is species specific, as human protein S potentiates the anticoagulant activity of human but not that of bovine APC, whereas bovine protein S is a cofactor to APC from both species. To elucidate which modules in protein S determine the species specificity, in vitro mutagenesis was used to construct six recombinant chimeric molecules between human and bovine protein S. Wild-type human and bovine protein S and the chimeras were expressed in 293 cells and the recombinant proteins purified by monoclonal antibody affinity chromatography. The recombinant proteins were found to be post-translationally modified, they bound C4b-binding protein and were functionally active as cofactors to APC. Chimeras having both the thrombin-sensitive region (TSR) and the first epidermal-growth-factor-(EGF)-like module of bovine origin expressed APC-cofactor activity similar to that of bovine protein S. Those chimeras, in which TSR or EGF1 derived from different species, manifested APC-cofactor activity similar to that of human protein S, i.e. they did not express cofactor activity to bovine APC. These data indicate that sequence differences in the TSR and EGF1 of human and bovine protein S cause the species specificity of the APC-cofactor activity. The data support the concept that these two modules of protein S interact with APC on the surface of negatively charged phospholipids.
Department of Clinical Chemistry, Lund University, Malmö General Hospital, Sweden.
X
X
He
L
L
Shen
B
B
Dahlbäck
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011486:Protein C / Q000378:metabolism*; D017293:Protein S / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D009838:Oligodeoxyribonucleotides; D011486:Protein C; D017293:Protein S; D011993:Recombinant Fusion Proteins