Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin-K-dependent protein S.
Vitamin-K-dependent protein S is an anticoagulant plasma protein which functions as cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa. In addition, it interacts with C4b-binding protein (C4BP), a regulator of the complement system. Using a human protein S cDNA clone as probe, cDNA clones for rabbit protein S were isolated from a rabbit liver cDNA library. The cDNA sequence encoded the mature protein S and 12 residues of the leader sequence. The amino acid sequence of the single-chain 634-amino-acid-residue-long rabbit protein S molecule was 82% and 81% identical to those of human and bovine protein S, respectively. Northern blotting demonstrated protein S mRNA not only in liver but also in reproductive organs (testis, ovary and uterus), in lung and brain. Recombinant rabbit protein S was expressed in eucaryotic cells and found to be post-translationally modified, i.e. it had the correct amino terminus, contained N-linked carbohydrate side chains, gamma-carboxyglutamic acid residues and beta-hydroxylated aspartic acid and asparagine residues. Recombinant rabbit protein S bound calcium like its human counterpart, as judged by its migration in the presence of calcium on agarose-gel electrophoresis. Rabbit protein S has been reported to be species specific with respect to its interaction with APC and not to function with bovine APC. However, we found it to act as cofactor to both human and bovine APC, albeit it was somewhat more efficient with human than with bovine APC. Rabbit protein S, like its human and bovine counterparts, bound human C4BP in a reaction which was associated with the loss of its APC-cofactor activity. However, unlike human plasma, rabbit plasma appeared to contain only the free form of protein S as a radiolabeled rabbit protein S tracer added to rabbit plasma migrated as free protein S on agarose-gel electrophoresis. Addition of human C4BP to rabbit plasma resulted in the formation of a C4BP-protein-S complex, suggesting an explanation for the absence of complexed protein S in rabbit plasma to be sought for in the structure of rabbit C4BP.
He
X
X
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Dahlbäck
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D016167:Integrin alphaXbeta2; D008969:Molecular Sequence Data; D017293:Protein S / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014812:Vitamin K / Q000378:metabolism
D002352:Carrier Proteins; D018076:DNA, Complementary; D016167:Integrin alphaXbeta2; D017293:Protein S; D011994:Recombinant Proteins; D014812:Vitamin K