Molecular cloning and functional characterization of rat plasma protein S.
Plasma protein S is a cofactor of activated protein C (APC) in the regulation of the blood coagulation system. Rat protein S homogeneously purified from plasma showed cofactor activity for rat APC, but not for human APC when the APC cofactor activity was assayed using protein S- and C4b-binding protein (C4BP)-depleted human plasma. Rat plasma protein S was separated by gel chromatography into two forms, a free form and a form complexed with C4BP. Rat protein S forms complexes with rat and human C4BP in a solid-phase model with apparent dissociation constants (Kds) of 6.7 x 10(-8) and 1.2 x 10(-8) M, respectively, in the presence of 5 mM Ca2+. Human protein S also forms a complex with solid-phase human and rat C4BP with Kds of 6.3 x 10(-9) and 2.7 x 10(-8) M, respectively. Human C4BP strongly inhibited the APC cofactor activity of both human and rat protein S, whereas rat C4BP was only weakly inhibitory. The degree of the inhibitory activity of C4BP appears to depend on the affinity between protein S and C4BP. In order to evaluate the structure-function relationship of the rat protein S, the complete cDNA sequence of rat protein S was determined. This cDNA of 3,315 bp was composed of a 103-bp 5'-noncoding region, a 2,028-bp coding region that encodes a preprosequence of 41 amino acids, a mature protein S of 634 amino acids and a stop codon, and a 1,184-bp 3'-noncoding region. The rat mature protein S consisted of domains with distinct functions similar to those of human protein S, and with two potential Asn-linked glycosylation sites. The amino acid sequence of the mature form of rat protein S showed 80.4, 78.7, and 79.7% identity with those of human, bovine, and rabbit mature protein S, respectively. These findings suggest that despite the species-specificity of the APC cofactor activity of rat protein S, it is structurally very similar to human protein S. Expression of rat protein S mRNA (approximately 3.5 kb) was demonstrated by RNA blot analysis not only in the liver, but also in the lung, spleen, testis, and uterus of rats.
Yasuda
F
F
Department of Molecular Pathobiology, Mie University School of Medicine.
Hayashi
T
T
Tanitame
K
K
Nishioka
J
J
Suzuki
K
K
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010314:Partial Thromboplastin Time; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000302:isolation & purification / Q000378:metabolism; D017293:Protein S / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D011486:Protein C; D017293:Protein S; D011994:Recombinant Proteins