THE EFFECT OF N-LINKED GLYCOSYLATION ON MOLECULAR-WEIGHT, THROMBIN CLEAVAGE, AND FUNCTIONAL-ACTIVITY OF HUMAN PROTEIN-S

Human protein S (HPS) has three potential N-linked glycosylation sites at Asn(458,468,489). To study the role of glycosylation at these site s, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn(458) --> Gln, Ser(460) --> Gly; Asn(4 68) --> Gln, Thr(470) --> Gly; Asn(489) --> Gln, Thr(491) --> Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-deri ved expression vectors, and conditioned medium was collected for recom binant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showi ng that each of the three potential N-glycosylation sites contain a si milar amount of carbohydrate. Mass spectral analysis yielded similar r esults and a molecular mass of similar to 78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mut ant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subse quently shown to migrate identically on SDS-PAGE gels. All forms of HP S have similar time courses for cleavage by alpha-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor spec ific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn(458) and Ser(460) mutant rHPS have only a slightly higher c ofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component syste m, glycosylation mutants of protein S showed a slightly enhanced abili ty to stimulate APC-mediated factor Va inactivation after an initial l ag phase. The interaction of rHPS glycosylation mutants with human C4b -binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn(458), Ser(460), have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the sam e apparent Kd as wild-type rHPS.