Summary
Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458’468’489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish
the consensus sequence of each N-linked glycosylation site (Asn458→Gln, Ser460→Gly; Asn468→Gln, Thr470→Gly; Asn489→Gln, Thr491→Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney
293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors,
and conditioned medium was collected for recombinant protein purification. SDS-PAGE
gels revealed that glycosylation mutants migrate identically and faster than the wild-type
rHPS, showing that each of the three potential N-glycosylation sites contain a similar
amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular
mass of ~78,000 for wild-type HPS. To demonstrate that the difference in mobility
between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived
HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown
to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses
for cleavage by α-thrombin. Functional studies indicate that wild-type rHPS possesses
the same cofactor specific activity as plasma-derived HPS, as tested by a standard
clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four
mutants have similar clotting activities, compared to wild-type rHPS. In a purified
component system, glycosylation mutants of protein S showed a slightly enhanced ability
to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction
of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied
by solution phase equilibrium binding assay. Two mutants (Asn458, Ser460) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have
the same apparent Kd as wild-type rHPS.
