The Effect of N-linked Glycosylation on Molecular Weight, Thrombin Cleavage, and Functional Activity of Human Protein S

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.

• References

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