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

Abstract

SummaryHuman 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.

Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein

“…The Heerlen polymorphism (S460P) also lacks glycosylation at Asn 458 . In the present work, the two glycosylation-deficient variants, N458A and S460A, were observed to bind to C4BP with an affinity similar to that of wild-type protein S, confirming the results on record (52,55). Hence, the carbohydrate at position 458 does not appear to influence the affinity for C4BP.…”

“…Protein S is glycosylated at three positions, N458, N468, and N489, all in the SHBG region (52). S460A is a naturally occurring mutation in phenotypic protein S deficiency (54).…”

Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

“…Human factor V was purified and converted to the active form of the cofactor (Va) as described by Katzmann et al (40). Human recombinant protein S (rHPS) was produced in human kidney 293 cells, purified, and chemically characterized as described elsewhere (1). Hirudin was obtained from Genentech (South San Francisco, CA).…”

“…EDTA was then added to 10 mM, and the digested protein S was loaded onto a Fast-Q-Flow column (0.2-ml bed volume) and washed with 10 column volumes of 20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM benzamidine, 10 mM EDTA (to disrupt the calcium-dependent interaction of protein S with phospholipid and to remove the phospholipid by elution. The column was then washed with 10 volumes of the above wash solution lacking EDTA, followed by elution of the purified protein S with 400 l of 20 mM Tris, pH 7.4, 150 mM NaCl, 20 mM CaCl, as described previously (1). Digestion of protein S was confirmed by SDS-PAGE of the eluted protein as described above, and protein concentration was determined by the micro-BCA protein assay (Pierce) following the supplier's instructions and using purified, untreated human rHPS as a standard.…”

“…The thrombin cleavage sites were first established for bovine protein S (at Arg 52 and Arg 70 ) by Dahlback et al (13) and recently at the corresponding residues (Arg 49 and Arg 70 ) for human protein S (1).…”