Construction, expression and preliminary characterization of glycosylation mutants of human protein S.
Human protein S (HPS) is a vitamin K dependent plasma glycoprotein involved in the regulation of activated protein C and possibly fibrinolysis. Its c-DNA sequence shows three N-glycosylation consensus sequences (Asn-X-Ser/Thr). In order to study influence of N-linked glycosylation on HPS function, set of mutants of HPS was constructed. Mutants were generated, starting from an SV40/Adeno derived pD5HPS2 expression vector, using PCR enabled, site specific methodology. They included single amino acid substitutions at each of three N-glycosylation consensus sequences: Asn458-->Gln, Ser460-->Gly, Asn468-->Gln, Thr470-->Gly, Asn489-->Gln, Thr491-->Gly. Variant HPS were expressed in stable 293 human kidney cell lines in the presence of vitamin K1 (we did not succeed in expressing variant Asn489-->Gln) and purified from conditioned media using pseudoaffinity chromatography on QAE-Sepharose. Variant Asn468-->Gln showed decreased gamma-carboxyglutamate content. All of the mutants were active in a clotting type assay based on factor Va inactivation, and they were compared to wt-HPS and plasma HPS. In conclusion, we have constructed, expressed and purified set of HPS mutants useful in studying the role of N-glycosylation in HPS function.
Rydzewski
A
A
Department of Nephrology, Medical Academy, Białystok, Poland.
Myśliwiec
M
M
Long
G L
GL
D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000138:chemical synthesis; D018076:DNA, Complementary / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D017293:Protein S / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D017293:Protein S