NOVEL ASN-LINKED OLIGOSACCHARIDES TERMINATING IN A(1-]4)[FUC-ALPHA(1-]3)]GLCNAC-BETA(1-]CENTER-DOT) ARE PRESENT IN RECOMBINANT HUMAN PROTEIN-C EXPRESSED IN HUMAN KIDNEY 293-CELLS

Recombinant human Protein C (rHPC), expressed in human kidney 293 cell s, has a higher anticoagulant activity than plasma HPC, while its in v ivo circulatory half-life is essentially unaltered compared to that of the natural protein. In seeking to elucidate the molecular basis for the improved efficacy of the recombinant antithrombotic drug, we focus ed on the carbohydrate moiety of rHPC. Protein C is a heavily post-tra nslationally modified serine protease with four N-glycosylation sites. Glycosyl composition analysis of rHPC revealed a 5-fold higher fucose content and a 2-fold lower sialic acid content compared to plasma HPC . In addition, we found that rHPC contains N-acetylgalactosamine (2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides, while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharides of rHPC were released by N-glycanase and separated into 25 fractions by high-pa ani on-exchange chromatography. The most abundant oligosaccharides were st ructurally characterized by glycosyl composition and linkage analysis, in conjunction with H-1-NMR spectroscopy at 600 MHz. The structure of the major neutral oligosaccharide in rHPC was determined to be: [GRAP HICS] Two representatives of the sialylated oligosaccharides in rHPC a re: