Novel Asn-linked oligosaccharides terminating in GaINAc (1->4)[Fuca(1->3)]GlcNAcB(1-> ) are present in recombinant human Protein C expressed in human kidney 293 cells
Recombinant human Protein C (rHPC), expressed in human kidney 293 cells, has a higher anticoagulant activity than plasma HPC, while its in vivo 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 focused on the carbohydrate moiety of rHPC. Protein C is a heavily post-translationally 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-acetylgaiac-tosamine (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-pH anion-exchange chromatography. The most abundant oligosaccharides were structurally characterized by glycosyl composition and linkage analysis, in conjunction with 1H-NMR spectroscopy at 600 MHz. The structure of the major neutral oligosaccharide in rHPC was determined to be: Two representatives of the sialylated oligosaccharides in rHPC are: and Thus, many of the Asn-linked oligosaccharides in rHPC were found to terminate in GaINAcβ(1→4)GlcNAcβ(1→•), in NeuAca(2→6)GalNAcβ(1→4)GlcNAcβ(1→•), and/or in GaINAcβ(1→4) [Fuca(1→3)]GlcNAcB(1→•). Since the latter trisaccharide was first [Yan, S.B., Chao, B.Y. and Van Halbeek, H. (1992) J. Cell. Biochem., 16D, 151] observed in the Asn-linked oligosaccharides of rHPC derived from human kidney 293 cells, we propose to label the GaINAcβ(1→4)[Fuca(1→3)]GlcNAcβ(1→•) terminal trisaccharide the PC-293 determinant. The PC-293-containing oligosac-charides may contribute to the higher anticoagulant activity of rHPC as compared to plasma HPC.