OLIGOSACCHARIDE STRUCTURES PRESENT ON ASPARAGINE-289 OF RECOMBINANT HUMAN PLASMINOGEN EXPRESSED IN A CHINESE-HAMSTER OVARY CELL-LINE

The oligosaccharide structures linked to Asn289 of a recombinant (r) variant (R561S) human plasminogen (HPg) expressed in Chinese hamster ovary (CHO) cells, after transfection of these cells with a plasmid containing the cDNA coding for the variant HPg, have been determined. Employing high-performance anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the protein by glycopeptidase F, compared with elution positions of standard oligosaccharides, coupled with monosaccharide compositional determinations and analyses of sequential exoglycosidase digestions and specific lectin binding, we find that considerable microheterogeneity in oligosaccharide structure exists at this sole potential N-linked glycosylation site on HPg. A variety of high-mannose structures, as well as bi-, tri-, and tetraantennary complex-type carbohydrate, has been found, in relative amounts of 1-25% of the total oligosaccharides. The complex-type structures contain variable amounts of sialic acid (Sia), ranging from 0 to 5 mol/mol of oligosaccharide in the different glycan structures. Neither hybrid-type moleucles, N-acetylglucosamine bisecting oligosaccharides, nor N-acetyllactosaminyl-repeat structures were found to be present in the complex-type carbohydrate pool in observable amounts. Of interest, a significant portion of the Sia exists an outer arm structures in an (alpha-2,6) linkage to the penultimate galactose, a novel finding in CHO cell-directed glycosylation. We conclude that considerable differences occur in the N-linked carbohydrate structures of CHO cell-expressed HPg, in insect cell-expressed HPg [Davidson, D. J., Fraser, M. J., & Castellino, F. J. (1990) Biochemistry 29, 5584-5590], and in human plasma HPg [Hayes, M. L., & Castellino, F. J. (1979) J. Biol. Chem. 254, 8668-8671, 8672-8676, 8677-8680], with both species-specific glycosylation and also protein-directed events of importance in the oligosaccharide trimming and processing that result.