Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells

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Abstract
Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]-glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-β-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5–8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(α1–3) substituents and/or sulfate groups linked to position six of β-galactosyl residues forming NeuAc(α2–3)[HO3S–6]Gal(β1–4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (α2–3)- or (α2–6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.