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

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6‐ 3 H]‐glucosamine or [ 35 S]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‐ N 4 ‐( 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 1 H‐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)[HO 3 S–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.