PMID: 2513186

 

    Legend: Sugar

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

Abstract :
  1. 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)
  2. Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo- beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and fractionated by high-performance liquid chromatography
  3. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy
  4. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing
  5. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species
  6. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans
  7. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides
  8. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[ HO3S-6]Gal ( beta 1-4 ) units
  9. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present
  10. Glycans at Asn184 were found to be less sialylated and sulfated