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 [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). 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. 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(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. 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. Glycans at Asn184 were found to be less sialylated and sulfated.
Pfeiffer
G
G
Biochemisches Institut am Klinikum, Universität Giessen, FRG.
Schmidt
M
M
Strube
K H
KH
Geyer
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000302:isolation & purification; D002236:Carbohydrate Conformation; D005260:Female; D006020:Glycopeptides / Q000302:isolation & purification; D006801:Humans; D008745:Methylation; D051379:Mice; D009844:Oligosaccharides / Q000302:isolation & purification; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D014599:Uterus
D001212:Asialoglycoproteins; D006020:Glycopeptides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator