title: Cell-type-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator.
abstract: Tissue plasminogen activator (t-PA) is an important initiator of fibrinolysis. The t-PA polypeptide has four potential N-glycosylation sites of which three are occupied in type I (Asn-117, -184, and -448) and two in type II (Asn-117 and -448). In an effort to elucidate the factors controlling the expression of N-linked oligosaccharides on this polypeptide, we have used a combination of sequential exoglycosidase digestion, methylation analysis, and controlled acetolysis to determine the oligosaccharide structures at each of the N-glycosylation sites of type I and type II t-PA when isolated from a human colon fibroblast cell strain and from a Bowes melanoma cell line. Our results suggest the following: (i) type I and type II t-PA are N-glycosylated in an identical way at Asn-117 and Asn-448, when isolated from the same cell line; (ii) Asn-117 is predominantly associated with oligomannose-type structures in all cases; (iii) Asn-184 and Asn-448 are predominantly associated with complex-type structures when t-PA is isolated from fibroblast cells, but with both complex- and oligomannose-type structures when isolated from melanoma cells; (iv) fibroblast cell derived t-PA is associated with both neutral and sialylated oligosaccharides, while melanoma cell derived t-PA is also associated with sulfated oligosaccharides, which are located exclusively at Asn-448 of type II t-PA; (v) no complex-type structures occur in common between t-PA from the two cell lines. These results indicate that the t-PA glycoprotein is secreted by each cell line as a set of glycoforms, each glycoform being unique with respect to the nature and disposition of oligosaccharides on a common polypeptide. Further, the two cell lines express no glycoform in common, despite expressing the same t-PA polypeptide. The implications of these results for both the control of oligosaccharide processing in different cell lines and the genetic engineering of mammalian glycoproteins are discussed.
authors.affiliation: Department of Biochemistry, University of Oxford, U.K.
authors.forename: R B
authors.initials: RB
authors.lastname: Parekh
authors.forename: R A
authors.initials: RA
authors.lastname: Dwek
authors.forename: J R
authors.initials: JR
authors.lastname: Thomas
authors.forename: G
authors.initials: G
authors.lastname: Opdenakker
authors.forename: T W
authors.initials: TW
authors.lastname: Rademacher
authors.forename: A J
authors.initials: AJ
authors.lastname: Wittwer
authors.forename: S C
authors.initials: SC
authors.lastname: Howard
authors.forename: R
authors.initials: R
authors.lastname: Nelson
authors.forename: N R
authors.initials: NR
authors.lastname: Siegel
authors.forename: M G
authors.initials: MG
authors.lastname: Jennings
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002850:Chromatography, Gel; D015870:Gene Expression; D006020:Glycopeptides / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000235:genetics / Q000302:isolation & purification / Q000493:pharmacokinetics; D011499:Protein Processing, Post-Translational; D010959:Tissue Plasminogen Activator / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000596:Amino Acids; D006020:Glycopeptides; D009844:Oligosaccharides; D010959:Tissue Plasminogen Activator