title: N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line.
abstract: To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.
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: P M
authors.initials: PM
authors.lastname: Rudd
authors.forename: J R
authors.initials: JR
authors.lastname: Thomas
authors.forename: T W
authors.initials: TW
authors.lastname: Rademacher
authors.forename: T
authors.initials: T
authors.lastname: Warren
authors.forename: T C
authors.initials: TC
authors.lastname: Wun
authors.forename: B
authors.initials: B
authors.lastname: Hebert
authors.forename: B
authors.initials: B
authors.lastname: Reitz
authors.forename: M
authors.initials: M
authors.lastname: Palmier
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line / Q000378:metabolism*; D002850:Chromatography, Gel; D006224:Cricetinae; D005260:Female; D006031:Glycosylation; D007700:Kinetics; D008745:Methylation; D051379:Mice; D009844:Oligosaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator