Cell-type-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator

• Published in: Biochemistry (Easton) 1989-09, Vol.28 (19), p.7644-7662
• Main Authors: Parekh, Raj B, Dwek, Raymond A, Thomas, Jerry R, Opdenakker, Ghislain, Rademacher, Thomas W, Wittwer, Arthur J, Howard, Susan C, Nelson, Rickey, Siegel, Ned R, Jennings, M, Harakas, Nikos, Feder, Joseph
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Animals; Biological and medical sciences; Blood coagulation. Blood cells; Carbohydrate Conformation; Carbohydrate Sequence; Cells, Cultured; Chromatography, Gel; Fundamental and applied biological sciences. Psychology; Gene Expression; General aspects, investigation methods, hemostasis, fibrinolysis; Glycopeptides - isolation & purification; Glycosylation; Humans; Hydrolysis; man; Methylation; Mice; Molecular and cellular biology; Molecular Sequence Data; Oligosaccharides - genetics; Oligosaccharides - isolation & purification; Oligosaccharides - pharmacokinetics; Protein Processing, Post-Translational; Tissue Plasminogen Activator - analysis; Tissue Plasminogen Activator - isolation & purification; Tissue Plasminogen Activator - metabolism; Tumor Cells, Cultured
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00445a021

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.