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.

The Glycosylation of the Complement Regulatory Protein, Human Erythrocyte CD59

“…5a and b). The differences in these profiles, which compare the overall glycosylation of CD59 expressed in two different cell types, confirm previous findings that glycosylation is cell type-specific (41). There are more than 40 peaks in each of these profiles indicating that platelet and erythrocyte CD59 are both expressed as a population of many different glycoforms.…”

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cells

“…The N-linked carbohydrate chains of K,tu-PA were completely a(l-6)-fucosylated at the innermost GlcNAc residue. The i-ecombinant t-PA [45] and variant recombinant t-PA [53], expressed in CHO cells, are also almost fully fucosylated, whereas human colon fibroblast t-PA is fucosylated for 58% [8]. Other structural differences occur in the ratio of tri-and tri'-antennary structures, being about 1 : 3 for K,tu-PA, 1 : 8 for human colon fibroblast t-PA, 1 : 1.3 for recombinant t-PA [45], and 1 : 4.4 for variant recombinant t-PA [53].…”

“…In t-PA from human colon fibroblast and Bowes melanoma cells, the oligosaccharides bound to Asnll7 are predominantly of the oligomannose type [8]. The oligosaccharides at Asn184 and Asn448 comprise predominantly Nacetyllactosamine type of structures in the case of human colon fibroblast cells [8], and contain predominantly GalNAcPl4GlcNAc elements in the case of Bowes melanoma cells [9]. The presence and the nature of the N-linked carbohydrate chains in t-PA have been reported to play a role in various biological phenomena [lo-121.…”

Low Density Lipoprotein Receptor-related Protein Modulates the Expression of Tissue-type Plasminogen Activator in Human Colon Fibroblasts

“…(Greenwich, CT). Monoclonal antibody 79 -7 against HCF-t-PA was obtained as described previously (25). Purified HCF t-PA and HCF t-PA⅐PAI-1 complexes were kindly provided by Dr. A. Wittwer (Monsanto Co., St. Louis, MO).…”