Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells

Abstract

Recombinant human uterine tissue plasminogen activator (PA), 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-fl-N-acetylglucosaminidase H or peptide-N4-(N-acetylfl-glucosaminy1)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 'H-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 Asnll7 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 charactcristic feature, part of the triantennary glycans at Am184 and Asn448 contain additional Gal(a1-3) substituents and/or sulfate groups linked to position six of /3-galactosyl residues forming NeuAc(a2 -3)[HO3S -6]Gal(P1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (a2-3)-or (a2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asnl84 were found to be less sialylated and sulfated.Human tissue plasminogen activator is a serine protease that converts the zymogen plasminogen into plasmin which degrades the fibrin network of thrombi formed in the vascular system. Thus, tPA plays a key role in physiological fibrinolysis (for reviews, see [l-61). Since this enzyme forms a ternary complex with fibrin and plasminogen leading to an increased plasminogen activation in proximity to the clot, tPA can be considered a promising candidate for thrombolytic therapy of acute myocardial infarction, cerebrovascular thrombosis and venous thromboembolism [7, 81. Human tPA is synthesized and secreted by vascular endothelial cells as well as a number of cultured cell lines such as normal human colon fibroblasts [9] and human melanoma cells [lo] and has also been isolated from uterine tissue [I 11. In all cases, a glycosylated single polypeptide of M , 68 000 is formed, which can be further cleaved by plasmin yielding the two-chain form of tPA, the chains of which are covalently linked by a disulfide bridge. Due to differences in N-terminal processing of the molecule, however, melanoma tPA and uterine tPA differ in their terminal amino acid starting position 1121.In order to obtain large quantities of active human tPA for clinical trials, recombinant melanoma or uterine tPA have been exprcssed in bacteria [13] Fig. 1). Analysis of tryptic melanoma tPA peptides demonstrated that Am184 is only partially glycosylatcd thus le...

Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N‐glycosylation site in the epidermal‐growth‐factor‐like domain

“…The origin and structures of oligosaccharide standards used for column calibration were described in detail previously (Pfeiffer et al, 1989(Pfeiffer et al, , 1990Geyer et al, 1984b).…”

“…Metabolic labelling of GS-tPA and YN-tPA with D-16-'H]glucosamine (2.5 pCi/ml; Amersham-Buchler) was carried out as described previously (Pfeiffer et al, 1989).…”

“…Radiolabelled and unlabelled recombinant GS-and YNtPA were isolated and purified as detailed elsewhere (Pfeiffer et al, 1989).…”

“…Previous studies revealed that human uterine recombinant tPA from C127 cells contains high-mannose, small amounts of hybrid and sialylated complex-type N-glycans, the latter of which carried, in part, a-galactosyl and/or sulphate residues (Pfeiffer et al, 1989(Pfeiffer et al, , 1992a. Since it has been demonstrated that alterations in the domain structure of tPA may influence its glycosylation pattern (Wilhelm et al, 1990), we have now studied the carbohydrate structure of these two glycosylation variants.…”

“…A d 8 4 is only partially glycosylated leading to glycoprotein variants with two or three oligosaccharide chains. Carbohydrate structure analyses of tPA from Bowes melanoma cells (Pohl et al, 1987;Parekh et al, 1989a;Chan et al, 1991) or human colon fibroblasts (Parekh et al, 1989a) as well as recombinant tPA from Chinese hamster ovary cells (Spellman et al, 1989;Chan et al, 1991) or C127 cells (Pfeiffer et al, 1989) demonstrated a site-specific glycosylation of this enzyme in so far as Asnll7 carries almost exclusively high-mannosetype glycans, whereas Asnl84 and Asn448 bear preponderantly complex-type sugar chains. Furthermore, melanomaderived tPA as well as recombinant tPA expressed in Chinese hamster ovary or human embryonic kidney cells were found to carry an O-glycosidically linked fucose residue in their domain which resembles epidermal-growth-factor (EGF) (Harris et al, 1991).…”