Characterization Studies of Human Tissue-type Plasminogen Activator Produced by Recombinant DNA Technology

Vehar, Gordon A.; Spellman, Michael W.; Keyt, Bruce A.; Ferguson, Carol; Keck, Rodney G.; Chloupek, Rosanne C.; Harris, Reed J.; Bennett, William F.; Builder, Stuart E.; Hancock, William S.

doi:10.1101/sqb.1986.051.01.067

Cited by 36 publications

(18 citation statements)

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“…In comparison to published data on the monosaccharide content of tPA from recombinant CHO cells (Vehar et al, 1986), the values obtained here are about 50% lower for each of the three neutral monosaccharides (galactose, mannose, and especially fucose; Table 2). In contrast, the values for N-acetylglucosamine and total sialic acids are similar to or greater than those of Vehar et al (1986). Since all three neutral monosaccharides were obtained from the same tPA sample and TFA hydrolysis step, it appears that the recovery of neutral monosaccharides from the TFA hydrolysate was suboptimal.…”

Section: Discussionsupporting

confidence: 46%

Glycosylation of CHO-Derived Recombinant tPA Produced under Elevated pCO2

Kimura¹,

Miller

1999

Biotechnol. Prog.

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Section: Discussionsupporting

confidence: 46%

Glycosylation of CHO-Derived Recombinant tPA Produced under Elevated pCO2

Kimura¹,

Miller

1999

Biotechnol. Prog.

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“…The two labeled peaks are two of the three glycopeptides expected for rt-PA. The T11 glycopeptide which contains the high mannose oligosaccharides is shown to be completely deglycosylated (compare Figure 2, A and B) (Vehar et al, 1986). Because two other peptides coelute with the T17 glycopeptide, a similar conclusion cannot be drawn directly from the chromatograms ( Figure 2A,B).…”

Section: Optimization Of Enzymatic Deglycosylation Conditionsmentioning

confidence: 48%

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

Papac¹

1998

Glycobiology

151

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“…Carbohydrate structure analyses of human tPA from melanoma cells [21] or recombinant tPA from Chinese hamster ovary cells [22,23] revealed a similar site-specific glycosylation at residues Asnll7, Asnl84 and Asn448, i.e. oligomannosidic substituents at Asnl17 and N-acetyllactosaminic glycans at Asn44X and Asn184.…”

Section: Discussionmentioning

confidence: 95%

“…Analysis of tryptic melanoma tPA peptides demonstrated that Am184 is only partially glycosylatcd thus leading to glycoprotein variants with two or three oligosaccharide side chains [20]. Carbohydrate structure analyses of tPA from melanoma cells [9,21], normal human colon fibroblasts [9] and recombinant tPA from chincse hamster ovary cells [22,23] further demonstrated that thcse glycoproteins carry at Asnl17 almost exclusively oligomannosidic glycans, while AsnlS4 and Asn448 were either substituted by N-acetyllactosaminic species [21-231 or by mixtures o l oligomannosidic, hybrid-type and N-acetyllactosaminic oligosaccharides [9].Carbohydrate moieties of a glycoprotein can influence its solubility, stability, protease resistance, antigenicity and also its tertiary structurc [24]. In the case of tPA, carbohydrates are also an important paraincter for its fibrinolytic and fibrinogenolytic activity [25] as well as the half-life of the…”

mentioning

confidence: 99%

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

Pfeiffer¹,

Schmidt

Strube

et al. 1989

European Journal of Biochemistry

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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...

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Characterization Studies of Human Tissue-type Plasminogen Activator Produced by Recombinant DNA Technology

Cited by 36 publications

References 0 publications

Glycosylation of CHO-Derived Recombinant tPA Produced under Elevated pCO2

Glycosylation of CHO-Derived Recombinant tPA Produced under Elevated pCO2

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

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

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