On the Composition and Function of the Carbohydrate Moiety of Tissue-Type Plasminogen Activator from Human Melanoma Cells

D C Rijken; J J Emeis; G J Gerwig

doi:10.1055/s-0038-1660134

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1985; 54(04): 788-791
DOI: 10.1055/s-0038-1660134

Original Article

Schattauer GmbH Stuttgart

On the Composition and Function of the Carbohydrate Moiety of Tissue-Type Plasminogen Activator from Human Melanoma Cells

D C Rijken

^*The Gaubius Institute, Health Research Division TNO, Leiden, The Netherlands,

,

J J Emeis

^*The Gaubius Institute, Health Research Division TNO, Leiden, The Netherlands,

,

G J Gerwig

^**The Department of Bio-Organic Chemistry, State University of Utrecht, Utrecht, The Netherlands

› Author Affiliations

Abstract

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Summary

Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography. Variant I contained 12.8 g and variant II 7.1 g of carbohydrate per 100 g protein. Both variants contained N-acetylgalactosamine, suggesting O-glycosylation in addition to N-glycosylation. The possible role of N-linked oligosaccharides for the biological activity of t-PA was studied using t-PA secreted by melanoma cells in the presence of tunicamycin, an inhibitor of N-glycosylation. The latter t-PA showed the same plasminogen activating and fibrin binding properties as normally glycosylated t-PA, indicating that N-linked carbohydrate is not involved in the fibrinolytic activity of t-PA.

Keywords

Plasminogen activator - Tissue plasminogen activator - Tissue - type plasminogen activator - Fibrinolysis - Carbohydrates - Tunicamycin

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References

References
1 Bachmann F, Kruithof E KO. Tissue plasminogen activator: chemical and physiological aspects. Semin Thromb Haemostas 1984; 10: 6-17
2 Astrup T, Permin PM. Fibrinolysis in the animals organism. Nature 1947; 159: 681-682
3 Wallen P, Bergsdorf N, Ranby M. Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin. Biochim Biophys Acta 1982; 719: 318-328
4 Rijken DC, Wijngaards G, Zaal-de JongM, Welbergen J. Purification and partial characterization of plasminogen activator from human uterine tissue. Biochim Biophys Acta 1979; 580: 140-153
5 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
6 Kluft C, Van Wezel AL, Van der Velden CAM, Emeis JJ, Verheijen JH, Wijngaards G. Large-scale production of extrinsic (tissue-type) plasminogen activator from human melanoma cells. In: Advances in Biotechnological Processes Mizrahi A, Van Wezel AL. (Eds) Alan R. Liss Inc; New York: 1983. 2 97-110
7 Wallen P, Pohl G, Bergsdorf N, Ranby M, Ny T, Jornvall H. Purification and characterization of a melanoma cell plasminogen activator. Eur J Biochem 1983; 132: 681-686
8 Nielsen LS, Hansen JG, Andreasen PA, Skriver L, Danø K, Zenthen J. Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification. EMBO J 1983; 2: 115-119
9 Kruithof E KO, Schleuning WD, Bachmann F. Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification. Biochem J 1985; 226: 631-636
10 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 1983; 301: 214-221
11 Edlund T, Ny T, Ranby M, Heden LO, Palm G, Holmgren E, Josephson S. Isolation of cDNA sequences coding for a part of human tissue plasminogen activator. Proc Natl Acad Sci 1983; 80: 349-352
12 Matsuo O, Rijken DC, Collen D. Comparison of the relative fibrinogenolytic, fibrinolytic and thrombolytic properties of tissue plasminogen activator and urokinase in vitro. Thromb Haemostas 1981; 45: 225-229
13 Mattsen C, Nyberg-Arrhenius V, Wallen P. Dissolution of thrombi by tissue plasminogen activator, urokinase and streptokinase in an artificial circulating system. Thromb Res 1981; 21: 535-545
14 Matsuo O, Rijken DC, Collen D. Thrombolysis by human tissue plasminogen activator and urokinase in rabbits with experimental pulmonary embolus. Nature 1981; 291: 590-591
15 Collen D, Lijnen HR. New approaches to thrombolytic therapy. Arteriosclerosis 1984; 4: 579-585
16 Ranby M, Bergsdorf N, Pohl G, Wallen P. Isolation of two variants of native one-chain tissue plasminogen activator. FEBS Lett 1982; 146: 289-292
17 Bennett WF. Two forms of tissue-type plasminogen activator (t-PA) differ at single specific glycosylation site. Thromb Haemostas 1983; 50: 106 (Abstr)
18 Pohl G, Kallstrom M, Bergsdorf N, Wallen P, Jornvall H. Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences. Biochemistry 1984; 23: 3701-3707
19 Haverkate F, Timan G. Preparation of plasminogen-free fibrinogen for fibrin plates. In: Progress in Chemical Fibrinolysis and Thrombolysis Davidson JF, Samama MM, Desnoyers PC. (Eds) Raven Press; New York: 1976. 2 73-77
20 Van Ruyven-Vermeer I AM, Nieuwenhuizen W. Purification of rat fibrinogen and its constituent chains. Biochem J 1978; 169: 277-285
21 Kamerling JP, Vliegenthart J FG. Sialic acids. Chemistry, metabolism and function. Cell Biol Monogr 1982; 10: 95-125
22 Verheijen JH, Mullaart E, Chang G TG, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
23 Rijken DC, Van Hinsbergh V WM, Sens EMC. Quantitation of tissue-type plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay. Thromb Res 1984; 33: 145-153
24 Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925
25 Cummings RD, Kornfeld S. Fractionation of asparagine-linked oligosaccharides by serial lectin-agarose affinity chromatography. A rapid, sensitive and specific technique. J Biol Chem 1982; 257: 11235-11240
26 Banyai L, Varadi A, Patthy L. Common evolutionary origin of the fibrin-binding structures of fibrinoectin and tissue-type plasminogen activator. FEBS Lett 1983; 163: 37-41
27 Takatsuki S, Kohno K, Tamura G. Inhibition of biosynthesis of polyisoprenol sugars in chick-embryo microsomes by tunicamycin. Agr Biol Chem 1975; 39: 2089-2091
28 Tkacz J, Lampen JO. Tunicamycin inhibition of polyisoprenul N-acetyl-glucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun 1975; 65: 248-257
29 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol Chem 1982; 257: 2912-2919
30 Little SP, Bang NU, Harms CS, Marks CA, Mattler LE. Functional properties of carbohydrate-depleted tissue plasminogen activator. Biochemistry 1984; 23: 6191-6195
31 Rijken DC. Identification and characterization of tissue-type and urokinase-type plasminogen activators. Thromb Haemostas 1983; 50: 94 (Abstr)
32 Emeis JJ, Van den Hoogen CM, Jense D. Hepatic clearance of tissue-type plasminogen activator in rats. Thromb Haemostas 1985; 54: 661-664