Download PDF
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
Further Information

Publication History

Received 20 May 1985

Accepted 05 September 1985

Publication Date:
19 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

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
 

  • References

  • 1 Bachmann F, Kruithof E KO. Tissue plasminogen activator: chemical and physiological aspects. Semin Thromb Haemostas 1984; 10: 6-17
    Article in Thieme ConnectPubMedGoogle Scholar
  • 2 Astrup T, Permin PM. Fibrinolysis in the animals organism. Nature 1947; 159: 681-682
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 15 Collen D, Lijnen HR. New approaches to thrombolytic therapy. Arteriosclerosis 1984; 4: 579-585
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 17 Bennett WF. Two forms of tissue-type plasminogen activator (t-PA) differ at single specific glycosylation site. Thromb Haemostas 1983; 50: 106 (Abstr)
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 21 Kamerling JP, Vliegenthart J FG. Sialic acids. Chemistry, metabolism and function. Cell Biol Monogr 1982; 10: 95-125
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 30 Little SP, Bang NU, Harms CS, Marks CA, Mattler LE. Functional properties of carbohydrate-depleted tissue plasminogen activator. Biochemistry 1984; 23: 6191-6195
    CrossrefPubMedGoogle Scholar
  • 31 Rijken DC. Identification and characterization of tissue-type and urokinase-type plasminogen activators. Thromb Haemostas 1983; 50: 94 (Abstr)
    PubMedGoogle Scholar
  • 32 Emeis JJ, Van den Hoogen CM, Jense D. Hepatic clearance of tissue-type plasminogen activator in rats. Thromb Haemostas 1985; 54: 661-664
    PubMedGoogle Scholar