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Characterization Studies on Human Melanoma Cell Tissue Plasminogen Activator

Bio/Technology volume 2, pages 1051–1057 (1984)Cite this article

Abstract

Purified tissue plasminogen activator synthesized by a human melanoma cell line has been partially characterized. On SDS gel electrophoresis, the purified samples exhibited a doublet at molecular weights 63,000 and 65,000. Staphylococcal V8 proteolytic digestion of the separated species yielded similar size fragments, indicating gross identity of the polypeptide chains. It was subsequently found that these species had differential binding to lysine-Sepharose, with the lower apparent molecular weight species binding with a higher affinity. Amino acid sequence determination of reverse phase HPLC tryptic fragments has resulted in the characterization of approximately 80 percent of the protein. The identification of a cDNA clone has allowed alignment of these tryptic peptides. The molecule was found to be composed of an amino terminal domain, two kringle structures, and a serine protease. The molecule is composed of 527 amino acids, and has potential glycosylation sites at asparagine positions 117, 184, 218, and 448. The peptide containing Asn 448 was sequenced and determined to be glycosylated, whereas a peptide containing Asn-218 was found not to be glycosylated. Comparison of the kringle and serine protease domains with other serine proteases is discussed.

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References

  1. Gaffney, P.J. 1981. The fibrinolytic system, p. 198–224. In: Haemostasis and Thrombosis, Bloom, A. L., and Thomas, D. P., (eds.), Churchill Livingstone, Inc., New York.

    Google Scholar 

  2. Robbins, K.C., Summaria, L., Hsieh, B. and Shah, R.J. 1967. The peptide chains of human plasmin. Mechanism of activation of human plasminogen to plasmin. J. Biol. Chem. 242: 2333–2342.

    CAS  PubMed  Google Scholar 

  3. Duckert, F. 1978. Urokinase, p. 209–237. In: Fibrinolytics and Antifibrinolytics, Markwardt, F., (ed.), Springer-Verlag, New York.

    Chapter  Google Scholar 

  4. Wun, T.-C., Schleuing, W.-D. and Reich, E. 1982. Isolation and characterization of urokinase from human plasma. J. Biol. Chem. 257: 3276–3283.

    CAS  PubMed  Google Scholar 

  5. Ratnoff, O.D. 1977. The surface-mediated initiation of blood coagulation and related phenomena, p. 25–55. In: Haemostasis: Biochemistry, Physiology, and Pathology, Ogston, D., Bennett, B., (eds.), John Wiley and Sons, London.

    Google Scholar 

  6. Kok, P. and Astrup, T. 1969. Isolation and purification of a tissue plasminogen activator and its comparison with urokinase. Biochemistry 8: 79–86.

    Article  CAS  Google Scholar 

  7. Cole, E.R. and Bachmann, F.W. 1977. Purification and properties of plasminogen activator from pig heart. J. Biol. Chem. 252: 3729–3737.

    CAS  PubMed  Google Scholar 

  8. Rijken, D.C., Wijngaards, G., Zaal-deJong, M. and Welbergen, J. 1979. Purification and partial characterization of plasminogen activator from human uterine tissue. Biochim. Biophys. Acta 580: 140–153.

    Article  CAS  Google Scholar 

  9. Aoki, N. and von Kaulla, K.N. 1971. The extraction of tissue plasminogen activator from human cadavers and a description of some of its properties. Am. J. Clin. Path. 55: 171–179.

    Article  CAS  Google Scholar 

  10. Rijken, D.C., Wijngaards, G. and Welbergen, J. 1979. Biochemical and immunological characterization of plasminogen activator from human tissue. Prog. Chem. Fibrinolysis Thrombolysis 4: 349–354.

    Google Scholar 

  11. Rijken, D.C. and Collen, D. 1981. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J. Biol. Chem. 256: 7035–7041.

    CAS  Google Scholar 

  12. Korninger, C. and Collen, D. 1981. Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in human blood and in various animal species in vitro. Thrombos. Haemostas. (Stuttgart) 46: 561–565.

    Article  CAS  Google Scholar 

  13. Hoylaerts, M., Rijken, D.C., Lijnen, H.R. and Collen, D. 1982. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J. Biol. Chem. 257: 2912–2919.

    CAS  PubMed  Google Scholar 

  14. Rijken, D.C., Hoylaerts, M. and Collen, D. 1982. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J. Biol. Chem. 257: 2920–2925.

    CAS  Google Scholar 

  15. Korninger, C., Stassen, J.M. and Collen, D. 1981. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb. Haemostas. (Stuttgart) 46: 658–661.

    Article  CAS  Google Scholar 

  16. Weimar, W., Stibbe, J., Van Seyer, A.J., Billiau, A., De Somer, B. and Collen, D. 1981. Specific lysis of an iliofemoral thrombus by administration of extrinsic (tissue-type) plasminogen activator. Lancet 2: 1018–1020.

    Article  CAS  Google Scholar 

  17. Korninger, C., Matsuo, O., Suy, R., Stassen, J.M. and Collen, D. 1982. Thrombolysis with human extrinsic (tissue-type) plasminogen activator in dogs with femoral vein thrombosis. J. Clin. Invest. 69: 573–580.

    Article  CAS  Google Scholar 

  18. Blix, S. 1962. The effectiveness of activators in clot lysis, with special reference to fibrinolytic therapy. A new method for determination of preformed clot lysis. Acta. Med. Scand. (Suppl 386) 1–24.

    Article  Google Scholar 

  19. Wallen, P. 1977. Activation of plasminogen with urokinase and tissue activator, p. 91–102. In: Thrombosis and Urokinase, Paoletti, R. and Sherry, (eds.), Academic Press, London.

    Google Scholar 

  20. Camiolo, S.M., Thorsen, S., Astrup, T. 1971. Fibrinogen olysis and fibrinolysis with tissue plasminogen activator, urokinase, streptokinase-activated human globulin, and plasmin. Proc. Soc. Exper. Biol. Med. 138: 277–280.

    Article  CAS  Google Scholar 

  21. Urokinase-Pulmonary Embolism Trial Study Group. 1974. Urokinase-streptokinase embolism trial. Phase II. J. Am. Med. Assn. 229: 1606–1613.

  22. Gurewich, V., Hyde, E. and Lipinski, B. 1975. The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs. Blood 46: 555–565.

    CAS  PubMed  Google Scholar 

  23. Paoletti, R. and Sherry, S. (eds.) 1977. Thrombosis and Urokinase, Academic Press, New York, New York.

    Google Scholar 

  24. Pennica, D., Holmes, W.E., Kahr, W.J., Harkins, R.N., Vehar, G.A., Ward, C.A., Yelverton, E., Seeburg, P.H., Heyneker, H.L., Goeddel, D.V. and Collen, D. 1983. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 301: 214–221.

    Article  CAS  Google Scholar 

  25. Astrup, T. and Mullertz, S. 1952. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys. 40: 346–351.

    Article  CAS  Google Scholar 

  26. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  Google Scholar 

  27. Morrissey, J.H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117: 307–310.

    Article  CAS  Google Scholar 

  28. Crestfield, A.M., Moore, S. and Stein, W.J. 1963. The preparation and enzymatic hydrolysis of reduced and s-carboxymethylated proteins. J. Biol. Chem. 238: 622–627.

  29. Kohr, W.J., Keck, R. and Harkins, R.N. 1982. Characterization of intact and trypsin-digested biosynthetic human growth hormone by high-pressure liquid chromatography. Anal. Biochem. 122: 348–359.

    Article  CAS  Google Scholar 

  30. Cleveland, D.W., Fisher, S.G., Kirschner, M.W. and Laemmli, U.K. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252: 1102–1106.

    CAS  PubMed  Google Scholar 

  31. Weber, K. and Osborn, M. 1969. The reliability of molecular weight determinations by dodecylsulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406–4412.

    CAS  Google Scholar 

  32. Radcliffe, R. and Heinze, T. 1978. Isolation of plasminogen activator from human plasma by chromatography on lysine-sepharose. Arch. Biochem. Biophys. 189: 185–194.

    Article  CAS  Google Scholar 

  33. Pepper, D.S. and Allen, R. 1978. Isolation of characterization of human cadaver vascular endothelial activator. In Progress in Chem. Fibrinolysis Thrombolysis 3: 91–98.

    Google Scholar 

  34. Jackson, R.L. and Hirs, C.H.W. 1970. The primary structure of porcine pancreatic ribonuclease. I. The distribution and sites of carbohydrate attachment. J. Biol. Chem. 245: 624–636.

    CAS  PubMed  Google Scholar 

  35. Marshall, R.D. 1974. Nature and metabolism of the carbohydrate/ peptide linkages of glycoproteins. Biochem. Soc. Symp. 40: 17–26.

    CAS  Google Scholar 

  36. Ronin, C., Bouchilloux, S., Granier, C. and Van Rietschoten, J. 1978. Enzymatic N-glycosylation of synthetic Asn-X-thr containing peptides. FEBS Letters 96: 179–182.

    Article  CAS  Google Scholar 

  37. Scheffer, A.J. and Beintema, J.J. 1974. Horse pancreatic ribonuclease. Eur. J. Biochem. 46: 221–233.

    Article  CAS  Google Scholar 

  38. Jackson, R.C. and Blobel, G. 1980. Post-translational processing of full-length presecretory proteins with canine pancreatic signal peptidase. Anals. N. Y. Acad. Sci. 343: 391–404.

    Article  CAS  Google Scholar 

  39. Strauss, A.W., Bennett, C.D., Donohue, A.M., Rodkey, J.A. and Alberts, A.W. 1977. Rat liver pre-proalbumin: complete amino acid sequence of the pre-piece. Analysis of the direct translation product of albumin messenger RNA. J. Biol. Chem. 252: 6846–6855.

    CAS  PubMed  Google Scholar 

  40. MacGillivray, R.T.A., Chung, D.W. and Davie, E.W. 1979. Biosynthesis of bovine plasma proteins in a cell-free system. Amino terminal sequence of preproalbumin. Eur. J. Biochem. 98: 477–485.

    Article  CAS  Google Scholar 

  41. Lawn, R.M., Adelman, J., Bock, S.C., Franke, A.E., Houck, C.M., Najarian, R.C., Seeburg, P.H. and Wion, K.L. 1981. The sequence of human serum albumin cDNA and its expression in E. coli. Nucleic Acids Research 9: 6103–6114.

    Article  CAS  Google Scholar 

  42. Wallen, P., Pohl, G., Bergsdorf, N., Ranby, M., Ny, T. and Jornvall, H. 1983. Purification and characterization of a melanoma cell plasminogen activator. Eur. J. Biochem. 132: 681–686.

    Article  CAS  Google Scholar 

  43. Dayhoff, M.O. 1978. Atlas of Protein Sequence and Structure 5, Suppl. 3: 79–81.

  44. Steffens, G.J., Gunzler, W.A., Oetting, F., Frankus, E. and Flohe, L. 1982. The complete amino acid sequence of low molecular mass urokinase from human urine. Hoppe-Seyler's Z. Physiol. Chem. 363: 1043–1058.

    Article  CAS  Google Scholar 

  45. Furie, B., Bing, D.H., Feldmann, R.J., Robison, D.J., Burnier, J.P. and Furie, B.C. 1982. Computer-generated models of blood coagulation factor Xa, factor IXa, and thrombin based upon structural homology with other serine proteases. J. Biol. Chem. 257: 3875–3882.

    CAS  PubMed  Google Scholar 

  46. Gregory, H. and Preston, B.M. 1977. The primary structure of human uro-gastrone. Int. J. Peptide and Protein Res. 9: 107–118.

    Article  CAS  Google Scholar 

  47. Magnusson, S., Petersen, T.E., Sottrup-Jensen, L. and Claeys, H. 1975. Complete primary structure of prothrombin: Isolation, structure and reactivity of ten carboxylaled glutamic acid residues and regulation of prothrombin activation by thrombin, p. 123–149. In: Proteases and Biological Control, Reich, E., Rifkin, D.B., and Shaw, E., (eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

    Google Scholar 

  48. Magnusson, S., Sottrup-Jensen, L., Peterson, T.E., Dudek-Wojcie-chowska, G. and Claeys, H. 1976. Homologous “kringle” structures common to plasminogen and prothrombin. Substrate specificity of enzymes activating prothrombin and plasminogen, p. 203–238. In: Proteolysis and Physiological Regulation. Ribbons, D. W. and Brew, K., (eds.), Academic Press, New York.

    Chapter  Google Scholar 

  49. Claeys, H., Sottrup-Jensen, L., Zajdel, M., Petersen, T.E. and Magnusson, S. 1976. Multiple gene duplication in the activation of plasminogen. Five regions of structural homology with the two internally homologous structures in prothrombin. FEBS Lett. 61: 20–24.

    Article  CAS  Google Scholar 

  50. Sottrup-Jensen, L., Claeys, H., Zajdel, M., Peterson, T.E. and Magnusson, S. 1978. The primary structure of human plasminogen: Isolation of two lysine-binding fragments and one “mini”-plasminogen (MW, 38,000) by elastase-catalyzed specific limited proteolysis. Progress in chemical fibrinolysis and thrombolysis. Davidson, J.F., Samama, M. and Denoyers, P., (eds). 3: 191–209.

  51. Gunzler, W.A., Steffens, G.J., Oetting, F., Kim, S.-M.A., Frankus, E. and Flohe, L. 1982. The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain. Hoppe-Seyler's Z. Physiol. Chem. 363: 1155–1165.

  52. Dayhoff, M.O. 1978. Atlas of Protein Sequence and Structure 5, Suppl. 3: 89.

  53. Lerch, P.G., Rickli, E.E., Lergier, W. and Gillessen, D. 1980. Localization of individual lysine-binding regions in human plasminogen and investigations on their complex-forming properties. Eur. J. Biochem. 107: 7–13.

    Article  CAS  Google Scholar 

  54. Heifetz, A., Keenan, R.W. and Elbein, A.D. 1979. Mechanism of action of tunicamycin on the UDP-Glc-NAc: dolychyl-phosphate Glc-NAc-1-phosphate transferase. Biochemistry 18: 2186–2192.

    Article  CAS  Google Scholar 

  55. Ranby, M., Bergsdorf, N., Pohl, G. and Wallen, P. 1982. Isolation of two variants of naiive one-chain tissue plasminogen activator. FFBS Letters 146: 289–292.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Protein Biochemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Gordon A. Vehar, William J. Kohr, William F. Bennett, Carole A. Ward & Richard N. Harkins

  2. Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Diane Pennica

  3. Center for Thrombosis and Vascular Research, Department of Medical Research, University of Leuven, B-3000, Leuven, Belgium

    Desire Collen

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  1. Gordon A. Vehar
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Vehar, G., Kohr, W., Bennett, W. et al. Characterization Studies on Human Melanoma Cell Tissue Plasminogen Activator. Nat Biotechnol 2, 1051–1057 (1984). https://doi.org/10.1038/nbt1284-1051

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