Advertisement

Structure of the Human Protease Nexin Gene and Expression of Recombinant Forms of PN-I

  • Michael McGrogan
  • Jackie Kennedy
  • Fred Golini
  • Nina Ashton
  • Frances Dunn
  • Kimberly Bell
  • Emily Tate
  • Randy W. Scott
  • Christian C. Simonsen
Part of the NATO ASI Series book series (NSSA, volume 191)

Abstract

Protease nexin (PN-I) is a member of the serpin family of serine protease inhibitors that are characterized by the formation of an irreversible complex with the catalytic site of their target serine proteases.1 PN-I is known to inhibit a number of biologically relevant serine proteases such as thrombin, urokinase, plasmin, and plasminogen activators.2–4 The significance of regulatory inhibitors in controlling the activity of the serine proteases has only begun to be appreciated in the areas of cell movement, blood coagulation, fibrinolysis, extracellular matrix modulation, and mitosis.5–8 Recently PN-I has been shown to be identical to glial derived nexin, which has been reported to possess neurite extension activity on peripheral nerve cells 3,9–11 Native PN-I is a glycoprotein of approximately 45,000 daltons that is secreted by various fibroblasts and extravascular cells.12 Multiple forms of PN-I have been described which differ in their behavior on SDS-PAGE, pH gradient gels, and heparin affinity chromatography.413 Although glycosylation differences can most certainly account for some of these differences, we have identified two species of human PN-I which we have designated αPN-I and βPN-I that differ by a net change of three amino acids.14

Keywords

Insect Cell Human Foreskin Fibroblast Serpin Gene Colon Fibroblast Human Foreskin Fibroblast Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    R.W. Carrell, and D.R. Boswell, Serpins: The super family of serine protease inhibitors. p. 403418. In: Protease Inhibitors. A. Barret and G. Salvesen (eds.). Elsevior, Amsterdam (1987).Google Scholar
  2. 2.
    J.B. Baker, DA. Low, R.L. Simmer, and D.D. Cunningham, Protease nexin: a cellular component that links thrombin plasminogen activator and mediates their binding to cells. Cell 21: 37–45 (1980).CrossRefPubMedGoogle Scholar
  3. 3.
    J.B. Baker, M. McGrogan, C.C. Simonsen, R.W. Scott, R.S. Gronke, and A. Honeyman, Protease nexin I. Structure and potential functions. In: The Pharmacology and Toxicology of Proteins. Holrenberg and Winklehalee, eds., U.C.LA. Symposium (1987).Google Scholar
  4. 4.
    R.W. Scott, B.L. Bergman, A. Bajpai, R.T. Hersh, H. Rodrigues, B.N. Jones, S. Watts, and J.B. Baker, Protease nexin. Properties and a modified purification procedure. J. Biol. Chem. 260: 70297034 (1985).Google Scholar
  5. 5.
    DA. Low, R.W. Scott, J.B. Baker, and D.D. Cunningham, Cells regulate their mitogenic response to thrombin through secretion of protease nexin. Nature 298: 476–478 (1982).CrossRefPubMedGoogle Scholar
  6. 6.
    J.B. Baker, DA. Low, D.L. Eaton, and D.D. Cunningham, Thrombin-mediated mitogenesis. The role of secreted protease nexin. J. Cell. PhysioL 112: 291–297 (1982).CrossRefGoogle Scholar
  7. 7.
    B.L. Bergman, R.W. Scott, A. Bajpai, S. Watts, and J.B. Baker, Protease nexin I inhibits destruction of extracellular matrix by human tumor cells. Proc. Natl. Acad. Sci. USA 83: 996–1000 (1986).CrossRefPubMedGoogle Scholar
  8. 8.
    R.S. Gronke, B.L. Bergman, and J.B. Baker, Thrombin interaction with platelets: influence of platelet protease nexin. J. Biol. Chem. 262: 3030–3036 (1987).PubMedGoogle Scholar
  9. 9.
    S.K. Gloor, K. Odink, J. Guenther, H. Nick, and D. Monard, A glial-derived neurite promoting factor with protease inhibitory activity belongs to the protease nexins. Cell 47: 687–593 (1986).CrossRefPubMedGoogle Scholar
  10. 10.
    D.J. Knauer, RA. Orlando, and D. Rosenblatt, The glioma cell-derived neurite promoting activity protein is functionally and immunologically related to human protease nexin-1. J. Cell Physiol. 132: 318–324 (1987).CrossRefGoogle Scholar
  11. 11.
    D. Gurwitz, and D.D. Cunningham, Thrombin modulates and reverses neuroblastoma neurite outgrowth. Proc. Natl. Acad. Sci. USA 85: 3440–3444 (1988).CrossRefPubMedGoogle Scholar
  12. 12.
    J.B. Baker, D.J. Knauer, and D.D. Cunningham, Protease nexins: secreted protease inhibitors that regulate protease actions at and near the cell surface, 153–172. In: The Receptors, Vol. 3. P.M. Conn, (ed.) Academic Press, N.Y. (1986).Google Scholar
  13. 13.
    R.W. Scott, and J.B. Baker, Purification of human protease nexin. J. Biol. Chem. 258: 10439–10444 (1983).PubMedGoogle Scholar
  14. 14.
    M.J. McGrogan, J. Kennedy, M.P. Li, C. Hsu, R.W. Scott, C.C. Simonsen, and J.B. Baker, Molecular cloning and expression of two forms of human protease nexin I. Bio/Tech. 6: 172–177 (1988).CrossRefGoogle Scholar
  15. 15.
    S.L. Berger, Isolation of cytoplasmic RNA: Ribonucleoside-vanadyl complexes. In: Methods of Enzymology. S.L. Berger, and A.R. Kimmel, (eds.) 152:227–234, Academic Press, N.Y. (1987).Google Scholar
  16. 16.
    J. Aviv, and P. Leder, Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69: 1408–1412 (1972).CrossRefPubMedGoogle Scholar
  17. 17.
    P. Thomas, Hybridization of denatured RNA and small DNA fragments to nitrocellulose. Proc. Natl. Acad. Sci. USA 77: 5201–5205 (1980).CrossRefPubMedGoogle Scholar
  18. 18.
    T. Maniatis, E.F. Fritsch, and J. Sambrook, Molecular cloning. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. (1982).Google Scholar
  19. 19.
    T.V. Hunyh, RA. Young, and R.W. Davis, In: DNA Cloning. A practical approach. D.M. Glover (ed)., Vol. 1 (1985).Google Scholar
  20. 20.
    R.W. Davis, D. Botstein, and J.R. Roth, Advanced bacterial genetics. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. (1980).Google Scholar
  21. 21.
    F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, JA. Smith, J.G. Seidman, and K. Struhl (ed), Current Protocols in Molecular Biology. Wiley Interscience, John Wiley and Sons. New York, N.Y. (1987).Google Scholar
  22. 22.
    J. Messing, and J. Viera, A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19: 269–276 (1982).CrossRefPubMedGoogle Scholar
  23. 23.
    J. Messing, New M13 vectors for cloning. Methods EnzymoL 101: 20–78 (1983).CrossRefPubMedGoogle Scholar
  24. 24.
    F. Sanger, S. Nicklen, and R. Coulson, DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467 (1977).CrossRefPubMedGoogle Scholar
  25. 25.
    G. Urlaub, and Chasin, Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA 77: 4216–4220 (1980).CrossRefGoogle Scholar
  26. 26.
    C.C. Simonsen, and A.D. Levinson, Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc. Natl. Acad. Sci. USA 80: 2495–2499 (1983a).CrossRefPubMedGoogle Scholar
  27. 27.
    C.C. Simonsen, and A.D. Levinson, Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen using SV40-HBV chimeric plasmids. Molec. and Cell. Biol. 3: 2250–2258 (1983b).Google Scholar
  28. 28.
    G.S. Gasser, C.C. Simonsen, J.W. Schilling, and R.T. Schimke, Expression of abbreviated mouse dihydrofolate reductase genes in cultured hamster cells. Proc. Natl. Acad. Sci. USA 79: 6522–6528 (1982).CrossRefPubMedGoogle Scholar
  29. 29.
    M.D. Summers, and G.E. Smith, A manual of methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agricultural Experiment Station Bulletin No. 1555 (1987).Google Scholar
  30. 30.
    VA. Luckow, and M.D. Summers, Trends in the development of baculovirus expression vectors. Bio/Tech. 6: 47–55 (1988).CrossRefGoogle Scholar
  31. 31.
    U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).CrossRefPubMedGoogle Scholar
  32. 32.
    E. Southern, Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Bio. 98: 503–517 (1975).CrossRefGoogle Scholar
  33. 33.
    D.J. Loskutoff, M. Linders, J. Keijer, H. Veerman, H. van Heerikhuizen, and H. Pannekoek, Structuree of the human plasminogen activator inhibitor 1 gene: nonrandom distribution of introns. Biochem. 26: 3763–3768 (1987).CrossRefGoogle Scholar
  34. 34.
    L. Strandberg, D. Lawrence, and T. Ny, The organization of the human plasminogen-activatorinhibitor-1 gene. Implications on the evolution of the serine-protease inhibitor family. Eur. J. Biochem. 176: 609–616 (1988).CrossRefPubMedGoogle Scholar
  35. 35.
    R. Breathnach, and P. Chamdon, Organization and expression of eukaryotic split genes coding for proteins. Ann. Rev. Biochem. 50: 349–383 (1981).CrossRefGoogle Scholar
  36. 36.
    H. Pannekoek, H. Veerman, H. Lambers, P. Diergaarde, C.L. Verwiej, A.-J. van Zonneveld, and JA. van Mourik, Endothelial plasminogen activator inhibitor (PM): a new member of the serpin gene family. EMBO J. 5: 2539–2544 (1986).Google Scholar
  37. 37.
    J. Sommer, S. Gloor, G.F. Rovelli, J. Hofsteenge, H. Nick, R. Meier, and D. Monard, cDNA sequence coding for a rat glial derived nexin and its homology to members of the serpin family. Biochem. 26: 6407–6410 (1987).CrossRefGoogle Scholar
  38. 38.
    R. Hubert, and R. Carrell, Implications of the three-dimensional structure of al-antitrypsin for structure and function of serpins. Biochem. 28: 8951–8966 (1989).CrossRefGoogle Scholar
  39. 39.
    S.E. Leff, M.G. Rosenfeld, and R.M. Evans, Complex transcriptional units: Diversity in gene expression by alternative DNA processing. Ann. Rev. Biochem. 55: 1091–1117 (1986).CrossRefPubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Michael McGrogan
    • 1
  • Jackie Kennedy
    • 1
  • Fred Golini
    • 1
  • Nina Ashton
    • 1
  • Frances Dunn
    • 1
  • Kimberly Bell
    • 1
  • Emily Tate
    • 1
  • Randy W. Scott
    • 1
  • Christian C. Simonsen
    • 1
  1. 1.INVITRON Corp.Redwood CityUSA

Personalised recommendations