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. 1987 May;84(9):2776–2780. doi: 10.1073/pnas.84.9.2776

Limited cleavage of cellular fibronectin by plasminogen activator purified from transformed cells.

J P Quigley, L I Gold, R Schwimmer, L M Sullivan
PMCID: PMC304741  PMID: 3033662

Abstract

The substrate specificity and direct catalytic activity of plasminogen activator (PA) was examined under conditions where its natural substrate, plasminogen, was missing or inhibited. PA, purified from cultures of transformed chicken fibroblasts, was incubated with purified preparations of potential substrates. The adhesive glycoprotein fibronectin, isolated from normal chicken fibroblast extracellular matrix, underwent limited but specific cleavage by PA in the absence of plasminogen. Analysis of the cleavage products by polyacrylamide gels under both reducing and nonreducing conditions indicated that PA-mediated cleavage occurred near the carboxyl terminus of fibronectin but on the amino-terminal side of the interchain disulfide bridge, thus disrupting the native dimeric fibronectin molecule. Under the identical conditions, chicken ovalbumin was not cleaved while the established substrate, chicken plasminogen, was extensively converted to plasmin. A monoclonal antibody, directed against avian PA and shown to inhibit plasminogen-free, cell-mediated matrix degradation, specifically inhibited the fibronectin cleavage. A human PA, urokinase, also cleaved fibronectin under plasminogen-free conditions yielding a limited number of high molecular weight cleavage products.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akiyama S. K., Yamada S. S., Yamada K. M. Characterization of a 140-kD avian cell surface antigen as a fibronectin-binding molecule. J Cell Biol. 1986 Feb;102(2):442–448. doi: 10.1083/jcb.102.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown P. J., Juliano R. L. Selective inhibition of fibronectin-mediated cell adhesion by monoclonal antibodies to a cell-surface glycoprotein. Science. 1985 Jun 21;228(4706):1448–1451. doi: 10.1126/science.4012302. [DOI] [PubMed] [Google Scholar]
  3. Culp L. A., Murray B. A., Rollins B. J. Fibronectin and proteoglycans as determinants of cell-substratum adhesion. J Supramol Struct. 1979;11(3):401–427. doi: 10.1002/jss.400110314. [DOI] [PubMed] [Google Scholar]
  4. Damsky C. H., Knudsen K. A., Bradley D., Buck C. A., Horwitz A. F. Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture. J Cell Biol. 1985 May;100(5):1528–1539. doi: 10.1083/jcb.100.5.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Danø K., Andreasen P. A., Grøndahl-Hansen J., Kristensen P., Nielsen L. S., Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res. 1985;44:139–266. doi: 10.1016/s0065-230x(08)60028-7. [DOI] [PubMed] [Google Scholar]
  6. Fairbairn S., Gilbert R., Ojakian G., Schwimmer R., Quigley J. P. The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants. J Cell Biol. 1985 Nov;101(5 Pt 1):1790–1798. doi: 10.1083/jcb.101.5.1790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Furie M. B., Rifkin D. B. Proteolytically derived fragments of human plasma fibronectin and their localization within the intact molecule. J Biol Chem. 1980 Apr 10;255(7):3134–3140. [PubMed] [Google Scholar]
  8. Goldberg B. Binding of soluble type I collagen molecules to the fibroblast plasma membrane. Cell. 1979 Feb;16(2):265–275. doi: 10.1016/0092-8674(79)90004-7. [DOI] [PubMed] [Google Scholar]
  9. Gospodarowicz D., Greenburg G., Birdwell C. R. Determination of cellular shape by the extracellular matrix and its correlation with the control of cellular growth. Cancer Res. 1978 Nov;38(11 Pt 2):4155–4171. [PubMed] [Google Scholar]
  10. Hayashi M., Yamada K. M. Domain structure of the carboxyl-terminal half of human plasma fibronectin. J Biol Chem. 1983 Mar 10;258(5):3332–3340. [PubMed] [Google Scholar]
  11. Humphries M. J., Akiyama S. K., Komoriya A., Olden K., Yamada K. M. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol. 1986 Dec;103(6 Pt 2):2637–2647. doi: 10.1083/jcb.103.6.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hynes R. Molecular biology of fibronectin. Annu Rev Cell Biol. 1985;1:67–90. doi: 10.1146/annurev.cb.01.110185.000435. [DOI] [PubMed] [Google Scholar]
  13. Jones P. A., DeClerck Y. A. Destruction of extracellular matrices containing glycoproteins, elastin, and collagen by metastatic human tumor cells. Cancer Res. 1980 Sep;40(9):3222–3227. [PubMed] [Google Scholar]
  14. Kefalides N. A., Alper R., Clark C. C. Biochemistry and metabolism of basement membranes. Int Rev Cytol. 1979;61:167–228. doi: 10.1016/s0074-7696(08)61998-1. [DOI] [PubMed] [Google Scholar]
  15. Keski-Oja J., Vaheri A. The cellular target for the plasminogen activator, urokinase, in human fibroblasts - 66 000 dalton protein. Biochim Biophys Acta. 1982 Apr 29;720(2):141–146. doi: 10.1016/0167-4889(82)90005-2. [DOI] [PubMed] [Google Scholar]
  16. Kjellén L., Oldberg A., Hök M. Cell-surface heparan sulfate. Mechanisms of proteoglycan-cell association. J Biol Chem. 1980 Nov 10;255(21):10407–10413. [PubMed] [Google Scholar]
  17. Kleinman H. K., Klebe R. J., Martin G. R. Role of collagenous matrices in the adhesion and growth of cells. J Cell Biol. 1981 Mar;88(3):473–485. doi: 10.1083/jcb.88.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kleinman H. K., Martin G. R., Fishman P. H. Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3367–3371. doi: 10.1073/pnas.76.7.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kraemer P. M. Heparin releases heparan sulfate from the cell surface. Biochem Biophys Res Commun. 1977 Oct 24;78(4):1334–1340. doi: 10.1016/0006-291x(77)91438-3. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Linsenmayer T. F., Gibney E., Toole B. P., Gross J. Cellular adhesion to collagen. Exp Cell Res. 1978 Oct 15;116(2):470–474. doi: 10.1016/0014-4827(78)90473-1. [DOI] [PubMed] [Google Scholar]
  22. Liotta L. A., Abe S., Robey P. G., Martin G. R. Preferential digestion of basement membrane collagen by an enzyme derived from a metastatic murine tumor. Proc Natl Acad Sci U S A. 1979 May;76(5):2268–2272. doi: 10.1073/pnas.76.5.2268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liotta L. A., Goldfarb R. H., Brundage R., Siegal G. P., Terranova V., Garbisa S. Effect of plasminogen activator (urokinase), plasmin, and thrombin on glycoprotein and collagenous components of basement membrane. Cancer Res. 1981 Nov;41(11 Pt 1):4629–4636. [PubMed] [Google Scholar]
  24. Liotta L. A., Goldfarb R. H., Terranova V. P. Cleavage of laminin by thrombin and plasmin: alpha thrombin selectively cleaves the beta chain of laminin. Thromb Res. 1981 Mar 15;21(6):663–673. doi: 10.1016/0049-3848(81)90268-1. [DOI] [PubMed] [Google Scholar]
  25. Malinoff H. L., Wicha M. S. Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells. J Cell Biol. 1983 May;96(5):1475–1479. doi: 10.1083/jcb.96.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McCarthy J. B., Hagen S. T., Furcht L. T. Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells. J Cell Biol. 1986 Jan;102(1):179–188. doi: 10.1083/jcb.102.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McDonald J. A., Kelley D. G. Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments. J Biol Chem. 1980 Sep 25;255(18):8848–8858. [PubMed] [Google Scholar]
  28. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  29. Mikuni-Takagaki Y., Toole B. P. Cell-substratum attachment and cell surface hyaluronate of Rous sarcoma virus-transformed chondrocytes. J Cell Biol. 1980 May;85(2):481–488. doi: 10.1083/jcb.85.2.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mosher D. F., Proctor R. A. Binding and factor XIIIa-mediated cross-linking of a 27-kilodalton fragment of fibronectin to Staphylococcus aureus. Science. 1980 Aug 22;209(4459):927–929. doi: 10.1126/science.7403857. [DOI] [PubMed] [Google Scholar]
  31. Petersen T. E., Thøgersen H. C., Skorstengaard K., Vibe-Pedersen K., Sahl P., Sottrup-Jensen L., Magnusson S. Partial primary structure of bovine plasma fibronectin: three types of internal homology. Proc Natl Acad Sci U S A. 1983 Jan;80(1):137–141. doi: 10.1073/pnas.80.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pierschbacher M. D., Hayman E. G., Ruoslahti E. Location of the cell-attachment site in fibronectin with monoclonal antibodies and proteolytic fragments of the molecule. Cell. 1981 Oct;26(2 Pt 2):259–267. doi: 10.1016/0092-8674(81)90308-1. [DOI] [PubMed] [Google Scholar]
  33. Pytela R., Pierschbacher M. D., Ruoslahti E. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell. 1985 Jan;40(1):191–198. doi: 10.1016/0092-8674(85)90322-8. [DOI] [PubMed] [Google Scholar]
  34. Quigley J. P. Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells. J Cell Biol. 1976 Nov;71(2):472–486. doi: 10.1083/jcb.71.2.472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Quigley J. P., Ossowski L., Reich E. Plasminogen, the serum proenzyme activated by factors from cells transformed by oncogenic viruses. J Biol Chem. 1974 Jul 10;249(13):4306–4311. [PubMed] [Google Scholar]
  36. Quigley J. P. Phorbol ester-induced morphological changes in transformed chick fibroblasts: evidence for direct catalytic involvement of plasminogen activator. Cell. 1979 May;17(1):131–141. doi: 10.1016/0092-8674(79)90301-5. [DOI] [PubMed] [Google Scholar]
  37. Rao C. N., Margulies I. M., Goldfarb R. H., Madri J. A., Woodley D. T., Liotta L. A. Differential proteolytic susceptibility of laminin alpha and beta subunits. Arch Biochem Biophys. 1982 Nov;219(1):65–70. doi: 10.1016/0003-9861(82)90134-5. [DOI] [PubMed] [Google Scholar]
  38. Sheela S., Barrett J. C. In vitro degradation of radiolabelled, intact basement membrane mediated by cellular plasminogen activator. Carcinogenesis. 1982;3(4):363–369. doi: 10.1093/carcin/3.4.363. [DOI] [PubMed] [Google Scholar]
  39. Sullivan L. M., Quigley J. P. An anticatalytic monoclonal antibody to avian plasminogen activator: its effect on behavior of RSV-transformed chick fibroblasts. Cell. 1986 Jun 20;45(6):905–915. doi: 10.1016/0092-8674(86)90565-9. [DOI] [PubMed] [Google Scholar]
  40. TEMIN H. M. The control of cellular morphology in embryonic cells infected with rous sarcoma virus in vitro. Virology. 1960 Feb;10:182–197. doi: 10.1016/0042-6822(60)90038-6. [DOI] [PubMed] [Google Scholar]
  41. Terranova V. P., Rao C. N., Kalebic T., Margulies I. M., Liotta L. A. Laminin receptor on human breast carcinoma cells. Proc Natl Acad Sci U S A. 1983 Jan;80(2):444–448. doi: 10.1073/pnas.80.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vartio T., Seppä H., Vaheri A. Susceptibility of soluble and matrix fibronectins to degradation by tissue proteinases, mast cell chymase and cathepsin G. J Biol Chem. 1981 Jan 10;256(1):471–477. [PubMed] [Google Scholar]
  43. Werb Z., Banda M. J., Jones P. A. Degradation of connective tissue matrices by macrophages. I. Proteolysis of elastin, glycoproteins, and collagen by proteinases isolated from macrophages. J Exp Med. 1980 Nov 1;152(5):1340–1357. doi: 10.1084/jem.152.5.1340. [DOI] [PMC free article] [PubMed] [Google Scholar]

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