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. 1990 Apr 1;110(4):1361–1367. doi: 10.1083/jcb.110.4.1361

Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin

PMCID: PMC2116088  PMID: 2139036

Abstract

Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis- (sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.

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Selected References

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

  1. Assoian R. K., Komoriya A., Meyers C. A., Miller D. M., Sporn M. B. Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization. J Biol Chem. 1983 Jun 10;258(11):7155–7160. [PubMed] [Google Scholar]
  2. Benoit R., Ling N., Esch F. A new prosomatostatin-derived peptide reveals a pattern for prohormone cleavage at monobasic sites. Science. 1987 Nov 20;238(4830):1126–1129. doi: 10.1126/science.2891188. [DOI] [PubMed] [Google Scholar]
  3. Brunner A. M., Gentry L. E., Cooper J. A., Purchio A. F. Recombinant type 1 transforming growth factor beta precursor produced in Chinese hamster ovary cells is glycosylated and phosphorylated. Mol Cell Biol. 1988 May;8(5):2229–2232. doi: 10.1128/mcb.8.5.2229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brunner A. M., Marquardt H., Malacko A. R., Lioubin M. N., Purchio A. F. Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins. J Biol Chem. 1989 Aug 15;264(23):13660–13664. [PubMed] [Google Scholar]
  5. Derynck R., Jarrett J. A., Chen E. Y., Eaton D. H., Bell J. R., Assoian R. K., Roberts A. B., Sporn M. B., Goeddel D. V. Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells. Nature. 1985 Aug 22;316(6030):701–705. doi: 10.1038/316701a0. [DOI] [PubMed] [Google Scholar]
  6. Frolik C. A., Wakefield L. M., Smith D. M., Sporn M. B. Characterization of a membrane receptor for transforming growth factor-beta in normal rat kidney fibroblasts. J Biol Chem. 1984 Sep 10;259(17):10995–11000. [PubMed] [Google Scholar]
  7. Gentry L. E., Lioubin M. N., Purchio A. F., Marquardt H. Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide. Mol Cell Biol. 1988 Oct;8(10):4162–4168. doi: 10.1128/mcb.8.10.4162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Keski-Oja J., Blasi F., Leof E. B., Moses H. L. Regulation of the synthesis and activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-beta. J Cell Biol. 1988 Feb;106(2):451–459. doi: 10.1083/jcb.106.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Laiho M., Saksela O., Andreasen P. A., Keski-Oja J. Enhanced production and extracellular deposition of the endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta. J Cell Biol. 1986 Dec;103(6 Pt 1):2403–2410. doi: 10.1083/jcb.103.6.2403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laiho M., Saksela O., Keski-Oja J. Transforming growth factor beta alters plasminogen activator activity in human skin fibroblasts. Exp Cell Res. 1986 Jun;164(2):399–407. doi: 10.1016/0014-4827(86)90038-8. [DOI] [PubMed] [Google Scholar]
  12. Lawrence D. A., Pircher R., Jullien P. Conversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions. Biochem Biophys Res Commun. 1985 Dec 31;133(3):1026–1034. doi: 10.1016/0006-291x(85)91239-2. [DOI] [PubMed] [Google Scholar]
  13. Lawrence D. A., Pircher R., Krycève-Martinerie C., Jullien P. Normal embryo fibroblasts release transforming growth factors in a latent form. J Cell Physiol. 1984 Oct;121(1):184–188. doi: 10.1002/jcp.1041210123. [DOI] [PubMed] [Google Scholar]
  14. Lyons R. M., Keski-Oja J., Moses H. L. Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium. J Cell Biol. 1988 May;106(5):1659–1665. doi: 10.1083/jcb.106.5.1659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miyazono K., Heldin C. H. Role for carbohydrate structures in TGF-beta 1 latency. Nature. 1989 Mar 9;338(6211):158–160. doi: 10.1038/338158a0. [DOI] [PubMed] [Google Scholar]
  16. Miyazono K., Hellman U., Wernstedt C., Heldin C. H. Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization. J Biol Chem. 1988 May 5;263(13):6407–6415. [PubMed] [Google Scholar]
  17. Pircher R., Jullien P., Lawrence D. A. Beta-transforming growth factor is stored in human blood platelets as a latent high molecular weight complex. Biochem Biophys Res Commun. 1986 Apr 14;136(1):30–37. doi: 10.1016/0006-291x(86)90872-7. [DOI] [PubMed] [Google Scholar]
  18. Purchio A. F., Cooper J. A., Brunner A. M., Lioubin M. N., Gentry L. E., Kovacina K. S., Roth R. A., Marquardt H. Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor. J Biol Chem. 1988 Oct 5;263(28):14211–14215. [PubMed] [Google Scholar]
  19. Roberts A. B., Frolik C. A., Anzano M. A., Sporn M. B. Transforming growth factors from neoplastic and nonneoplastic tissues. Fed Proc. 1983 Jun;42(9):2621–2626. [PubMed] [Google Scholar]
  20. Sato Y., Rifkin D. B. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. J Cell Biol. 1989 Jul;109(1):309–315. doi: 10.1083/jcb.109.1.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tucker R. F., Branum E. L., Shipley G. D., Ryan R. J., Moses H. L. Specific binding to cultured cells of 125I-labeled type beta transforming growth factor from human platelets. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6757–6761. doi: 10.1073/pnas.81.21.6757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tucker R. F., Shipley G. D., Moses H. L., Holley R. W. Growth inhibitor from BSC-1 cells closely related to platelet type beta transforming growth factor. Science. 1984 Nov 9;226(4675):705–707. doi: 10.1126/science.6093254. [DOI] [PubMed] [Google Scholar]
  24. Wakefield L. M., Smith D. M., Flanders K. C., Sporn M. B. Latent transforming growth factor-beta from human platelets. A high molecular weight complex containing precursor sequences. J Biol Chem. 1988 Jun 5;263(16):7646–7654. [PubMed] [Google Scholar]

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