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. 2002 May 15;364(Pt 1):227–234. doi: 10.1042/bj3640227

Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-alpha-converting enzyme.

Meng-Huee Lee 1, Vandana Verma 1, Klaus Maskos 1, Deepa Nath 1, Vera Knäuper 1, Philippa Dodds 1, Augustin Amour 1, Gillian Murphy 1
PMCID: PMC1222565  PMID: 11988096

Abstract

We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (K(app)(i)) and association rates (k(on)) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60 pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-alpha demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-alpha. Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-alpha shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.

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Selected References

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  1. Amour A., Knight C. G., Webster A., Slocombe P. M., Stephens P. E., Knäuper V., Docherty A. J., Murphy G. The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3. FEBS Lett. 2000 May 19;473(3):275–279. doi: 10.1016/s0014-5793(00)01528-3. [DOI] [PubMed] [Google Scholar]
  2. Amour A., Slocombe P. M., Webster A., Butler M., Knight C. G., Smith B. J., Stephens P. E., Shelley C., Hutton M., Knäuper V. TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett. 1998 Sep 11;435(1):39–44. doi: 10.1016/s0014-5793(98)01031-x. [DOI] [PubMed] [Google Scholar]
  3. Apte S. S., Olsen B. R., Murphy G. The gene structure of tissue inhibitor of metalloproteinases (TIMP)-3 and its inhibitory activities define the distinct TIMP gene family. J Biol Chem. 1995 Jun 16;270(24):14313–14318. doi: 10.1074/jbc.270.24.14313. [DOI] [PubMed] [Google Scholar]
  4. Beutler B., Cerami A. Tumor necrosis, cachexia, shock, and inflammation: a common mediator. Annu Rev Biochem. 1988;57:505–518. doi: 10.1146/annurev.bi.57.070188.002445. [DOI] [PubMed] [Google Scholar]
  5. Black R. A., Durie F. H., Otten-Evans C., Miller R., Slack J. L., Lynch D. H., Castner B., Mohler K. M., Gerhart M., Johnson R. S. Relaxed specificity of matrix metalloproteinases (MMPS) and TIMP insensitivity of tumor necrosis factor-alpha (TNF-alpha) production suggest the major TNF-alpha converting enzyme is not an MMP. Biochem Biophys Res Commun. 1996 Aug 14;225(2):400–405. doi: 10.1006/bbrc.1996.1186. [DOI] [PubMed] [Google Scholar]
  6. Black R. A., Rauch C. T., Kozlosky C. J., Peschon J. J., Slack J. L., Wolfson M. F., Castner B. J., Stocking K. L., Reddy P., Srinivasan S. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 1997 Feb 20;385(6618):729–733. doi: 10.1038/385729a0. [DOI] [PubMed] [Google Scholar]
  7. Borland G., Murphy G., Ager A. Tissue inhibitor of metalloproteinases-3 inhibits shedding of L-selectin from leukocytes. J Biol Chem. 1999 Jan 29;274(5):2810–2815. doi: 10.1074/jbc.274.5.2810. [DOI] [PubMed] [Google Scholar]
  8. Butler G. S., Hutton M., Wattam B. A., Williamson R. A., Knäuper V., Willenbrock F., Murphy G. The specificity of TIMP-2 for matrix metalloproteinases can be modified by single amino acid mutations. J Biol Chem. 1999 Jul 16;274(29):20391–20396. doi: 10.1074/jbc.274.29.20391. [DOI] [PubMed] [Google Scholar]
  9. Fitzgerald M. L., Wang Z., Park P. W., Murphy G., Bernfield M. Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase. J Cell Biol. 2000 Feb 21;148(4):811–824. doi: 10.1083/jcb.148.4.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garton K. J., Gough P. J., Blobel C. P., Murphy G., Greaves D. R., Dempsey P. J., Raines E. W. Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1). J Biol Chem. 2001 Aug 8;276(41):37993–38001. doi: 10.1074/jbc.M106434200. [DOI] [PubMed] [Google Scholar]
  11. Gearing A. J., Beckett P., Christodoulou M., Churchill M., Clements J., Davidson A. H., Drummond A. H., Galloway W. A., Gilbert R., Gordon J. L. Processing of tumour necrosis factor-alpha precursor by metalloproteinases. Nature. 1994 Aug 18;370(6490):555–557. doi: 10.1038/370555a0. [DOI] [PubMed] [Google Scholar]
  12. Hargreaves P. G., Wang F., Antcliff J., Murphy G., Lawry J., Russell R. G., Croucher P. I. Human myeloma cells shed the interleukin-6 receptor: inhibition by tissue inhibitor of metalloproteinase-3 and a hydroxamate-based metalloproteinase inhibitor. Br J Haematol. 1998 Jun;101(4):694–702. doi: 10.1046/j.1365-2141.1998.00754.x. [DOI] [PubMed] [Google Scholar]
  13. Kashiwagi M., Tortorella M., Nagase H., Brew K. TIMP-3 is a potent inhibitor of aggrecanase 1 (ADAM-TS4) and aggrecanase 2 (ADAM-TS5). J Biol Chem. 2001 Jan 23;276(16):12501–12504. doi: 10.1074/jbc.C000848200. [DOI] [PubMed] [Google Scholar]
  14. Langton K. P., Barker M. D., McKie N. Localization of the functional domains of human tissue inhibitor of metalloproteinases-3 and the effects of a Sorsby's fundus dystrophy mutation. J Biol Chem. 1998 Jul 3;273(27):16778–16781. doi: 10.1074/jbc.273.27.16778. [DOI] [PubMed] [Google Scholar]
  15. Lee M. H., Knäuper V., Becherer J. D., Murphy G. Full-length and N-TIMP-3 display equal inhibitory activities toward TNF-alpha convertase. Biochem Biophys Res Commun. 2001 Jan 26;280(3):945–950. doi: 10.1006/bbrc.2000.4192. [DOI] [PubMed] [Google Scholar]
  16. Loechel F., Fox J. W., Murphy G., Albrechtsen R., Wewer U. M. ADAM 12-S cleaves IGFBP-3 and IGFBP-5 and is inhibited by TIMP-3. Biochem Biophys Res Commun. 2000 Nov 30;278(3):511–515. doi: 10.1006/bbrc.2000.3835. [DOI] [PubMed] [Google Scholar]
  17. Maskos K., Fernandez-Catalan C., Huber R., Bourenkov G. P., Bartunik H., Ellestad G. A., Reddy P., Wolfson M. F., Rauch C. T., Castner B. J. Crystal structure of the catalytic domain of human tumor necrosis factor-alpha-converting enzyme. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3408–3412. doi: 10.1073/pnas.95.7.3408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McGeehan G. M., Becherer J. D., Bast R. C., Jr, Boyer C. M., Champion B., Connolly K. M., Conway J. G., Furdon P., Karp S., Kidao S. Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor. Nature. 1994 Aug 18;370(6490):558–561. doi: 10.1038/370558a0. [DOI] [PubMed] [Google Scholar]
  19. Milla M. E., Leesnitzer M. A., Moss M. L., Clay W. C., Carter H. L., Miller A. B., Su J. L., Lambert M. H., Willard D. H., Sheeley D. M. Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE). J Biol Chem. 1999 Oct 22;274(43):30563–30570. doi: 10.1074/jbc.274.43.30563. [DOI] [PubMed] [Google Scholar]
  20. Montero J. C., Yuste L., Díaz-Rodríguez E., Esparís-Ogando A., Pandiella A. Differential shedding of transmembrane neuregulin isoforms by the tumor necrosis factor-alpha-converting enzyme. Mol Cell Neurosci. 2000 Nov;16(5):631–648. doi: 10.1006/mcne.2000.0896. [DOI] [PubMed] [Google Scholar]
  21. Moss M. L., Jin S. L., Milla M. E., Bickett D. M., Burkhart W., Carter H. L., Chen W. J., Clay W. C., Didsbury J. R., Hassler D. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature. 1997 Feb 20;385(6618):733–736. doi: 10.1038/385733a0. [DOI] [PubMed] [Google Scholar]
  22. Murphy G., Houbrechts A., Cockett M. I., Williamson R. A., O'Shea M., Docherty A. J. The N-terminal domain of tissue inhibitor of metalloproteinases retains metalloproteinase inhibitory activity. Biochemistry. 1991 Aug 20;30(33):8097–8102. doi: 10.1021/bi00247a001. [DOI] [PubMed] [Google Scholar]
  23. Murphy G., Willenbrock F., Ward R. V., Cockett M. I., Eaton D., Docherty A. J. The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases. Biochem J. 1992 May 1;283(Pt 3):637–641. doi: 10.1042/bj2830637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Muskett F. W., Frenkiel T. A., Feeney J., Freedman R. B., Carr M. D., Williamson R. A. High resolution structure of the N-terminal domain of tissue inhibitor of metalloproteinases-2 and characterization of its interaction site with matrix metalloproteinase-3. J Biol Chem. 1998 Aug 21;273(34):21736–21743. doi: 10.1074/jbc.273.34.21736. [DOI] [PubMed] [Google Scholar]
  25. Nath D., Williamson N. J., Jarvis R., Murphy G. Shedding of c-Met is regulated by crosstalk between a G-protein coupled receptor and the EGF receptor and is mediated by a TIMP-3 sensitive metalloproteinase. J Cell Sci. 2001 Mar;114(Pt 6):1213–1220. doi: 10.1242/jcs.114.6.1213. [DOI] [PubMed] [Google Scholar]
  26. Nguyen Q., Willenbrock F., Cockett M. I., O'Shea M., Docherty A. J., Murphy G. Different domain interactions are involved in the binding of tissue inhibitors of metalloproteinases to stromelysin-1 and gelatinase A. Biochemistry. 1994 Mar 1;33(8):2089–2095. doi: 10.1021/bi00174a015. [DOI] [PubMed] [Google Scholar]
  27. Rio C., Buxbaum J. D., Peschon J. J., Corfas G. Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4. J Biol Chem. 2000 Apr 7;275(14):10379–10387. doi: 10.1074/jbc.275.14.10379. [DOI] [PubMed] [Google Scholar]
  28. Tuuttila A., Morgunova E., Bergmann U., Lindqvist Y., Maskos K., Fernandez-Catalan C., Bode W., Tryggvason K., Schneider G. Three-dimensional structure of human tissue inhibitor of metalloproteinases-2 at 2.1 A resolution. J Mol Biol. 1998 Dec 11;284(4):1133–1140. doi: 10.1006/jmbi.1998.2223. [DOI] [PubMed] [Google Scholar]
  29. Wei P., Zhao Y. G., Zhuang L., Ruben S., Sang Q. X. Expression and enzymatic activity of human disintegrin and metalloproteinase ADAM19/meltrin beta. Biochem Biophys Res Commun. 2001 Jan 26;280(3):744–755. doi: 10.1006/bbrc.2000.4200. [DOI] [PubMed] [Google Scholar]
  30. Williams J. W., Morrison J. F., Duggleby R. G. Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase. Biochemistry. 1979 Jun 12;18(12):2567–2573. doi: 10.1021/bi00579a021. [DOI] [PubMed] [Google Scholar]
  31. Wu B., Arumugam S., Gao G., Lee G. I., Semenchenko V., Huang W., Brew K., Van Doren S. R. NMR structure of tissue inhibitor of metalloproteinases-1 implicates localized induced fit in recognition of matrix metalloproteinases. J Mol Biol. 2000 Jan 14;295(2):257–268. doi: 10.1006/jmbi.1999.3362. [DOI] [PubMed] [Google Scholar]
  32. Yu W. H., Yu S., Meng Q., Brew K., Woessner J. F., Jr TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix. J Biol Chem. 2000 Oct 6;275(40):31226–31232. doi: 10.1074/jbc.M000907200. [DOI] [PubMed] [Google Scholar]

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