Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Jul 1;333(Pt 1):159–165. doi: 10.1042/bj3330159

Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves the recombinant aggrecan substrate rAgg1mut at the 'aggrecanase' and the MMP sites. Characterization of MT1-MMP catabolic activities on the interglobular domain of aggrecan.

F H Büttner 1, C E Hughes 1, D Margerie 1, A Lichte 1, H Tschesche 1, B Caterson 1, E Bartnik 1
PMCID: PMC1219568  PMID: 9639575

Abstract

The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704-709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor 'aggrecanase' catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269-20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the 'aggrecanase' site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS...) and BC-14 (anti-FFGV...) to distinguish between 'aggrecanase' and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong 'aggrecanase' activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for 'aggrecanase' activity in vivo.

Full Text

The Full Text of this article is available as a PDF (408.0 KB).

Selected References

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

  1. Arner E. C., Decicco C. P., Cherney R., Tortorella M. D. Cleavage of native cartilage aggrecan by neutrophil collagenase (MMP-8) is distinct from endogenous cleavage by aggrecanase. J Biol Chem. 1997 Apr 4;272(14):9294–9299. doi: 10.1074/jbc.272.14.9294. [DOI] [PubMed] [Google Scholar]
  2. Aruffo A., Stamenkovic I., Melnick M., Underhill C. B., Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell. 1990 Jun 29;61(7):1303–1313. doi: 10.1016/0092-8674(90)90694-a. [DOI] [PubMed] [Google Scholar]
  3. Büttner F. H., Chubinskaya S., Margerie D., Huch K., Flechtenmacher J., Cole A. A., Kuettner K. E., Bartnik E. Expression of membrane type 1 matrix metalloproteinase in human articular cartilage. Arthritis Rheum. 1997 Apr;40(4):704–709. doi: 10.1002/art.1780400415. [DOI] [PubMed] [Google Scholar]
  4. Caterson B., Hughes C. E., Roughley P., Mort J. S. Anabolic and catabolic markers of proteoglycan metabolism in osteoarthritis. Acta Orthop Scand Suppl. 1995 Oct;266:121–124. [PubMed] [Google Scholar]
  5. Cole A. A., Chubinskaya S., Schumacher B., Huch K., Szabo G., Yao J., Mikecz K., Hasty K. A., Kuettner K. E. Chondrocyte matrix metalloproteinase-8. Human articular chondrocytes express neutrophil collagenase. J Biol Chem. 1996 May 3;271(18):11023–11026. doi: 10.1074/jbc.271.18.11023. [DOI] [PubMed] [Google Scholar]
  6. Doege K. J., Sasaki M., Kimura T., Yamada Y. Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms. J Biol Chem. 1991 Jan 15;266(2):894–902. [PubMed] [Google Scholar]
  7. Flannery C. R., Lark M. W., Sandy J. D. Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage. J Biol Chem. 1992 Jan 15;267(2):1008–1014. [PubMed] [Google Scholar]
  8. Fosang A. J., Last K., Knäuper V., Murphy G., Neame P. J. Degradation of cartilage aggrecan by collagenase-3 (MMP-13). FEBS Lett. 1996 Feb 12;380(1-2):17–20. doi: 10.1016/0014-5793(95)01539-6. [DOI] [PubMed] [Google Scholar]
  9. Fosang A. J., Last K., Knäuper V., Neame P. J., Murphy G., Hardingham T. E., Tschesche H., Hamilton J. A. Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain. Biochem J. 1993 Oct 1;295(Pt 1):273–276. doi: 10.1042/bj2950273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fosang A. J., Last K., Maciewicz R. A. Aggrecan is degraded by matrix metalloproteinases in human arthritis. Evidence that matrix metalloproteinase and aggrecanase activities can be independent. J Clin Invest. 1996 Nov 15;98(10):2292–2299. doi: 10.1172/JCI119040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fosang A. J., Last K., Neame P. J., Murphy G., Knäuper V., Tschesche H., Hughes C. E., Caterson B., Hardingham T. E. Neutrophil collagenase (MMP-8) cleaves at the aggrecanase site E373-A374 in the interglobular domain of cartilage aggrecan. Biochem J. 1994 Dec 1;304(Pt 2):347–351. doi: 10.1042/bj3040347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fosang A. J., Neame P. J., Hardingham T. E., Murphy G., Hamilton J. A. Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins. J Biol Chem. 1991 Aug 25;266(24):15579–15582. [PubMed] [Google Scholar]
  13. Fosang A. J., Neame P. J., Last K., Hardingham T. E., Murphy G., Hamilton J. A. The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B. J Biol Chem. 1992 Sep 25;267(27):19470–19474. [PubMed] [Google Scholar]
  14. Hasty K. A., Pourmotabbed T. F., Goldberg G. I., Thompson J. P., Spinella D. G., Stevens R. M., Mainardi C. L. Human neutrophil collagenase. A distinct gene product with homology to other matrix metalloproteinases. J Biol Chem. 1990 Jul 15;265(20):11421–11424. [PubMed] [Google Scholar]
  15. Hughes C. E., Büttner F. H., Eidenmüller B., Caterson B., Bartnik E. Utilization of a recombinant substrate rAgg1 to study the biochemical properties of aggrecanase in cell culture systems. J Biol Chem. 1997 Aug 8;272(32):20269–20274. doi: 10.1074/jbc.272.32.20269. [DOI] [PubMed] [Google Scholar]
  16. Hughes C. E., Caterson B., Fosang A. J., Roughley P. J., Mort J. S. Monoclonal antibodies that specifically recognize neoepitope sequences generated by 'aggrecanase' and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro. Biochem J. 1995 Feb 1;305(Pt 3):799–804. doi: 10.1042/bj3050799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hunkapiller M. W., Hood L. E. Protein sequence analysis: automated microsequencing. Science. 1983 Feb 11;219(4585):650–659. doi: 10.1126/science.6687410. [DOI] [PubMed] [Google Scholar]
  18. Ilic M. Z., Handley C. J., Robinson H. C., Mok M. T. Mechanism of catabolism of aggrecan by articular cartilage. Arch Biochem Biophys. 1992 Apr;294(1):115–122. doi: 10.1016/0003-9861(92)90144-l. [DOI] [PubMed] [Google Scholar]
  19. Ilic M. Z., Mok M. T., Williamson O. D., Campbell M. A., Hughes C. E., Handley C. J. Catabolism of aggrecan by explant cultures of human articular cartilage in the presence of retinoic acid. Arch Biochem Biophys. 1995 Sep 10;322(1):22–30. doi: 10.1006/abbi.1995.1431. [DOI] [PubMed] [Google Scholar]
  20. Imai K., Ohta S., Matsumoto T., Fujimoto N., Sato H., Seiki M., Okada Y. Expression of membrane-type 1 matrix metalloproteinase and activation of progelatinase A in human osteoarthritic cartilage. Am J Pathol. 1997 Jul;151(1):245–256. [PMC free article] [PubMed] [Google Scholar]
  21. Knäuper V., Cowell S., Smith B., López-Otin C., O'Shea M., Morris H., Zardi L., Murphy G. The role of the C-terminal domain of human collagenase-3 (MMP-13) in the activation of procollagenase-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction. J Biol Chem. 1997 Mar 21;272(12):7608–7616. doi: 10.1074/jbc.272.12.7608. [DOI] [PubMed] [Google Scholar]
  22. Knäuper V., Will H., López-Otin C., Smith B., Atkinson S. J., Stanton H., Hembry R. M., Murphy G. Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme. J Biol Chem. 1996 Jul 19;271(29):17124–17131. doi: 10.1074/jbc.271.29.17124. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Lark M. W., Bayne E. K., Flanagan J., Harper C. F., Hoerrner L. A., Hutchinson N. I., Singer I. I., Donatelli S. A., Weidner J. R., Williams H. R. Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. J Clin Invest. 1997 Jul 1;100(1):93–106. doi: 10.1172/JCI119526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lark M. W., Gordy J. T., Weidner J. R., Ayala J., Kimura J. H., Williams H. R., Mumford R. A., Flannery C. R., Carlson S. S., Iwata M. Cell-mediated catabolism of aggrecan. Evidence that cleavage at the "aggrecanase" site (Glu373-Ala374) is a primary event in proteolysis of the interglobular domain. J Biol Chem. 1995 Feb 10;270(6):2550–2556. doi: 10.1074/jbc.270.6.2550. [DOI] [PubMed] [Google Scholar]
  26. Lichte A., Kolkenbrock H., Tschesche H. The recombinant catalytic domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) induces activation of progelatinase A and progelatinase A complexed with TIMP-2. FEBS Lett. 1996 Nov 18;397(2-3):277–282. doi: 10.1016/s0014-5793(96)01206-9. [DOI] [PubMed] [Google Scholar]
  27. Lohmander L. S., Hoerrner L. A., Lark M. W. Metalloproteinases, tissue inhibitor, and proteoglycan fragments in knee synovial fluid in human osteoarthritis. Arthritis Rheum. 1993 Feb;36(2):181–189. doi: 10.1002/art.1780360207. [DOI] [PubMed] [Google Scholar]
  28. Lohmander L. S., Neame P. J., Sandy J. D. The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum. 1993 Sep;36(9):1214–1222. doi: 10.1002/art.1780360906. [DOI] [PubMed] [Google Scholar]
  29. Loulakis P., Shrikhande A., Davis G., Maniglia C. A. N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage. Biochem J. 1992 Jun 1;284(Pt 2):589–593. doi: 10.1042/bj2840589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ohuchi E., Imai K., Fujii Y., Sato H., Seiki M., Okada Y. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem. 1997 Jan 24;272(4):2446–2451. doi: 10.1074/jbc.272.4.2446. [DOI] [PubMed] [Google Scholar]
  31. Pei D., Weiss S. J. Transmembrane-deletion mutants of the membrane-type matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. J Biol Chem. 1996 Apr 12;271(15):9135–9140. doi: 10.1074/jbc.271.15.9135. [DOI] [PubMed] [Google Scholar]
  32. Prickett K. S., Amberg D. C., Hopp T. P. A calcium-dependent antibody for identification and purification of recombinant proteins. Biotechniques. 1989 Jun;7(6):580–589. [PubMed] [Google Scholar]
  33. Sandy J. D., Flannery C. R., Neame P. J., Lohmander L. S. The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain. J Clin Invest. 1992 May;89(5):1512–1516. doi: 10.1172/JCI115742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sandy J. D., Neame P. J., Boynton R. E., Flannery C. R. Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain. J Biol Chem. 1991 May 15;266(14):8683–8685. [PubMed] [Google Scholar]
  35. Sato H., Takino T., Okada Y., Cao J., Shinagawa A., Yamamoto E., Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature. 1994 Jul 7;370(6484):61–65. doi: 10.1038/370061a0. [DOI] [PubMed] [Google Scholar]
  36. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  37. Tortorella M. D., Pratta M. A., Fox J. W., Arner E. C. The interglobular domain of cartilage aggrecan is cleaved by hemorrhagic metalloproteinase HT-d (atrolysin C) at the matrix metalloproteinase and aggrecanase sites. J Biol Chem. 1998 Mar 6;273(10):5846–5850. doi: 10.1074/jbc.273.10.5846. [DOI] [PubMed] [Google Scholar]
  38. Valhmu W. B., Palmer G. D., Rivers P. A., Ebara S., Cheng J. F., Fischer S., Ratcliffe A. Structure of the human aggrecan gene: exon-intron organization and association with the protein domains. Biochem J. 1995 Jul 15;309(Pt 2):535–542. doi: 10.1042/bj3090535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Walakovits L. A., Moore V. L., Bhardwaj N., Gallick G. S., Lark M. W. Detection of stromelysin and collagenase in synovial fluid from patients with rheumatoid arthritis and posttraumatic knee injury. Arthritis Rheum. 1992 Jan;35(1):35–42. doi: 10.1002/art.1780350106. [DOI] [PubMed] [Google Scholar]
  40. Wilhelm S. M., Collier I. E., Kronberger A., Eisen A. Z., Marmer B. L., Grant G. A., Bauer E. A., Goldberg G. I. Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6725–6729. doi: 10.1073/pnas.84.19.6725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ye Q. Z., Johnson L. L., Hupe D. J., Baragi V. Purification and characterization of the human stromelysin catalytic domain expressed in Escherichia coli. Biochemistry. 1992 Nov 17;31(45):11231–11235. doi: 10.1021/bi00160a038. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES