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. 1996 Nov 15;98(10):2292–2299. doi: 10.1172/JCI119040

Aggrecan is degraded by matrix metalloproteinases in human arthritis. Evidence that matrix metalloproteinase and aggrecanase activities can be independent.

A J Fosang 1, K Last 1, R A Maciewicz 1
PMCID: PMC507679  PMID: 8941646

Abstract

Proteolytic degradation of aggrecan is a hallmark of the pathology of arthritis, yet the identity of the enzyme(s) in cartilage responsible for this degradation is unknown. Previous studies have suggested that the matrix metalloproteinases (MMPs) may be involved but there has been no definitive evidence for their direct action in the proteolysis of aggrecan in human arthritis. We now show unequivocally that aggrecan fragments derived from the specific action of MMPs can be detected in synovial fluids from patients with both inflammatory and noninflammatory arthritis, with a neoepitope monoclonal antibody AF-28 that detects the NH2-terminal sequence F342FGVG.... The synovial fluid MMP fragments were of low buoyant density and distributed exclusively at the top of cesium chloride density gradients, suggesting that these fragments lacked chondroitin sulfate chains. AF-28 immunoblotting of synovial fluid aggrecan fragments revealed a population of small AF-28 fragments of 30-50 kD. Based on their size relative to characterized products of an MMP-8 digest (Fosang, A.J., K. Last, P. Gardiner, D.C. Jackson, and L. Brown. 1995, Biochem. J. 310:337-343), these AF-28 fragments were derived from proteinase cleavage at, or near, the ...ITEGE373 / ARGSV... aggrecanase site. Immunodetection with polyclonal anti-ITEGE antiserum revealed that these fragments lacked the ...ITEGE374 COOH terminus and were not therefore products of aggrecanase action. The same fluid samples contained a broad 68-90-kD G1 fragment that contained the COOH-terminal ...ITEGE374 neoepitope. The results suggest that in some circumstances, despite extensive proteolysis of the core protein, aggrecan molecules may be cleaved by MMPs or aggrecanase in the interglobular domain, but not both.

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Selected References

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  1. Altman R., Asch E., Bloch D., Bole G., Borenstein D., Brandt K., Christy W., Cooke T. D., Greenwald R., Hochberg M. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum. 1986 Aug;29(8):1039–1049. doi: 10.1002/art.1780290816. [DOI] [PubMed] [Google Scholar]
  2. Arner E. C. Effect of animal age and chronicity of interleukin-1 exposure on cartilage proteoglycan depletion in vivo. J Orthop Res. 1994 May;12(3):321–330. doi: 10.1002/jor.1100120304. [DOI] [PubMed] [Google Scholar]
  3. Barry F. P., Rosenberg L. C., Gaw J. U., Gaw J. U., Koob T. J., Neame P. J. N- and O-linked keratan sulfate on the hyaluronan binding region of aggrecan from mature and immature bovine cartilage. J Biol Chem. 1995 Sep 1;270(35):20516–20524. doi: 10.1074/jbc.270.35.20516. [DOI] [PubMed] [Google Scholar]
  4. Buttle D. J., Handley C. J., Ilic M. Z., Saklatvala J., Murata M., Barrett A. J. Inhibition of cartilage proteoglycan release by a specific inactivator of cathepsin B and an inhibitor of matrix metalloproteinases. Evidence for two converging pathways of chondrocyte-mediated proteoglycan degradation. Arthritis Rheum. 1993 Dec;36(12):1709–1717. doi: 10.1002/art.1780361210. [DOI] [PubMed] [Google Scholar]
  5. Carlson C. S., Loeser R. F., Johnstone B., Tulli H. M., Dobson D. B., Caterson B. Osteoarthritis in cynomolgus macaques. II. Detection of modulated proteoglycan epitopes in cartilage and synovial fluid. J Orthop Res. 1995 May;13(3):399–409. doi: 10.1002/jor.1100130314. [DOI] [PubMed] [Google Scholar]
  6. Carroll G. Measurement of sulphated glycosaminoglycans and proteoglycan fragments in arthritic synovial fluid. Ann Rheum Dis. 1989 Jan;48(1):17–24. doi: 10.1136/ard.48.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cawston T. E., Weaver L., Coughlan R. J., Kyle M. V., Hazleman B. L. Synovial fluids from infected joints contain active metalloproteinases and no inhibitory activity. Br J Rheumatol. 1989 Oct;28(5):386–392. doi: 10.1093/rheumatology/28.5.386. [DOI] [PubMed] [Google Scholar]
  8. Cawston T. Blocking cartilage destruction with metalloproteinase inhibitors: a valid therapeutic target? Ann Rheum Dis. 1993 Nov;52(11):769–770. doi: 10.1136/ard.52.11.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cawston T., McLaughlan P., Coughlan R., Kyle V., Hazleman B. Synovial fluids from infected joints contain metalloproteinase--tissue inhibitor of metalloproteinase (TIMP) complexes. Biochim Biophys Acta. 1990 Jan 29;1033(1):96–102. doi: 10.1016/0304-4165(90)90200-g. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Cole A. A., Kuettner K. E. MMP-8 (neutrophil collagenase) mRNA and aggrecanase cleavage products are present in normal and osteoarthritic human articular cartilage. Acta Orthop Scand Suppl. 1995 Oct;266:98–102. [PubMed] [Google Scholar]
  12. DIAGNOSTIC criteria for rheumatoid arthritis: 1958 revision by a committee of the American Rheumatism Association. Ann Rheum Dis. 1959 Mar;18(1):49–53. [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Dudhia J., Davidson C. M., Wells T. M., Vynios D. H., Hardingham T. E., Bayliss M. T. Age-related changes in the content of the C-terminal region of aggrecan in human articular cartilage. Biochem J. 1996 Feb 1;313(Pt 3):933–940. doi: 10.1042/bj3130933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  16. Farndale R. W., Sayers C. A., Barrett A. J. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res. 1982;9(4):247–248. doi: 10.3109/03008208209160269. [DOI] [PubMed] [Google Scholar]
  17. Fosang A. J., Last K., Gardiner P., Jackson D. C., Brown L. Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids. Biochem J. 1995 Aug 15;310(Pt 1):337–343. doi: 10.1042/bj3100337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Hanley J. A., Lippman-Hand A. If nothing goes wrong, is everything all right? Interpreting zero numerators. JAMA. 1983 Apr 1;249(13):1743–1745. [PubMed] [Google Scholar]
  24. Heinegård D., Axelsson I. Distribution of keratan sulfate in cartilage proteoglycans. J Biol Chem. 1977 Mar 25;252(6):1971–1979. [PubMed] [Google Scholar]
  25. Heinegård D., Inerot S., Wieslander J., Lindblad G. A method for the quantification of cartilage proteoglycan structures liberated to the synovial fluid during developing degenerative joint disease. Scand J Clin Lab Invest. 1985 Sep;45(5):421–427. doi: 10.1080/00365518509155238. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Hughes C. E., Caterson B., White R. J., Roughley P. J., Mort J. S. Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin. J Biol Chem. 1992 Aug 15;267(23):16011–16014. [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Lark M. W., Williams H., Hoernner L. A., Weidner J., Ayala J. M., Harper C. F., Christen A., Olszewski J., Konteatis Z., Webber R. Quantification of a matrix metalloproteinase-generated aggrecan G1 fragment using monospecific anti-peptide serum. Biochem J. 1995 Apr 1;307(Pt 1):245–252. doi: 10.1042/bj3070245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lewthwaite J., Blake S. M., Hardingham T. E., Warden P. J., Henderson B. The effect of recombinant human interleukin 1 receptor antagonist on the induction phase of antigen induced arthritis in the rabbit. J Rheumatol. 1994 Mar;21(3):467–472. [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Mehmet H., Scudder P., Tang P. W., Hounsell E. F., Caterson B., Feizi T. The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated hepta- or larger oligosaccharides of the poly(N-acetyllactosamine) series. Eur J Biochem. 1986 Jun 2;157(2):385–391. doi: 10.1111/j.1432-1033.1986.tb09680.x. [DOI] [PubMed] [Google Scholar]
  35. Mok M. T., Ilic M. Z., Handley C. J., Robinson H. C. Cleavage of proteoglycan aggregate by leucocyte elastase. Arch Biochem Biophys. 1992 Feb 1;292(2):442–447. doi: 10.1016/0003-9861(92)90014-n. [DOI] [PubMed] [Google Scholar]
  36. Mort J. S., Dodge G. R., Roughley P. J., Liu J., Finch S. J., DiPasquale G., Poole A. R. Direct evidence for active metalloproteinases mediating matrix degradation in interleukin 1-stimulated human articular cartilage. Matrix. 1993 Mar;13(2):95–102. doi: 10.1016/s0934-8832(11)80068-5. [DOI] [PubMed] [Google Scholar]
  37. Plaas A. H., Sandy J. D. A cartilage explant system for studies on aggrecan structure, biosynthesis and catabolism in discrete zones of the mammalian growth plate. Matrix. 1993 Mar;13(2):135–147. doi: 10.1016/s0934-8832(11)80072-7. [DOI] [PubMed] [Google Scholar]
  38. Poole A. R., Ionescu M., Swan A., Dieppe P. A. Changes in cartilage metabolism in arthritis are reflected by altered serum and synovial fluid levels of the cartilage proteoglycan aggrecan. Implications for pathogenesis. J Clin Invest. 1994 Jul;94(1):25–33. doi: 10.1172/JCI117314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ratcliffe A., Doherty M., Maini R. N., Hardingham T. E. Increased concentrations of proteoglycan components in the synovial fluids of patients with acute but not chronic joint disease. Ann Rheum Dis. 1988 Oct;47(10):826–832. doi: 10.1136/ard.47.10.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ratcliffe A., Shurety W., Caterson B. The quantitation of a native chondroitin sulfate epitope in synovial fluid lavages and articular cartilage from canine experimental osteoarthritis and disuse atrophy. Arthritis Rheum. 1993 Apr;36(4):543–551. doi: 10.1002/art.1780360416. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Saxne T., Castro F., Rydholm U., Svantesson H. Cartilage derived proteoglycans in body fluids of children. Inverse correlation with age. J Rheumatol. 1989 Oct;16(10):1341–1344. [PubMed] [Google Scholar]
  44. Saxne T., Heinegård D. Synovial fluid analysis of two groups of proteoglycan epitopes distinguishes early and late cartilage lesions. Arthritis Rheum. 1992 Apr;35(4):385–390. doi: 10.1002/art.1780350404. [DOI] [PubMed] [Google Scholar]
  45. Saxne T., Heinegård D., Wollheim F. A., Pettersson H. Difference in cartilage proteoglycan level in synovial fluid in early rheumatoid arthritis and reactive arthritis. Lancet. 1985 Jul 20;2(8447):127–128. doi: 10.1016/s0140-6736(85)90229-6. [DOI] [PubMed] [Google Scholar]
  46. Saxne T., Heinegård D., Wollheim F. A. Therapeutic effects on cartilage metabolism in arthritis as measured by release of proteoglycan structures into the synovial fluid. Ann Rheum Dis. 1986 Jun;45(6):491–497. doi: 10.1136/ard.45.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Seed M. P., Ismaiel S., Cheung C. Y., Thomson T. A., Gardner C. R., Atkins R. M., Elson C. J. Inhibition of interleukin 1 beta induced rat and human cartilage degradation in vitro by the metalloproteinase inhibitor U27391. Ann Rheum Dis. 1993 Jan;52(1):37–43. doi: 10.1136/ard.52.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Singer I. I., Kawka D. W., Bayne E. K., Donatelli S. A., Weidner J. R., Williams H. R., Ayala J. M., Mumford R. A., Lark M. W., Glant T. T. VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis. J Clin Invest. 1995 May;95(5):2178–2186. doi: 10.1172/JCI117907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Thonar E. J., Lenz M. E., Klintworth G. K., Caterson B., Pachman L. M., Glickman P., Katz R., Huff J., Kuettner K. E. Quantification of keratan sulfate in blood as a marker of cartilage catabolism. Arthritis Rheum. 1985 Dec;28(12):1367–1376. doi: 10.1002/art.1780281209. [DOI] [PubMed] [Google Scholar]
  50. Vilim V., Fosang A. J. Proteoglycans isolated from dissociative extracts of differently aged human articular cartilage: characterization of naturally occurring hyaluronan-binding fragments of aggrecan. Biochem J. 1994 Dec 15;304(Pt 3):887–894. doi: 10.1042/bj3040887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Willkens R. F., Arnett F. C., Bitter T., Calin A., Fisher L., Ford D. K., Good A. E., Masi A. T. Reiter's syndrome. Evaluation of preliminary criteria for definite disease. Bull Rheum Dis. 1982;32(4):31–34. [PubMed] [Google Scholar]
  52. Witter J., Roughley P. J., Webber C., Roberts N., Keystone E., Poole A. R. The immunologic detection and characterization of cartilage proteoglycan degradation products in synovial fluids of patients with arthritis. Arthritis Rheum. 1987 May;30(5):519–529. doi: 10.1002/art.1780300506. [DOI] [PubMed] [Google Scholar]

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