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. 1997 Aug 15;326(Pt 1):235–241. doi: 10.1042/bj3260235

Aggrecan degradation in human intervertebral disc and articular cartilage.

R Sztrolovics 1, M Alini 1, P J Roughley 1, J S Mort 1
PMCID: PMC1218660  PMID: 9337874

Abstract

Aggrecan degradation in human intervertebral disc and articular cartilage has been studied by using anti-neoepitope antibodies specific for the N-terminal degradation products generated by cleavage within the interglobular domain at the metalloproteinase and aggrecanase sites. Immunoblot analysis of extracts of annulus fibrosus, nucleus pulposus and articular cartilage demonstrated age-related patterns in the abundance of both degradation products. In all three tissues the metalloproteinase-generated fragment was present at very low levels in young individuals but increased in abundance with age. In the disc tissues, the abundance of this degradation product levelled off in the juvenile; for cartilage this occurred in early adulthood. Despite these temporal differences, the levels attained in adults were comparable for the three tissues. In contrast, the aggrecanase-generated degradation product exhibited tissue-specific differences in the variation of its abundance with age. Whereas this degradation product increased with age in annulus fibrosus and articular cartilage and had levelled off by adulthood, in nucleus pulposus it was present in greatest abundance in young individuals and decreased to very low levels with age. Examination of discs exhibiting various degrees of degeneration did not reveal any differences in the levels of the metalloproteinase and aggrecanase-generated cleavage products that could not be accounted for by differences in age. In adults the product of aggrecanase action was much more abundant in articular cartilage than in either of the disc tissues, despite the age-related increase also observed for annulus fibrosus. Analysis of tissue extracts with an antibody recognizing the G1 domain of aggrecan identified two major degradation products whose abundance and size were correlated with the fragments detected by the anti-neoepitope antibodies. Taken together, these results indicate that cleavage at the metalloproteinase and aggrecanase sites are quantitatively important events in aggrecan catabolism in both articular cartilage and intervertebral disc in vivo. Moreover the two enzyme systems act independently and exhibit differences in the degree to which they contribute to aggrecan degradation in these tissues.

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Selected References

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  1. 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]
  2. Bernatowicz M. S., Matsueda G. R. Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent. Anal Biochem. 1986 May 15;155(1):95–102. doi: 10.1016/0003-2697(86)90231-9. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Doege K., Sasaki M., Horigan E., Hassell J. R., Yamada Y. Complete primary structure of the rat cartilage proteoglycan core protein deduced from cDNA clones. J Biol Chem. 1987 Dec 25;262(36):17757–17767. [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. Hardingham T. E., Fosang A. J. Proteoglycans: many forms and many functions. FASEB J. 1992 Feb 1;6(3):861–870. [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. King J., Laemmli U. K. Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol. 1971 Dec 28;62(3):465–477. doi: 10.1016/0022-2836(71)90148-3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. Mach L., Schwihla H., Stüwe K., Rowan A. D., Mort J. S., Glössl J. Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself. Biochem J. 1993 Jul 15;293(Pt 2):437–442. doi: 10.1042/bj2930437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Oegema T. R., Jr Biochemistry of the intervertebral disc. Clin Sports Med. 1993 Jul;12(3):419–439. [PubMed] [Google Scholar]
  21. Roughley P. J., White R. J. Age-related changes in the structure of the proteoglycan subunits from human articular cartilage. J Biol Chem. 1980 Jan 10;255(1):217–224. [PubMed] [Google Scholar]
  22. Roughley P. J., White R. J., Magny M. C., Liu J., Pearce R. H., Mort J. S. Non-proteoglycan forms of biglycan increase with age in human articular cartilage. Biochem J. 1993 Oct 15;295(Pt 2):421–426. doi: 10.1042/bj2950421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roughley P. J., White R. J., Mort J. S. Presence of pro-forms of decorin and biglycan in human articular cartilage. Biochem J. 1996 Sep 15;318(Pt 3):779–784. doi: 10.1042/bj3180779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Thompson J. P., Pearce R. H., Schechter M. T., Adams M. E., Tsang I. K., Bishop P. B. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila Pa 1976) 1990 May;15(5):411–415. doi: 10.1097/00007632-199005000-00012. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Walcz E., Deák F., Erhardt P., Coulter S. N., Fülöp C., Horvath P., Doege K. J., Glant T. T. Complete coding sequence, deduced primary structure, chromosomal localization, and structural analysis of murine aggrecan. Genomics. 1994 Jul 15;22(2):364–371. doi: 10.1006/geno.1994.1396. [DOI] [PubMed] [Google Scholar]

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