Advertisement

Proteoglycans pp 219-237 | Cite as

Analysis of Aggrecan Catabolism by Immunoblotting and Immunohistochemistry

  • Peter J. RoughleyEmail author
  • John S. Mort
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 836)

Abstract

Aggrecan is essential for the normal function of articular cartilage and intervertebral disc, where it provides the ability for the tissues to withstand compressive loading. This property depends on both the high charge density endowed by its numerous chondroitin sulfate and keratan sulfate chains and its ability to form large molecular aggregates via interaction with hyaluronan. Degradation of aggrecan via the action of proteases takes place throughout life and the degradation products accumulate in the tissue and impair its function. Such degradation is exacerbated in degenerative or inflammatory joint disorders. The use of antibodies recognizing the various regions of aggrecan and the neoepitopes generated upon proteolytic cleavage has shown that matrix metalloproteinases and aggrecanases, members of the ADAMTS family, are responsible for aggrecan degradation, both throughout life and in disease. By using immunoblotting techniques, it is possible to determine the extent of aggrecan degradation and to identify the degradation products that have accumulated in the tissue, and immunohistochemistry allows the location of the aggrecan degradation to be established.

Key words

Aggrecan Cartilage Intervertebral disc Degradation Matrix metalloproteinase Aggrecanase Neoepitope Immunoblotting Immunohistochemistry 

Notes

Acknowledgments

The authors would like to thank Lisa Lamplugh for technical assistance with the immunoblotting and immunohistochemistry and Guylaine Bédard for the artwork. The work was supported by the Shriners of North America and the Canadian Institutes of Health Research.

References

  1. 1.
    Hascall, V. C. (1988) Proteoglycans: the chondroitin sulfate/keratan sulfate proteoglycan of cartilage. ISI Atlas Sci. Biochem. 1, 189–198.Google Scholar
  2. 2.
    Watanabe, H., Yamada, Y., and Kimata, K. (1998) Roles of aggrecan, a large chondroitin sulfate proteoglycan, in cartilage structure and function. J. Biochem. (Tokyo) 124, 687–693.Google Scholar
  3. 3.
    Johnson, W. E. B., Caterson, B., Eisenstein, S. M., and Roberts, S. (2005) Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro. Spine 30, 1139–1147.PubMedCrossRefGoogle Scholar
  4. 4.
    Doege, K. J., Sasaki, M., Kimura, T., and Yamada, Y. (1991) 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. 266, 894–902.PubMedGoogle Scholar
  5. 5.
    Neame, P. J. and Barry, F. P. (1993) The link proteins. Experientia 49, 393–402.PubMedCrossRefGoogle Scholar
  6. 6.
    Fosang, A. J. and Hardingham, T. E. (1989) Isolation of the N-terminal globular protein domains from cartilage proteoglycans. Identification of G2 domain and its lack of interaction with hyaluronate and link protein. Biochem. J. 261, 801–809.PubMedGoogle Scholar
  7. 7.
    Zheng, J., Luo, W., and Tanzer, M. L. (1998) Aggrecan synthesis and secretion - A paradigm for molecular and cellular coordination of multiglobular protein folding and intracellular trafficking. J. Biol. Chem. 273, 12999–13006.PubMedCrossRefGoogle Scholar
  8. 8.
    Day, J. M., Olin, A. I., Murdoch, A. D., Canfield, A., Sasaki, T., Timpl, R., Hardingham, T. E., and Aspberg, A. (2004) Alternative splicing in the aggrecan G3 domain influences binding interactions with tenascin-C and other extracellular matrix proteins. J. Biol. Chem. 279, 12511–12518.PubMedCrossRefGoogle Scholar
  9. 9.
    Doege, K. J., Coulter, S. N., Meek, L. M., Maslen, K., and Wood, J. G. (1997) A human-specific polymorphism in the coding region of the aggrecan gene - Variable number of tandem repeats produce a range of core protein sizes in the general population. J. Biol. Chem. 272, 13974–13979.PubMedCrossRefGoogle Scholar
  10. 10.
    Roughley, P., Martens, D., Rantakokko, J., Alini, M., Mwale, F., and Antoniou, J. (2006) The involvement of aggrecan polymorphism in degeneration of human intervertebral disc and articular cartilage. Eur. Cell Mater. 11, 1–7.PubMedGoogle Scholar
  11. 11.
    Roughley, P. J. and Mort, J. S. (1986) Ageing and the aggregating proteoglycans of human articular cartilage. Clin. Sci. 71, 337–344.PubMedGoogle Scholar
  12. 12.
    Nagase, H., Visse, R., and Murphy, G. (2006) Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc. Res. 69, 562–573.PubMedCrossRefGoogle Scholar
  13. 13.
    Jones, G. C. and Riley, G. P. (2005) ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis. Arthritis Res. Ther. 7, 160–169.PubMedCrossRefGoogle Scholar
  14. 14.
    Fosang, A. J., Neame, P. J., Last, K., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1992) The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B. J. Biol. Chem. 267, 19470–19474.PubMedGoogle Scholar
  15. 15.
    Tortorella, M. D., Pratta, M., Liu, R. Q., Austin, J., Ross, O. H., Abbaszade, I., et al. (2000) Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566–18573.PubMedCrossRefGoogle Scholar
  16. 16.
    Sivan, S. S., Tsitron, E., Wachtel, E., Roughley, P. J., Sakkee, N., Van der Ham, F., et al. (2006) Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid. J. Biol. Chem. 281, 13009–13014.PubMedCrossRefGoogle Scholar
  17. 17.
    Margolis, R. U. and Margolis, R. K. (1994) Aggrecan-versican-neurocan family of proteoglycans. Methods Enzymol. 245, 105–128.Google Scholar
  18. 18.
    Spicer, A. P., Joo, A., and Bowling, R. A., Jr. (2003) A hyaluronan binding link protein gene family whose members are physically linked adjacent to chondroitin sulfate proteoglycan core protein genes - The missing links. J. Biol. Chem. 278, 21083–21091.PubMedCrossRefGoogle Scholar
  19. 19.
    Schwartz, N. B. and Domowicz, M. (2004) Proteoglycans in brain development. Glycoconjugate J. 21, 329–341.CrossRefGoogle Scholar
  20. 20.
    Barry, F. P., Neame, P. J., Sasse, J., and Pearson, D. (1994) Length variation in the keratan sulfate domain of mammalian aggrecan. Matrix 14, 323–328.CrossRefGoogle Scholar
  21. 21.
    Mort, J. S. and Roughley, P. J. (2004) Production of antibodies against degradative neoepitopes in aggrecan. Methods Mol. Med. 100, 237–250.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Research UnitShriners Hospital for ChildrenMontrealCanada

Personalised recommendations