Skip to main content
PMC home page
Molecular Pathology : MP logoLink to Molecular Pathology : MP
. 1999 Dec;52(6):323–331. doi: 10.1136/mp.52.6.323

Differential detection of type II collagen N-terminal and C-terminal denaturation epitopes in degrading cartilage.

L J Croucher 1, A P Hollander 1
PMCID: PMC395717  PMID: 10748865

Abstract

AIMS: To investigate the relative stability of collagen metabolites in degrading cartilage. METHODS: New antipeptide antibodies to denaturation epitopes located in the N-terminal and C-terminal regions of the type II collagen helix have been made and characterized. Type II collagen fragments in the conditioned medium from cultures of degrading bovine nasal cartilage were detected by immunoblotting with the new antisera as well as by N-terminal sequencing. The antibodies were also used in immunohistochemical studies of normal and osteoarthritic human cartilage. RESULTS: Type II collagen fragments with an apparent molecular mass of approximately 30 kDa were detected in cartilage conditioned media using antibody AH12L3, which recognizes N-terminal epitope AH12. The N-terminal sequence of one of these fragments matched exactly a sequence in the N-terminal region of type II collagen. Antibody AH9L2, which recognizes C-terminal epitope AH9, did not bind to any protein bands in the immunoblotted culture medium. In immunohistochemical studies, antibody AH12L3 detected extensive regions of degraded collagen in osteoarthritic cartilage and a more restricted pattern of staining in nonarthritic cartilage. Far less immunostaining was apparent in all cartilage specimens with antibody AH9L2. CONCLUSIONS: These results indicate that the N-terminal region of type II collagen is more resistant to proteolysis than the C-terminal region, an observation that has important implications for the choice of epitopes that are likely to be good markers of damage to cartilage collagen in patients with arthritis.

Full Text

The Full Text of this article is available as a PDF (231.2 KB).

Selected References

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

  1. Bateman J. F., Chan D., Moeller I., Hannagan M., Cole W. G. A 5' splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated pro alpha 1(I) chains with a non-collagenous insertion destabilizing the triple helix. Biochem J. 1994 Sep 15;302(Pt 3):729–735. doi: 10.1042/bj3020729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Billinghurst R. C., Dahlberg L., Ionescu M., Reiner A., Bourne R., Rorabeck C., Mitchell P., Hambor J., Diekmann O., Tschesche H. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest. 1997 Apr 1;99(7):1534–1545. doi: 10.1172/JCI119316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birkedal-Hansen H., Moore W. G., Bodden M. K., Windsor L. J., Birkedal-Hansen B., DeCarlo A., Engler J. A. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4(2):197–250. doi: 10.1177/10454411930040020401. [DOI] [PubMed] [Google Scholar]
  4. Danielsen C. C. Thermal stability of human-fibroblast-collagenase-cleavage products of type-I and type-III collagens. Biochem J. 1987 Nov 1;247(3):725–729. doi: 10.1042/bj2470725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dodge G. R., Pidoux I., Poole A. R. The degradation of type II collagen in rheumatoid arthritis: an immunoelectron microscopic study. Matrix. 1991 Nov;11(5):330–338. doi: 10.1016/s0934-8832(11)80204-0. [DOI] [PubMed] [Google Scholar]
  6. Dodge G. R., Poole A. R. Immunohistochemical detection and immunochemical analysis of type II collagen degradation in human normal, rheumatoid, and osteoarthritic articular cartilages and in explants of bovine articular cartilage cultured with interleukin 1. J Clin Invest. 1989 Feb;83(2):647–661. doi: 10.1172/JCI113929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunbar B., Wilson S. B. A buffer exchange procedure giving enhanced resolution to polyacrylamide gels prerun for protein sequencing. Anal Biochem. 1994 Jan;216(1):227–228. doi: 10.1006/abio.1994.1029. [DOI] [PubMed] [Google Scholar]
  8. Ellis A. J., Curry V. A., Powell E. K., Cawston T. E. The prevention of collagen breakdown in bovine nasal cartilage by TIMP, TIMP-2 and a low molecular weight synthetic inhibitor. Biochem Biophys Res Commun. 1994 May 30;201(1):94–101. doi: 10.1006/bbrc.1994.1673. [DOI] [PubMed] [Google Scholar]
  9. Fields G. B. A model for interstitial collagen catabolism by mammalian collagenases. J Theor Biol. 1991 Dec 21;153(4):585–602. doi: 10.1016/s0022-5193(05)80157-2. [DOI] [PubMed] [Google Scholar]
  10. Freije J. M., Díez-Itza I., Balbín M., Sánchez L. M., Blasco R., Tolivia J., López-Otín C. Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J Biol Chem. 1994 Jun 17;269(24):16766–16773. [PubMed] [Google Scholar]
  11. Gross J., Nagai Y. Specific degradation of the collagen molecule by tadpole collagenolytic enzyme. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1197–1204. doi: 10.1073/pnas.54.4.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Highberger J. H., Corbett C., Gross J. Isolation and characterization of a peptide containing the site of cleavage of the chick skin collagen alpha 1[I] chain by animal collagenases. Biochem Biophys Res Commun. 1979 Jul 12;89(1):202–208. doi: 10.1016/0006-291x(79)90964-1. [DOI] [PubMed] [Google Scholar]
  13. Hollander A. P., Heathfield T. F., Webber C., Iwata Y., Bourne R., Rorabeck C., Poole A. R. Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay. J Clin Invest. 1994 Apr;93(4):1722–1732. doi: 10.1172/JCI117156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hollander A. P., Pidoux I., Reiner A., Rorabeck C., Bourne R., Poole A. R. Damage to type II collagen in aging and osteoarthritis starts at the articular surface, originates around chondrocytes, and extends into the cartilage with progressive degeneration. J Clin Invest. 1995 Dec;96(6):2859–2869. doi: 10.1172/JCI118357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Kafienah W., Brömme D., Buttle D. J., Croucher L. J., Hollander A. P. Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix. Biochem J. 1998 May 1;331(Pt 3):727–732. doi: 10.1042/bj3310727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kobayashi I., Ziff M. Electron microscopic studies of the cartilage-pannus junction in rheumatoid arthritis. Arthritis Rheum. 1975 Sep-Oct;18(5):475–483. doi: 10.1002/art.1780180507. [DOI] [PubMed] [Google Scholar]
  18. Kozaci L. D., Buttle D. J., Hollander A. P. Degradation of type II collagen, but not proteoglycan, correlates with matrix metalloproteinase activity in cartilage explant cultures. Arthritis Rheum. 1997 Jan;40(1):164–174. doi: 10.1002/art.1780400121. [DOI] [PubMed] [Google Scholar]
  19. Mankin H. J., Dorfman H., Lippiello L., Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am. 1971 Apr;53(3):523–537. [PubMed] [Google Scholar]
  20. Miller E. J., Finch J. E., Jr, Chung E., Butler W. T., Robertson P. B. Specific cleavage of the native type III collagen molecule with trypsin. Similarity of the cleavage products to collagenase-produced fragments and primary structure at the cleavage site. Arch Biochem Biophys. 1976 Apr;173(2):631–637. doi: 10.1016/0003-9861(76)90300-3. [DOI] [PubMed] [Google Scholar]
  21. Mitchell P. G., Magna H. A., Reeves L. M., Lopresti-Morrow L. L., Yocum S. A., Rosner P. J., Geoghegan K. F., Hambor J. E. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest. 1996 Feb 1;97(3):761–768. doi: 10.1172/JCI118475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murphy G. Matrix metalloproteinases and their inhibitors. Acta Orthop Scand Suppl. 1995 Oct;266:55–60. [PubMed] [Google Scholar]
  23. 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]
  24. Sakai T., Gross J. Some properties of the products of reaction of tadpole collagenase with collagen. Biochemistry. 1967 Feb;6(2):518–528. doi: 10.1021/bi00854a021. [DOI] [PubMed] [Google Scholar]
  25. Vankemmelbeke M., Dekeyser P. M., Hollander A. P., Buttle D. J., Demeester J. Characterization of helical cleavages in type II collagen generated by matrixins. Biochem J. 1998 Mar 1;330(Pt 2):633–640. doi: 10.1042/bj3300633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Venn M., Maroudas A. Chemical composition and swelling of normal and osteoarthrotic femoral head cartilage. I. Chemical composition. Ann Rheum Dis. 1977 Apr;36(2):121–129. doi: 10.1136/ard.36.2.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vitagliano L., Némethy G., Zagari A., Scheraga H. A. Structure of the type I collagen molecule based on conformational energy computations: the triple-stranded helix and the N-terminal telopeptide. J Mol Biol. 1995 Mar 17;247(1):69–80. doi: 10.1006/jmbi.1994.0123. [DOI] [PubMed] [Google Scholar]
  28. Wu J. J., Lark M. W., Chun L. E., Eyre D. R. Sites of stromelysin cleavage in collagen types II, IX, X, and XI of cartilage. J Biol Chem. 1991 Mar 25;266(9):5625–5628. [PubMed] [Google Scholar]
  29. Xu C., Oyajobi B. O., Frazer A., Kozaci L. D., Russell R. G., Hollander A. P. Effects of growth factors and interleukin-1 alpha on proteoglycan and type II collagen turnover in bovine nasal and articular chondrocyte pellet cultures. Endocrinology. 1996 Aug;137(8):3557–3565. doi: 10.1210/endo.137.8.8754787. [DOI] [PubMed] [Google Scholar]

Articles from Molecular Pathology are provided here courtesy of BMJ Publishing Group

RESOURCES