• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Feb 1, 1995; 305(Pt 3): 799–804.
PMCID: PMC1136329

Monoclonal antibodies that specifically recognize neoepitope sequences generated by 'aggrecanase' and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro.


Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, 'aggrecanase', and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of 'aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune-reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that 'aggrecanase' activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than 'aggrecanase' are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited 'aggrecanase' activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Paulsson M, Mörgelin M, Wiedemann H, Beardmore-Gray M, Dunham D, Hardingham T, Heinegård D, Timpl R, Engel J. Extended and globular protein domains in cartilage proteoglycans. Biochem J. 1987 Aug 1;245(3):763–772. [PMC free article] [PubMed]
  • Hardingham TE, Muir H. The specific interaction of hyaluronic acid with cartillage proteoglycans. Biochim Biophys Acta. 1972 Sep 15;279(2):401–405. [PubMed]
  • Heinegård D, Hascall VC. Aggregation of cartilage proteoglycans. 3. Characteristics of the proteins isolated from trypsin digests of aggregates. J Biol Chem. 1974 Jul 10;249(13):4250–4256. [PubMed]
  • Hardingham TE. The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem J. 1979 Jan 1;177(1):237–247. [PMC free article] [PubMed]
  • Flannery CR, Lark MW, Sandy JD. 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]
  • Sandy JD, Flannery CR, Neame PJ, Lohmander LS. 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. [PMC free article] [PubMed]
  • Lohmander LS, Neame PJ, Sandy JD. 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. [PubMed]
  • Sandy JD, Boynton RE, Flannery CR. Analysis of the catabolism of aggrecan in cartilage explants by quantitation of peptides from the three globular domains. J Biol Chem. 1991 May 5;266(13):8198–8205. [PubMed]
  • Sandy JD, Neame PJ, Boynton RE, Flannery CR. 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]
  • Loulakis P, Shrikhande A, Davis G, Maniglia CA. 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. [PMC free article] [PubMed]
  • Ilic MZ, Handley CJ, Robinson HC, Mok MT. Mechanism of catabolism of aggrecan by articular cartilage. Arch Biochem Biophys. 1992 Apr;294(1):115–122. [PubMed]
  • Doege KJ, 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]
  • Fosang AJ, Neame PJ, Last K, Hardingham TE, Murphy G, Hamilton JA. 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]
  • Fosang AJ, Last K, Knäuper V, Neame PJ, Murphy G, Hardingham TE, Tschesche H, Hamilton JA. Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain. Biochem J. 1993 Oct 1;295(Pt 1):273–276. [PMC free article] [PubMed]
  • Hughes CE, Caterson B, White RJ, Roughley PJ, Mort JS. 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]
  • Caterson B, Christner JE, Baker JR. Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan. J Biol Chem. 1983 Jul 25;258(14):8848–8854. [PubMed]
  • Farndale RW, Sayers CA, Barrett AJ. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res. 1982;9(4):247–248. [PubMed]
  • Ratcliffe A, Doherty M, Maini RN, Hardingham TE. 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. [PMC free article] [PubMed]
  • Roughley PJ, Barrett AJ. The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis. Biochem J. 1977 Dec 1;167(3):629–637. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Fosang AJ, Hardingham TE. 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. 1989 Aug 1;261(3):801–809. [PMC free article] [PubMed]
  • Schnierer S, Kleine T, Gote T, Hillemann A, Knäuper V, Tschesche H. The recombinant catalytic domain of human neutrophil collagenase lacks type I collagen substrate specificity. Biochem Biophys Res Commun. 1993 Mar 15;191(2):319–326. [PubMed]
  • Fairbanks G, Steck TL, Wallach DF. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. [PubMed]
  • Fosang AJ, Neame PJ, Hardingham TE, Murphy G, Hamilton JA. Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins. J Biol Chem. 1991 Aug 25;266(24):15579–15582. [PubMed]
  • Roughley PJ, White RJ, Poole AR. Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage. Biochem J. 1985 Oct 1;231(1):129–138. [PMC free article] [PubMed]
  • Fosang AJ, Last K, Neame PJ, Murphy G, Knäuper V, Tschesche H, Hughes CE, Caterson B, Hardingham TE. 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. [PMC free article] [PubMed]

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


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links