). Computer analysis of our sequence revealed an open reading frame coding for a protein of 1595 amino acid residues with a predicted molecular mass of 175,814 Dalton and a 3' untranslated region (218 bp) followed by a poly(A) tail. The catalytic domain includes the consensus sequence HEXXHXXGXXHD (at positions 369–381) involved in the coordination of the catalytic zinc atom at the active site of metalloproteinases.
2. ADAMTS-7 exhibited higher expression in musculoskeletal tissues, and its concentration was found to be up-regulated in the cartilage and synovium of patients with rheumatoid arthritis
An RT-PCR assay was performed to examine the expression of ADAMTS-7 mRNA in eight specimens of normal (N) human musculoskeletal tissue. A 167 bp hADAMTS-7 fragment was amplified from bone, cartilage, synovium, tendon, and ligament, in which COMP was also present. ADAMTS-7 is also detectable, although at lower levels, in meniscus, skeletal muscle, and fat. These results demonstrate that although ADAMTS-7 is coexpressed in COMP-producing musculoskeletal tissues, it has a wider tissue distribution. Quantitative real-time PCR was performed to determine whether the expression of ADAMTS-7 in cartilage and synovium is altered in osteoarthritis (OA) and/or rheumatoid arthritis (RA). ADAMTS-7 mRNA was significantly up-regulated in RA cartilage and only slightly up-regulated in OA cartilage, compared to N controls. Further analysis of synovium samples revealed that the concentration of ADAMTS-7 was also significantly up-regulated in RA synovium compared to N synovium.
3. COMP binds to ADAMTS-7 in vitro and in vivo
The interaction between COMP and ADAMTS-7 first revealed in our Y2H assay was confirmed using an in vitro glutathione S-transferase (GST) pulldown assay. Briefly, affinity-purified GST or purified rADAMTS-7 C-terminal (aa 1139–1595) as a GST fusion protein (GST7-CT) was immobilized on beads and incubated with purified hCOMP; after being washed, bound protein was detected by Western blotting. Purified GST did not pull down hCOMP, whereas GST7-CT efficiently pulled down hCOMP (Fig. 1
A), indicating that hCOMP directly binds to the C-terminal of ADAMTS-7; in addition, this association was significantly enhanced by the addition of divalent cations (5 mM Ca2+). The interaction of COMP and ADAMTS-7 was also characterized by titration experiments in which COMP showed dose-dependent binding and saturation to the dilution series of solid-phase His-TS7C4TSP (Fig. 1B
).
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Figure 1. COMP associates with ADAMTS-7. A) GST pulldown assay. Purified GST or GST7-CT fusion protein immobilized on beads was incubated with purified hCOMP in the presence or absence of 5 mM Ca2+. Proteins trapped were examined by immunoblotting with anti-COMP antibodies. Purified COMP (lane 1) was used as a positive control. B) Solid-phase assay. Various amounts of His-TS7C4TSP were immobilized on solid-phase plates. After being blocked, COMP was added to each well, followed by addition of 10 mM CaCl2. Bound protein from the liquid phase was detected using anti-COMP antibodies. C) CO-IP assay. Cartilage extracts were incubated with either anti-COMP antibodies or control IgG, and immunoprecipitated protein complex and cartilage extracts (serving as a positive control) were examined by Western blotting with an anti-ADAMTS-7 antibody.
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In vivo interaction between COMP and ADAMTS-7 was verified using a coimmunoprecipitation assay. Cartilage extracts were first incubated with either anti-COMP antiserum or control IgG, and the immunoprecipitated complexes were subjected to reducing SDS-PAGE and detected with anti-ADAMTS-7 antibodies. A specific ADAMTS-7 band was present in the immunoprecipitated complexes brought down by anti-COMP but not control IgG antibodies, demonstrating that ADAMTS-7 specifically binds to COMP in vivo (Fig. 1C
).
4. The EGF-like domain of COMP selectively associates with the four C-terminal TSP motifs of ADAMTS-7
After ADAMTS-7 was identified as a COMP-binding protein using the Y2H screen, we sought to establish whether domains of COMP in addition to the EGF domain associate with ADAMTS-7. A filter-based ßbeta;-galactosidase assay showed that ADAMTS-7 selectively interacts with the EGF-like domain of COMP. The same assay was performed to identify the COMP-binding motif in ADAMTS-7 using various constructs that expressed various ADAMTS-7 deletion mutants in yeast. Our conclusion from this set of experiments is that four C-terminal TSP repeats of ADAMTS-7 are required and sufficient for its interaction with COMP.
5. COMP is cleaved by the recombinant ADAMTS-7
The catalytic domain of ADAMTS-20 produced in bacteria was found to digest its substrates in vitro. Using a similar method, we purified the catalytic domain (aa 217–468) of hADAMTS-7 as a GST fusion protein (GST-TS7-CT) in bacteria. A GST moiety was further removed by factor X cleavage, and the purity of the proteins was confirmed by visualization using silver staining. The recombinant catalytic domain of ADAMTS-7 was incubated with purified human COMP in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 2 mM ZnCl2, and 0.05% Brij-35, pH 7.5. Our data shows that the catalytic domain of ADAMTS-7 digested COMP in a dose- and time-dependent manner.
The COMP-degrading activity of ADAMTS-7 was also demonstrated by using the recombinant intact enzyme: control cell lysates did not show any enzymatic activity, whereas cell lysates prepared from ADAMTS-7 bacluovirus-infected insect cells did cleave COMP in the presence of both lower (0.1 mM) and higher (2.0 mM) concentrations of Zn. In addition, the cleavage of COMP by ADAMTS-7 was blocked in the presence of 5 mM EDTA chelator. Two additional fragments between 95 and 51 kDa were seen in addition to the 100 kDa fragment that was predominantly produced with the catalytic domain alone, suggesting that intact enzyme may cleave COMP at more than one site. The COMP productsdegraded by ADAMTS-7 appear to have a molecular mass similar to some of the more prominent fragments that have been resolved from the synovial fluid of patients with arthritic diseases.
Divalent cations are crucial for the enzymatic activity of metalloproteinases. To further determine whether ADAMTS-7-mediated COMP cleavage requires the involvement of cations, purified COMP substrate and ADAMTS-7 enzyme were incubated in digestion buffer in the presence or absence of various cations (Ca2+, Zn2+, and/or Mg2+). A degraded COMP fragment was detectable in the digestion buffer with Zn2+ but was undetectable in the digestion buffer with either Ca2+ or Mg2+ used alone. In the presence of Zn2+, the addition of Ca2+ changed the electrophoretic mobility of the COMP-digested fragment, probably due to a conformation change in COMP. Mg2+ seemed not to affect COMP digestion by ADAMTS-7, since it did not affect the electrophoretic mobility of the COMP-digested fragment when used alone or in combination with other cations.
Digestions were performed at various values of pH to determine its effect in regulating ADAMTS-7 activity. ADAMTS-7 generated the largest amount of COMP fragments in the range of physiological pH (pH 7.5) up to pH 9.5; the enzyme did not produce visible COMP fragments at pH values <6.5 or >10.5, indicating that the digestive activity of ADAMTS-7 is pH dependent.
CONCLUSIONS AND SIGNIFICANCE
A Y2H screen was used to identify a protein interaction partner of COMP, an extracellular matrix (ECM) protein that is prominent in cartilage and undergoes degradation in arthritic diseases. The present study demonstrated that ADAMTS-7 binds to the EGF repeat domain of COMP via its four C-terminal TSP motifs and that recombinant ADAMTS-7 can digest COMP in a dose-, time-, cation-, and pH-dependent manner. In view of the fact that prominent COMP degradative fragments in arthritic conditions are produced by cleavage within the EGF-like molecular domain of COMP, the binding of ADAMTS-7 to this same region with subsequent COMP cleavage is strong evidence that ADAMTS-7 plays an important role in COMP degradation. Based on these findings, we propose a working model to explain how COMP is degraded by ADAMTS-7: in vivo COMP forms a pentamer (apparent molecular mass of 
550 kDa) via its N-terminal pentamerizing domain, and the enzyme (five molecules of ADAMTS-7) captures this pentamer via the direct binding of its tail (four C-terminal TSP-1 repeats) to the EGF-like domain of COMP and cleaves this region through its head catalytic domain (Fig. 2
).
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Figure 2. Schematic of proposed model of cleavage of COMP by ADAMTS-7. The catalytic domain of ADAMTS-7 is indicated by arrowheads.
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Prominent degradative fragments of COMP have been observed in patients with OA and RA; the enzyme(s) responsible for this degradation, however, were previously unknown. Purified COMP has been reported to be digested by several metalloproteinases, including interstitial collagenase (MMP-1), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), gelatinase-B (MMP-9), MMP-19, enamelysin (MMP-20), and aggrecanase-1(ADAMTS-4); in all cases, however, the assays were performed using an in vitro digestion system in which both enzymes and substrates were at higher concentrations than those in physiological/pathological conditions. None of these metalloproteinases has been found capable of associating with COMP, which is probably important for COMP degradation in vivo. To our knowledge, ADAMTS-7 is the first enzyme found to directly interact with and degrade COMP.
The identification of ADAMTS-7 as a COMP-binding protein and subsequent characterization of the enzyme/substrate association and of COMP degradation mediated by ADAMTS-7 significantly extend our understanding of the degradative events that occur in arthritic disorders and promise to increase our ability to monitor the biological and physical properties of the cartilage ECM.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3877fje
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K. Xu, Y. Zhang, K. Ilalov, C. S. Carlson, J. Q. Feng, P. E. Di Cesare, and C.-j. Liu
Cartilage Oligomeric Matrix Protein Associates with Granulin-Epithelin Precursor (GEP) and Potentiates GEP-stimulated Chondrocyte Proliferation
J. Biol. Chem.,
April 13, 2007;
282(15):
11347 - 11355.
[Abstract]
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
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