ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein

Abstract

Degradative fragments of cartilage oligomeric matrix protein (COMP) have been observed in arthritic patients. The physiological enzyme(s) that degrade COMP, however, remain unknown. We performed a yeast two-hybrid screen (Y2H) to search for proteins that associate with COMP to identify an interaction partner that might degrade it. One screen using the epidermal growth factor (EGF) domain of COMP as bait led to the discovery of ADAMTS-7. Rat ADAMTS-7 is composed of 1595 amino acids, and this protein exhibits higher expression in the musculoskeletal tissues. COMP binds directly to ADAMTS-7 in vitro and in native articular cartilage. ADAMTS-7 selectively interacts with the EGF repeat domain but not with the other three functional domains of COMP, whereas the four C-terminal TSP motifs of ADAMTS-7 are required and sufficient for association with COMP. The recombinant catalytic domain and intact ADAMTS-7 are capable of digesting COMP in vitro. The enzymatic activity of ADAMTS-7 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the concentration of ADAMTS-7 in cartilage and synovium of patients with rheumatoid arthritis is significantly increased as compared to normal cartilage and synovium. ADAMTS-7 is the first metalloproteinase found to bind directly to and degrade COMP.

Figure 1

ADAMTS-7 is expressed in human musculoskeletal tissues. Amplification products are consistent with a predicted size of 167 bp for hADAMTS-7 and 983 bp for hGAPDH. SKM = skeletal muscle; Lanes marked “M” contain DNA marker (Amersham Pharmacia Biotech, Piscataway, NJ).

Figure 2

Binding of COMP to ADAMTS-7 in assay to test interaction of proteins fused to the VP16 AD and proteins fused to the Gal4 DBD. Each pair of plasmids, as indicated, encoding proteins fused to VP16 (below the line) in vector pPC86 (i.e., pPC86-c-jun, pPC86-ADAMTS-7, and pPC86-Rb) and those encoding proteins fused to Gal4 (above the line) in the vector pDBleu (i.e., pDB-c-fos, pDB-COMP, and pDB-lamin) were cotransfected into yeast strain MAV203. Yeast transformants were selected on sd- leu^-/trp^- plates and tested for β-galactosidase activity (left panel), for growth on plates lacking histidine and uracil and containing 3AT (middle panel, sd-leu^-/trp^-/his^-/ura^-/3AT⁺), and for growth on plates containing 5-fluoroorotic acid (5FOA) (right panel, sd- leu^-/trp^-/ 5FOA⁺). The known interaction between c-Jun and c-Fos is used as a positive control, and the lack of interaction between Rb and lamin serves as a negative control.

Figure 3

COMP associates with ADAMTS-7 both in vitro and in vivo. A) GST pulldown assay. Purified GST (lane 2) or GST-TS7-CT fusion protein (lane 3 and 4) immobilized on GSH-Sepharose beads were incubated with purified hCOMP in the presence (lane 4) or absence (lane 3) of 5 mM Ca²⁺. Proteins trapped by C terminal of ADAMTS-7 fused to GST were examined by immunoblotting with anti-COMP antibodies. Purified COMP (lane 1) was used as a positive control. Arrow indicates full-length COMP band. B) Solid-phase assay. Various amounts of recombinant His tagged C-terminal 4 TSP motifs of ADAMTS7 (His-TS7C4TSP) were immobilized on solid-phase 96-well microtiter plates. After being blocked, COMP was added to each well, followed by the addition of 10 mM CaCl₂. Samples were then allowed to bind overnight at 4°C. Bound protein from liquid phase was detected using monoclonal antibodies to the bound COMP. C) Characterization of anti-ADAMTS-7 Ab. Cell lysates prepared from Sf9 insect cells infected with control (lane 1) or ADAMTS-7 bacluovirus (lane 2), from HEK293 cells stably transfected with a COMP expression construct (lane 3), and from MG-63 osteoblastic cells (lane 4) were subjected to SDS-PAGE and immunoblotted with affinity-purified anti-ADAMTS-7 antibodies. D) CO-IP assay. Cartilage extracts were incubated with either anti-COMP (lane 2) antiserum or control IgG (lane 3), followed by protein A/G agarose. Immunoprecipitated protein complex and cartilage extracts (lane 1, which provides a positive control) were examined by Western blotting with an anti-ADAMTS-7 Ab.

Figure 4

ADAMTS-7 selectively binds to the EGF-like domain of COMP. A) Schematic structure of COMP constructs used to map those domains (N-terminal, EGF-like, type III, and C-terminal) that bind to ADAMTS-7. Presence or absence of binding between COMP domains and ADAMTS-7 is indicated a “+” or “-,” respectively. B) β-Galactosidase activity was used to test interaction between the C-terminal domain of rADAMTS-7 and 1 of 4 domains of COMP. Three independent yeast transformants for each pair of plasmids were transferred onto a nitrocellulose membrane, and β-galactosidase activity was determined. The known interaction between c-Jun and c-Fos was used as a positive control, and the lack of interaction between Rb and lamin served as a negative control[b].

Figure 5

Four C-terminal TSP motifs are required and sufficient for interaction with COMP. A) Schematic diagram of ADAMTS-7 constructs used to map those of its fragments that bind to COMP. Numbers refer to amino acid residues in the ADAMTS-7; ovals = TSP motifs. Interactions between COMP and ADAMTS-7 derivatives are summarized and indicated by “Yes” or “No.” B) β-Galactosidase activity was used to test interaction between ADAMTS-7 derivatives and COMP. Three independent yeast transformants for each pair of plasmids were transferred onto a nitrocellulose membrane and the β-galactosidase activity determined.

Figure 6

ADAMTS-7 digests COMP in vitro. A) Purified GST-TS7-CD fusion protein (lane 1) and TS-7-CD without a GST moiety (lane 2) were separated by SDS-PAGE and visualized with silver staining. B) Catalytic domain of ADAMTS-7 digests COMP in a dose-dependent manner. Purified hCOMP was incubated with various amounts of catalytic domain of ADAMTS-7 (TS7-CD), as indicated; cleaved products were separated by nonreduced SDS-PAGE, and intact COMP and fragments were visualized with Coomassie brilliant blue G-colloidal solution. Resultant fragment is indicated by arrow. C) Catalytic domain of ADAMTS-7 digests COMP in a time-dependent manner. Purified hCOMP (200 nM) was incubated with recombinant catalytic domain of ADAMTS-7 (TS7-CD, 30 nM), and resultant products were analyzed as in B. D) Recombinant full-length ADAMTS-7 digests COMP in vitro. Purified COMP (200 nM) was incubated with the cell lystes prepared from Sf9 insect cells infected with either control (lane 1) or ADAMTS-7 bacluovirus in digestion buffer in the presence of indicated amounts of Zn²⁺ or 5 mM EDTA (lanes 2, 3, and 4), and nonreduced cleaved products were resolved by SDS-PAGE and detected by Western blotting with anti-COMP polyclonal antiserum.

Figure 7

Zn²⁺- and pH-dependence of ADAMTS-7-mediated COMP digestion. A) Zn²⁺ is essential for the enzymatic activity of ADAMTS-7. Purified COMP (250 nM) was incubated with purified catalytic domain of ADAMTS-7 (25 nM) in digestion buffer supplemented with 5 mM CaCl₂, 2 mM ZnCl₂, 2.5 mM MgCl₂, or a combination of these or in the presence of 5 mM EDTA, as indicated; the digested proteins were resolved by 10% nonreduced SDS-PAGE and the gel was stained with Coomassie brilliant blue G-colloidal solution. Resultant fragment is indicated by large arrow; small arrow indicates additional (lower) band in lane 5, which is probably due to the conformation change in the presence of Ca²⁺. B) Activity of ADAMTS-7 is pH dependent. The same digestion was performed in a buffer supplemented with 5 mM CaCl₂, 2 mM ZnCl₂, and 2.5 mM MgCl₂ at various pH, as indicated; digested proteins were resolved as in A.

Figure 8

Increased expression of ADAMTS-7 in cartilage and synovium of RA patients. A) Expression of ADAMTS-7 in normal, OA, and RA cartilage assayed by real-time PCR; expression of ADAMTS-7 in each sample was normalized against 18S rRNA endogenous control. Normalized values were then calibrated against the normal cartilage value. B) Expression of ADAMTS-7 in normal and RA synovium. Samples were processed and data analyzed as in A. Units are arbitrary, and leftmost bar in each panel indicates a relative concentration of ADAMTS-7 of 1. ^***P < 0.001 vs. N control.