ADAMTS4 (Aggrecanase-1) Interaction with the C-terminal Domain of Fibronectin Inhibits Proteolysis of Aggrecan
2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês
10.1074/jbc.m314216200
ISSN1083-351X
AutoresGakuji Hashimoto, Masayuki Shimoda, Yasunori Okada,
Tópico(s)Platelet Disorders and Treatments
ResumoADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (adisintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including ΔSp (ΔArg693–Lys837, lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC50 = 110 nm), whereas no inhibition was observed with ΔSp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC50 = 170 nm). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage. ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (adisintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including ΔSp (ΔArg693–Lys837, lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC50 = 110 nm), whereas no inhibition was observed with ΔSp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC50 = 170 nm). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage. Members of the ADAMTS (adisintegrin and metalloproteinase with thrombospondin motifs) gene family are composed of at least 19 molecules (1Llamazares M. Cal S. Quesada V. Lopez-Otin C. J. Biol. Chem. 2003; 278: 13382-13389Google Scholar) and are involved in various biological and biochemical events such as fertilization, proteoglycan degradation, processing of fibrillar collagens, and intravascular coagulation (2Shindo T. Kurihara H. Kuno K. Yokoyama H. Wada T. Kurihara Y. Imai T. Wang Y. Ogata M. Nishimatsu H. Moriyama N. Oh-hashi Y. Morita H. Ishikawa T. Nagai R. Yazaki Y. Matsushima K. J. Clin. Investig. 2000; 105: 1345-1352Google Scholar, 3Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R.-Q. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Newton R.C. Magolda R.L. Trzaskos J.M. Arner E.C. 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Among them, ADAMTS1, ADAMTS4 (also referred to as aggrecanase-1), ADAMTS5 (aggrecanase-2), and ADAMTS9 cleave specific Glu–X bonds (where X is most often Ala or Gly) of the core protein of aggrecan, a major proteoglycan in articular cartilage (3Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R.-Q. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Newton R.C. Magolda R.L. Trzaskos J.M. Arner E.C. Science. 1999; 284: 1664-1666Google Scholar, 6Tortorella M.D. Malfait A.M. Deccico C. Arner E. Osteoarthritis Cartilage. 2001; 9: 539-552Google Scholar, 7Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Google Scholar). Previous studies suggested that the major aggrecan fragments found in vitro in response to cytokine-stimulated cartilage degradation and in vivo in arthritic joint fluids are generated through cleavage at Glu–X bonds by the glutamyl endopeptidase activity of these AD-AMTS members (8Lark M.W. Gordy J.T. Weidner J.R. Ayala J. Kimura J.H. Williams H.R. Mumford R.A. Flannery C.R. Carlson S.S. Iwata M. Sandy J.D. J. Biol. Chem. 1995; 270: 2550-2556Google Scholar, 9Lohmander L.S. Neame P.J. Sandy J.D. Arthritis Rheum. 1993; 36: 1214-1222Google Scholar). Recent studies further demonstrated that ADAMTS4 is induced by stimulation of chondrocytes and synovial cells with interleukin-1, tumor necrosis factor-α, or transforming growth factor-β, although ADAMTS5 is constitutively expressed (6Tortorella M.D. Malfait A.M. Deccico C. Arner E. Osteoarthritis Cartilage. 2001; 9: 539-552Google Scholar, 10Yamanishi Y. Boyle D.L. Clark M. Maki R.A. Tortorella M.D. Arner E.C. Firestein G.S. J. Immunol. 2002; 168: 1405-1412Google Scholar). In addition, ADAMTS4 is overexpressed by synovial cells and chondrocytes in osteoarthritis and rheumatoid arthritis (10Yamanishi Y. Boyle D.L. Clark M. Maki R.A. Tortorella M.D. Arner E.C. Firestein G.S. J. Immunol. 2002; 168: 1405-1412Google Scholar). Thus, ADAMTS4 is considered to play an important role in the aggrecan degradation of articular cartilage in osteoarthritis and rheumatoid arthritis. The aggrecanase activity of ADAMTS4 is inhibited by TIMP-3 (tissue inhibitor of metalloproteinases-3) among the four TIMP proteins (TIMP-1–4) (11Kashiwagi M. Tortorella M. Nagase H. Brew K. J. Biol. Chem. 2001; 276: 12501-12504Google Scholar, 12Hashimoto G. Aoki T. Nakamura H. Tanzawa K. Okada Y. FEBS Lett. 2001; 494: 192-195Google Scholar), all of which were originally cloned as inhibitors of matrix metalloproteinases (MMPs). 1The abbreviations used are: MMPs, matrix metalloproteinases; TSP, thrombospondin-1; CR, cysteine-rich; ECM, extracellular matrix; X-α-gal, 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside; PBS, phosphate-buffered saline; Fn-f120, central cell-binding 120-kDa fragment of fibronectin; DSS, disuccinimidyl suberate; DMEM, Dulbecco's modified Eagle's medium; Fn-f40, C-terminal 40-kDa fragment of fibronectin; BSA, bovine serum albumin; MT, membrane-type. However, it is not known whether this is the only regulatory mechanism of ADAMTS4 activity. ADAMTS4 consists of a prodomain, a furin cleavage site, a catalytic domain, a disintegrin-like motif, a thrombospondin-1 (TSP) motif, a cysteine-rich (CR) domain, and a C-terminal spacer domain. As shown with ADAMTS1 (13Kuno K. Matsushima K. J. Biol. Chem. 1998; 273: 13912-13917Google Scholar), ADAMTS4 has an affinity for extracellular matrix (ECM) molecules, being deposited in the ECM after synthesis (14Gao G. Westling J. Thompson V.P. Howell T.D. Gottschall P.E. Sandy J.D. J. Biol. Chem. 2002; 277: 11034-11041Google Scholar). In fact, the binding activity of C-terminal CR and/or spacer domains of ADAMTS4 for sulfated glycosaminoglycans of aggrecan has been reported (15Flannery C.R. Zeng W. Corcoran C. Collins-Racie L.A. Chockalingam P.S. Hebert T. Mackie S.A. McDonagh T. Crawford T.K. Tomkinson K.N. LaVallie E.R. Morris E.A. J. Biol. Chem. 2002; 277: 42775-42780Google Scholar). Interestingly, the aggrecanase activity of full-length active ADAMTS4 is blocked probably through interaction with ECM molecules, and activity appears after removal of the C-terminal spacer domain (14Gao G. Westling J. Thompson V.P. Howell T.D. Gottschall P.E. Sandy J.D. J. Biol. Chem. 2002; 277: 11034-11041Google Scholar). Thus, these data suggest that ADAMTS4 may have binding molecules by which the activity is regulated. However, no information is available for proteins interacting with ADAMTS4. In this study, by screening a human chondrocyte cDNA library, we sought binding proteins that may be involved in regulating the activity. Since the spacer domain of ADAMTS4 is not conserved among ADAMTS members, we used the domain as bait in a yeast two-hybrid system and found fibronectin to be a candidate for a regulator of ADAMTS4 activity. The data in this study demonstrate that fibronectin inhibits the aggrecanase activity of ADAMTS4 through the interaction between the C-terminal regions of each molecule. Yeast Two-hybrid System—MATCHMAKER Gal4 two-hybrid system 3 and the MATCHMAKER human chondrocyte cDNA library were purchased from Clontech (Palo Alto, CA). cDNA derived from the C-terminal spacer domain of ADAMTS4 was amplified by PCR using a set of primers (forward primer, 5′-GGAATTCCATATGAAGCAGTCAGGCTCCTTCAG-3′; and reverse primer, 5′-TTTGAATTCTTTCCTGCCCGCCCAGGG-3′) from the ADAMTS4 expression plasmid pSG0688 as described previously (16Nakamura H. Fujii Y. Inoki I. Sugimoto K. Tanzawa K. Matsuki H. Miura R. Yamaguchi Y. Okada Y. J. Biol. Chem. 2000; 275: 38885-38890Google Scholar). The amplified PCR product corresponding to Lys687–Lys837 was digested with NdeI and EcoRI (Takara Bio Inc., Otsu, Japan) and cloned into the pGBKT7 vector (Clontech), generating the pGBKT7/TS4sp plasmid. The plasmids were co-introduced into Saccharomyces cerevisiae strain AH109 with the human chondrocyte cDNA library according to the manufacturer's instructions. Yeast transformants were plated and selected on medium lacking leucine, tryptophan, histidine, and adenine. Robust colonies >2 mm in diameter were restreaked onto the same agar plates and allowed to grow for 1 week. This restreaking step was repeated twice more, and plasmids were isolated and introduced into Escherichia coli strain DH5α according to the manufacturer's instructions. Clones harboring target cDNA were isolated, and cDNA sequences were determined using a MegaBase 1000 DNA sequencer (Amersham Biosciences). Yeast Two-hybrid Assay—cDNA fragments encoding six different parts of fibronectin (see Fig. 1A) were amplified by PCR using the chondrocyte cDNA library as a template and the following primer sets: 5′-TTTGGATCCGTTATGACAATGGAAAACACTATC-3′ (forward) and 5′-TTTGAATTCAGCTTGGATAGGTCTGTAAAG-3′ (reverse) for fragment I, 5′-TTTGGATCCCAAGTGGTCCTGTCGAAGTA-3′ (forward) and 5′-TTTGAATTCCAGTGTGGTAAAGACTCCAG-3′ (reverse) for fragment II, 5′-TTTGGATCCCTGGGAGCTCTATTCCACC-3′ (forward) and 5′-TTTGAATTCAGTGATGGTGGCTCGAGGAG-3′ (reverse) for fragment III, 5′-TTTGGATCCCCCTCACCAACCTCACTCCA-3′ (forward) and 5′-TTTGAATTCTTAATGGAAATTGGCTTGCT-3′ (reverse) for fragment IV, 5′-TTTGGATCCACCGAACAGAAATTGACAA-3′ (forward) and 5′-TTTGAATTCCTGTGGACTGGGTTCCAATC-3′ (reverse) for fragment V, and 5′-TTTGGATCCCTATTCCTGCACCAACTGAC-3′ (forward) and 5′-TTTCTCGAGCTCTCGGGAATCTTCTCTGT-3′ (reverse) for fragment VI. The amplified products were digested with BamHI and EcoRI for fragments I–V, and with BamHI and XhoI for fragment VI and then cloned into the pACT2 vector (Clontech). The pGBKT7/TS4sp plasmid and each fibronectin expression plasmid were co-introduced into strain AH109. They were then selected on medium lacking tryptophan and leucine, and raised colonies were streaked onto medium lacking tryptophan, leucine, histidine, and adenine in the presence of 20 μg/ml X-α-gal and cultured at 27 °C for 2 days. The α-galactosidase activity of each transformant was measured using p-nitrophenyl-α-d-galactoside according to the method described (36Clontech Yeast Protocols Handbook. Clontech, Palo Alto, CA2001Google Scholar). The activity measured by the absorbance at 410 nm was normalized by cell density. Chemical Cross-linking—To verify the binding of ADAMTS4 to fibronectin, 125I-labeled ADAMTS4 (13 nm) was incubated with human plasma fibronectin (0, 9.1, or 91 nm; Chemicon International, Inc., Temecula, CA) or with a 120-kDa fibronectin fragment with the central cell-binding domain (Fn-f120; 100 nm; Invitrogen) in phosphate-buffered saline (PBS) for 16 h at 4 °C and cross-linked by treating the mixture with 1 mm disuccinimidyl suberate (DSS) for 2 h on ice. The reaction was stopped by incubation with 100 mm Tris-HCl (pH 7.5) for 15 min at room temperature, and the sample was subjected to SDSPAGE (12.5% total acrylamide) under reducing conditions. The gel was dried and analyzed by with a Fuji Film BAS 2000 analyzer. Construction of ADAMTS4 and Its Deletion Mutants—To construct the expression plasmid of wild-type ADAMTS4, pSG0688 was digested with EcoRI and KpnI, and ADAMTS4 cDNA with the FLAG tag sequence was subcloned into pcDNA3.1/Zeo(–) (Invitrogen). Construction of ADAMTS4 and its C-terminally truncated mutants, i.e. ΔSp lacking the spacer domain (Arg693–Lys837), ΔCR/Sp lacking most of the CR domain and the spacer domain (Pro603–Lys837), and ΔT/CR/Sp lacking the TSP, CR, and spacer domains (Gly521–Lys837) (see Fig. 2A), was carried out by PCR using pSG0688 as a template. Amplified products were digested with EcoRI and XhoI and cloned into pCMVtag4a (Stratagene, La Jolla, CA). The primer sets used for PCR were as follows: 5′-TAATACGACTCACTATAGGG-3′ (common forward) and 5′-TTTCTCGAGGAAGGAGCCTGACTGCTTG-3′ (reverse) for ΔSp (Met1–Phe692), the common forward primer and 5′-TTTCTCGAGCCCTGGGAAGCTCTTGA-3′ (reverse) for ΔCR/Sp (Met1–Gly602), and the common forward primer and 5′-TTTCTCGAGAGCCTGTGGAATATTGAAG-3′ (reverse) for ΔT/CR/Sp (Met1–Ala520). These plasmids were digested with EcoRI and KpnI, and each truncated ADAMTS4 cDNA with a FLAG tag was subcloned into pcDNA3.1/Zeo(–). Purification of Recombinant ADAMTS4 Proteins—293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and transfected with the above-mentioned pcDNA3.1/Zeo(–) plasmid containing cDNA encoding ADAMTS4 or its mutants using DOTAP liposomal transfection reagent (Roche Diagnostics). Transfected cells were cultured for 2 days and selected with 150 μg/ml Zeocin (Invitrogen) for 3 weeks. Stable transfectants expressing full-length ADAMTS4 or its truncated mutants were established by expansion from a single cell, and the conditioned culture media from these transfectants were harvested after a 48-h incubation in serum-free DMEM. For the purification of full-length ADAMTS4 and ΔT/CR/Sp, the conditioned media were concentrated using an Amicon Diaflo apparatus fitted with a YM-10 membrane; mixed with 4 volumes of 50 mm Tris-HCl (pH 7.5), 10 mm CaCl2, 0.05% Brij 35, and 0.02% NaN3 (TCB buffer); and applied to an SP-Sepharose fast flow column (2.5 × 10 cm; Amersham Biosciences) equilibrated with TCB buffer. The recombinant proteins were eluted by a linear gradient of 0–1 m NaCl, and the combined fractions containing ADAMTS4 or ΔT/CR/Sp were applied to a column of anti-FLAG antibody M2 affinity gel (1 × 6 cm; Sigma) equilibrated with 50 mm Tris-HCl (pH 7.5), 0.15 m NaCl, 10 mm CaCl2, 0.05% Brij 35, and 0.02% NaN3 (TNCB buffer). ADAMTS4 and ΔT/CR/Sp were eluted with 6 m urea in TNCB buffer containing 1 m NaCl after washing the column with the buffer containing 1 m NaCl and dialyzed against TNCB buffer to remove urea. For the purification of ΔSp and ΔCR/Sp, which did not show a strong affinity for the anti-FLAG antibody gel, concentrated culture media were applied to a DEAE-Sepharose fast flow column (2.5 × 10 cm; Amersham Biosciences) equilibrated with TCB buffer, and unbound fractions mixed with 9 volumes of TCB buffer were applied to an SP-Sepharose fast flow column. The mutants were eluted by a linear gradient of 0–1 m NaCl as described above, dialyzed, and then applied to a chelating Sepharose fast flow column (Amersham Biosciences) charged with ZnCl2 according to our previous method (17Imai K. Yokohama Y. Nakanishi I. Ohuchi E. Fujii Y. Nakai N. Okada Y. J. Biol. Chem. 1995; 270: 6691-6697Google Scholar). ΔSp and ΔCR/Sp were eluted by a linear gradient of 0–1 m NaCl and dialyzed against TNCB buffer. The concentrations of these ADAMTS4 species were determined using a BCA protein assay kit (Pierce). The purity of recombinant proteins was evaluated by SDS-PAGE, followed by silver staining of the gels and/or autoradiography of iodinated proteins according to our previous methods (18Hashimoto G. Inoki I. Fujii Y. Aoki T. Ikeda E. Okada Y. J. Biol. Chem. 2002; 277: 36288-36295Google Scholar). Binding of ADAMTS4 to Immobilized Fibronectin and Its Fragments—Microtiter plates with 96 wells (Immunomodule, Nalge Nunc, Rochester, NY) were coated by incubation with 50 μl of intact fibronectin, the C-terminal 40-kDa fibronectin fragment (Fn-f40; 25 nm; Invitrogen), or 25 nm Fn-f120 for 16 h at 4 °C. The plates were washed twice with TNCB buffer and subsequently blocked with 1% bovine serum albumin (BSA) in TNCB buffer for 2 h at room temperature. They were then incubated with 125I-labeled full-length ADAMTS4 or its deletion mutants (∼5 × 105 cpm, 20 ng/well) for 24 h at 4 °C. To confirm the specific binding, 125I-labeled ADAMTS4 was mixed with a 10- or 50-fold excess amount of unlabeled ADAMTS4 or buffer and then subjected to the binding assay using microtiter plates coated with intact fibronectin. After washing twice with TNCB buffer, the bound proteins were dissociated by treatment with 1 n NaOH, and the radioactivity of the bound fractions was counted using a γ-counter (ARC-600, Aloka, Tokyo, Japan). Similarly, the possibility of interaction between fibronectin and aggrecan was examined in a binding assay by incubating 125I-labeled fibronectin in the aggrecan-coated wells and measuring the bound radioactivity. Laser Scanning Confocal Microscopy of Transfected Cells—Stable transfectants expressing full-length or truncated ADAMTS4 species were established as described above and detached from the dishes by incubation with 0.25% trypsin and 0.02% EDTA for 3 min at 37 °C. After blocking the activity of trypsin with 10% fetal bovine serum, the cell suspensions were washed twice with PBS and incubated with 1 μg/ml fibronectin in PBS for 10 min at 37 °C. The cells were suspended in serum-free DMEM containing 1% insulin/transferrin/selenium/X supplement (Invitrogen) after washing with serum-free DMEM. Fibronectin-treated cells were then cultured on Lab-Tek chamber slides (1 × 105 cells/well; Nalge Nunc) for 1 day. The cells were incubated with 3% normal goat serum in PBS to block nonspecific binding and then reacted with anti-FLAG antibody (1:100), anti-fibronectin antibody (1: 20; Santa Cruz Biotechnology Inc., Santa Cruz, CA), or non-immune mouse IgG (Dako Corp., Glostrup, Denmark) for 1 h at room temperature. They were fixed with methanol/acetone/formaldehyde (19:19:2, v/v) and incubated with fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies (1:50; Dako Corp.) after washing with PBS. Transfected cells expressing the pcDNA3.1/Zeo(–) vector (mock transfectants) and parental 293T cells were also subjected to double immunostaining as a negative control. All preparations were viewed under an Olympus laser scanning confocal microscope at a similar sensitivity (550 V for fluorescein isothiocyanate and 600 V for rhodamine), and differential interference contrast images were also viewed for comparison. Detection of Aggrecanase Activity and Its Inhibition by Fibronectin— Aggrecan (100 μg) prepared from bovine nasal cartilage (12Hashimoto G. Aoki T. Nakamura H. Tanzawa K. Okada Y. FEBS Lett. 2001; 494: 192-195Google Scholar) was incubated with purified full-length ADAMTS4 (8 nm) or truncated ADAMTS4 species (ΔSp, ΔCR/Sp, or ΔT/CR/Sp; 8 nm each) for 16 h at 37 °C and deglycosylated after termination of the reaction with 20 mm EDTA as described previously (12Hashimoto G. Aoki T. Nakamura H. Tanzawa K. Okada Y. FEBS Lett. 2001; 494: 192-195Google Scholar). The digestion products were then subjected to SDS-PAGE (10% total acrylamide) under reducing conditions. Proteins in the gel were transferred onto polyvinylidene fluoride membranes, and aggrecanase activity was evaluated by detecting aggrecan fragments with the neoepitope (NITEGE373) by immunoblotting using the neoepitope-specific antibody I19C (2 μg/ml), which was kindly provided by Drs. Kotaro Sugimoto and Kazuhiko Tanzawa (Sankyo Co., Ltd., Tokyo) (19Sugimoto K. Takahashi M. Yamamoto Y. Shimada K. Tanzawa K. J. Biochem. (Tokyo). 1999; 126: 449-455Google Scholar). After reaction with horseradish peroxidase-linked anti-IgG antibody (1:5000; Amersham Biosciences), immunoreactive proteins on the membranes were detected using the ECL™ Western blot detection system (Amersham Biosciences) according to the manufacturer's instructions. For the study of fibronectin inhibition, wild-type ADAMTS4 and ΔSp (8 nm each), which showed sufficient aggrecanase activity, were incubated with increasing concentrations of fibronectin (0, 10, 20, 40, 100, 200, 500, and 1000 nm final concentrations) for 2 h at 37 °C prior to the reaction. Aggrecan was then digested by incubation with the mixtures for 16 h at 37 °C, and aggrecanase activity was monitored as described above. Since the aggrecanase activity of wild-type ADAMTS4 was inhibited by intact fibronectin, the inhibitory effects of the fibronectin fragments (Fn-f40 and Fn-f120) on the activity were also examined using ADAMTS4 preincubated with the fragments at final concentrations of 0, 10, 20, 40, 100, 200, 500, 750, and 1000 nm in the presence of 1 mm phenylmethanesulfonyl fluoride, which was added to completely block a trace amount of serine proteinase(s) contaminating the preparations of fibronectin fragments. The densities of immunoreactive bands were measured by scanning densitometry using NIH Image Version 1.62 according to our previous method (12Hashimoto G. Aoki T. Nakamura H. Tanzawa K. Okada Y. FEBS Lett. 2001; 494: 192-195Google Scholar). Statistical Analysis—Measured values are expressed as the mean ± S.D. In the solid-phase binding assay, the difference in radioactivity was analyzed by the Bonferroni/Dunn test. Tests were performed using StatView Version 5.0. p < 0.05 was considered significant. Screening of Proteins That Interact with ADAMTS4 —To identify the proteins that interact with ADAMTS4, 3 × 106 clones from the human chondrocyte cDNA library were screened by the yeast two-hybrid system using a cDNA fragment encoding the C-terminal spacer domain (Lys687–Lys837, 151 amino acids) of ADAMTS4 as bait. Among the 156 clones grown on medium lacking tryptophan, leucine, histidine, and adenine, 31 clones (20%) were identified as human fibronectin. These 31 clones had different 5′-ends, but all of them except for the shortest clone covered the whole region of the C-terminal heparin-binding domain/fibrin-binding domain of fibronectin (Fig. 1A). Interaction of ADAMTS4 with Fibronectin Fragments in Yeast—The region of binding of ADAMTS4 to fibronectin was examined by yeast two-hybrid assays. Yeast strain AH109 was cotransformed with the pGBKT7/TS4sp and pACT2/FnI–VI plasmids, containing cDNA encoding fragment I, II, III, IV, V, or VI of fibronectin (Fig. 1A). As shown in Fig. 1B, clones cotransformed with pGBKT7/TS4sp and pACT2/FnVI (referred to as Sp/VI) as well as positive clones expressing p53 and SV40 (referred to as p53/SV40) could grow to form blue-stained colonies on medium lacking the three amino acids and adenine. In contrast to these transformants, other clones expressing ADAMTS4 and fibronectin fragments I–V showed only negligible background growth (Fig. 1B). Although positive control p53/SV40 transformants grew faster than Sp/VI transformants, the intensity of blue color for X-α-gal staining was much higher in the Sp/VI transformants than in the control (Fig. 1B). When the α-galactosidase activity of each transformant was evaluated by measuring the absorbance at 410 nm, Sp/VI transformants showed remarkably higher activity compared with other transformants and control p53/SV40 transformants (Fig. 1C). Cross-linking Study of ADAMTS4 and Fibronectin—To further study the interaction of ADAMTS4 with fibronectin, a cross-linking experiment was carried out by incubating 125I-labeled ADAMTS4 with increased amounts of intact fibronectin. As shown in Fig. 1D, 125I-labeled ADAMTS4, which migrated at 73 kDa, shifted to the site of ∼400 kDa when reacted with intact fibronectin and cross-linked with DSS. On the other hand, reaction of an ADAMTS4 and Fn-f120 mixture with DSS showed negligible cross-linked products. Although ADAMTS4 became a broad band ranging from 70 to 80 kDa in the presence of DSS, dimerization of the proteinase was not detected. Thus, the molecular shift was considered to be caused by a cross-linked complex of ADAMTS4 and intact fibronectin. Binding of ADAMTS4 to the C-terminal Region of Fibronectin—To identify the region of fibronectin that interacts with ADAMTS4, a solid-phase binding assay was performed using immobilized intact fibronectin, Fn-f120, and Fn-f40. A large amount of 125I-labeled ADAMTS4 could bind to intact fibronectin-coated wells, whereas BSA-coated wells showed only back-ground binding. The radioactivity bound to Fn-f40 (but not Fn-f120) was significantly higher than that to BSA (p < 0.01 versus BSA) (Fig. 1E). Purification and Aggrecanase Activity of ADAMTS4 and Its Mutants—Full-length ADAMTS4 and its C-terminally truncated mutants (ΔSp, ΔCR/Sp, and ΔT/CR/Sp, which lack the C-terminal spacer domain; most of the CR and whole spacer domain; and the TSP, CR, and spacer domains, respectively) (Fig. 2A) were expressed in stably transfected 293T cells. These ADAMTS4 species were purified from the conditioned media. As shown in Fig. 2B, purified recombinant ADAMTS4, ΔSp, ΔCR/Sp, and ΔT/CT/Sp showed major protein bands of 73, 58, 48, and 39 kDa, respectively, on silver-stained gels after SDS-PAGE. All bands were recognized by immunoblotting with anti-FLAG antibody (Fig. 2C). When the aggrecanase activity of each recombinant ADAMTS4 species was examined by immunoblotting of aggrecan digestion products using the neoepitope-specific antibody, an immunoreactive aggrecan fragment of ∼80 kDa appeared after digestion with wild-type ADAMTS4 and ΔSp (Fig. 2D), indicating that these two recombinant ADAMTS4 species have potent aggrecanase activity. On the other hand, ΔCR/Sp showed only weak activity, and ΔT/CR/Sp had no activity (Fig. 2D). Binding of the C-terminal Spacer Domain of ADAMTS4 to Fibronectin—To determine which domain of ADAMTS4 is involved in binding to fibronectin, a solid-phase binding assay was performed by incubating 125I-labeled ADAMTS4, ΔSp, ΔCR/Sp, or ΔT/CR/Sp in fibronectin-coated wells. As shown in Fig. 3A, the binding activity of these ADAMTS4 species was higher than that of BSA, which had only background signals. However, the binding activity of wild-type ADAMTS4 was remarkably ∼3-fold higher than that of C-terminally truncated ADAMTS4 mutants (Fig. 3A). The specific binding between 125I-labeled ADAMTS4 and fibronectin was confirmed since the binding was competitively inhibited by unlabeled ADAMTS4 in a dose-dependent manner (Fig. 3B). Thus, these results suggest that the C-terminal spacer domain (Arg693–Lys837) of ADAMTS4 is involved in binding to intact fibronectin. Docking of ADAMTS4 on the Cell Membrane of Fibronectin-coated Cells—The pericellular interaction of ADAMTS4 species with fibronectin was examined by double immunostaining of stable transfectants expressing wild-type ADAMTS4, ΔSp, ΔCR/Sp, ΔT/CR/Sp, or vector alone. As shown in Fig. 4, fibronectin was immunolocalized on the cell surface of all the transfectants. Although wild-type ADAMTS4 was strongly colocalized with fibronectin on the cell surface, negligible or no immunoreaction was observed with ΔSp, ΔCR/Sp, ΔT/CR/Sp, or mock transfectants (Fig. 4). Inhibition of the Aggrecanase Activity of ADAMTS4 by Fibronectin and Its Fragments—To study the effect of fibronectin on wild-type ADAMTS4 and ΔSp, both of which have definite aggrecanase activity, these recombinant proteinases were incubated with intact fibronectin (0, 10, 20, 40, 100, 200, 500, and 1000 nm final concentrations), and aggrecanase activity was determined by immunoblotting using the neoepitope-specific antibody. As shown in Fig. 5A, fibronectin c
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