Altered Proteolytic Activities of ADAMTS-4 Expressed by C-terminal Processing
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m312123200
ISSN1083-351X
AutoresMasahide Kashiwagi, Jan J. Enghild, Christi M. Gendron, Clare Hughes, Bruce Caterson, Yoshifumi Itoh, Hideaki Nagase,
Tópico(s)Cell Adhesion Molecules Research
ResumoADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu373-Ala374 bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu1480-Gly1481 bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu373-Ala374 and Glu1480-Gly1481 bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1α-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered. ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu373-Ala374 bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu1480-Gly1481 bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu373-Ala374 and Glu1480-Gly1481 bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1α-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered. ADAMTS-4 1The abbreviations used are: ADAMTS, a disintegrin and metalloprotease with thrombospondin motifs; Dis, disintegrin; TS, thrombospondin type I; CysR, cysteine-rich; Sp, spacer; IL-1, interleukin-1; GAG, glycosaminoglycan; TIMP, tissue inhibitor of metalloproteinases; MMP, matrix metalloproteinases; MMP-3 (ΔC), matrix metalloproteinase-3 lacking the hemopexin domain; Cm-Tf, reduced, carboxymethylated transferrin; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; CS-2, chondroitin sulfate-rich region 2; PNGase F, peptide N-glycosidase F; ECM, extracellular matrix; IGD, interglobular domain; Ni-NTA, nickel-nitrilotriacetic acid. is a zinc metalloproteinase that belongs to the reprolysin subfamily of metallopeptidase M12 family as the metalloproteinase domain is related to snake venom metalloproteinases, reprolysins (1Rawlings N.D. O'Brien E. Barrett A.J. Nucleic Acids Res. 2002; 30: 343-346Crossref PubMed Scopus (177) Google Scholar). It has also been designated as "aggrecanase 1" based on its ability to cleave the Glu373-Ala374 bond located within the interglobular domain (IGD) of the aggrecan core protein (2Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. 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. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (625) Google Scholar), an original characteristic property proposed for aggrecanases (3Sandy J.D. Neame P.J. Boynton R.E. Flannery C.R. J. Biol. Chem. 1991; 266: 8683-8685Abstract Full Text PDF PubMed Google Scholar). The enzyme is considered to participate in early stages of cartilage destruction in rheumatoid arthritis and in osteoarthritis as well as normal turnover of aggrecan (4Arner E.C. Curr. Opin. Pharmacol. 2002; 2: 322-329Crossref PubMed Scopus (137) Google Scholar, 5Nagase H. Kashiwagi M. Arthritis Res. Ther. 2003; 5: 94-103Crossref PubMed Google Scholar). Bovine and porcine articular cartilage (6Curtis C.L. Hughes C.E. Flannery C.R. Little C.B. Harwood J.L. Caterson B. J. Biol. Chem. 2000; 275: 721-724Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 7Tortorella M.D. Malfait A.M. Deccico C. Arner E. Osteoarthritis Cartilage. 2001; 9: 539-552Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 8Little C.B. Hughes C.E. Curtis C.L. Jones S.A. Caterson B. Flannery C.R. Arthritis Rheum. 2002; 46: 124-129Crossref PubMed Scopus (48) Google Scholar), human chondrocytes (9Bau B. Gebhard P.M. Haag J. Knorr T. Bartnik E. Aigner T. Arthritis Rheum. 2002; 46: 2648-2657Crossref PubMed Scopus (349) Google Scholar), and bovine chondrocytes (6Curtis C.L. Hughes C.E. Flannery C.R. Little C.B. Harwood J.L. Caterson B. J. Biol. Chem. 2000; 275: 721-724Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) treated with IL-1 all have increased the levels of ADAMTS-4 mRNA. Human synovial fibroblasts also increase both mRNA and protein levels of ADAMTS-4 when treated with transforming growth factor β (10Yamanishi Y. Boyle D.L. Clark M. Maki R.A. Tortorella M.D. Arner E.C. Firestein G.S. J. Immunol. 2002; 168: 1405-1412Crossref PubMed Scopus (155) Google Scholar). The enzyme is a multidomain metalloproteinase, consisting of a large propeptide, a catalytic metalloproteinase domain, a disintegrin (Dis), a thrombospondin type I (TS), a cysteine-rich (CysR), and a spacer (Sp) domain (2Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. 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. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (625) Google Scholar). Besides the TEGE373∼374ARGS bond, recombinant ADAMTS-4 expressed in insect cells cleaves the core protein at GELE1480∼1481GR-GT, KEEE1666∼1667GLGS, TQAE1771∼1772AGEG, and VSQE1871∼1872LGQR sites ("∼" indicates the bond cleaved) in the chondroitin sulfate-rich CS-2 region of bovine aggrecan core protein (11Tortorella M.D. Pratta M. Liu R.Q. Austin J. Ross O.H. Abbaszade I. Burn T. Arner E. J. Biol. Chem. 2000; 275: 18566-18573Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). These sites are more readily cleaved than the originally characterized cleavage Glu373-Ala374 bond (11Tortorella M.D. Pratta M. Liu R.Q. Austin J. Ross O.H. Abbaszade I. Burn T. Arner E. J. Biol. Chem. 2000; 275: 18566-18573Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). On the other hand, the aggrecanase activity was not detected when the catalytic domain of ADAMTS-4 was expressed, although it retained the activity against a 41-residue synthetic peptide encompassing the Glu373-Ala374 bond (12Tortorella M. Pratta M. Liu R.Q. Abbaszade I. Ross H. Burn T. Arner E. J. Biol. Chem. 2000; 275: 25791-25797Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 13Miller J.A. Liu R.Q. Davis G.L. Pratta M.A. Trzaskos J.M. Copeland R.A. Anal. Biochem. 2003; 314: 260-265Crossref PubMed Scopus (29) Google Scholar). It has also been reported that ADAMTS-4 is unable to cleave the core protein when aggrecan was deglycosylated by chondroitinase ABC and keratanase (12Tortorella M. Pratta M. Liu R.Q. Abbaszade I. Ross H. Burn T. Arner E. J. Biol. Chem. 2000; 275: 25791-25797Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Interaction of the TS domain of the enzyme and anionic polysaccharide chains of aggrecan is considered to be important for the expression of aggrecanase activity, because a peptide harboring a potential heparan sulfate binding motif W(S/G)XW (14Guo N.H. Krutzsch H.C. Negre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (142) Google Scholar) inhibited the aggrecanase activity with an IC50 value in the low micromolar range (12Tortorella M. Pratta M. Liu R.Q. Abbaszade I. Ross H. Burn T. Arner E. J. Biol. Chem. 2000; 275: 25791-25797Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). More recently, however, Gao et al. (15Gao G. Westling J. Thompson V.P. Howell T.D. Gottschall P.E. Sandy J.D. J. Biol. Chem. 2002; 277: 11034-11041Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) have reported that the full-length ADAMTS-4 expressed in mammalian cells exhibits little aggrecanase activity as determined by the detection of fragments with the newly generated N-terminal 374ARGSV378 sequence using a neoepitope antibody, but that this activity was greatly increased when the full-length ADAMTS-4 was processed to 60- and 50-kDa forms by removing the C-terminal part of the enzyme. Similar C-terminal processing was also reported that the recombinant ADAMTS-4 expressed in Chinese hamster ovary/A2 cells was autolytically processed to 53 and 40 kDa by cleaving Lys694-Phe695 and Thr581-Phe582 bonds, and both forms cleave aggrecan at the Glu373-Ala374 bond (16Flannery 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: 42775-42780Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Thus, it is not clear which domains of the enzyme play a key role in aggrecanase activity. The aim of this study is to investigate the role of the noncatalytic ancillary domains of ADAMTS-4 in aggrecan degradation. For this purpose, we have systematically deleted the ancillary domains from the C terminus and measured the aggrecanase activity of the truncated forms using four different assay methods: i) an aggrecan-polyacrylamide beads assay (17Nagase H. Woessner Jr., J.F. Anal. Biochem. 1980; 107: 385-392Crossref PubMed Scopus (66) Google Scholar); (ii) detection of core protein fragments using the antibody 2-B-6 that recognizes the chondroitinase-generated chondroitin-4-sulfate "stubs" for general aggrecan degradation products (18Caterson B. Christner J.E. Baker J.R. Couchman J.R. Fed. Proc. 1985; 44: 386-393PubMed Google Scholar); (iii) detecting the cleavage at the Glu373-Ala374 bond in the IGD (19Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (197) Google Scholar); and (iv) at the Glu1480-Gly1481 bond in the CS-2 region using specific neoepitope antibodies. We have also examined other protein substrates, including reduced, carboxymethylated transferrin (Cm-Tf). The results show that considerable changes occur in substrate specificity upon deletion of each domain. Full-length ADAMTS-4 binds to the extracellular matrix (ECM) and the cell surface, but the deletion of the C-terminal Sp domain releases the enzyme from these sites. Direct extraction of ADAMTS-4 from porcine articular cartilage treated with interleukin-1α (IL-1α) indicated that the majority of the enzyme in such tissues lacks a portion of the C-terminal domains. Our studies suggest that ADAMTS-4 in the tissue may have a broader substrate spectrum than has previously been considered and trafficking of the enzyme is also regulated in the extracellular space by C-terminal processing. Materials—pCEP4 plasmid vector, pMT/V5HisA vector, and 293-EBNA cells were from Invitrogen (Groningen, The Netherlands). Restriction enzymes, T4 DNA ligase, and peptide N-glycosidase F (PNGase F) were from New England Biolabs (Hitchin, UK). Pfu Turbo DNA polymerase was from Stratagene Europe (Amsterdam, The Netherlands). HTB-94 human chondrosarcoma cell line was from American Type Cell Collection. FuGENE 6 transfection reagent was from Roche Applied Science. Alkaline phosphatase-conjugated goat anti-(mouse IgG) IgG, alkaline phosphatase-conjugated goat anti-(rabbit IgG) IgG, and Western Blue stabilized substrate for alkaline phosphatase were from Promega (Southampton, UK). Precision protein standard was from the Bio-Rad Laboratory Ltd. (Hemel Hempstead, UK). Recombinant interleukin-1α (IL-1α) was a gift from Prof. J. Saklatvala (Imperial College London, UK). Anti-FLAG M2 antibody (mouse monoclonal antibody), anti-FLAG M2-agarose, decorin, biglycan, and fibromodulin were from Sigma (Dorset, UK). Goat serum was from DAKO (Ely, UK). The Alexa-488-conjugated goat anti-(mouse IgG) IgG, Alexa-568-conjugated goat anti-(rabbit IgG) IgG, and Alexa-660-labeled phalloidin were from Molecular Probes (Leiden, The Netherlands). Chondroitinase ABC and keratanase were from Seikagaku Kogyo (Tokyo, Japan). Sephacryl S-200, and N-hydroxysuccinimide-activated Sepharose were from Amersham Biosciences (Little Chalfont, UK). Cm-Tf was prepared as described previously (20Nagase H. Methods Enzymol. 1995; 248: 449-470Crossref PubMed Scopus (70) Google Scholar). Aggrecan monomers were purified from bovine nasal cartilage under dissociative conditions according to Hascall and Sajdera (21Hascall V.C. Sajdera S.W. J. Biol. Chem. 1969; 244: 2384-2396Abstract Full Text PDF PubMed Google Scholar). Human recombinant TIMP-1 and TIMP-2 were expressed in mammalian cells and purified as described previously (22Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (98) Google Scholar, 23Troeberg L. Tanaka M. Wait R. Shi Y.E. Brew K. Nagase H. Biochemistry. 2002; 41: 15025-15035Crossref PubMed Scopus (63) Google Scholar). The N-terminal domain of human TIMP-3 with C-terminal His tag (N-TIMP-3-His) was expressed in Escherichia coli, folded in vitro and purified (24Kashiwagi M. Tortorella M. Nagase H. Brew K. J. Biol. Chem. 2001; 276: 12501-12504Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). N-TIMP-3-Sepharose was prepared by coupling 1 mg of N-TIMP-3 to 2 ml of NHS-activated Sepharose according to the manufacturer's instruction. Ni-NTA-agarose was from Qiagen Ltd. (Crawley, UK). Anti-ADAMTS-4 Antibody and Neoepitope Antibodies against Aggrecan—The antiserum against human ADAMTS-4 was raised in a rabbit using the purified recombinant catalytic domain of the enzyme. The antibody was rendered specific to ADAMTS-4 after purification by an affinity chromatography using Sepharose coupled with the catalytic domain of ADAMTS-4. The antibody that recognizes the aggrecanase-cleaved C-terminal neoepitope –TAGELE1480 was raised in a rabbit using the peptide CGGTAGELE linked to keyhole limpet hemocyanin. Monoclonal antibodies, BC-3 that recognizes the N-terminal neoepitope 374ARGSV–of aggrecan core protein and 2-B-6 that recognizes the chondroitinase-generated chondroitin-4-sulfate stubs attached to the aggrecan core protein were prepared and characterized as described by Hughes et al. (19Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (197) Google Scholar) and Caterson et al. (18Caterson B. Christner J.E. Baker J.R. Couchman J.R. Fed. Proc. 1985; 44: 386-393PubMed Google Scholar), respectively. Construction of cDNA Coding for ADAMTS-4 and the C-terminal Deletion Mutants—cDNA encoding human ADAMTS-4 was obtained by reverse transcription-polymerase chain reaction using the total RNA extracted from human primary chondrocytes. The PCR was carried out for 35 cycles of denaturation (30 s at 94 °C), annealing (30 s at 60 °C), and extension (1 min at 72 °C) using Pfu Turbo DNA polymerase with specific primers, derived from human ADAMTS-4 DNA sequence (NCBI accession number: NM005099). The two primers used were: 5′ forward primer, 5′-ATGTCCCAGACAGGCTCGCATCCC-3′; and 3′ reverse primer, 5′-TTATTTCCTGCCCGCCCTCC-3′. A PCR fragment (2500 bp) was ligated into pUC19 plasmid vector (pUC19-ADAMTS-4), and the nucleotide sequence determined and confirmed as human ADAMTS-4. To construct the vector expressing the full-length ADAMTS-4 (TS4-1), PCR was first performed with the forward primer, 5′-GGAATTCGCCACCATGTCCCAGACAGGCTCG-3′ (ADAMTS-4/FW/EcoRI) containing EcoRI site (underlined), Kozak consensus sequence (in italic) (25Kozak M. J. Mol. Biol. 1987; 196: 947-950Crossref PubMed Scopus (996) Google Scholar) and ADAMTS-4 N-terminal sequence, and the reverse primer, 5′-GTCATCGTCATCTTTATAATCTTTCCTGCCCGCCCAGGG-3′ (TS4-1/R-V/FLAG) containing the FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-stop) (in italic) and the C-terminal sequence of ADAMTS-4 using the pUC19-ADAMTS-4 vector as a template. The second PCR was carried out using the first PCR product as a template with the same 5′ forward primer as the first PCR (ADAMTS-4/FW/EcoRI) and the 3′ reverse primer 5′-TAGACTCGAGTTACTTGTCATCGTCATCTTTATA-3′ (FLAG/RV/XhoI) containing XhoI site (underlined) and the FLAG epitope sequence (in italic). The resulting cDNA (2.5 kb) was ligated into pMT/V5 His A vector and cloned into the pCEP4 vector. The domain deletion mutants were also constructed by PCR using the ADAMTS-4 FW EcoRI primer and the following 3′ reverse primers containing FLAG sequence (in italic): 5′-GTCATCGTCATCTTTATAATCGCTGCAACCAGAACCGTC-3′ (TS4-2/RV/FLAG); 5′-GTCATCGTCATCTTTATAATCTGGGCAGTCCTCAGTGTT-3′ (TS4-3/RV/FLAG); 5′-GTCATCGTCATCTTTATAATCAGCCTGTGGAATATTGAA-3′ (TS4-4/RV/FLAG); or 5′-GTCATCGTCATCTTTATAATCCACAGGCAGATGCAATGG-3′ (TS4-5/RV/FLAG) (see Fig. 1 for schematic representation of domain deletion mutants). The PCR products were cloned into the pCEP4 vector as described above. The inactive ADAMTS-4(E362A) mutant was constructed by PCR "megaprimer" method (26Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar) with primers 5′-GCTCATGCCCTGGGTCATGTCTTCAACATG-3′ (forward) and 5′-ACCCAGGGCATGAGCAGCAGTGAAGGCTGA-3′ (reverse), where the original codon GAA encoding Glu362 was mutated to GCC encoding Ala (underlined). Expression and Purification of the Recombinant ADAMTS-4 and Its Variants—The pCEP4 vector harboring ADAMTS-4 or its variants was transfected into 293-EBNA cells by lipofection with FuGENE6. The stably transfected cells were selected for hygromycin B resistance (200 μg/ml) in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum, penicillin (100 units/ml), and streptomycin (100 units/ml). To obtain the recombinant protein, the culture media were replaced with DMEM containing 0.2% lactalbumin hydrolysate, penicillin, and streptomycin, and the conditioned media were harvested after 1 week. Full-length ADAMTS-4 (TS4-1) secreted from the cells bound to the cell surface and ECM, and little was detected in the medium. Thus, 100 μg/ml heparin was added during the culture, which released TS4-1 into the medium. The collected conditioned media (1 liter) were centrifuged to remove cell debris and applied to a column of anti-FLAG M2-agarose (2 ml) for purification. The material bound to the column was eluted with 100 μg/ml FLAG peptide in 20 mm Tris-HCl (pH 7.5), 100 mm NaCl, 10 mm CaCl2, and 0.02% NaN3. The eluted recombinant ADAMTS-4 was further purified by gel filtration on S-200 Sephacryl equilibrated with 50 mm Tris-HCl (pH 7.5) containing 100 mm NaCl, 10 mm CaCl2, 0.02% Brij-35, and 0.02% NaN3 (TNC buffer). About 80% of aggrecanase activity in the conditioned medium of TS4-1 did not bind to anti-FLAG M2 agarose. To purify TS4-1 in the flow-through fraction, it was applied to a column of N-TIMP-3-Sepharose. The bound material was eluted with 6 m urea in 50 mm Tris-HCl (pH 8.0) and 500 mm NaCl. The eluate was then dialyzed extensively against TNC buffer. The domain deletion variants were secreted into the medium. They were purified by anti-FLAG affinity chromatography and gel filtration as described above. The concentration of active recombinant ADAMTS-4 and variants were determined by titration with the known concentrations of N-TIMP-3. SDS-PAGE and Western Blotting—SDS-PAGE was carried out according to Bury (27Bury A.F. J. Chromatogr. 1981; 213: 491-500Crossref Scopus (203) Google Scholar), and proteins were stained by silver (28Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar). Precision protein standards were used as molecular weight markers. For Western blotting analyses, proteins separated by SDS-PAGE were electrotransferred to polyvinylidene difluoride membranes. The membranes were blocked by 5% dry milk solution and reacted with the primary antibody anti-FLAG M2 antibody (1:2000 dilution), anti-ADAMTS-4 catalytic domain antibody (1:1000 dilution), BC-3 neoepitope antibody (1:200 dilution), anti-GELE antibody (1:2000 dilution), or 2-B-6 antibody (1: 1000 dilution). The antigen·antibody complexes were then reacted with the second antibody conjugated with alkaline phosphatase, and the protein bands were visualized using Western Blue stabilized substrate. Immunolocalization of ADAMTS-4 —Human chondrosarcoma HTB-94 cells were seeded and grown on gelatin-coated glass coverslips and transfected with the pCEP4 expression vector containing ADAMTS-4 cDNA or its variants by lipofection with FuGENE6. In the following day the cells were washed once with serum-free DMEM and incubated in serum-free DMEM for 48 h. The cell layers were washed once with phosphate-buffered saline (PBS), fixed using 4% (w/v) paraformaldehyde in PBS for 7 min, washed with PBS four times and incubated in blocking solution (3% (w/v) bovine albumin and 5% (v/v) goat serum in PBS) for 1 h at room temperature. The cell layers were then reacted with the primary antibodies, the mouse anti-FLAG M2 antibody (1: 2000 dilution), and rabbit anti-(catalytic domain of human ADAMTS-4) IgG (1:500 dilution) in the blocking solution for 2 h at room temperature. The specimens were then washed twice with PBS and incubated with fluorescence-labeled secondary antibodies (Alexa-488-conjugated goat anti-(mouse IgG) IgG (1:500 dilution) and Alexa-568-conjugated goat anti-(rabbit IgG) IgG (1:500 dilution)) in blocking solution for 1 h at room temperature. The specimens were washed twice with PBS and fixed again with 4% (w/v) paraformaldehyde in PBS for 10 min. After washing twice with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS, and samples were incubated with Alexa-660-conjugated phalloidin in 0.1% Triton X-100 in PBS to visualize the actin filaments within the cell. The samples were viewed using a Nikon Eclipse TE2000-U microscope equipped with a PerkinElmer Life Sciences Ultraview Live Cell Imaging System using excitation and emission wavelengths at 488 and 525 nm for Alexa-488, at 568 and 607 nm for Alexa-568, and at 647 and 700 nm for Alexa-660, respectively. Aggrecanase Assays—To quantitate aggrecan-degrading activity of ADAMTS-4 and variants, polyacrylamide beads co-polymerized with aggrecan monomers were used as described by Nagase and Woessner (17Nagase H. Woessner Jr., J.F. Anal. Biochem. 1980; 107: 385-392Crossref PubMed Scopus (66) Google Scholar). Dry aggrecan beads (1 mg of beads containing 180 μg of aggrecan) were hydrated in an Eppendorf tube by adding 100 μl of enzyme solution in TNC buffer and incubated at 37 °C for various periods of time. The beads were removed by brief centrifugation, and 10 μl of the supernatant reacted with 200 μl of dimethylmethylene blue dye solution to quantify the released glycosaminoglycans (GAG) according to Farndale et al. (29Farndale R.W. Buttle D.J. Barrett A.J. Biochim. Biophys. Acta. 1986; 883: 173-177Crossref PubMed Scopus (2907) Google Scholar). One unit of aggrecan-degrading activity is defined as release of 1 μg of GAG in 1 min at 37 °C. To determine aggrecanase activity by neoepitope antibodies, bovine aggrecan (500 nm) was incubated with 100 μl of ADAMTS-4 or its variants in TNC buffer at 37 °C for the indicated period of time. The reaction was terminated with 10 mm EDTA, and the digestion products were deglycosylated by chondroitinase ABC (0.01 unit/10 μg of aggrecan) and keratanase (0.01 unit/10 μg of aggrecan) in Tris acetate (pH 6.5), 5 mm EDTA at 37 °C for 3 h. The digestion products were then precipitated with 10 volumes of acetone and subjected to Western blotting analysis with the BC-3 antibody, anti-GELE, or 2-B-6 antibody as primary antibody as described by Little et al. (30Little C.B. Hughes C.E. Curtis C.L. Janusz M.J. Bohne R. Wang-Weigand S. Taiwo Y.O. Mitchell P.G. Otterness I.G. Flannery C.R. Caterson B. Matrix Biol. 2002; 21: 271-288Crossref PubMed Scopus (113) Google Scholar). N-terminal Sequence Analysis of Cm-Tf and Fibromodulin Fragments—Cm-Tf (2.5 mg/ml) was incubated with 50 nm of TS4-2 in TNC buffer at 37 °C for 3, 6, 12, and 24 h, and the products were analyzed by SDS-PAGE to determine the relative rate of peptide bond cleavage of Cm-Tf. For N-terminal sequence analysis, Cm-Tf digested for 24 h was subjected to SDS-PAGE analysis, and the fragments were electrotransferred to a polyvinylidene difluoride membrane and visualized by Coomassie Brilliant Blue R-250. The bands were excised, placed directly onto a Polybrene-treated glass filter, and sequenced by automated Edman degradation in an Applied Biosystems 477A sequencer with "on line" 120A PTH analysis. Fibromodulin (0.1 mg/ml) was reacted with 50 nm TS4-2 for 48 h, and the products were deglycosylated by PNGase F and subjected to SDS-PAGE. The fragment was sequenced as for Cm-Tf fragments. Identification of ADAMTS-4 in Porcine Articular Cartilage—Porcine articular cartilage (2 g) was sliced (approx 1 × 5 × 5 mm) and cultured in DMEM containing 10% (v/v) fetal calf serum for 2 days. The tissue was then washed once with serum-free DMEM and cultured with or without IL-1α (10 ng/ml) in the same medium. The cartilage was harvested, and proteins were extracted with 10 ml of 4 m guanidine hydrochloride in 50 mm sodium acetate (pH 5.8) containing a mixture of proteinase inhibitors (5 mm EDTA, 1 mm p-aminoethylbenzene sulfonyl fluoride, and 10 μm E-64) for 48 h at 4 °C. The cartilage extracts were mixed with 100 nm recombinant human TIMP-1 and 100 nm recombinant N-TIMP-3-His, and dialyzed extensively against TNC buffer. The dialyzed sample (3-ml aliquot) was mixed with 30 μl of Ni-NTA-agarose by inversion at 4 °C for 2 h. After washing the agarose beads with TNC buffer, the bound protein was eluted with 30 μl of 50 mm Tris-HCl (pH 7.5) containing 6 m urea and 500 mm NaCl and analyzed by SDS-PAGE and Western blotting with anti-ADAMTS-4 catalytic domain antibody. Expression of the Recombinant ADAMTS-4 and C-terminal Domain Deletion Variants—Full-length mature form of ADAMTS-4 and the C-terminal domain deletion enzymes were purified from conditioned media by anti-FLAG affinity chromatography and gel filtration on Sephacryl S-200. All preparations were homogenous on SDS-PAGE when stained with silver (Fig. 2A). Purified materials were also detected by Western blotting analysis with an anti-FLAG antibody (Fig. 2B) and anti-catalytic domain antibody (Fig. 2C). Approximately 50 μg of the 70-kDa TS4-1 (full-length), 520 μg of the 53-kDa TS4-2 (Sp deletion), 620 μg of the 40-kDa TS4-3 (Sp and CysR deletion), 1.8 mg of the 35-kDa TS4-4 (Sp, CysR, and TS deletion), 1.2 mg of the 26-kDa TS4-5 (the catalytic domain), and 110 μg of the 70-kDa TS4-1(E362A) were purified from 1 liter of conditioned media. The N-terminal sequence analysis of the isolated ADAMTS-4 was Phe-Ala-Ser-Leu-Ser, indicating that the propeptide was processed intracellularly and mature forms were secreted from the cell. Full-length TS4-1 was largely found on the cell surface or bound to the ECM (see below), but the addition of heparin to the culture released the majority of TS4-1 into the medium. However, about 80% of the aggrecanase activity in the conditioned medium of TS4-1 did not bind to the anti-FLAG column, indicating a loss of the C-terminal FLAG during culture. When the unbound fraction was applied to a column of N-TIMP-3-Sepharose and eluted with 6 m urea in 50 mm Tris-HCl (pH 8.0) containing 500 mm NaCl, tw
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