Abstract: Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0–9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology. Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0–9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology. Destruction of articular cartilage is a feature of various arthritides, including rheumatoid and osteoarthritis, that results in joint impairment and disability. It is caused primarily by an elevation in proteolytic enzymes that degrade macromolecules of the cartilage extracellular matrix. Aggrecan degradation is initially observed followed by essentially irreversible collagen degradation. The proteinases that are responsible for aggrecan degradation in cartilage are matrix metalloproteinases (MMPs) 4The abbreviations used are: MMP, matrix metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin type 1 motifs; anti-TS5cat, rabbit anti-ADAMTS-5 catalytic domain; Cm-Tf, reduced, carboxymethylated transferrin; CysR, cysteine-rich; Dis, disintegrin; DMEM, Dulbecco's modified Eagle's medium; IGD, interglobular domain of aggrecan; IL-1, interleukin-1; MT-MMP, membrane-type matrix metalloproteinase; N-TIMP, amino-terminal domain of tissue inhibitor of metalloproteinases; Sp, spacer; TIMP, tissue inhibitor of metalloproteinases; TS, thrombospondin type 1; MES, 4-morpholineethanesulfonic acid; ECM, extracellular matrix. and "aggrecanases," members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) family (1Nagase H. Kashiwagi M. Arthritis Res. Ther. 2003; 5: 94-103Crossref PubMed Google Scholar, 2Porter S. Clark I.M. Kevorkian L. Edwards D.R. Biochem. J. 2005; 386: 15-27Crossref PubMed Scopus (625) Google Scholar). Aggrecanase activity was first defined as the ability to cleave the Glu373-Ala374 bond in the interglobular domain (IGD) of the aggrecan core protein (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, 4Ilic M.Z. Handley C.J. Robinson H.C. Mok M.T. Arch. Biochem. Biophys. 1992; 294: 115-122Crossref PubMed Scopus (164) Google Scholar). The first two proteinases shown to be capable of cleaving aggrecan at this site were ADAMTS-4 (aggrecanase 1) (5Tortorella 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. Newton R.C. Magolda R.L. Trzaskos J.M. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (625) Google Scholar), and ADAMTS-5 5ADAMTS-5 was originally referred to as "ADAMTS-11." (aggrecanase 2) (6Abbaszade I. Liu R.Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Hollis G.F. Arner E.C. Burn T.C. J. Biol. Chem. 1999; 274: 23443-23450Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). More recently, ADAMTS-1, -8, -9, -15, -16, and -18 were shown to cleave the Glu373-Ala374 bond in the IGD at a high enzyme-substrate ratio (2Porter S. Clark I.M. Kevorkian L. Edwards D.R. Biochem. J. 2005; 386: 15-27Crossref PubMed Scopus (625) Google Scholar, 7Zeng W. Corcoran C. Collins-Racie L.A. LaVallie E.R. Morris E.A. Flannery C.R. Biochim. Biophys. Acta. 2006; 1760: 517-524Crossref PubMed Scopus (91) Google Scholar). Although this bond is also cleaved by MMP-8 (8Fosang A.J. Last K. Neame P.J. Murphy G. Knäuper V. Tschesche H. Hughes C.E. Caterson B. Hardingham T.E. Biochem. J. 1994; 304: 347-351Crossref PubMed Scopus (113) Google Scholar) and MMP-14 (9Buttner F.H. Hughes C.E. Margerie D. Lichte A. Tschesche H. Caterson B. Bartnik E. Biochem. J. 1998; 333: 159-165Crossref PubMed Scopus (58) Google Scholar) at high concentrations in vitro, the primary MMP cleavage site in the IGD is considered to be at the Asn341-Phe342 bond (1Nagase H. Kashiwagi M. Arthritis Res. Ther. 2003; 5: 94-103Crossref PubMed Google Scholar). The cleavage of the Glu373-Ala374 bond in IL-1-stimulated pig articular cartilage explants is blocked by tissue inhibitor of metalloproteinases 3 (TIMP-3) but not by other TIMPs (10Gendron C. Kashiwagi M. Hughes C. Caterson B. Nagase H. FEBS Lett. 2003; 555: 431-436Crossref PubMed Scopus (76) Google Scholar). This is a further implication of ADAMTS metalloproteinases as the proteinases responsible for the observed aggrecanase activity. Among the ADAMTSs that have aggrecanase activity, ADAMTS-4 has received more attention than other ADAMTSs because the elevation of its mRNA is readily observed in chondrocytes treated with IL-1, whereas ADAMTS-5 mRNA is not (11Tortorella M.D. Malfait A.M. Deccico C. Arner E. Osteoarthritis Cartilage. 2001; 9: 539-552Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 12Bau B. Gebhard P.M. Haag J. Knorr T. Bartnik E. Aigner T. Arthritis Rheum. 2002; 46: 2648-2657Crossref PubMed Scopus (349) Google Scholar). However, ADAMTS-4-null mice did not exhibit any significant protective effect on cartilage aggrecan loss compared with the wild-type mice when challenged in an osteoarthritis model induced by surgical joint destabilization (13Glasson S.S. Askew R. Sheppard B. Carito B.A. Blanchet T. Ma H.L. Flannery C.R. Kanki K. Wang E. Peluso D. Yang Z. Majumdar M.K. Morris E.A. Arthritis Rheum. 2004; 50: 2547-2558Crossref PubMed Scopus (257) Google Scholar). Similarly ADAMTS-1-null mice were not protected against joint destruction in an antigen-induced arthritis model (14Little C.B. Mittaz L. Belluoccio D. Rogerson F.M. Campbell I.K. Meeker C.T. Bateman J.F. Pritchard M.A. Fosang A.J. Arthritis Rheum. 2005; 52: 1461-1472Crossref PubMed Scopus (95) Google Scholar). These studies suggest that neither ADAMTS-4 nor ADAMTS-1 may be the major enzyme that causes aggrecan loss during the progression of arthritis. On the other hand, studies with ADAMTS-5-null mice showed that the lack of ADAMTS-5 protected against cartilage destruction when osteoarthritis (15Glasson S.S. Askew R. Sheppard B. Carito B. Blanchet T. Ma H.L. Flannery C.R. Peluso D. Kanki K. Yang Z. Majumdar M.K. Morris E.A. Nature. 2005; 434: 644-648Crossref PubMed Scopus (1017) Google Scholar) or inflammatory arthritis (16Stanton H. Rogerson F.M. East C.J. Golub S.B. Lawlor K.E. Meeker C.T. Little C.B. Last K. Farmer P.J. Campbell I.K. Fourie A.M. Fosang A.J. Nature. 2005; 434: 648-652Crossref PubMed Scopus (764) Google Scholar) was induced. This suggests that ADAMTS-5 plays a key role in aggrecan degradation at least in these models of arthritis in mice. ADAMTS-4 and ADAMTS-5 are multidomain metalloproteinases secreted from the cell into the extracellular space. Both enzymes have a similar domain arrangement consisting of a prodomain, a catalytic metalloproteinase domain, a disintegrin (Dis) domain, a thrombospondin type I (TS) domain, a cysteine-rich (CysR) domain, and a spacer (Sp) domain. In addition, ADAMTS-5 contains an extra TS domain after the spacer domain (see Fig. 1 for domain arrangements). We have shown previously that the non-catalytic ancillary domains of ADAMTS-4 play a major role in regulating aggrecanase activity (17Kashiwagi M. Enghild J.J. Gendron C. Hughes C. Caterson B. Itoh Y. Nagase H. J. Biol. Chem. 2004; 279: 10109-10119Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). In particular, full-length ADAMTS-4 digests the aggrecan core protein most effectively but has little activity to cleave the initially characterized aggrecanase cleavage site, Glu373-Ala374 (17Kashiwagi M. Enghild J.J. Gendron C. Hughes C. Caterson B. Itoh Y. Nagase H. J. Biol. Chem. 2004; 279: 10109-10119Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 18Gao G. Plaas A. Thompson V.P. Jin S. Zuo F. Sandy J.D. J. Biol. Chem. 2004; 279: 10042-10051Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), or other non-aggrecan protein substrates (17Kashiwagi M. Enghild J.J. Gendron C. Hughes C. Caterson B. Itoh Y. Nagase H. J. Biol. Chem. 2004; 279: 10109-10119Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). When the carboxyl-terminal Sp domain is removed, the enzyme gains activity for the Glu373-Ala374 bond as well as new proteolytic activities against proteins such as reduced, carboxymethylated transferrin (Cm-Tf), fibromodulin, and decorin (17Kashiwagi M. Enghild J.J. Gendron C. Hughes C. Caterson B. Itoh Y. Nagase H. J. Biol. Chem. 2004; 279: 10109-10119Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). ADAMTS-4 is converted to a molecular size similar to that of our Sp deletion mutant by MMP-17 (MT4-MMP) in chondrocytes (18Gao G. Plaas A. Thompson V.P. Jin S. Zuo F. Sandy J.D. J. Biol. Chem. 2004; 279: 10042-10051Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and cartilage explants (19Patwari P. Gao G. Lee J.H. Grodzinsky A.J. Sandy J.D. Osteoarthritis Cartilage. 2005; 13: 269-277Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). This suggests that the removal of the ADAMTS-4 spacer domain and hence increased ADAMTS-4 proteolytic capabilities occur in vivo. However, little proteolytic activity was detected with the metalloproteinase domain alone (17Kashiwagi M. Enghild J.J. Gendron C. Hughes C. Caterson B. Itoh Y. Nagase H. J. Biol. Chem. 2004; 279: 10109-10119Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Unlike ADAMTS-4, the biochemical characterization of ADAMTS-5 is limited. The only available studies investigate its ability to degrade aggrecan, brevican, and α2-macroglobulin (7Zeng W. Corcoran C. Collins-Racie L.A. LaVallie E.R. Morris E.A. Flannery C.R. Biochim. Biophys. Acta. 2006; 1760: 517-524Crossref PubMed Scopus (91) Google Scholar, 20Vankemmelbeke M.N. Holen I. Wilson A.G. Ilic M.Z. Handley C.J. Kelner G.S. Clark M. Liu C. Maki R.A. Burnett D. Buttle D.J. Eur. J. Biochem. 2001; 268: 1259-1268Crossref PubMed Scopus (112) Google Scholar, 21Tortorella M.D. Liu R.Q. Burn T. Newton R.C. Arner E. Matrix Biol. 2002; 21: 499-511Crossref PubMed Scopus (120) Google Scholar, 22Tortorella M.D. Arner E.C. Hills R. Easton A. Korte-Sarfaty J. Fok K. Wittwer A. Liu R.Q. Malfait A.M. J. Biol. Chem. 2004; 279: 17554-17561Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 23Held-Feindt J. Paredes E.B. Blomer U. Seidenbecher C. Stark A.M. Mehdorn H.M. Mentlein R. Int. J. Cancer. 2006; 118: 55-61Crossref PubMed Scopus (108) Google Scholar). We therefore investigated the functional significance of the non-catalytic domains of ADAMTS-5 by expressing recombinant enzymes where we have systematically deleted the ancillary domains from the carboxyl terminus. We then measured their enzymatic activities for the aggrecan core protein, Cm-Tf, and other cartilage matrix macromolecules. These studies indicated that ADAMTS-5 is able to cleave aggrecan core protein about 1,000 times more effectively than ADAMTS-4. Furthermore this work revealed that the roles of the non-catalytic domains for aggrecanolytic activities and the salt sensitivity between the two enzymes are significantly different. Our results provide biochemical evidence to support that ADAMTS-5 is the major aggrecanolytic metalloproteinase. Materials—Human embryonic kidney cells (HEK 293-EBNA), pCEP4 vector, and the Zero Blunt TOPO PCR kit were from Invitrogen. G418 and hygromycin B were from PAA Laboratories (Sommerset, UK). Macro-Prep 25 S resin and prestained and unstained Precision Protein Standards for SDS-PAGE were from Bio-Rad. Anti-rabbit alkaline phosphatase-linked antibody, anti-mouse alkaline phosphatase-linked antibody, and alkaline phosphatase substrate (5-bromo-4-choloro-3-indolyl 1-phosphate and nitroblue tetrazolium) were from Promega (Southampton, UK). Decorin, biglycan, fibromodulin, anti-FLAG M2-agarose, FLAG peptide, and human transferrin were from Sigma-Aldrich. Restriction enzymes were from New England Biolabs (Hitchin, UK). Pfu DNA polymerase, GFX Gel Band PCR Purification kit, and GFX Microplasmid Purification kit were from Amersham Biosciences. pCR-Script and pSG5 were from Stratagene (Amsterdam, The Netherlands). FuGENE 6 was from Roche Applied Science. Recombinant human N-TIMP-1 (the amino-terminal domain of tissue inhibitor of metalloproteinase-1), human TIMP-2, and human N-TIMP-3 were prepared as described previously (see Ref. 10Gendron C. Kashiwagi M. Hughes C. Caterson B. Nagase H. FEBS Lett. 2003; 555: 431-436Crossref PubMed Scopus (76) Google Scholar). Aggrecan was purified from bovine nasal cartilage according to the method of Hascall and Sajdera (24Hascall V.C. Sajdera S.W. J. Biol. Chem. 1969; 244: 2384-2396Abstract Full Text PDF PubMed Google Scholar). Gelatin was prepared by heat denaturing guinea pig type I collagen (25Itoh Y. Binner S. Nagase H. Biochem. J. 1995; 308: 645-651Crossref PubMed Scopus (99) Google Scholar). Human fibronectin was purified from expired human plasma as described previously (26Ruoslahti E. Engvall E. Ann. N. Y. Acad. Sci. 1978; 312: 178-191Crossref PubMed Scopus (70) Google Scholar). Monoclonal antibodies BC-3 that recognizes the newly generated amino-terminal 374ARGSV epitope of the aggrecan core protein and 2-B-6 that recognizes the chondroitinase-resistant chondroitin 4-sulfate stubs of aggrecan core protein were generated as described previously (27Caterson B. Christner J.E. Baker J.R. Couchman J.R. Fed. Proc. 1985; 44: 386-393PubMed Google Scholar, 28Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (197) Google Scholar). The rabbit anti-GELE antibody that recognizes the new GELE1480 carboxyl terminus generated by aggrecanase cleavage of the aggrecan core protein was raised in a rabbit against the aggrecan peptide sequence CGGTAGELE (amino acids 1475–1480) (the amino acids in italics are not part of the aggrecan sequence but added for the purpose of linking the peptide to keyhole limpet hemocyanin). The rabbit anti-ADAMTS-5 catalytic domain (anti-TS5cat) antibody was raised in a rabbit against the peptide sequence CEETFGSTEDKRL (amino acids 410–422) and linked to keyhole limpet hemocyanin. Both peptides were kindly provided by Prof. G. B. Fields (Florida Atlantic University). Construction of cDNA Coding for Human ADAMTS-5 and the Carboxyl-terminal Domain Deletion Mutants—Full-length ADAMTS-5 (TS5-1) was amplified from the cDNA of primary human chondrocytes and subcloned into pCR-Script. Carboxyl-terminal FLAG-tagged full-length ADAMTS-5 (ADAMTS5-1) and its domain deletion mutants were created using the PCR method. The PCR was performed with pCR-Script-ADAMTS-5 as a template and amplified by Pfu Turbo DNA polymerase using two primers: forward primer, 5′-ACTGGTACCACCATGCTGCTCGGGTGGGC-3′ (ADAMTS5 FW) containing a KpnI restriction site (underlined) and a Kozak consensus sequence (italic); reverse primer, 5′-AGCAGATCTCTATTTATCATCATCATCTTTATAATCACATTTCTTCAACAAGCATTGC-3′ (ADAMTS5-1 RV FLAG), 5′-AGCAGATCTCTATTTATCATCATCATCTTTATAATCAGTGTGTGATCCCACTTTATTG-3′ (ADAMTS5-2 RV FLAG), 5′-AGCAGATCTCTATTTATCATCATCATCTTTATAATCTGTACAGCTGGAGTTGTCTC-3′ (ADAMTS5-3 RV FLAG), 5′-AGCAGATCTCTATTTATCATCATCATCTTTATAATCGGGTGGGCAGGGCATGA-3′ (ADAMTS5-4 RV FLAG), 5′-AGCAGATCTCTATTTATCATCATCATCTTTATAATCGCTTGACGTTGAATAATATTTTTTC-3′ (ADAMTS5-5 RV FLAG), or 5′-AGCAGATCTCTATTTATCATCATCATCTTTATAATCTTCTTCGGGGCCCAGGAT-3′ (ADAMTS5-6 RV FLAG) containing a BglII restriction enzyme site (underlined), a stop codon, and the FLAG epitope (coding Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys in italics). The PCR was carried out for 35 cycles of denaturation (60 s at 94 °C), annealing (60 s at 55 °C), and extension (3 min 30 s at 72 °C). The PCR products were ligated into the pCR-BLUNT II-TOPO vector using the Zero Blunt TOPO PCR Cloning kit according to the manufacturer's instructions, sequenced, and then subcloned into both the pCEP4 and pSG5 vectors using the KpnI and BglII restriction enzyme sites that were introduced into the ADAMTS-5 DNA products at the PCR step. A diagram of the ADAMTS-5 constructs (TS5-1 to TS5-6) created is shown in Fig. 1. Expression and Purification of the Recombinant Human ADAMTS-5 and Its Domain Deletion Mutants—The pCEP4 vector containing full-length human ADAMTS-5 (TS5-1) or its domain deletion mutants was transfected into HEK 293-EBNA cells with FuGENE 6. The stably transfected cells were selected for hygromycin B resistance (100 μg/ml) in DMEM, 10% fetal calf serum, 250 μg/ml G418, 100 units/ml penicillin, and 100 units/ml streptomycin over a period of 3 weeks. To obtain the recombinant protein, the culture medium was replaced with serum-free DMEM containing 0.2% lactalbumin hydrolysate, penicillin, and streptomycin. In the case of TS5-1-, -2-, and -3-producing cells, 100 μg/ml heparin was added into the culture to obtain mature enzyme in the conditioned media. After 3 days, the conditioned media were harvested and stored at –20 °C until purification. A stable HEK 293-EBNA cell line was not successfully established for TS5-6; therefore this enzyme was purified from transiently transfected HTB-94 cells. The collected conditioned media (1 liter) were filtered to remove any cell debris and passed over a 3-ml anti-FLAG M2-agarose column at 4 °C. The column was first washed extensively with 50 mm Tris-HCl (pH 7.5) containing 1 m NaCl, 10 mm CaCl2, 0.02% NaN3, and 0.02% Brij-35. The bound material was eluted with 200 μg/ml FLAG peptide in 50 mm Tris-HCl (pH 7.5) containing 100 mm NaCl, 10 mm CaCl2, 0.02% NaN3, and 0.02% Brij-35. FLAG-eluted TS5-2, TS5-3, TS5-4, TS5-5, and TS5-6 were further purified on a Sephacryl S-200 gel filtration column (1.6 × 100 cm) to remove the FLAG peptide. Due to the low abundance of TS5-1 in the starting material (∼30 μg/liter of conditioned media), a Macro-Prep 25 S resin was used to remove the FLAG peptide and to concentrate the protein. FLAG-eluted fractions were dialyzed against 20 mm Tris acetate (pH 6.4) and applied to a column of Macro-Prep S resin (300 μl). The column was washed with 50 mm Tris-HCl (pH 7.5) containing 250 mm NaCl, 10 mm CaCl2, 0.02% NaN3, and 0.02% Brij-35. ADAMTS5-1 was eluted from the column with 50 mm Tris-HCl (pH 7.5) containing 1 m NaCl, 10 mm CaCl2, 0.02% NaN3, and 0.02% Brij-35. The TS5-1-containing fractions, monitored using SDS-PAGE, were pooled; dialyzed against aggrecanase reaction buffer (50 mm Tris-HCl (pH 7.5) containing 100 mm NaCl, 10 mm CaCl2, 0.02% NaN3, and 0.02% Brij-35); snap frozen; and stored at –80 °C. Active Site Titration of ADAMTS-5 and Its Mutant—The concentrations of active ADAMTS-5 and its domain deletion mutants were determined by titration with N-TIMP-3. The concentration of TS4-5 and TS5-6 (both are the catalytic domain only) was determined with Coomassie Brilliant Blue R-250 using bovine serum albumin as standard due to low enzymatic activity. Transient Transfection of the Human Chondrosarcoma HTB-94 Cells—Human chondrosarcoma cells (HTB-94) were seeded at 5 × 104 cells/well of a 12-well plate in media containing 5% fetal calf serum. The cells were grown overnight and transfected the following day with 0.5 μg of DNA using FuGENE 6 according to the manufacturer's instructions. The next day, the conditioned media were removed, the cells were washed once with serum-free DMEM to remove any remaining serum, and 500 μl of serum-free DMEM with or without 100 μg/ml heparin was added to each culture and incubated for 48 h (the total transfection time was 72 h). The conditioned media were then harvested, and detached cells were removed by centrifugation at 3,000 × g for 10 min. Proteins in the conditioned media were precipitated using a final concentration of 3.3% trichloroacetic acid and subjected to Western blot analysis with the anti-FLAG M2 antibody to detect ADAMTS-5 and its mutants. Immunolocalization of ADAMTS-5 in Human Chondrosarcoma HTB-94 Cells—The procedures were essentially the same as those described for ADAMTS-4 studies (17Kashiwagi M. Enghild J.J. Gendron C. Hughes C. Caterson B. Itoh Y. Nagase H. J. Biol. Chem. 2004; 279: 10109-10119Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). HTB-94 cells were transiently transfected with the pCEP4 expression vector containing cDNA encoding ADAMTS-5 or its variants and cultured in serum-free DMEM with or without heparin (100 μg/ml) for 48 h. To localize each recombinant ADAMTS-5 protein, the cells were washed, fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline for 7 min, washed again, and then incubated with blocking solution. The samples were then incubated with the mouse anti-FLAG M2 antibody followed by Alexa-488-conjugated goat anti-(mouse IgG) IgG. The specimens were then washed with phosphate-buffered saline, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline, and incubated with Alexa-660-conjugated phalloidin to visualize actin filaments within the cells. The samples were viewed using a Nikon Eclipse TE 2000-U microscope equipped with a PerkinElmer Life Sciences Ultraview Live Cell Imaging System with excitation and emission wavelengths at 488 and 525 nm for Alexa-488 and at 647 and 700 nm for Alexa-660, respectively. SDS-PAGE and Western Blot Analysis—SDS-PAGE was run under reducing conditions using a modification of the Ammediol/glycine/HCl buffer system of Wyckoff et al. (29Wyckoff M. Rodbard D. Chrambach A. Anal. Biochem. 1977; 78: 459-482Crossref PubMed Scopus (226) Google Scholar). Proteins were stained either with Coomassie Brilliant Blue R-250 or with silver (30Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar). For Western blot analysis after SDS-PAGE, proteins were electrotransferred onto a polyvinylidene difluoride membrane, and the membrane was processed as described previously (17Kashiwagi M. Enghild J.J. Gendron C. Hughes C. Caterson B. Itoh Y. Nagase H. J. Biol. Chem. 2004; 279: 10109-10119Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Aggrecanase Assays—Aggrecan (750 nm) was incubated with ADAMTS4-2 (TS4-2), full-length ADAMTS-5, or an ADAMTS-5 domain deletion mutant in 100 μl of aggrecanase reaction buffer (50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 10 mm CaCl2, 0.02% NaN3, and 0.02% Brij 35) at 37 °C for the indicated period of time. The reactions were stopped by adding an equal volume of double strength glycosaminoglycan buffer (200 mm sodium acetate, 50 mm Tris-HCl (pH 6.8), and 100 mm EDTA). Aggrecan was then deglycosylated by incubating with 0.01 units of chondroitinase ABC/10 μg of aggrecan and 0.01 units of keratanase/10 μg of aggrecan for 16–18 h at 37 °C. The samples were precipitated by adding 5 volumes of acetone, incubated at –20 °C for 15 min, and then centrifuged at 3,000 × g for 10 min. The pellet was dried and dissolved in 20 μl of reducing sample buffer. The products were analyzed by Western blot analysis with the 2-B-6, BC-3, or anti-GELE antibody as described previously (17Kashiwagi M. Enghild J.J. Gendron C. Hughes C. Caterson B. Itoh Y. Nagase H. J. Biol. Chem. 2004; 279: 10109-10119Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). For the comparative studies, all polyvinylidene difluoride membranes were processed simultaneously, and Western analyses were carried out under identical conditions. Relative activities among different forms of ADAMTS-5 and TS4-2 were estimated by visual inspection of Western blots for equivalent amount of products formed based on the concentration of the enzyme and the time of incubation. Gels and blots were scanned using a Bio-Rad GS-710 scanning densitometer, and the band intensity was quantified using the 1D Phoretix quantification software (Nonlinear Dynamics, Newcastle upon Tyne, UK). Amino-terminal Sequence Analysis of Cm-Tf and Fibromodulin Fragments—Cm-Tf (10 mg/ml) was incubated with TS4-2 or TS5-4 in aggrecanase reaction buffer at 37 °C for 72 h, and the products were analyzed by SDS-PAGE. The fragments were electrotransferred to a polyvinylidene difluoride membrane and visualized with Coomassie Brilliant Blue R-250. The bands were excised and placed directly onto a Polybrene-treated glass filter, and samples were analyzed by automated Edman degradation in an Applied Biosystems Procise 494HT sequencer with on-line phenylthiohydantoin high pressure liquid chromatography analysis. Fibromodulin (0.1 mg/ml) was incubated with TS5-4 for 4 h, and the products were deglycosylated with peptide N-glycosidase F prior to SDS-PAGE. The 29-kDa fibromodulin fragment was sequenced in a manner similar to that described above for the Cm-Tf fragments. Full-length ADAMTS-5 and its carboxyl-terminal domain-deleted mutants were purified from the conditioned media of stably transfected HEK 293-EBNA or transiently transfected HTB-94 cells by anti-FLAG M2 affinity chromatography. The FLAG peptide used to elute the bound material was removed either by Macro-Prep 25 S or gel filtration chromatography. All of the preparations exhibited a single band by SDS-PAGE analysis with silver staining except TS5-5, which appeared as two bands due to N-linked oligosaccharides (Fig. 2; see below). The purified proteins were verified to be ADAMTS-5 by Western blot analysis using anti-TS5cat antibody (Fig. 2C). Approximately 2 μg of TS5-1, 70 μg of TS5-2, 260 μg of TS5-3, 150 μg of TS5-4, 250 μg of TS5-5, and 10 μg of TS5-6 were purified from 1 liter of conditioned media. All forms of ADAMTS-5 were stable after freezing, but some autolysis was observed after repeated freezing and thawing. The molecular masses of the recombinant enzymes estimated by SDS-PAGE were slightly higher than that predicted from their amino acid composition (see Fig. 1), consistent with glycosylation of the protein (7Zeng W. Corcoran C. Collins-Racie L.A. LaVallie E.R. Morris E.A. Flannery C.R. Biochim. Biophys. Acta. 2006; 1760: 517-524Crossref PubMed Scopus (91) Google Scholar). ADAMTS-5 contains four potential N-glycosylation sites and one potential mucin-type O-glycosylation site. The four N-glycosylation sites were predicted using NetNGlyc 1.0 in the following domains: one in the Dis domain, one in the CysR do