Title: Characterization of ADAMTS-9 and ADAMTS-20 as a Distinct ADAMTS Subfamily Related to Caenorhabditis elegans GON-1
Abstract: We demonstrate that in humans, two metalloproteases, ADAMTS-9 (1935 amino acids) and ADAMTS-20 (1911 amino acids) are orthologs of GON-1, an ADAMTS protease required for gonadal morphogenesis in Caenorhabditis elegans. ADAMTS-9 and ADAMTS-20 have an identical modular structure, are distinct in possessing 15 TSRs and a unique C-terminal domain, and have a similar gene structure, suggesting that they comprise a new subfamily of human ADAMTS proteases. ADAMTS20 is very sparingly expressed, although it is detectable in epithelial cells of the breast and lung. However, ADAMTS9 is highly expressed in embryonic and adult tissues, and therefore we characterized the ADAMTS-9 protein further. Although the ADAMTS-9 zymogen has many proprotein convertase processing sites, pulse-chase analysis, site-directed mutagenesis, and amino acid sequencing demonstrated that maturation to the active form occurs by selective proprotein convertase (e.g. furin) cleavage of the Arg287–Phe288 bond. Although lacking a transmembrane sequence, ADAMTS-9 is retained near the cell surface as well as in the ECM of transiently transfected COS-1 and 293 cells. COS-1 cells transfected with ADAMTS9 (but not vector-transfected cells) proteolytically cleaved bovine versican and aggrecan core proteins at the Glu441–Ala442bond of versican V1 and the Glu1771–Ala1772bond of aggrecan, respectively. In contrast, the ADAMTS-9 catalytic domain alone was neither localized to the cell surface nor able to confer these proteolytic activities on cells, demonstrating that the ancillary domains of ADAMTS-9, including the TSRs, are required both for specific extracellular localization and for its versicanase and aggrecanase activities. We demonstrate that in humans, two metalloproteases, ADAMTS-9 (1935 amino acids) and ADAMTS-20 (1911 amino acids) are orthologs of GON-1, an ADAMTS protease required for gonadal morphogenesis in Caenorhabditis elegans. ADAMTS-9 and ADAMTS-20 have an identical modular structure, are distinct in possessing 15 TSRs and a unique C-terminal domain, and have a similar gene structure, suggesting that they comprise a new subfamily of human ADAMTS proteases. ADAMTS20 is very sparingly expressed, although it is detectable in epithelial cells of the breast and lung. However, ADAMTS9 is highly expressed in embryonic and adult tissues, and therefore we characterized the ADAMTS-9 protein further. Although the ADAMTS-9 zymogen has many proprotein convertase processing sites, pulse-chase analysis, site-directed mutagenesis, and amino acid sequencing demonstrated that maturation to the active form occurs by selective proprotein convertase (e.g. furin) cleavage of the Arg287–Phe288 bond. Although lacking a transmembrane sequence, ADAMTS-9 is retained near the cell surface as well as in the ECM of transiently transfected COS-1 and 293 cells. COS-1 cells transfected with ADAMTS9 (but not vector-transfected cells) proteolytically cleaved bovine versican and aggrecan core proteins at the Glu441–Ala442bond of versican V1 and the Glu1771–Ala1772bond of aggrecan, respectively. In contrast, the ADAMTS-9 catalytic domain alone was neither localized to the cell surface nor able to confer these proteolytic activities on cells, demonstrating that the ancillary domains of ADAMTS-9, including the TSRs, are required both for specific extracellular localization and for its versicanase and aggrecanase activities. The ADAMTS (Adisintegrin-likeand metalloprotease (reprolysin type) withthrombospondin type I motif) family consists of secreted zinc metalloproteases with a precisely ordered modular organization that includes at least one thrombospondin type I repeat (TSR) 1The abbreviations used are: TSR, thrombospondin type I repeat; DMEM, Dulbecco's modified Eagle's medium; GAG, glycosaminoglycan; ORF, open reading frame; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (1Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Google Scholar, 2Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Google Scholar). Important functions have been established for several members of the family. ADAMTS-4, ADAMTS-5, and (less efficiently) ADAMTS-1 degrade the cartilage proteoglycan aggrecan and are referred to as aggrecanases (3Tortorella 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. et al.Science. 1999; 284: 1664-1666Google Scholar, 4Abbaszade 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. Burn T.C. et al.J. Biol. Chem. 1999; 274: 23443-23450Google Scholar, 5Kuno K. Okada Y. Kawashima H. Nakamura H. Miyasaka M. Ohno H. Matsushima K. FEBS Lett. 2000; 478: 241-245Google Scholar). They play a major role in aggrecan loss in arthritis (6Sandy J.D. Flannery C.R. Neame P.J. Lohmander L.S. J. Clin. Invest. 1992; 89: 1512-1516Google Scholar, 7Lohmander L.S. Neame P.J. Sandy J.D. Arthritis Rheum. 1993; 36: 1214-1222Google Scholar). ADAMTS-1 and ADAMTS-4 participate in the turnover of the aggrecan-related proteoglycans versican and brevican in blood vessels (8Sandy J.D. Westling J. Kenagy R.D. Iruela-Arispe M.L. Verscharen C. Rodriguez-Mazaneque J.C. Zimmermann D.R. Lemire J.M. Fischer J.W. Wight T.N. Clowes A.W. J. Biol. Chem. 2001; 276: 13372-13378Google Scholar) and the nervous system, respectively (9Matthews R.T. Gary S.C. Zerillo C. Pratta M. Solomon K. Arner E.C. Hockfield S. J. Biol. Chem. 2000; 275: 22695-22703Google Scholar). ADAMTS2mutations cause dermatosparaxis, a recessively inherited disorder characterized by severe skin fragility that results from incomplete proteolytic removal of the procollagen I amino propeptide (N-propeptide) (10Colige A. Sieron A.L. Li S.W. Schwarze U. Petty E. Wertelecki W. Wilcox W. Krakow D. Cohn D.H. Reardon W. Byers P.H. Lapiere C.M. Prockop D.J. Nusgens B.V. Am. J. Hum. Genet. 1999; 65: 308-317Google Scholar). ADAMTS-3 and ADAMTS-14 are procollagen N-propeptidases with probable roles in procollagen II processing in cartilage or procollagen I processing in tissues other than skin, respectively (11Colige A. Vandenberghe I. Thiry M. Lambert C.A. Van Beeumen J. Li S.W. Prockop D.J. Lapiere C.M. Nusgens B.V. J. Biol. Chem. 2002; 277: 5756-5766Google Scholar, 12Fernandes R.J. Hirohata S. Engle J.M. Colige A. Cohn D.H. Eyre D.R. Apte S.S. J. Biol. Chem. 2001; 276: 31502-31509Google Scholar). ADAMTS13 mutations lead to inherited thrombocytopenic purpura, a coagulation disorder caused by deficient proteolytic processing of von Willebrand factor (13Levy G.G. Nichols W.C. Lian E.C. Foroud T. McClintick J.N. McGee B.M. Yang A.Y. Siemieniak D.R. Stark K.R. Gruppo R. Sarode R. Shurin S.B. Chandrasekaran V. Stabler S.P. Sabio H. Bouhassira E.E. Upshaw Jr., J.D. Ginsburg D. Tsai H.M. Nature. 2001; 413: 488-494Google Scholar).Adamts1-null mice have abnormal adipogenesis, defective angiogenesis in the adrenal gland, and a defect of ureteric ECM turnover, leading to hydronephrosis (14Shindo 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. Invest. 2000; 105: 1345-1352Google Scholar). Adamts2-null mice have fragile skin, and males are infertile (15Li S.W. Arita M. Fertala A. Bao Y. Kopen G.C. Langsjo T.K. Hyttinen M.M. Helminen H.J. Prockop D.J. Biochem. J. 2001; 355: 271-278Google Scholar). Many other ADAMTS enzymes have been discovered through molecular cloning, and their functions are presently unknown. Altogether, 19 human ADAMTS symbols identifying 18 distinct genes and their products have been assigned (note that ADAMTS5 (1Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Google Scholar) and ADAMTS11 (4Abbaszade 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. Burn T.C. et al.J. Biol. Chem. 1999; 274: 23443-23450Google Scholar) designate the same gene). 2Gene nomenclature (ADAMTS9 andADAMTS20) was assigned after consultation with the Human Gene Nomenclature Committee. Adamts9 and Adamts20are the respective mouse orthologs. The protein products of these genes are designated as ADAMTS-9 and ADAMTS-20. Similar nomenclature is used for other ADAMTS genes and their products. GON-1 refers to the product of the C. elegans gon-1 gene. ADAMTS are also present in invertebrates, which contain fewer ADAMTS genes than mammalian genomes. A Caenorhabditis elegansADAMTS gene, gon-1, has an essential role in reproduction (16Blelloch R. Anna-Arriola S.S. Gao D. Li Y. Hodgkin J. Kimble J. Dev. Biol. 1999; 216: 382-393Google Scholar). The protease (GON-1) encoded by gon-1 is required for migration of distal tip cells during gonadal morphogenesis. It may have a role in degradation of basement membrane or for processing of extracellular cues required for cell migration (16Blelloch R. Anna-Arriola S.S. Gao D. Li Y. Hodgkin J. Kimble J. Dev. Biol. 1999; 216: 382-393Google Scholar). GON-1 is the largest of all ADAMTS enzymes described to date and contains 18 TSRs (16Blelloch R. Anna-Arriola S.S. Gao D. Li Y. Hodgkin J. Kimble J. Dev. Biol. 1999; 216: 382-393Google Scholar). In addition, it has a presumed globular domain at the C terminus without similarity to known proteins. Human ADAMTS-9, as previously described (17Clark M.E. Kelner G.S. Turbeville L.A. Boyer A. Arden K.C. Maki R.A. Genomics. 2000; 67: 343-350Google Scholar) contains four TSRs. Despite being a much smaller enzyme than GON-1, it had greater sequence similarity to it than to any other human ADAMTS (17Clark M.E. Kelner G.S. Turbeville L.A. Boyer A. Arden K.C. Maki R.A. Genomics. 2000; 67: 343-350Google Scholar). Here, we characterize a considerably longer form of ADAMTS-9 (designated ADAMTS-9B, but referred to subsequently in this paper as ADAMTS-9) that we propose is the authentic full-length product of ADAMTS9. In addition, we have discovered a novel enzyme, ADAMTS-20, and determined its complete primary sequence. ADAMTS-9 and ADAMTS-20 have an identical domain organization and exon structure and a very similar primary sequence, showing that they comprise a distinct subfamily of GON-1-related ADAMTS proteases in the mammalian genome. We have characterized the zymogen maturation and cellular localization of the more highly expressed of these two proteins, ADAMTS-9, and have investigated its role in proteolysis of the large aggregating proteoglycans versican and aggrecan. Our data demonstrate the critical requirement of the ancillary domains for the proteolytic function and localization of ADAMTS-9. BLAST (Basic Local Alignment Search Tool) programs from the National Center for Biotechnology Information were used to search the data base of expressed sequence tags (dBEST), using the protein sequences of ADAMTS proteases previously discovered by us (1Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Google Scholar, 18Georgiadis K.E. Hirohata S. Seldin M.F. Apte S.S. Genomics. 1999; 62: 312-315Google Scholar). To extend the initially identified ADAMTS9 cDNA to the 5′-end, human chondrocyte, muscle, heart, or fetal brain mRNA (Marathon cDNA, Clontech, Palo Alto, CA) was used as the template for rapid amplification of cDNA ends as previously described (1Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Google Scholar). To confirm that the overlapping cDNA clones obtained represented a contiguous mRNA, the complete ORF was amplified by PCR. The oligonucleotide primers 5′-AAGCGGCCGCACCATGCAGTTTGTATCC-3′ (NotI site underlined and start codon italicized) and 5′-CTCGAGAATAAAACTCGCACCTCCAGGC-3′ (XhoI site underlined and modified stop codon italicized) were used for PCR with human fetal skeletal muscle cDNA as template and Advantage 2 polymerase (Clontech, Palo Alto, CA). The 5.8-kb PCR product was cloned into pGEM-T Easy (Promega, Madison, WI) and sequenced completely. cDNA cloning ofAdamts9 will be reported elsewhere. 3K. A. Jungers and S. S. Apte, unpublished data. To ask whether there existed additional ADAMTS proteases with a domain organization similar to GON-1 and ADAMTS-9, the human genome sequence (Celera, Rockville, MD) was searched using the amino acid sequence of the unique C-terminal domain of ADAMTS-9. GENSCAN (available on the World Wide Web at genes.mit.edu/GENSCAN.html) analysis of genomic DNA upstream and downstream of the initially identified ADAMTS20sequence was used to identify putative ADAMTS20 exons. Oligonucleotide primers based on the sequences of these putativeADAMTS20 exons were used for PCR spanning adjacent exons using cDNA derived from the human K562 (erythroleukemia) and A549 (lung cancer) cell lines. The exon-intron structures of ADAMTS9 andADAMTS20 were deduced by comparison of the respective cDNAs with human genome sequences using BLAST searches of private (Celera) and public (GenBankTM) databases. Multiple tissue northern blots containing 1 μg/lane poly(A+) RNA from mouse embryos and individual adult mouse and human tissues (Clontech, Palo Alto, CA) were hybridized to [α-32P]dCTP-labeled ADAMTS9,ADAMTS20, or Adamts9 probes, followed by autoradiographic exposure for 3–7 days. cDNA panels derived from human adult and fetal organs normalized with respect toGAPDH mRNA levels were purchased from Clontech. Real time PCR of these cDNA templates was performed in an ABI Prism 7700 sequence detector using SYBR Green PCR Core Reagents (Applied Biosystems, Foster City, CA), as previously described (12Fernandes R.J. Hirohata S. Engle J.M. Colige A. Cohn D.H. Eyre D.R. Apte S.S. J. Biol. Chem. 2001; 276: 31502-31509Google Scholar). PCR amplifications were performed in triplicate for all templates, along with parallel measurements of GAPDHcDNA for normalization. The GAPDH-normalized quantitative data forADAMTS9 and ADAMTS20 were used to determine theADAMTS9/ADAMTS20 transcript ratio in all templates examined. The following primers were used for amplification at a concentration of 300 nm each: ADAMTS9forward, 5′-GGACAAGCGAAGGACATCC-3′; ADAMTS9 reverse, 5′-ATCCATCCATAATGGCTTCC-3′; ADAMTS20 forward, 5-GGTGGCATGTTATTGGCAAAA-3′; ADAMTS20 reverse, 5′-CACAGTTACCATGGCATAGTTCTTG-3′; GAPDH primers were described previously (12Fernandes R.J. Hirohata S. Engle J.M. Colige A. Cohn D.H. Eyre D.R. Apte S.S. J. Biol. Chem. 2001; 276: 31502-31509Google Scholar). RT-PCR performed in the absence of template was negative with all primer pairs. RNA in situ hybridization was performed essentially as previously described (19Albrecht U. Eichele G. Helms J.A. Lu H.C. Daston G.P. Molecular and Cellular Methods in Developmental Toxicology. CRC Press, Inc., Boca Raton, FL1997: 23-48Google Scholar), using 35S-labeled antisense and sense cRNA probes transcribed from a 600-nt cDNA template encoding the unique domain of ADAMTS-20. Normal human breast and lung samples as well as samples of squamous cell carcinoma of breast and adenocarcinoma of lung were obtained under a Cleveland Clinic Foundation Institutional Review Board-approved protocol and fixed in formalin (tissue samples were provided by the Cooperative Human Tissue Network). 5-μm-thick paraffin sections were hybridized to the probes prior to dipping in photographic emulsion (Eastman Kodak Co.) and followed by autoradiographic exposure for 7 days. Nuclei were stained with 4′,6-diamidino-2-phenylindole. TheADAMTS9 cDNA was excised as aNotI-XhoI fragment and cloned into theNotI and SalI sites of pFLAG-CMV-5a (Sigma) to introduce an in-frame C-terminal FLAG tag (ADAMTS-9FLAG). For expression of ADAMTS-91–508 (the signal peptide, prodomain, and catalytic domain), PCR amplification was done using the same forward primer as for the full-length ADAMTS9 cDNA, the reverse primer 5′-AACTCGAGTTAGGCAAAGGGTAGGGTCTG-3′ (XhoI site underlined), and fetal heart cDNA (Clontech) as template. The resulting amplicon was cloned in pFLAG-CMV-5a (Sigma) and pcDNA3.1 MYC/HIS B+ (Invitrogen) to generate proteins with in-frame C-terminal FLAG ormyc-His tags, respectively, ADAMTS-91–508FLAG, and ADAMTS-91–508MYC/HIS. Site-directed mutagenesis of the convertase (e.g. furin) sites (Arg33 → Ala, Arg74 → Ala, Arg280 → Ala, and Arg287 → Ala) in ADAMTS-91–508MYC/HISwas done using the QuikChange site-directed mutagenesis kit (Stratagene). The insert of the KIAA0688 gene (20Ishikawa K. Nagase T. Suyama M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 169-176Google Scholar) encoding ADAMTS-4 (3Tortorella 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. et al.Science. 1999; 284: 1664-1666Google Scholar) in pBluescript SK (Stratagene) was excised with EcoRI andXhoI and inserted into the corresponding sites of pcDNA3.1MYC/HIS A- (Invitrogen) to generate a mammalian expression vector producing untagged ADAMTS-4. The ADAMTS4 andADAMTS5 ORFs from the convertase-processing site to the stop codon were PCR-amplified and cloned into p3XFLAG-CMV-9 (Sigma) for expression in frame with a preprotrypsin leader sequence and three tandem FLAG tags present just downstream of the signal peptidase cleavage site. These proteins are therefore secreted with N-terminal FLAG tag (3×FLAGADAMTS-4 or 3×FLAGADAMTS-5). All expression plasmids and site-directed mutations were verified by DNA sequencing. COS-1 and 293-HEK cells were maintained and transfected with ADAMTS-9FLAG, ADAMTS-91–508FLAG,3×FLAGADAMTS-4, or 3×FLAGADAMTS-5, as described previously (21Hirohata S. Wang L.W. Miyagi M. Yan L. Seldin M.F. Keene D.R. Crabb J.W. Apte S.S. J. Biol. Chem. 2002; 22: 22Google Scholar). Transfected cell lysates and culture medium were harvested separately after 48 h and were separated by reducing SDS-PAGE followed by Western blot analysis using the FLAG M2 monoclonal antibody (Sigma). For immunolocalization of extracellular ADAMTS-9FLAG, ADAMTS-91–508FLAG,3×FLAGADAMTS-4, and 3×FLAGADAMTS-5, cells were stained with anti-FLAG M2 monoclonal antibody 48 h post-transfection without permeabilization as previously described (21Hirohata S. Wang L.W. Miyagi M. Yan L. Seldin M.F. Keene D.R. Crabb J.W. Apte S.S. J. Biol. Chem. 2002; 22: 22Google Scholar). Alternatively, transfected cells were stained following fixation in 4% paraformaldehyde (staining with permeabilization). Nuclei were stained with 4′,6-diamidino-2-phenylindole. As controls, COS-1 and 293 cells were transfected with the empty FLAG vector alone, followed by the immunostaining procedure, or the primary antibody was omitted for FLAG staining. To release ADAMTS-9 from the cell surface, transfected 293 cells and ECM were harvested by scraping and resuspended in phosphate-buffered saline (10 mm phosphate buffer, pH 7.4, 2.7 mmKCl, 137 mm NaCl). Cells and ECM were gently agitated by end-over end rotation in PBS alone or in PBS plus 100 mm or 200 mm NaCl at 4 °C for 30 min. 508MYC/HIS Purification and Analysis—To obtain stably transfected 293 cells expressing ADAMTS-91–508MYC/HIS, selection with G418 (750 μg/ml) was applied after transfection, and selected clones were maintained in culture medium containing 5% serum and 250 μg/ml G418. Conditioned medium was dialyzed into binding buffer (20 mm sodium phosphate, 500 mm NaCl, pH 7.8, containing 0.03% Brij-35 (Sigma)) prior to binding on a 5-ml Ni2+-Sepharose column (ProBond™; Invitrogen). The column was washed with 3 column volumes of binding buffer. A gradient of 0–42.5 mm imidazole in binding buffer was used to remove nonspecifically bound molecules from the column. Stepwise elution was done using one-column volume batches of 0–250 mm imidazole in binding buffer. Elution was monitored by Western blotting using antibody 9E10. The majority of protein was determined to elute at 50 mm imidazole. ADAMTS-91–508MYC/HIS was electrophoresed on 10% SDS-PAGE, electrotransferred to polyvinylidene difluoride membrane, and lightly stained with modified Coomassie Blue (Simply Blue Safe Stain; Invitrogen). The 28-kDa band was excised and subjected to Edman degradation on an Applied Biosystems Procise 492 sequencer in the Molecular Biotechnology Core Facility of the Lerner Research Institute. Deglycosylation of lysate and conditioned medium from ADAMTS-91–508FLAG-transfected cells was done using 10 units of PNGase F (Roche Molecular Biosciences) for 3 h at 37 °C in 150 mm sodium phosphate, pH 7.4, 50 mm EDTA, 0.1% SDS, 1% 2-mercaptoethanol, 0.5% Triton X-100, followed by immunoprecipitation with anti-FLAG M2 as described below. Stably transfected cells were cultured with and without tunicamycin A homolog (Sigma) as previously described (21Hirohata S. Wang L.W. Miyagi M. Yan L. Seldin M.F. Keene D.R. Crabb J.W. Apte S.S. J. Biol. Chem. 2002; 22: 22Google Scholar), followed by Western blotting of conditioned medium and cell lysates. Unless specified, reagents were purchased from Sigma. QBI 293A cells (Quantum Biotechnologies, Montreal, Canada) were maintained in complete DMEM containing 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cells were transiently transfected with ADAMTS-91–508FLAG or ADAMTS-91–508MYC/HISusing Fugene 6 (Roche Molecular Biosciences). 24 h following transfection, cells were washed twice with warm phosphate-buffered saline and incubated in Met/Cys-free medium (MEM SelectAmine kit; Invitrogen), supplemented with 10% dialyzed FCS, 1 mmglutamine, and a [35S]methionine/cysteine mixture (EXPRE35S35S™; PerkinElmer Life Sciences). A 15- or 30-min labeling (pulse) was followed by incubation in complete nonradioactive medium (chase) for the indicated times. The cell layer was washed with PBS, and cells were lysed with 1 ml of radioimmune precipitation buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, and 4 mm EDTA) containing protease inhibitors (1 μmaprotinin, 10 μm pepstatin, 10 μmleupeptin, and 1 μm phenylmethylsulfonyl fluoride). Samples were centrifuged to remove insoluble material. FLAG M2 antibody or penta-His antibody (Qiagen, Mississauga, Canada) was added to cell lysate and medium followed by overnight incubation at 4 °C. Protein A/G Plus-agarose beads were added and incubated with samples for 1 h at 4 °C. Beads were washed three times with 1 ml of radioimmune precipitation buffer, and labeled proteins were resolved by reducing SDS-PAGE. Gels were treated with ENTENSIFY reagent (PerkinElmer Life Sciences), dried, and exposed for fluorography. CHO.RPE 40 cells (22Spence M.J. Sucic J.F. Foley B.T. Moehring T.J. Somat. Cell. Mol. Genet. 1995; 21: 1-18Google Scholar) were maintained as previously described (32Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Google Scholar). They were transfected with ADAMTS-91–508MYC/HIS alone or in combination with furin. QBI 293A cells were transiently transfected either with ADAMTS- 91–508MYC/HIS, or its derivatives obtained by site-directed mutagenesis. Cells were metabolically labeled as above for 3 h, and immunoprecipitation and fluorography were done as above. Versican monomer (a mixture of the V1 and V0 forms) was isolated from bovine aorta, as previously described (8Sandy J.D. Westling J. Kenagy R.D. Iruela-Arispe M.L. Verscharen C. Rodriguez-Mazaneque J.C. Zimmermann D.R. Lemire J.M. Fischer J.W. Wight T.N. Clowes A.W. J. Biol. Chem. 2001; 276: 13372-13378Google Scholar). COS-1 cells were transfected with ADAMTS-9FLAG, ADAMTS-91–508FLAG, or ADAMTS-4 expression plasmids or with empty vector pFLAG-CMV-5a (as negative control) in six-well plates in DMEM plus 10% fetal bovine serum. Cells from a single well were used for each experiment. 48 h after transfection, cells were scraped off and suspended in serum-free DMEM followed by six washes in fresh serum-free DMEM. Cells were resuspended in a final volume of 75 μl of serum-free DMEM. Versican (5 μg in a volume of 25 μl) was added, and the reaction was incubated at 37 °C for 18 h. The reaction was centrifuged briefly, and the cell pellets were retained for Western blotting of ADAMTS-9FLAG using the anti-FLAG M2 monoclonal antibody (Sigma). An equal volume of 2× glycosaminoglycan digestion buffer (200 mm Tris, pH 6.5, 100 mm sodium acetate) containing 0.5 units of chondroitinase ABC (Seikagaku) was added to the supernatant, followed by incubation for 16–18 h at 37 °C. Protein was precipitated with 5 volumes of acetone at −20 °C for 15 min, dissolved in 50 μl of Laemmli sample buffer, and electrophoresed on 7% SDS-PAGE prior to blotting to nitrocellulose. A rabbit polyclonal antiserum to the versican Asp-Pro-Glu-Ala-Ala-Glu (DPEAAE) neoepitope (8Sandy J.D. Westling J. Kenagy R.D. Iruela-Arispe M.L. Verscharen C. Rodriguez-Mazaneque J.C. Zimmermann D.R. Lemire J.M. Fischer J.W. Wight T.N. Clowes A.W. J. Biol. Chem. 2001; 276: 13372-13378Google Scholar) (provided by Dr. John Sandy) was used at 1:1000 dilution for Western blotting, followed by enhanced chemiluminescence detection of antibody binding. Anti-DPEAAE recognizes the new C terminus resulting from cleavage of versican at the Glu441–Ala442 bond (this enumeration describes the site in the V1 isoform); the corresponding peptide bond is Glu1428–Ala1429 in the V0 isoform, since this form contains an additional GAG-bearing region, GAG-α, as a result of alternative splicing (8Sandy J.D. Westling J. Kenagy R.D. Iruela-Arispe M.L. Verscharen C. Rodriguez-Mazaneque J.C. Zimmermann D.R. Lemire J.M. Fischer J.W. Wight T.N. Clowes A.W. J. Biol. Chem. 2001; 276: 13372-13378Google Scholar). Aggrecan monomer was isolated from bovine articular cartilage as previously described (23Sajdera S.W. Hascall V.C. J. Biol. Chem. 1969; 244: 77-87Google Scholar). Aggrecan (20 μg) was incubated with transfected cells as described above. Neoepitope Western blot analysis was as performed for versican (above), except that the proteolytic cleavage at the Glu1771–Ala1772 bond of aggrecan was detected using anti-Ala1772-Gly-Glu-Gly (AGEG) antiserum (24Tortorella M.D. Pratta M. Liu R.Q. Austin J. Ross O.H. Abbaszade I. Burn T. Arner E. J. Biol. Chem. 2000; 275: 18566-18573Google Scholar) (provided by Micky Tortorella). Our search for novel ADAMTS proteases identified a human expressed sequence tag (GenBankTM accession number AA205581 encoded by IMAGE clone 646675) from neuroepithelium-derived NT2 cells treated with retinoic acid. The ORF of this expressed sequence tag was homologous to ADAMTS proteases and encoded four TSRs followed by a C-terminal domain containing 10 cysteines that was similar to the C terminus of a polypeptide predicted by the C. elegans F25H8.3 cosmid (C. elegans protein data base Wormpep, www.sanger.ac.uk/Projects/C_elegans/wormpep) and subsequently identified as GON-1. The novel human ORF was designated ADAMTS-9. Completion of the full-length protein coding sequence to the putative start codon required several rounds of rapid amplification of cDNA ends. Together, the cloned cDNA sequences represent an mRNA of 8 kb (Fig. 1 a). The 3′-untranslated region in IMAGE clone 646675 contained a consensus polyadenylation signal (AATAAA) 15 nucleotides upstream of the poly(A) tail. The most 5′ clone obtained (TS9-B10) contained 32 bp of the 5′-untranslated region. The putative signal peptide coding sequence was preceded by a methionine codon within a satisfactory Kozak consensus sequence (A at −3 relative to ATG), but there was no upstream, in-frame stop codon. The search for ADAMTS-9-related proteins led to identification of a polypeptide (Celera hCP1629711) predicted by exons on human chromosome 12. The complete 5733-nt-long ADAMTS-20 ORF was assembled from overlapping cDNA clones (Fig. 1 a). TheADAMTS20 mRNA was found in low quantities, routinely requiring 35 cycles of PCR or nested PCR for visualization of the PCR products on a gel. Because of the rarity of ADAMTS20transcripts as well as the presence of numerous regions that are difficult to PCR-amplify, we have been so far unable to obtain the complete ORF in a single PCR reaction. ADAMTS-9 and ADAMTS-20 are similar in length, containing 1935 and 1911 amino acids, respectively (Figs.1 b and 2). Each contains a C-terminal array of 14 TSRs (15 TSRs/enzyme) that is interrupted by short “linker” peptides located between TSR-6 and -7 and TSR-8 and -9 that do not have similar sequences. ADAMTS-9 and ADAMTS-20 are very similar to each other, with 48% identity and 64% similarity. The cysteine signatures of individual modules in ADAMTS-9 and ADAMTS-20 are identical to those of most other ADAMTS enzymes, with the exception of the procollagen aminopropeptidases (ADAMTS-2, ADAMTS-3, ADAMTS-14) and ADAMTS-13, which have distinctive prodomains and catalytic domains (12Fernandes R.J. Hirohata S. Engle J.M. Colige A. Cohn D.H. Eyre D.R. Apte S.S. J. Biol. Chem. 2001; 276: 31502-31509Google Scholar). Each module in ADAMTS-9 and ADAMTS-20 (with one exception, described below) contains an even number of cysteines, suggesting participation in internal disulfide bonds. There are 126 cysteines in mature ADAMTS-9, predicting 63 intrachain disulfide bonds. ADAMTS-20 has a Cys to Tyr substitutio