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ADAMTS7B, the Full-length Product of the ADAMTS7 Gene, Is a Chondroitin Sulfate Proteoglycan Containing a Mucin Domain

2004; Elsevier BV; Volume: 279; Issue: 34 Linguagem: Inglês

10.1074/jbc.m402380200

ISSN

1083-351X

Autores

Robert Somerville, Jean-Michel Longpré, Elizabeth D. Apel, Renate Lewis, Lauren W. Wang, Joshua R. Sanes, Richard Leduc, Suneel Apte,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg220 (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave α2-macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family. We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg220 (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave α2-macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family. The extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; TSR, thrombospondin type 1 repeat; ORF, open reading frame; PNGase F, peptide N-glycosidase F; CHO, Chinese hamster ovary; GAG, glycosaminoglycan; PBS, phosphate-buffered saline; CS, chondroitin sulfate.1The abbreviations used are: ECM, extracellular matrix; TSR, thrombospondin type 1 repeat; ORF, open reading frame; PNGase F, peptide N-glycosidase F; CHO, Chinese hamster ovary; GAG, glycosaminoglycan; PBS, phosphate-buffered saline; CS, chondroitin sulfate. is an information-rich assembly influencing cell proliferation, apoptosis, and cell migration. Proteases have an essential role in modulating the environmental cues that ECM provides for tissue morphogenesis, homeostasis, and disease progression. Metalloproteases, especially matrix metalloproteases, have a conspicuous role in ECM degradation as well as in proteolysis of cell-surface and soluble proteins (1Somerville R.P. Oblander S.A. Apte S.S. Genome Biology. 2003; (http://genomebiology.com/2003/4/1/reviews/216)PubMed Google Scholar, 2Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3172) Google Scholar). Another metalloprotease family, ADAM (adisintegrin and metalloprotease domain), contains transmembrane enzymes with a major role in ectodomain shedding of cell-surface molecules, but a negligible function in ECM proteolysis (3Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The active site of ADAM proteases, unlike that of the matrix metalloproteases, is of the reprolysin (snake venom zinc metalloprotease) type (3Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The recent discovery of the ADAMTS (adisintegrin-like and metalloprotease domain with thrombospondin type 1 motif) family brought to light metalloproteases that contain a reprolysin-type catalytic site, but, unlike the ADAM proteases, are secreted enzymes with a prominent role in ECM proteolysis. ADAMTS proteases have a characteristic modular structure whose hallmark is the presence of one or more thrombospondin type 1 repeats (TSRs) (4Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). In the short period of time since the discovery of ADAMTS1 in 1997 (4Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar), important functions have been attributed to a number of family members, and mutations of two of these enzymes have been shown to cause human genetic disorders. Inactivating mutations of ADAMTS13 cause inherited thrombocytopenic purpura due to a failure to process von Willebrand factor (5Levy 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: 488-494Crossref PubMed Scopus (1410) Google Scholar). Mutations of ADAMTS2, a procollagen aminopropeptidase, cause skin fragility in a variety of mammals, including humans, through failure of incompletely processed collagen to form proper fibrils (6Colige 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: 308-317Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). ADAMTS2, ADAMTS3, and ADAMTS14 form a subfamily of procollagen aminopropeptidases (7Colige 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: 5756-5766Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 8Fernandes R.J. Hirohata S. Engle J.M. Colige A. Cohn D.H. Eyre D.R. Apte S.S. J. Biol. Chem. 2001; 276: 31502-31509Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Other ADAMTS enzymes (e.g. ADAMTS1, ADAMTS4, ADAMTS5, and ADAMTS9) may mediate aggrecan degradation in arthritis and the turnover of the related proteoglycans versican and brevican in blood vessels and the nervous system, respectively (9Abbaszade 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.T. Arner E.C. Burn T.C. J. Biol. Chem. 1999; 274: 23443-23450Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 10Matthews R.T. Gary S.C. Zerillo C. Pratta M. Solomon K. Arner E.C. Hockfield S. J. Biol. Chem. 2000; 275: 22695-22703Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11Rodríguez-Manzaneque J.C. Westling J. Thai S.N. Luque A. Knauper V. Murphy G. Sandy J.D. Iruela-Arispe M.L. Biochem. Biophys. Res. Commun. 2002; 293: 501-508Crossref PubMed Scopus (199) Google Scholar, 12Sandy 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: 13372-13378Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 13Somerville R.P. Longpré 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: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 14Tortorella 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 (612) Google Scholar). Adamts1 null mice have abnormal adipogenesis, defective angiogenesis in the adrenal gland, and altered ureteric ECM turnover (15Shindo 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: 1345-1352Crossref PubMed Scopus (268) Google Scholar). ADAMTS1 inhibits angiogenesis by its ability to sequester the vascular endothelial growth factor (16Luque A. Carpizo D.R. Iruela-Arispe M.L. J. Biol. Chem. 2003; 278: 23656-23665Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). A mutation in Adamts20 gave rise to the mouse mutant Belted, which has defects in skin pigmentation owing to presumed effects on melanoblast migration (17Rao C. Foernzler D. Loftus S.K. Liu S. McPherson J.D. Jungers K.A. Apte S.S. Pavan W.J. Beier D.R. Development (Camb.). 2003; 130: 4665-4672Crossref PubMed Scopus (71) Google Scholar). Although progress in this field has been rapid, the properties and functions of the majority of the 19 ADAMTS proteases remain unknown, and we refer to these as orphan enzymes (i.e. without a cognate substrate). ADAMTS7 2Adamts7 and ADAMTS7 denote mouse and human genes, respectively. ADAMTS7B is the full-length protein product of these genes in both species. ADAMTS7A is a shorter version of this protein that was described previously (18Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The same nomenclature is used for other ADAMTS proteases.2Adamts7 and ADAMTS7 denote mouse and human genes, respectively. ADAMTS7B is the full-length protein product of these genes in both species. ADAMTS7A is a shorter version of this protein that was described previously (18Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The same nomenclature is used for other ADAMTS proteases. was originally identified as an enzyme containing two TSRs and having a similar domain structure to ADAMTS5, ADAMTS6, and ADAMTS8 (18Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). In this work, we describe a longer form of ADAMTS7 (designated ADAMTS7B) that we believe is the authentic full-length version of this enzyme and that suggests that our previous characterization of the C terminus of ADAMTS7 may have been incomplete. The primary structure of ADAMTS7B presented here places this enzyme in a distinct phylogenetic clade with ADAMTS12 (19Cal S. Arguelles J.M. Fernandez P.L. Lopez-Otin C. J. Biol. Chem. 2001; 276: 17932-17940Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In addition, we have undertaken a detailed characterization of ADAMTS7B with emphasis on its unusual post-translational modification. ADAMTS7B is shown to contain a mucin domain within which is attached one or more chondroitin sulfate chains. This suggests that ADAMTS7B may exist in some cells and tissues as a proteoglycan, with potentially important functional consequences. In addition, we have characterized the maturation of the ADAMTS7B zymogen, demonstrated that the zymogen and mature enzyme may be cell-associated through different mechanisms, and shown that the mature enzyme and possibly the zymogen are active proteases. Materials—Reagents and chemicals were purchased from Sigma unless otherwise stated. cDNA Cloning of Mouse ADAMTS7B—A partial cDNA encoding the 290 C-terminal amino acids of ADAMTS7B along with ∼200 bp of 3′-untranslated sequence was isolated from an embryonic day 17 mouse cDNA library (Clontech). This fragment was used as a starting point to clone cDNAs encoding the entire 1641-amino acid open reading frame (ORF) using the original library plus 5′-rapid amplification of cDNA ends of RNA from 13- and 17-day-old mouse embryos and Sol8 mouse myotubes. All sequences were verified by isolation of independent duplicate clones. To confirm the continuity of the clones and to generate an expression plasmid, primers from the 5′- and 3′-ends were used to amplify the entire ORF using cDNA obtained from C2C12 mouse myotubes. Northern Blot Analysis—Northern blots containing poly(A)+ RNA (1 μg/lane) from mouse embryos and adult human tissues (BD Biosciences) were hybridized to a [α-32P]dCTP-labeled Adamts7b probe, followed by autoradiographic exposure for 7 days. Human and Mouse ADAMTS7 Expression Plasmids—For PCR amplification of full-length mouse ADAMTS7B, we used the above mouse plasmid as template and oligonucleotide 5′-gaattccaccacattggtgtgcacc-3′ (with the EcoRI site underlined) as the forward primer and oligonucleotide 5′-gcggccgcccgtctggctaccccgctga-3′ (with the NotI site underlined) as the reverse primer. The fidelity of PCR amplification was confirmed by nucleotide sequencing. For expression of mouse ADAMTS7PRO-CAT-Myc/His (encoding the signal peptide, prodomain, and catalytic domain, constituting residues 1–444 in mouse ADAMTS7B), PCR amplification was performed utilizing the same forward primer and the reverse primer 5′-gtcgacttgagtgcaatgacatccttgg-3′ (with the SalI site underlined). A critical P1 Arg residue within each of the two major furin cleavage sites in ADAMTS7PRO-CAT-Myc/His was substituted with Ala (Arg60 → Ala and Arg220 → Ala) by site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene). These cDNAs were all cloned into pcDNA3.1(+)-Myc/HisB for expression in-frame with tandem Myc and His6 tags. The corresponding human catalytic domain construct (encoding residues 1–459) was made by excision from an IMAGE clone (expressed sequence tag N48032) using EcoRI and HindIII and cloning into the matching sites of pcDNA3.1(+)-Myc/HisB. cDNA encoding the mucin domain (mouse Pro980–Arg1366, named ADAMTS7BFLAG-MUC) was amplified using mouse ADAMTS7B cDNA as a template with primers 5′-aaagcttcccatgtacatagtggaca-3′ (with the HindIII site underlined) and 5′-aggatcctcacctggcaggcagtggat-3′ (with the BamHI site underlined) and cloned into pCMV9–3XFLAG for expression with a preprotrypsin leader sequence and three tandem N-terminal FLAG tags. Transfection and Selection of Stable Cell lines—DNA for transfection was prepared using the Wizard™ purification system (Promega Corp.). 80% confluent HEK293F cells (Invitrogen) in 6-well plates were transfected with 100 ng of ADAMTS7B, ADAMTS7BFLAG-MUC, or human or mouse ADAMTS7PRO-CAT-Myc/His plasmid using FuGENE 6 (Roche Diagnostics) following the manufacturer's instructions. After 24 h, the medium was changed and supplemented with 1 mg/ml active G418 (Mediatech, Herndon, VA). Discrete clones were isolated using cloning discs (PGC Scientifics, Frederick, MD) and expanded in 24-well plates. Western blotting with anti-c-Myc monoclonal antibody 9E10 or anti-FLAG monoclonal antibody M2 was used to determine the level of protein expression in the media of these clones as appropriate for the plasmid used. The highest expressing clones were expanded to purify human or mouse ADAMTS7PRO-CAT-Myc/His, full-length mouse ADAMTS7B protein, or mouse ADAMTS7BFLAG-MUC. Protein Purification and N-terminal Sequence Analysis—HEK293F cells expressing human or mouse ADAMTS7PRO-CAT-Myc/His were cultured in three-tier flasks (Nunc, Rochester, NY) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. When cultures were 80% confluent, their medium was replaced with serum-free medium (293 SFM medium, Invitrogen) with subsequent culture at 37 °C in the presence of 8% CO2 for 5 days. The medium was collected, centrifuged to remove cellular debris, and supplemented with NaCl to a final concentration of 0.5 m. ProBond resin (Invitrogen) was prepared by washing with 1 bed volume of binding buffer (0.5 m NaCl and 20 mm sodium phosphate, pH 7.8). The medium and resin were mixed overnight at 4 °C (100 μl of resin/100 ml of medium). The resin was pelleted by centrifugation at 1500 rpm and then washed five times with 10 volumes of binding buffer. Bound protein was eluted by sequential washes with binding buffer containing 50, 100, 150, 200, and 250 mm imidazole. The washes and eluted protein fractions were assayed for the presence and purity of protein by Coomassie Blue staining. Gels were electroblotted onto polyvinylidene difluoride membrane (Millipore Corp.) for Western blotting. The recombinant protein was detected using anti-c-Myc epitope monoclonal antibody 9E10 or anti-pentahistidine antibody (QIAGEN Inc., Valencia, CA) in conjunction with horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Antibody binding was detected by enhanced chemiluminescence (Amersham Biosciences). Purification and analysis of full-length mouse ADAMTS7B were essentially similar, except that the cell layer was first washed with 0.5 m NaCl to release the protein from the cell surface; this was pooled with the medium from these cultures and purified and analyzed as described above. Purification of full-length ADAMTS7B was challenging and provided extremely small amounts of substantially purified enzyme such that quantitation could not be done. Since most of the studies with this enzyme were qualitative in nature for determination of its intrinsic characteristics, most assays used sufficient enzyme to permit visualization on Western blots. ADAMTS7BFLAG-MUC was isolated from the conditioned medium of stably transfected cells by affinity purification with anti-FLAG antibody M2-Sepharose beads, following which the beads were extracted in Laemmli sample buffer prior to electrophoresis. For N-terminal sequencing of human and mouse ADAMTS7PRO-CAT-Myc/His, purified protein was electroblotted onto polyvinylidene difluoride membrane and lightly stained with Simply Blue Safe Stain (Invitrogen). The 50- and 29-kDa major bands were excised for amino acid sequencing by Edman degradation on an Applied Biosystems Procise 492 sequencer in the Molecular Biotechnology Core Facility of the Lerner Research Institute. In all the subsequent studies, we used full-length mouse ADAMTS7B or mouse ADAMTS7PRO-CAT-Myc/His as indicated. No further studies with human ADAMTS7PRO-CAT-Myc/His beyond the N-terminal sequencing described above were conducted. Analysis of N- and O-Glycosylation of ADAMTS7B—For enzymatic removal of N-linked carbohydrate with peptide N-glycosidase F (PNGase F), HEK293 cell lysate expressing ADAMTS7B or purified ADAMTS7PRO-CAT-Myc/His was denatured in 0.5% SDS and 0.1 m β-mercaptoethanol at 100 °C for 10 min and cooled to room temperature, and Nonidet P-40 was added to a concentration of 1% (v/v) to neutralize the PNGase F inhibitory effects of SDS. The reduced samples were incubated with 100 units of PNGase F (New England Biolabs Inc., Beverly, MA) in 50 mm sodium phosphate, pH 7.5, for 2 h at 37 °C. SDS-PAGE and Western blotting (with antibody 9E10) were used to compare in-gel migration with an untreated sample. For ligand blot analysis, ADAMTS7BFLAG-MUC was electrophoresed on denaturing 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride. Horseradish peroxidase-conjugated peanut agglutinin (from Arachis hypogaea) was incubated with the membrane, followed by washes with Tris-buffered saline containing 0.1% Tween 20 and detection by ECL (20Christian S. Ahorn H. Koehler A. Eisenhaber F. Rodi H.P. Garin-Chesa P. Park J.E. Rettig W.J. Lenter M.C. J. Biol. Chem. 2001; 276: 7408-7414Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 21Lotan R. Skutelsky E. Danon D. Sharon N. J. Biol. Chem. 1975; 250: 8518-8523Abstract Full Text PDF PubMed Google Scholar). Enzymatic removal of O-linked carbohydrate was done using a mixture of 10 milliunits of sialidase and 0.5 milliunits of O-glycosidase (Roche Diagnostics) in 50 mm sodium phosphate, pH 7.5, and 0.5% Triton X-100, followed by immunoblotting with anti-FLAG antibody M2. Undigested ADAMTS7BFLAG-MUC was used as a control. Glycosaminoglycan Analysis—ADAMTS7B and ADAMTS7BFLAG-MUC were treated with 0.5 units of protease-free chondroitinase ABC (Seikagaku America, East Falmouth, MA) in 100 mm Tris and 50 mm sodium acetate, pH 6.5, for 2 h at 37 °C. HEK293F, COS-1, and CHO-K1 cell lysates obtained from transiently transfected cells expressing ADAMTS7B were treated with 2.0 units of chondroitinase ABC, 5 units each of keratanase I and II, or 2.5 units each of heparinase I and III in serum-free Dulbecco's modified Eagle's medium for 4 h at 37 °C. The proteins from the digests were precipitated with acetone and electrophoresed on reducing 7% SDS-polyacrylamide gel. ADAMTS7B was detected by Western blotting using monoclonal antibodies 9E10 (for ADAMTS7B) and M2 (for ADAMTS7BFLAG-MUC). Samples untreated with the respective enzymes were used as negative controls. The blots were subsequently stripped by treatment with 62.5 mm Tris-HCl, pH 6.8, 2% SDS, and 100 mm 2-mercaptoethanol at 55 °C for 30 min. The membrane was reprobed using mouse anti-chondroitin 6-sulfate monoclonal antibody 2035 (Chemicon International, Inc., Temecula, CA), which specifically recognizes stubs with an unsaturated glucuronic acid terminus derived from chondroitin 6-sulfate in chondroitin sulfate proteoglycans following chondroitinase digestion. These stubs are not present in the native undigested chondroitin sulfate proteoglycans. To further verify the attachment of glycosaminoglycan (GAG) to the ADAMTS7B core protein, we transfected the ADAMTS7B expression plasmid into CHO-K1 cells with a null mutation in β-d-xylosyltransferase (CHO-K1 pgsA-745; American Type Culture Collection, CRL-2242) (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (482) Google Scholar) and analyzed the extracts of these cells by Western blotting using monoclonal antibody 9E10. Transfected parental CHO-K1 cells (CCL-61) were analyzed in parallel as a control. The conditioned medium from HEK293F clones expressing full-length ADAMTS7B was batch-incubated overnight with heparin-Sepharose (Bio-Rad) at 4 °C with end-over-end rotation. The resin was washed five times with phosphate-buffered saline (PBS) and eluted with increasing concentrations of NaCl (0.2–0.5 m). Uneluted protein was removed by boiling the beads in Laemmli sample buffer. Unbound protein was precipitated with cold acetone. Aliquots of the medium before binding, unbound proteins, and proteins bound to the beads were analyzed by Western blotting with antibody 9E10. Biosynthesis of ADAMTS7B—QBI 293A cells (Quantum Biotechnologies, Montreal, Canada) were maintained, transiently transfected with mouse ADAMTS7PRO-CAT-Myc/His using FuGENE 6, and metabolically labeled with a [35S]methionine/cysteine mixture (Expre35S35S, PerkinElmer Life Sciences) essentially as described previously (13Somerville R.P. Longpré 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: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). A 15-min label (pulse) was followed by incubation in complete nonradioactive medium (chase) for various times. Immunoprecipitation with anti-pentahistidine monoclonal antibody was done as described previously (13Somerville R.P. Longpré 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: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). For inhibition of furin, decanoyl-RVKR chloromethyl ketone, a lipid-permeable inhibitor (furin-1 inhibitor; Calbiochem) (23Stieneke-Grober A. Vey M. Angliker H. Shaw E. Thomas G. Roberts C. Klenk H.D. Garten W. EMBO J. 1992; 11: 2407-2414Crossref PubMed Scopus (482) Google Scholar), was added to HEK293F cells stably expressing ADAMTS7PRO-CAT-Myc/His at concentrations ranging from 1 to 100 μm for 24 h, and the culture medium was analyzed by Western blotting using monoclonal antibody 9E10. Cotransfection of ADAMTS7B and Proprotein Convertases—CHO RPE.40 cells lacking furin were maintained as described previously (24Rodríguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). They were transfected with mouse ADAMTS7PRO-CAT-Myc/His alone (as a control) or in combination with plasmids encoding the proprotein convertases furin, PACE4, PC6B, and PC7. QBI 293A cells were transiently transfected with ADAMTS7PRO-CAT-Myc/His, also as a control. Cells were metabolically labeled for 3 h, and immunoprecipitation and fluorography of the cell lysate and medium were performed as described (13Somerville R.P. Longpré 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: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Cell-surface Biotinylation—Stably transfected HEK293F cells expressing ADAMTS7PRO-CAT-Myc/His were scraped from plates; washed six times with cold PBS, pH 8.0; and diluted to 2.5 × 107 cells/ml. As a control to eliminate all cell-surface proteins, an equal number of cells were treated with trypsin/EDTA for 15 min and washed once with medium containing serum to inactivate trypsin and then six times with cold PBS. 1 ml of each cell suspension was mixed with 0.5 mg of sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce Biotechnology, Inc.) and incubated for 30 min at room temperature. The cells were pelleted and washed once with 50 mm Tris-Cl, pH 8.0, and three times with PBS, pH 8.0. The cell pellet was lysed in radioimmune precipitation assay buffer (1× PBS, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS) at 4 °C for 30 min and centrifuged. The soluble portion of the lysate was transferred to a fresh tube and incubated overnight with 50 μl of streptavidin-agarose at 4 °C with rotation. The streptavidin-agarose was pelleted by centrifugation at 3000 rpm in a microcentrifuge and washed three times in radioimmune precipitation assay buffer and PBS. The supernatant was discarded, and the streptavidin-agarose was resuspended in 50 μl of Laemmli buffer and boiled for 5 min. The agarose was pelleted, and the supernatant was loaded onto a 10% SDS-polyacrylamide gel for Western blotting with anti-c-Myc antibody 9E10. Pulse-Chase Analysis of Cell-surface Biotinylated Proteins— HEK293F monolayers expressing mouse ADAMTS7PRO-CAT-Myc/His were biotinylated as described above for cell suspensions. The monolayers were washed three times with Tris-buffered saline to inactivate the biotinylation reagent. The monolayers were returned to 10 ml of 293 SFM medium and further cultured for 3 h. 1 ml of medium was taken for analysis at sequential time points, and biotinylated protein was purified from the medium using streptavidin-agarose, followed by Western blotting as described above. Cell lysates were taken at fixed time points to investigate the fate of surface-biotinylated proteins by Western blotting as described above. α2-Macroglobulin, Versican, and Aggrecan Processing Assays—To determine whether purified ADAMTS7PRO-CAT-Myc/His and ADAMTS7B were catalytically active, 5 μl of purified full-length ADAMTS7B or ADAMTS7PRO-CAT-Myc/His was incubated with 5 μg of α2-macroglobulin (Calbiochem) in 50 mm Tris-Cl, pH 7.8, 100 mm NaCl, and 5 mm CaCl2 for 2 h at 37 °C. Laemmli sample buffer was added, and the sample was electrophoresed without heating on 10% SDS-polyacrylamide gel, followed by Western blotting with antibody 9E10 to visualize ADAMTS7B. In similar digests, Coomassie Blue staining was used after reducing SDS-PAGE to visualize α2-macroglobulin. Processing of specific ADAMTS-susceptible peptide bonds in versican and aggrecan was analyzed using transfected HEK293F cells essentially as described previously (13Somerville R.P. Longpré 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: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) as well as in solution using the same volume of enzyme used in α2-macroglobulin digests. Briefly, ADAMTS7B-transfected and ADAMTS4-transfected (as a positive control) cells were incubated in suspension culture with versican and aggrecan. Subsequently, aggrecan and versican were precipitated from the medium, deglycosylated, and analyzed by Western blotting using anti-neoepitope antibodies specific for the cleaved Glu1771–Ala1772 and Glu441–Ala442 peptide bonds of aggrecan and versican, respectively (13Somerville R.P. Longpré 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: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Cell lysates were analyzed by Western blotting with antibodies 9E10 and M2 to determine the expression levels of ADAMTS7B and ADAMTS4, respectively. Primary Structure of ADAMTS7B—Shortly after the acceptance of our or

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