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Discovery and Characterization of a Novel, Widely Expressed Metalloprotease, ADAMTS10, and Its Proteolytic Activation

2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês

10.1074/jbc.m409036200

ISSN

1083-351X

Autores

Robert Somerville, Katherine A. Jungers, Suneel Apte,

Tópico(s)

Complement system in diseases

Resumo

We describe the discovery and characterization of ADAMTS10, a novel metalloprotease encoded by a locus on human chromosome 19 and mouse chromosome 17. ADAMTS10 has the typical modular organization of the ADAMTS family, with five thrombospondin type 1 repeats and a cysteine-rich PLAC (protease and lacunin) domain at the carboxyl terminus. Its domain organization and primary structure is similar to a novel long form of ADAMTS6. In contrast to many ADAMTS proteases, ADAMTS10 is widely expressed in adult tissues and throughout mouse embryo development. In situ hybridization analysis showed widespread expression of Adamts10 in the mouse embryo until 12.5 days of gestation, after which it is then expressed in a more restricted fashion, with especially strong expression in developing lung, bone, and craniofacial region. Mesenchymal, not epithelial, expression in the developing lung, kidney, gonad, salivary gland, and gastrointestinal tract is a consistent feature of Adamts10 regulation. N-terminal sequencing and treatment with decanoyl-Arg-Val-Lys-Arg-chloromethylketone indicate that the ADAMTS10 zymogen is processed by a subtilisin-like proprotein convertase at two sites (Arg64↓Gly and Arg233↓Ser). The widespread expression of ADAMTS10 suggests that furin, a ubiquitously expressed proprotein convertase, is the likely processing enzyme. ADAMTS10 expressed in HEK293F and COS-1 cells is N-glycosylated and is secreted into the medium, as well as sequestered at the cell surface and extracellular matrix, as demonstrated by cell surface biotinylation and immunolocalization in nonpermeabilized cells. ADAMTS10 is a functional metalloprotease as demonstrated by cleavage of α2-macroglobulin, although physiological substrates are presently unknown. We describe the discovery and characterization of ADAMTS10, a novel metalloprotease encoded by a locus on human chromosome 19 and mouse chromosome 17. ADAMTS10 has the typical modular organization of the ADAMTS family, with five thrombospondin type 1 repeats and a cysteine-rich PLAC (protease and lacunin) domain at the carboxyl terminus. Its domain organization and primary structure is similar to a novel long form of ADAMTS6. In contrast to many ADAMTS proteases, ADAMTS10 is widely expressed in adult tissues and throughout mouse embryo development. In situ hybridization analysis showed widespread expression of Adamts10 in the mouse embryo until 12.5 days of gestation, after which it is then expressed in a more restricted fashion, with especially strong expression in developing lung, bone, and craniofacial region. Mesenchymal, not epithelial, expression in the developing lung, kidney, gonad, salivary gland, and gastrointestinal tract is a consistent feature of Adamts10 regulation. N-terminal sequencing and treatment with decanoyl-Arg-Val-Lys-Arg-chloromethylketone indicate that the ADAMTS10 zymogen is processed by a subtilisin-like proprotein convertase at two sites (Arg64↓Gly and Arg233↓Ser). The widespread expression of ADAMTS10 suggests that furin, a ubiquitously expressed proprotein convertase, is the likely processing enzyme. ADAMTS10 expressed in HEK293F and COS-1 cells is N-glycosylated and is secreted into the medium, as well as sequestered at the cell surface and extracellular matrix, as demonstrated by cell surface biotinylation and immunolocalization in nonpermeabilized cells. ADAMTS10 is a functional metalloprotease as demonstrated by cleavage of α2-macroglobulin, although physiological substrates are presently unknown. Proteolytic processing of structural components of the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; α2-M, α2-macroglobulin; ORF, open reading frame; IMAGE, Integrated Mapping of Genomes and their Expression; BLAST, Basic Local Alignment Search Tool; aa, amino acid(s); TSR, thrombospondin type 1 repeat; EST, expressed sequence tag; dec, decanoyl; cmk, chloromethylketone; PNGase F, peptide:N-glycanase F. and cell signaling-related molecules such as cytokines, growth factors, their binding proteins, receptors, and adhesion molecules has important biological consequences (1Somerville R.P. Oblander S.A. Apte S.S. Genome Biol. 2003; 4: 216Crossref PubMed Scopus (248) Google Scholar, 2Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3256) Google Scholar). Proteases that cleave such molecules thus play important roles in tissue remodeling, morphogenesis, inflammatory and degenerative diseases, and cancer. Zinc-metalloendopeptidases (metalloproteases) comprise an important superfamily of such enzymes. Specific roles for distinct metalloprotease families and their individual members have emerged over the past decade through genetic studies in humans and mice. Matrix metalloproteases are the major ECM-degrading enzymes, but they also have a role in proteolysis of other secreted molecules and cell surface proteins (1Somerville R.P. Oblander S.A. Apte S.S. Genome Biol. 2003; 4: 216Crossref PubMed Scopus (248) Google Scholar, 2Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3256) Google Scholar). ADAMs are primarily "sheddases," proteases that process cell surface molecules and are thought to have little, if any, direct role in ECM catabolism (3Kheradmand F. Werb Z. BioEssays. 2002; 24: 8-12Crossref PubMed Scopus (111) Google Scholar, 4Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). 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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 (303) Google Scholar, 16Wang W.M. Lee S. Steiglitz B.M. Scott I.C. Lebares C.C. Allen M.L. Brenner M.C. Takahara K. Greenspan D.S. J. Biol. Chem. 2003; 278: 19549-19557Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). These two processing activities appear to be highly specialized, and the enzymes responsible for them have distinct sequence and structural features not shared by the other ADAMTS proteases (17Zheng X. Chung D. Takayama T.K. Majerus E.M. Sadler J.E. Fujikawa K. J. Biol. Chem. 2001; 276: 41059-41063Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 18Colige 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 (161) Google Scholar). On the other hand, a number of ADAMTS with disparate domain and sequence features (such as ADAMTS1, ADAMTS4, ADAMTS5, and ADAMTS9) are known to process large aggregating proteoglycans such as aggrecan and versican (10Tortorella 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, 12Abbaszade 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. J. Biol. Chem. 1999; 274: 23443-23450Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 20Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 21Rodriguez-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 (209) Google Scholar). Nevertheless, this is not a general property of all ADAMTS proteases, since we have shown recently that ADAMTS7 cannot process versican or aggrecan at sites cleaved by the other proteoglycan-degrading ADAMTS (22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). ADAMTS proteases are modular, consisting of a protease domain and an ancillary domain (23Apte S.S. Int. J. Biochem. Cell Biol. 2004; 36: 981-985Crossref PubMed Scopus (216) Google Scholar). The protease domain of these enzymes, like that of ADAMs, but not MMPs, is of the reprolysin (snake venom) type. The hallmark of the ADAMTS proteases is the presence of at least one thrombospondin type 1 repeat (TSR). Other highly conserved modules are arranged around this central TSR in a specific organization, and there are additional TSRs near the carboxyl terminus in all members of the ADAMTS family with the exception of ADAMTS4 (23Apte S.S. Int. J. Biochem. Cell Biol. 2004; 36: 981-985Crossref PubMed Scopus (216) Google Scholar). ADAMTS proteases are synthesized as zymogens that are targeted to the secretory pathway and activated by proprotein convertases. Zymogen processing leads to removal of a 200–220-amino acid-long prodomain in the secretory pathway or at the cell surface. 19 mammalian ADAMTS proteases are known, and all except ADAMTS10, the subject of this article, have previously been described in the literature. Within the ADAMTS family, subsets of proteases have highly conserved domain organization, primary sequence and gene structure, suggestive of a close evolutionary and perhaps functional relationship (7Fernandes 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 (199) Google Scholar, 20Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 23Apte S.S. Int. J. Biochem. Cell Biol. 2004; 36: 981-985Crossref PubMed Scopus (216) Google Scholar). In this context, determination of the primary structure of ADAMTS10 led to realization of a putative long form of ADAMTS6, whose domain organization and primary structure support the contention that it forms a phylogenetic subset with ADAMTS10. Unlike most other ADAMTS proteases, including ADAMTS6 (24Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), ADAMTS10 is widely expressed. We investigated the developmental regulation of the Adamts10 gene in mice and the activation mechanism and localization of the enzyme in cultured cells. ADAMTS10 is shown to be a functional metalloprotease, although its physiological substrates are presently unknown. cDNA Cloning of Human and Mouse ADAMTS10 —Using the tBLASTn (Basic Local Alignment Search Tool) program at the National Center for Biotechnology Information, we searched the data base of expressed sequence tags (dBEST), using the protein sequences of a number of ADAMTS proteases, and identified similarities in a human EST (GenBank™ accession number AA588434) derived from the human prostate-derived IMAGE clone 1101403. The IMAGE clone was purchased (Research Genetics, Huntsville, AL), and the insert was sequenced in its entirety. Oligonucleotide primers based on the sequences at the ends of this clone were used with human fetal brain cDNA (Marathon cDNA, Clontech, Palo Alto, CA) as a template to perform iterative rapid amplification of cDNA ends by PCR at 5′- and 3′-ends, essentially as previously described (24Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 25Georgiadis K.E. Hirohata S. Seldin M.F. Apte S.S. Genomics. 1999; 62: 312-315Crossref PubMed Scopus (28) Google Scholar). Nucleotide sequencing was done at the National Institutes of Health-supported Molecular Biotechnology Core of the Lerner Research Institute (Cleveland Clinic Foundation), and nucleotide sequence data were analyzed using Lasergene software (DNAStar Inc., Madison, WI). Integration of the overlapping sequences provided the complete ORF and primary sequence of human ADAMTS10. To confirm that the overlapping human cDNAs were derived from a single transcript, we designed PCR primers incorporating the most 5′ cloned human sequence and the stop codon of the ADAMTS10 ORF (forward primer, 5′-AAGAATTCAGAGACATGTGGACACGTGG-3′ (EcoRI site underlined, start codon in boldface type); reverse primer, 5′-AAGTCGACCGAGTGGCCCTGGCAGGTTTTGC-3′ (SalI site underlined, modified stop codon (to Ser) in boldface type)). PCR was done using human fetal lung cDNA or human lung cancer cell line A549 cDNA as templates (Clontech, Palo Alto, CA) and using the following conditions: 95 °C for 1 min and then 30 cycles of 95 °C for 30 s, 58 °C for 30 s, and 68 °C for 5 min. The resulting 3.2-kbp amplicon was gel-purified, ligated into pGEM-T Easy (Promega, Madison, WI), and sequenced. The mouse IMAGE clone 1077653 (EST AA822090) was detected in GenBank™ as a presumptive Adamts10 clone, 2Gene nomenclature has been assigned in agreement with the Human Gene Nomenclature Committee. ADAMTS10 and Adamts10 are human and mouse orthologs. The protein products of both genes are designated as ADAMTS10. Similar nomenclature is used for other ADAMTS genes and their products. purchased from Research Genetics (Huntsville, AL), and its 1.6-kbp insert was sequenced in its entirety. Additional 5′ mouse cDNA sequence was deduced from mouse genomic sequences (available with GenBank™ accession numbers AC073802 and AC073766), using the GENSCAN program at the Massachusetts Institute of Technology (available on the World Wide Web at CCR-081.mit.edu/GENSCAN.html) to predict the exons in these sequences. The complete mouse ADAMTS10 ORF was amplified by PCR of mouse 17.5-day-old embryo cDNA in similar fashion to that described above for the human cDNA. Northern Analysis—Mouse embryo Northern blots and multiple tissue Northern blots from adult human and mouse tissues and from human cancer cell lines (Clontech, Palo Alto, CA, and Seegene Inc.) were hybridized to the [α-32P]dCTP-labeled inserts of human and mouse ADAMTS10 IMAGE clones, as per the manufacturer's recommendations, followed by autoradiographic exposure for 4 days. In Situ Hybridization—Adamts10 IMAGE clone 1077653 was digested with StuI and XhoI to delete 792 bp of the 1642-bp insert. The plasmid containing the remainder was blunt-ended with Klenow fragment of DNA polymerase I (New England Biolabs, Beverly, MA) and religated to obtain an 850-bp Adamts10 cDNA encoding part of the cysteine-rich domain, spacer domain, and first two TSRs (plasmid 1077653X1). This plasmid was used to transcribe sense and antisense cRNA probes continuously labeled with [35S]UTP. Paraffin sections of formaldehyde-fixed mouse embryos of age 9.5, 12.5, 14.5, 15.5, and 17.5 days were hybridized to the Adamts10 probes as previously described (20Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), followed by dipping in photographic emulsion for autoradiography. Adamts10 autoradiographic signal was visualized with dark field microscopy, whereas cell nuclei were stained with 4,6-diamidino-2-phenylindole (Hoechst 33258 dye; Sigma), which fluoresces blue under UV light. ADAMTS10 Expression Plasmids—The preprocatalytic coding region of human ADAMTS10 (ADAMTS10-(1–463)) was amplified by PCR using the oligonucleotide primers 5′-AAGAATTCGGCCTCTATGGCTCCCGCC-3′ (forward primer) and 5′-AAGTCGACCACAAAGTCCTGTCTGGG-3′ (reverse primer; introduced SalI site is underlined) and Advantage 2 high fidelity polymerase (Clontech, Palo Alto, CA). The PCR products were gel-purified and ligated to the pGEM-T easy vector (Promega Corp., Madison, WI). The insert of a sequence-verified clone was then ligated into the EcoRI and XhoI site of pcDNAmyc His A+ (Invitrogen) for expression of ADAMTS10-(1–463) with a C-terminal tandem myc and His6 tag. The full-length ADAMTS10 cDNA described above was cloned in frame with a C-terminal tandem myc and His6 tag Transfection and Selection of Stable Cell Lines—HEK293F cells (Invitrogen) at 80% confluence were transfected in 6-well plates with 100 ng of full-length ADAMTS10 or ADAMTS10-(1–463) expression plasmid DNA using Fugene 6 (Roche Applied Science) as per manufacturer's instructions. At the first medium change, it was supplemented with 1 mg/ml G418 (Mediatech, Herndon, VA). Discrete colonies were isolated using cloning discs (PGC Scientific, Frederick, MD) and expanded. Western blotting with anti-myc monoclonal antibody 9E10 (Invitrogen) was used to determine the level of protein expression in the media of these clones. Expression and Characterization of ADAMTS10 and ADAMTS10-(1–463)—Stably transfected cells expressing full-length ADAMTS10 and ADAMTS10-(1–463) 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, the serum-containing medium was replaced with serum-free 293CD medium (Invitrogen) followed by further culture at 37 °C in the presence of 8% CO2 for 5 days. Conditioned medium was collected, centrifuged briefly 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, 20 mm sodium phosphate, pH 7.8). The media and resin were mixed overnight at 4 °C in a 1:1 (v/v) ratio in binding buffer. After this binding step, the resin was pelleted by centrifugation at 1000 × g and then washed five times with 10 resin bed volumes of binding buffer. Bound proteins were 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 desired proteins by Western blotting (using anti-myc monoclonal antibody) and by reducing SDS-PAGE with Coomassie Blue staining, respectively. Maximal yield was obtained on elution in 100–250 mm imidazole. Following purification of ADAMTS10-(1–463), major bands of ∼52, ∼50, and ∼29 kDa were excised after electroblotting to a polyvinylidene difluoride membrane. N-terminal sequence was determined by Edman degradation at the National Institutes of Health-supported Biotechnology Core of the Lerner Research Institute. For identification of the zymogen-processing enzyme, ADAMTS10-(1–463)-expressing cells were treated with increasing concentrations (1–100 μm) of the lipid-permeable furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) (Calbiochem) for 24 h, and secreted protein was detected by Western blot analysis of conditioned medium as previously described (22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Purified ADAMTS10-(1–463) was deglycosylated with PNGase F (New England Biolabs, Beverly, MA) and detected by Western blotting with anti-myc antibody 9E10 as previously described (20Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Characterization of full-length ADAMTS10 was done using stably transfected HEK293F cells or transiently transfected COS cells or substantially purified protein. Western blotting was done with anti-myc antibody 9E10. Protein deglycosylation was done as previously described using purified protein (20Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Processing of α2-macroglobulin (α2-M) was tested by incubation with purified protein as described previously (22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Proteolysis of the aggrecan core protein using ADAMTS10- and ADAMTS4-transfected cells was evaluated as described previously (20Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, equal numbers of transfected cells were incubated with 20 μg of aggrecan, and the presence of a cleaved peptide bond detected by the anti-AGEG neoepitope antibody (26Tortorella 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) was sought by Western blotting of the aggrecan as previously described (20Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). ADAMTS10 Localization in Transfected Cells—These studies examined the distribution of full-length ADAMTS10 in vitro, in regard to the cells expressing it. COS-1 cells (ATCC, Manassas, VA) were transiently transfected with 1 μg of full-length ADAMTS10 prior to immunofluorescent localization of secreted protein in nonpermeabilized cells, essentially as previously described (27Hirohata 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: 12182-12189Abstract Full Text Full Text PDF Scopus (73) Google Scholar). ADAMTS10 was detected using antibody 9E10 and Alexa-488-conjugated goat anti-mouse secondary antibody (Molecular Probes, Inc., Eugene, OR) in an indirect immunofluorescence method that does not detect intracellular protein. Following staining for tagged ADAMTS10, cells were permeabilized and nuclei were stained with 4′,6-diamidino-2-phenylindole as previously described (27Hirohata 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: 12182-12189Abstract Full Text Full Text PDF Scopus (73) Google Scholar), followed by fluorescent microscopy. Medium, cell lysate, and extracellular matrix from these cultures and from stably transfected HEK293F cells were collected as previously described and subjected to immunoblotting with antibody 9E10 following reducing SDS-PAGE (27Hirohata 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: 12182-12189Abstract Full Text Full Text PDF Scopus (73) Google Scholar). Stably transfected HEK293F cells expressing ADAMTS10 in suspension were biotinylated as previously described (22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Isolation of biotinylated proteins from the cell surface and their analysis by electrophoresis was as previously described (22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). As a control, cells were treated with trypsin to eliminate all cell surface proteins prior to biotinylation, essentially as previously described (22Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang W.L. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). cDNA Cloning of Human and Mouse ADAMTS10 —Using the tBLASTn algorithm to scan dBEST for novel ESTs that were homologous to cognate ADAMTS proteins, we identified the EST 1101403. This EST was identified when the data base was screened with the sequence of ADAMTS6 but not with other ADAMTS proteins. Following extension of the EST by rapid amplification of cDNA ends in both directions, we generated an amplicon of 3.2 kbp from human fetal lung and the A549 cell line. The cDNA encoded an open reading frame of 1103 amino acids with the typical ADAMTS mo

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