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Identification, Characterization, and Intracellular Processing of ADAM-TS12, a Novel Human Disintegrin with a Complex Structural Organization Involving Multiple Thrombospondin-1 Repeats

2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês

10.1074/jbc.m100534200

ISSN

1083-351X

Autores

Santiago Cal, José M. Argüelles, Pedro L. Fernández, Carlos López-Otı́n,

Tópico(s)

Biological Stains and Phytochemicals

Resumo

We have identified and cloned a human fetal lung cDNA encoding a new protein of the ADAM-TS family (adisintegrin and metalloproteinase domain, with thrombospondin type-1 modules) that has been called ADAM-TS12. This protein exhibits a domain organization similar to the remaining family members including a propeptide and metalloproteinase-like, disintegrin-like, and cysteine-rich domains. However, the number and organization of the TS repeats is unique with respect to other human ADAM-TSs. A total of eight TS-1 repeats arranged in three groups are present in this novel ADAM-TS. Analysis of intracellular processing of ADAM-TS12 revealed that it is synthesized as a precursor molecule that is first activated by cleavage of the prodomain in a furin-mediated process and subsequently processed into two fragments of different size: a 120-kDa N-terminal proteolytically active fragment containing the metalloproteinase and disintegrin domains, and a 83-kDa C-terminal fragment containing most of the TS-1 repeats. Somatic cell hybrid and radiation hybrid mapping experiments showed that the human ADAM-TS12 gene maps to 5q35, a location that differs from all ADAM genes mapped to date. Northern blot analysis of RNAs from human adult and fetal tissues demonstrated that ADAM-TS12 transcripts are only detected at significant levels in fetal lung but not in any other analyzed tissues. In addition, ADAM-TS12 transcripts were detected in gastric carcinomas and in tumor cell lines from diverse sources, being induced by transforming growth factor-β in KMST human fibroblasts. These data suggest that ADAM-TS12 may play roles in pulmonary cells during fetal development or in tumor processes through its proteolytic activity or as a molecule potentially involved in regulation of cell adhesion. We have identified and cloned a human fetal lung cDNA encoding a new protein of the ADAM-TS family (adisintegrin and metalloproteinase domain, with thrombospondin type-1 modules) that has been called ADAM-TS12. This protein exhibits a domain organization similar to the remaining family members including a propeptide and metalloproteinase-like, disintegrin-like, and cysteine-rich domains. However, the number and organization of the TS repeats is unique with respect to other human ADAM-TSs. A total of eight TS-1 repeats arranged in three groups are present in this novel ADAM-TS. Analysis of intracellular processing of ADAM-TS12 revealed that it is synthesized as a precursor molecule that is first activated by cleavage of the prodomain in a furin-mediated process and subsequently processed into two fragments of different size: a 120-kDa N-terminal proteolytically active fragment containing the metalloproteinase and disintegrin domains, and a 83-kDa C-terminal fragment containing most of the TS-1 repeats. Somatic cell hybrid and radiation hybrid mapping experiments showed that the human ADAM-TS12 gene maps to 5q35, a location that differs from all ADAM genes mapped to date. Northern blot analysis of RNAs from human adult and fetal tissues demonstrated that ADAM-TS12 transcripts are only detected at significant levels in fetal lung but not in any other analyzed tissues. In addition, ADAM-TS12 transcripts were detected in gastric carcinomas and in tumor cell lines from diverse sources, being induced by transforming growth factor-β in KMST human fibroblasts. These data suggest that ADAM-TS12 may play roles in pulmonary cells during fetal development or in tumor processes through its proteolytic activity or as a molecule potentially involved in regulation of cell adhesion. Withdrawal: Identification, characterization, and intracellular processing of ADAM-TS12, a novel human disintegrin with a complex structural organization involving multiple thrombospondin-1 repeatsJournal of Biological ChemistryVol. 294Issue 4PreviewVOLUME 276 (2001) PAGES 17932–17940 Full-Text PDF Open Access expressed sequence tag base pair(s) polymerase chain reaction thrombospondin transforming growth factor rapid amplification of cDNA ends kilobase(s) hemagglutinin interleukin Cell-cell and cell-extracellular matrix interactions are essential for the development and maintenance of an organism. Likewise, proteolysis of the extracellular matrix is of vital importance for a series of tissue-remodeling processes occurring during both normal and pathological conditions, such as tissue morphogenesis, wound healing, inflammation, or tumor cell invasion and metastasis. These events are mediated by a variety of cell surface adhesion proteins and proteases, with different structural and functional characteristics (1Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar). Among them, a group of recently described proteins called ADAMs (adisintegrin andmetalloproteinase domain) have raised considerable interest because of their potential ability to perform both functions, adhesion and proteolysis (2Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Crossref PubMed Scopus (441) Google Scholar, 3Blobel C.P. Cell. 1997; 91: 589-592Abstract Full Text Full Text PDF Scopus (332) Google Scholar). ADAMs were first associated with reproductive processes like spermatogenesis and heterotypic sperm-egg binding and fusion (4Wolfsberg T.G. White J.M. Dev. Biol. 1997; 180: 389-401Crossref Scopus (217) Google Scholar). However, over the last few years, the spectrum of functional roles for ADAMs has considerably expanded to processes such as myogenesis (5Gilpin B.J. Loeche F. Mattei M.G. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), osteoblast differentiation (6Inoue D. Reid M. Lum L. Krätzschmar J. Weskamp G. Myung Y.M. Baron R. Blobel C.P. J. Biol. Chem. 1998; 273: 4180-4187Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and host defense (7Mueller C.G. Rissoan M.C. Salinas B. Ait-Yahia S. Ravel O. Bridon J.M. Briere F. Lebecque S. Liu Y.J. J. Exp. Med. 1997; 189: 655-663Crossref Scopus (75) Google Scholar). Furthermore, some ADAM family members, including TACE (tumor necrosis factor-α-converting enzyme), ADAM-12 (meltrin α), and ADAM-23, have been suggested to play important roles in the development and progression of inflammatory and tumor processes (8Emi M. Katagiri T. Harada Y. Saito H. Inazawa J. Ito I. Kasumi F. Nakamura Y. Nat. Genet. 1993; 5: 151-157Crossref PubMed Scopus (98) Google Scholar, 9Krätzschmar J. Lum L. Blobel C.P. J. Biol. Chem. 1995; 271: 4593-4598Abstract Full Text Full Text PDF Scopus (155) Google Scholar, 10Yavari R. Adida C. Bray-Ward P. Brines M. Xu T. Hum. Mol. Genet. 1998; 7: 1161-1167Crossref PubMed Scopus (56) Google Scholar, 11Cal S. Pérez-Freije J.M. López J.M. Takada Y. López-Otı́n C. Mol. Biol. Cell. 2000; 11: 1457-1469Crossref PubMed Scopus (107) Google Scholar, 12Iba K Albrechtsen R. Gilpin B.J. Loechel F. Wewer U.M. Am. J. Pathol. 1999; 154: 1489-1501Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 13Wu E.N. Croucher P.I. McKie N. Biochem. Biophys. Res. Commum. 1997; 235: 437-442Crossref PubMed Scopus (99) Google Scholar, 14Black R.A. Rauch C.T. Kozlosky J.J.P. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Bolani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2715) Google Scholar, 15Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (474) Google Scholar). The structural and functional complexity of the ADAM family of cellular disintegrins has considerably grown after the finding of a series of new members characterized by the presence of thrombospondin repeats in their amino acid sequence. The first member of this subfamily, ADAM-TS1, was identified as a consequence of its association with the development of cancer cachexia as well as with various inflammatory processes (16Kuno 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 (440) Google Scholar). Subsequently, the cloning of the cDNA for procollagen I amino-proteinase, whose deficiency cause Ehlers-Danlos syndrome type VIIC in humans, revealed a significant degree of structural similarities with ADAM-TS1, and it was called ADAM-TS2 (17Colige 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 (302) Google Scholar). ADAM-TS4 and ADAM-TS5/TS11, other members of this subfamily of disintegrins containing thrombospondin motifs, have been characterized as aggrecanases responsible for the degradation of cartilage aggrecan in arthritic diseases (18Tortorella 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 R.M. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (621) Google Scholar, 19Abbaszade 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 (445) Google Scholar). ADAM-TS4 has been found to be responsible for brevican cleavage in glioma cells, a proteolytic cleavage that has been proposed to be critical in mediating the invasiveness of these tumors (20Matthews 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 (172) Google Scholar). On the other hand, ADAM-TS8 (also called METH-2) and ADAM-TS1 have been characterized as proteins with angio-inhibitory activity (21Vazquez F. Hastings G. Ortega M.A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Finally, other family members identified in human tissues such as ADAM-TS3, ADAM-TS5, ADAM-TS6, ADAM-TS7, and ADAM-TS9 have been only characterized at the structural level, and their putative functional significance is still unclear (22Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar,23Melody E.C. Gregory S.K. Laurie A.T. Antonia B. Karen C.A. Richard A.M. Genomics. 2000; 67: 343-350Crossref PubMed Scopus (81) Google Scholar). These recent findings have stimulated the search for new ADAMs potentially associated with some of the conditions involving cell-cell interactions or extracellular matrix degradation taking place during both normal or pathological conditions (1Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar, 2Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Crossref PubMed Scopus (441) Google Scholar, 3Blobel C.P. Cell. 1997; 91: 589-592Abstract Full Text Full Text PDF Scopus (332) Google Scholar). In this work, we have examined the possibility that additional yet uncharacterized ADAMs could be produced by human tissues, with the finding of a novel family member, belonging to the ADAM-TS subfamily that we have called ADAM-TS12. We describe the molecular cloning and complete nucleotide sequence of a cDNA coding for this protein. We also report an analysis of the intracellular processing and enzymatic activity of ADAM-TS12. Finally, we describe the chromosomal location of the ADAM-TS12 gene and analyze its expression and regulation in normal and tumor tissues. A human fetal lung cDNA library constructed in λgt11, a human matched tumor/normal expression array, and Northern blots containing polyadenylated RNAs from different adult and fetal human tissues were from CLONTECH (Palo Alto, CA). Human tumors were obtained from patients who had undergone surgery for diverse malignancies at the Hospital Clinico, Barcelona, Spain (Banco de Tejidos y Tumores/Servicio Anatomı́a Patológica). Restriction endonucleases and other reagents used for molecular cloning were from Roche Molecular Biochemicals. All media and supplements for cell culture were obtained from Sigma, except for fetal calf serum, which was from Roche Molecular Biochemicals. A search of the GenBankTMdata base of human ESTs1 for sequences with homology to members of the ADAM family led us to identify a sequence (AI039653; WashU-Merck EST Project) derived from a fetal lung cDNA clone and showing significant similarity with sequences of previously described ADAMs. To obtain this DNA fragment, we performed PCR amplification of a human fetal lung cDNA (CLONTECH) with two specific primers 5′-CAACCCAGGAGGACATGTGA and 5′-TTCTCACGAGGAGAAGGACC, derived from theAI039653 sequence. The PCR reaction was carried out in a GeneAmp 2400 PCR system from PerkinElmer Life Sciences for 40 cycles of denaturation (94 °C, 15 s), annealing (64 °C, 20 s), and extension (68 °C, 30 s). The 390-bp PCR product amplified from human fetal lung cDNA was cloned, and its identity was confirmed by nucleotide sequencing using the kit DR terminator Taq FS and the automatic DNA sequencer ABI-PRISM 310 (PerkinElmer Life Sciences). This cDNA was then excised from the vector, radiolabeled, and used to screen the same fetal lung cDNA library according to standard procedures. The 3′-ends of cloned cDNAs were extended by successive cycles of rapid amplification of cDNA ends (RACE) using RNA from human fetal lung and the MarathonTM cDNA amplification kit (CLONTECH), essentially as described by the manufacturer. Each cycle of RACE allowed the extension of ∼100–200 bp of cDNA toward the 3′-end. Finally, the full-length cDNA was obtained by PCR amplification using the Expand Long PCR kit (Roche Molecular Biochemicals). The PCR reactions were performed for 35 cycles of denaturation (15 s at 94 °C), annealing (15 s at 64 °C), and extension (3 min at 68 °C), with primers 5′-ATGCCATGTGCCCAGAGGAGCT and 5′-GGGCTTAGAGTTCTTTTGAC. Following gel purification, the amplification product was cloned and sequenced. DNA from a panel of monochromosomal somatic cell hybrids containing a single human chromosome in a mouse or hamster cell line background was PCR screened for the presence of the genomic sequence flanked by the primers: 5′-TCCAGTCCGAGTAGATGCCAGTG and 5′-GTGCCACTGAGATGGCAGAGGGG. Amplification conditions were as follows: 35 cycles of denaturation (94 °C, 15 s), annealing (65 °C, 15 s), and extension (68 °C, 2 min) using the Expand Long PCR kit. Radiation hybrid mapping was carried out using the Genebridge 4 panel (Human Genome Mapping Resource Center, Cambridgeshire, UK). DNA samples from this panel (25 ng) were PCR screened for the presence of the genomic sequences flanked by the primers 5′-CTTGAGCTCAGGGAGCTCATTCAT and 5′-GGGGAGGCTCTGATTTCTC AGCAA. Amplification conditions were as follows: 35 cycles of denaturation (94 °C, 15 s), annealing (68 °C, 15 s), and extension (72 °C, 1 min). PCR results were converted to a vector of 93 0 values (no amplification), 1 values (amplification), and 2 values (blanks and uncertainties) and submitted to the mapping server of the Whitehead Institute/MIT Center for Genome Research, with a minimum LOD (log of the odds) score of 15. Human cancer cells from different sources were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2. Cells were subcultured weekly by incubation at 37 °C for 2 min with 0.0125% trypsin in 0.002% EDTA, followed by the addition of complete medium, washing, and resuspension in fresh medium. For most experiments, ∼5 × 105 cells/well were plated out in 100-mm dishes, transferred to serum-free Dulbecco's modified Eagle's medium for 24 h, and then exposed to the different cytokines and growth factors at the concentrations and for the times indicated. Extracellular matrix remaining on the dishes was extracted with Laemmli sample buffer (60 mm Tris-HCl, pH 6.8, 2% SDS, 5% mercaptoethanol, and 10% glycerol) and analyzed by SDS-polyacrylamide gel electrophoresis. Nylon filters containing 2 μg of poly(A)+ RNA of a wide variety of human tissues were prehybridized at 42 °C for 3 h in 50% formamide, 5× SSPE (1× SSPE = 150 mm NaCl, 10 mmNaH2PO4, 1 mm EDTA, pH 7.4), 10× Denhardt's solution, 2% SDS, and 100 μg/ml of denatured herring sperm DNA and then hybridized with a radiolabeled ADAM-TS12-specific 1.8-kb-long probe, corresponding to the 5′-region of the cDNA. Hybridization was performed for 20 h under the same conditions. Filters were washed with 0.1× SSC, 0.1% SDS for 2 h at 50 °C and exposed to autoradiography. RNA integrity and equal loading was assessed by hybridization with an actin probe. To assay the presence of ADAM-TS12 in human tumor specimens, total RNA was isolated from malignant tumors by guanidium thiocyanate-phenol-chloroform extraction and used for cDNA synthesis with the RNA PCR kit from PerkinElmer Life Sciences. After reverse transcription using 1 μg of total RNA and random hexamers as primers according to the instructions of the manufacturer, the whole mixture was used for PCR with two ADAM-TS12-specific oligonucleotides (5′-AAGCATGCTCGGCGACATGCG and 5′-ACTGCGAATCCGCACTCCACC), as described above. The PCR products were analyzed in 1.5% agarose gels. cDNA quality was verified by performing control reactions with primers derived from the sequence of actin. Negative controls were also performed in all cases by omitting the template or reverse transcriptase. The H465Q and E466A mutations in the metalloproteinase domain of ADAM-TS12 were carried out by PCR-based methods. An oligonucleotide containing a MumI sequence and two mutations 5′-TTCACAATTGCCCA AG CGCTAGGACACAGC (with A and C indicating changes T to A and A to C in the original sequence) and a second oligonucleotide (5′-TTGCAGAGCCTCTCTCTGCGCTC) were first used to PCR amplify a DNA fragment with the following conditions: 94 °C for 2 min (1 cycle) and 94 °C for 0.1 s, 60 °C for 0.1 s, and 68 °C for 30 s (20 cycles). The PCR product of the expected size was digested with MumI and BstEII and cloned in pcDNA3. The presence of the mutations in the plasmid (pcDNA3-ADAM-TS12-MUT) was confirmed by nucleotide sequencing. A full-length cDNA encoding ADAM-TS12 was PCR amplified with oligonucleotides 5′-ATGCCATGTGCCCAGAGGAGCT and 5′-GGGCTTAGAGTTCTTTTGAC and cloned in the EcoRV site of a modified pcDNA3 vector containing a 24-bp linker coding for the hemagglutinin (HA) epitope of human influenza virus. Thus, the resulting ADAM- TS12 protein was HA-tagged at the C terminus. Similarly, two oligonucleotides (5′-GTCACTTGACTACAAGGACGACGATGACAAGGG and 5′-AACTGATGTTCCTGCTGCTACT GTTCCCCAGTG) were used to introduce the FLAG epitope at the BstEII site at position 1642 of the ADAM-TS12 cDNA. COS-7, HT-1080, or LoVo cells were transfected with 1 μg of plasmids pcDNA3-ADAM-TS12-HA, pcDNA3-ADAM- TS12-FLAG, pcDNA3-ADAM-TS12-MUT, or pcDNA3 alone, using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Transfected cells were used for preparing protein extracts, which were then analyzed by Western blot. Blots were visualized by ECL according to the manufacturer's instructions (Amersham Pharmacia Biotech). One microgram of pcDNA3 containing the full-length ADAM-TS12 cDNA was transcribed and translated using the coupled reticulocyte TNT T7 kit (Promega) in the presence of [35S]methionine (Amersham Pharmacia Biotech), following the manufacturer's instructions. Parallel experiments were conducted using empty pcDNA3 as control. Protein translation products were analyzed by SDS-polyacrylamide gel electrophoresis followed by overnight autoradiography. The proteolytic activity of ADAM-TS12 was determined using the α2-macroglobulin complex formation assay. Lysates from cells transfected with ADAM-TS12 were solubilized in 25 mm Tris-HCl, pH 7.4, 0.5% sodium deoxycolate, 0.1% SDS, 100 mm NaCl, and 1% Triton X-100. The α2-macroglobulin substrate was added at a final concentration of 0.25 units/ml and incubated at 37 °C for 16 h in the presence or absence of 1 mm EDTA. To identify putative novel members of the ADAM family expressed in human tissues, we used the BLAST algorithm to scan the GenBankTM data base of ESTs looking for sequences with significant similarity to previously described family members. This analysis allowed us to identify a 469-bp EST that, after conceptual translation, generated an open reading frame with significant amino acid sequence similarity to sequences found in disintegrins purified from snake venom. A cDNA containing part of this EST was obtained by PCR amplification of total λ-phage DNA prepared from a human fetal lung cDNA library. The 390-bp PCR amplified product was cloned, and its identity was confirmed by nucleotide sequence analysis. Then the cloned fragment was radiolabeled and used as a probe to screen the same fetal lung cDNA library employed for the previous PCR amplification experiment. Upon screening of ∼1 × 106 plaque forming units, a single positive clone was identified and characterized. DNA was isolated from this positive clone (called FL1), and its nucleotide sequence was determined by automatic DNA sequencing. This analysis revealed that the length of FL1 was 1.8 kb and contained the entire 469-bp sequence corresponding to the AI039653 EST. A comparative analysis of the identified sequence with that corresponding to other ADAMs suggested that it was incomplete at the 3′-end. To extend the partial cDNA sequence toward the 3′-end, we performed 3′-RACE experiments using a specific oligonucleotide deduced from the end of the FL1 clone and RNA from human fetal lung as a template. Successive rounds of 3′-RACE experiments performed in similar conditions led us finally to obtain a fragment containing an in frame stop codon. Nucleotide sequencing of several independently isolated subclones of the RACE reaction did not reveal any differences between them. Computer analysis of the obtained sequence (Fig. 1) revealed an open reading frame coding for a protein of 1,543 amino acids with a predicted molecular mass of 177.5 kDa (EMBL accession numberAJ250725). Further structural analysis of the identified amino acid sequence showed a significant similarity with other human ADAMs, the maximum percentage of identities being with members of the ADAM-TS subfamily (57% with ADAM-TS7). An alignment of the deduced amino acid sequence confirmed that this protein possesses all characteristic domains of the ADAM-TS family members including signal sequence, propeptide, and metalloproteinase-like, disintegrin-like and cysteine-rich domains, as well as a series of TS-1 repeats (Fig. 1). However, the organization of the TS-1 repeats of the identified sequence is unique to this protein (Fig. 2). Thus, all previously described human family members exhibit a first TS repeat 52 amino acids in length that is separated from a C-terminal TS module through a cysteine-rich domain and a spacer sequence. This second module is composed of a single TS repeat in ADAM-TS5, ADAM-TS6, ADAM-TS7, and ADAM-TS8, two TS repeats in ADAM-TS1, and three TS repeats in ADAM-TS2, ADAM-TS3, and ADAM-TS9, whereas ADAM-TS4 lacks any additional repeat. The fetal lung ADAM-TS described herein contains three TS repeats in this second TS module, but, in addition, an extra C-terminal TS module can be identified in its sequence. This third TS module is separated from the second one by a spacer sequence (spacer-2) 319 amino acids long, and it is formed by four TS repeats. This latter domain is followed by a C-terminal extension rich in cysteine residues that shows similarities with the C-terminal region of other ADAM-TSs. The additional domains present in ADAM-TS12 are the cause of the large size of this novel human disintegrin when compared with all remaining family members. The alignment of the amino acid sequence deduced for ADAM-TS12 with that of other human ADAM-TSs also revealed the presence of additional structural hallmarks of the ADAM-TS family members (Figs. 1 and 2). Thus, the putative proregion contains two conserved Cys residues (at positions 139 and 160) as well as an additional one located in the sequence PTCGLKD (positions 206–212) that resembles the Cys switch motif (PRCG XPD) present in the matrix metalloproteinases and involved in maintaining enzyme latency. The prodomain ends in a dibasic motif that could correspond to a furin activation sequence for generation of the mature enzyme. The catalytic domain includes the consensus sequence HEXXHXXGXXHD (at positions 392–403) involved in the coordination of the catalytic zinc atom at the active site of metalloproteinases and bearing the Asp residue that distinguishes reprolysins from matrix metalloproteinases. This domain also contains the eight cysteine residues present in the catalytic region of all ADAM-TS family members as well as a conserved methionine residue located 16 amino acids C-terminal to the zinc-binding site that contributes to form the Met turn structure present in both reprolysins and matrix metalloproteinases. Furthermore, the disintegrin-like domain is very similar in size (79 residues) to those found in most ADAM-TSs and contains the eight cysteines present in this region of ADAM-TS proteins with the exception of ADAM-TS6. Finally, the cysteine-rich domain shows a high percentage of sequence identity with the equivalent domain in other ADAM-TSs (59.6% with ADAM-TS7), including the 10 conserved cysteine residues present in all of them. Taking together all of these structural comparisons, it seems that the newly identified human protein is a member of the ADAM-TS family of disintegrins, albeit with a more complex organization of thrombospondin repeats. Following the nomenclature system for cellular disintegrins, the officially approved name for this enzyme is ADAM-TS12. To determine the chromosomal location of the human ADAM-TS12 gene, we used a PCR-based strategy to screen a panel of somatic cell hybrid lines containing a single human chromosome in a rodent background. The sequence-tagged site specific for the ADAM-TS12 gene was generated by using two specific oligonucleotides whose sequence was derived from a noncoding sequence flanking the second exon of the gene and from a coding sequence of this same exon. As can be seen in Fig.3 A, positive amplification results were only obtained in the hybrid containing the autosome number 5. Southern blot analysis confirmed that the amplified band corresponded to ADAM-TS12 (Fig. 3 A). Because no amplification products of the expected size were observed in the hybrids containing the remaining human chromosomes, we can conclude that the ADAM-TS12 gene maps to chromosome 5. To further determine the chromosomal location of the human gene encoding ADAM-TS12, we used the same sequence-tagged site oligonucleotides to PCR screen a panel of radiation hybrids containing human chromosome fragments in a rodent background. Computer analysis of positive amplification results indicated that the ADAM-TS12 gene is located in chromosome 5q35 at 54.47 cR from marker WI-6737 (Fig. 3 B and data not shown). Interestingly, ADAM-TS2 and ADAM-TS6 also lie on human chromosome 5, although they are not necessarily clustered. Thus, ADAM-TS2 is located at 5q23, and ADAM-TS6 is located at an undefined locus at this chromosome. However, all of the remaining ADAM-TS genes are dispersed in the human genome. Thus, ADAM-TS1 and ADAM-TS5/TS11 are linked on 21q21-q22, ADAM-TS3 maps to 4q21, ADAM-TS4 maps to 1q31, ADAM-TS8 (METH-2) maps to 11q25, ADAM-TS9 maps to 3p14, and ADAM-TS7 maps to an undefined locus at chromosome 15 (22, 23). Members of the ADAM family of metalloproteinases are synthesized as precursor molecules that are subjected to proteolytic processing-mediated events to generate the final active molecules. ADAM-TS12 contains putative Cys switch and furin cleavage motifs that could be involved in the activation mechanism of this enzyme by removal of the inhibitory prodomain. To analyze the intracellular processing of ADAM-TS12, we first prepared a pcDNA3 expression vector (ADAM-TS12-HA) containing the full-length cDNA for ADAM-TS12 with an HA epitope at the 3′-terminal end. Western blot analysis of COS-7 cell extracts transiently transfected with this construct revealed the presence of a band of about 83 kDa, immunoreactive against anti-HA and absent in cells transfected with an empty vector (Fig.4 A). This size is considerably lower than that of 175 kDa, which was derived for ADAM-TS12 afterin vitro transcription and translation experiments (Fig.4 B). According to these results, it seems that ADAM-TS12 is extensively processed at the N-terminal end leading to the removal of a considerable part of this region. Amino acid sequence analysis of ADAM-TS12 allowed us to estimate that the putative processing site to generate a protein of about 83 kDa would be necessarily located after the cysteine-rich domain of the protein and around its first TS-1 domain. To further analyze this question, we prepared an additional construct (pcDNA3-ADAM-TS12-FLAG) also containing the HA epitope at the C-terminal end but including a FLAG e

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