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• W2128487191 abstract "We report the primary structure of three novel, putative zinc metalloproteases designated ADAM-TS5, ADAM-TS6, and ADAM-TS7. All have a similar domain organization, comprising a preproregion, a reprolysin-type catalytic domain, a disintegrin-like domain, a thrombospondin type-1 (TS) module, a cysteine-rich domain, a spacer domain without cysteine residues, and a COOH-terminal TS module. These genes are differentially regulated during mouse embryogenesis and in adult tissues, with Adamts5 highly expressed in the peri-implantation period in embryo and trophoblast. These proteins are similar to four other cognate gene products, defining a distinct family of human reprolysin-like metalloproteases, the ADAM-TS family. The other members of the family are ADAM-TS1, an inflammation-induced gene, the procollagen I/II amino-propeptide processing enzyme (PCINP, ADAM-TS2), and proteins predicted by the KIAA0366 and KIAA0688 genes (ADAM-TS3 and ADAM-TS4). Individual ADAM-TS members differ in the number of COOH-terminal TS modules, and some have unique COOH-terminal domains. The ADAM-TS genes are dispersed in human and mouse genomes. We report the primary structure of three novel, putative zinc metalloproteases designated ADAM-TS5, ADAM-TS6, and ADAM-TS7. All have a similar domain organization, comprising a preproregion, a reprolysin-type catalytic domain, a disintegrin-like domain, a thrombospondin type-1 (TS) module, a cysteine-rich domain, a spacer domain without cysteine residues, and a COOH-terminal TS module. These genes are differentially regulated during mouse embryogenesis and in adult tissues, with Adamts5 highly expressed in the peri-implantation period in embryo and trophoblast. These proteins are similar to four other cognate gene products, defining a distinct family of human reprolysin-like metalloproteases, the ADAM-TS family. The other members of the family are ADAM-TS1, an inflammation-induced gene, the procollagen I/II amino-propeptide processing enzyme (PCINP, ADAM-TS2), and proteins predicted by the KIAA0366 and KIAA0688 genes (ADAM-TS3 and ADAM-TS4). Individual ADAM-TS members differ in the number of COOH-terminal TS modules, and some have unique COOH-terminal domains. The ADAM-TS genes are dispersed in human and mouse genomes. extracellular matrix adisintegrin-likeand metalloprotease domain withthrombospondin type I motifs matrix metalloproteinase procollagen I/II amino-propeptide processing enzyme Ehlers-Danlos syndrome type VIIC rapidamplification of cDNA ends polymerase chain reaction kilobase pairs base pairs centimorgan restriction fragment length variants expressed sequence tag integrated mapping ofgenomes and their expression open reading frame Proteolysis of extracellular matrix (ECM)1 plays a critical role in establishing tissue architecture during development and in tissue degradation in diseases such as cancer, arthritis, Alzheimer's disease, and a variety of inflammatory conditions (1Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar, 2Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 3Black R.A. White J.M. Curr. Opin. Cell Biol. 1998; 10: 654-659Crossref PubMed Scopus (428) Google Scholar). The proteolytic enzymes responsible include members of diverse protease families and they may work in concert or in cascades to degrade or process molecules. Two groups of zinc metalloproteases in particular, ADAMs (adisintegrin andmetalloprotease) (2Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 3Black R.A. White J.M. Curr. Opin. Cell Biol. 1998; 10: 654-659Crossref PubMed Scopus (428) Google Scholar, 4Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Crossref PubMed Scopus (439) Google Scholar) and MMPs (matrixmetalloproteinases) (1Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar), appear broadly relevant to extracellular proteolysis. These families include a large number of enzymes (over 20 gene products each) with demonstrated cleavage activity for matrix molecules as well as nonmatrix, bioactive molecules such as tumor necrosis factor-α (reviewed in Ref. 3Black R.A. White J.M. Curr. Opin. Cell Biol. 1998; 10: 654-659Crossref PubMed Scopus (428) Google Scholar). In other instances of extracellular proteolysis, such as cleavage of aggrecan at the Glu373-Ala374 peptide bond (5Flannery C.R. Lark M.W. Sandy J. J. Biol. Chem. 1992; 267: 1008-1014Abstract Full Text PDF PubMed Google Scholar) or the shedding of l-selectin from leukocytes (6Borland G. Murphy G. Ager A. J. Biol. Chem. 1999; 274: 2810-2815Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), the responsible proteases have not yet been reported. Such activities may eventually be attributed to cognate proteases but they may well be due to one or more hitherto unknown enzymes. For these reasons, it is important to define the full repertoire of enzymes possessed by cells, their regulation, and their substrate preferences.ADAMs, also referred to as MDC (metalloprotease-disintegrins withcysteine-rich domains, 2) have catalytic domains with zinc-binding signatures and disintegrin domains that are very similar to the snake venom metalloproteinases (reviewed in Ref. 7Bjarnason J.B. Fox J.W. Methods Enzymol. 1995; 248: 345-368Crossref PubMed Scopus (244) Google Scholar); together, the ADAMs and snake venom metalloproteinases are referred to as reprolysins (7Bjarnason J.B. Fox J.W. Methods Enzymol. 1995; 248: 345-368Crossref PubMed Scopus (244) Google Scholar). Most ADAM members are quite similar in domain organization (2Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 4Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Crossref PubMed Scopus (439) Google Scholar, 7Bjarnason J.B. Fox J.W. Methods Enzymol. 1995; 248: 345-368Crossref PubMed Scopus (244) Google Scholar), bearing from amino to carboxyl termini, a signal peptide, a proregion, a zinc-metalloprotease catalytic domain with the typical reprolysin signature HE X 1 X 2H X 3 X 1G X 1 X HD (X is typically: a hydrophobic residue (superscript 1), glycine or a hydrophobic residue (superscript 2), asparagine (superscript 3)), a disintegrin domain, a cysteine-rich domain, an epidermal growth factor-like domain, and in many cases a membrane-spanning region and a cytoplasmic domain with signaling potential. A recently described murine gene encoded a secreted protein that differed substantially from the prototypic ADAM structure and was designated ADAM-TS1 2Nomenclature: gene nomenclature has been assigned in agreement with the Human Gene Nomenclature Committee.ADAMTS5, ADAMTS6, and ADAMTS7 etc. are human genes; Adamts5, Adamts6, and Adamts7, etc. are the mouse orthologs. The protein products of the respective genes are designated as ADAM-TS5, ADAM-TS6, and ADAM-TS7.2Nomenclature: gene nomenclature has been assigned in agreement with the Human Gene Nomenclature Committee.ADAMTS5, ADAMTS6, and ADAMTS7 etc. are human genes; Adamts5, Adamts6, and Adamts7, etc. are the mouse orthologs. The protein products of the respective genes are designated as ADAM-TS5, ADAM-TS6, and ADAM-TS7. (8Kuno 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 (433) Google Scholar). ADAM-TS1 lacks the epidermal growth factor-like repeat, does not have a canonical disintegrin sequence, and possesses three modules with similar thrombospondin type-1 (TS) repeats (8Kuno 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 (433) Google Scholar). This unique structure, the existence of three other similar gene products (ADAM-TS2–4) in the public domain, and our discovery of three novel, related gene products led us to recognize and to describe here the essential features of the ADAM-TS family.The existence of procollagen I/II amino-propeptide processing enzyme (PCINP) has been known for decades (9Lapière C.M. Lenaers A. Cohn L.D. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 3054-3058Crossref PubMed Scopus (184) Google Scholar), and the recent cDNA cloning of bovine PCINP (10Colige A. Li S.W. Sieron A.L. Nusgens B.V. Prockop D.J. Lapière C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2374-2379Crossref PubMed Scopus (157) Google Scholar) demonstrated similarities of this enzyme with ADAM-TS1. The gene is now designated ADAMTS2 in keeping with nomenclature recommended by the Human Gene Nomenclature Committee. Lack of ADAM-TS2 is known to cause dermatosparaxis in cattle (11Hanset R. Ansay M. Ann. Med. Vet. 1967; 7: 451-470Google Scholar) or Ehlers-Danlos syndrome type VIIC (EDS-VIIC) in humans (12Lapière C.M. Nusgens B.V. Arch. Dermatol. 1993; 129: 1316-1319Crossref PubMed Scopus (18) Google Scholar, 13Smith L.T. Wertelecki W. Milstone L.M. Petty E.M. Seashore M.R. Braverman I.M. Jenkins T.G. Byers P.H. Am. J. Hum. Genet. 1992; 51: 235-244PubMed Google Scholar). EDS-VIIC is a recessively inherited disorder, characterized clinically by severe skin fragility and biochemically by the presence in skin of procollagen incompletely processed at the amino terminus. The precise mutations in ADAMTS2 have not yet been reported in the literature. ADAM-TS3 and ADAM-TS4 designate the proteins predicted by mRNAs transcribed from the KIAA0366 (14Nagase T. Ishikawa K. Nakajima D. Ohira M. Seki N. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1997; 4: 141-150Crossref PubMed Scopus (212) Google Scholar) and KIAA0688 (15Ishikawa K. Nagase T. Suyama M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 169-176Crossref PubMed Scopus (169) Google Scholar) genes, respectively. These genes have been deposited in GenBankTM by the Kazusa DNA Institute and are designated ADAMTS3 and ADAMTS4, respectively.We now describe here: 1) the discovery of three novel genes, ADAM-TS5–7 belonging to this family; 2) the relationship of their predicted protein products to essential structural features of the ADAM-TS family; 3) analysis of expression of our novel genes in various tissues; and 4) the distribution of the ADAM-TS genes in human and mouse genomes.RESULTSCloning of Novel ADAM-TS cDNAsUsing the BLAST algorithm to scan dBEST for novel ESTs that were homologous to cognate ADAM-TS genes, we identified a number of nonoverlapping sequences deposited in dBEST. Full-length sequencing of the inserts of these clones confirmed that they were not identical to any other gene sequences deposited in GenBankTM. Using nested oligonucleotide primers based on the 5′ and 3′ ends of the sequences of the I.M.A.G.E. clones, we did successive rounds of RACE to extend the sequences as shown in Fig. 1(a–c). RACE primers were generated 50–200 bp from the ends of the sequences so that the contiguity of RACE clones with I.M.A.G.E. clones could be clearly established. The predicted domain organization relative to the cloned cDNA and the relationship of the products of these novel genes to the four cognate ADAM-TS members are shown in Fig.1.Cloning of Adamts5 and ADAMTS5A single round of 5′ and 3′ RACE sufficed for cloning of the entire coding sequence of Adamts5 and part of the catalytic zinc binding site through to the stop codon of ADAMTS5. The ADAMTS5 clone 345484-52 contained an incompletely processed mRNA (Fig.1 a) revealing a splice junction splitting the zinc binding sequence in the ADAM-TS5 cDNA. Although the complete primary structure of ADAM-TS5 could be deduced from the mouse cDNA the corresponding human cDNA sequence has not yet been completely cloned. At its 5′ end, clone TH5 contains a methionine codon (ATG) within a consensus Kozak sequence for initiation of translation (25Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar), but we have been unable to extend the 5′ sequence further to see if this is the first methionine in the predicted open reading frame (ORF). The predicted amino acid sequences of Adamts5 and ADAMTS5 are shown in alignment in Fig.2 a.Cloning of ADAMTS6The I.M.A.G.E. clone 742630 contained an ORF flanked by consensus splice sequences, indicating the presence of introns (Fig. 1 b). Two successive rounds of RACE at the 5′ end and a single round of RACE at the 3′ end provided the complete coding sequence of ADAM-TS6. The putative ATG codon is within a Kozak consensus sequence and encodes the first methionine within the ORF.Cloning of ADAMTS7The I.M.A.G.E. clone 272098 encoded a putative preproregion and was extended in the 3′-direction by two successive rounds of RACE (Fig. 1 c). Attempts to extend the sequence at its 5′ end have not been successful. However, we identified a typical signal peptide sequence downstream of the first methionine in the translated ORF; this methionine codon lay within a satisfactory Kozak consensus for translation initiation, and we believe it likely that it encodes the start codon.Comparison of the Predicted Structure of ADAM-TS5, ADAM-TS6, and ADAM-TS7Fig. 2 (a–c) displays the deduced primary sequence of each protein. An alignment of these sequences (including ADAM-TS1–4) may be viewed as supplemental data on-line.The three novel conceptual gene products described here share a common domain organization. From amino to carboxyl termini, we describe them as follows.A PreproregionA typical signal sequence of variable length is followed by a putative proregion of variable length but demonstrating short stretches of sequence similarity (see alignment on-line). Three cysteine residues are predicted within each novel prodomain. The COOH-terminal most of these lies within a sequence context similar to the cysteine “switch” of the MMPs (26Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-228Crossref PubMed Scopus (688) Google Scholar) (Fig. 2, a–c). All three novel genes predict consensus cleavage signals for furin, three in the case of ADAM-TS5 and one each in the case of ADAM-TS6 and ADAM-TS7. The most carboxyl-terminal furin cleavage site in ADAM-TS5 likely predicts the processing site for generation of the mature protease. The amino terminus of the mature proteins is predicted to start at the residue immediately following the cleavage site (Fig. 2, a–c).A Catalytic DomainThe catalytic domains are very similar to each other and contain eight cysteine residues and a typical reprolysin-type zinc binding signature (Fig. 2, a–c, and Fig. 3 a). Five cysteine residues are upstream of the zinc binding sequence, while three residues are downstream, an arrangement that is shared with other ADAM-TS members. Like all MMPs and reprolysins, the zinc binding signature is followed in all ADAM-TS proteins by a methionine residue within a conserved sequence context (Fig. 3 a). We designate this as being the methionine of the “Met-turn” a structural landmark present in all the MMPs and ADAMs; the Met-turn is a tight turn arranged as a right handed screw in the adamalysin and MMP polypeptides COOH-terminal to the third zinc-binding histidine (32Stocker W. Bode W. Curr. Opin. Struct. Biol. 1995; 5: 383-390Crossref PubMed Scopus (184) Google Scholar). The methionine of the Met-turn is at a similar, but not constant, interval from the ADAM-TS zinc-binding signature, and in all the ADAM-TS members, a constant cysteine residue is present in that interval.Figure 3a, sequence alignment of the catalytic zinc binding sites and Met-turn of two snake venom metalloproteases, four ADAMs, and the seven cognate members of the ADAM-TS family. The asterisk indicates a glycine residue which is replaced by asparagine in ADAM-TS1 and ADAM-TS4, and the solid circle indicates the methionine residue of the Met-turn. b, the first thrombospondin type-1 module of ADAMTS1–7. Residues identical to those in ADAM-TS1 are boxed. Residues indicating a potential linear heparin-binding sequence are italicized. c, Dendrogram illustrating the phyllogenetic relationships between the individual members of the ADAM-TS family prepared by MegAlign.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A Disintegrin-like DomainThe catalytic domain is followed by a domain of 60–90 residues with 35–45% similarity to snake venom disintegrins, but without the canonical cysteine arrangement seen in the latter. We term this the disintegrin-like domain; while of comparable length in ADAM-TS5 and ADAM-TS7, it is considerably shorter in ADAM-TS6. The disintegrin-like domain contains eight cysteine residues (except for ADAM-TS6 which has six).A TS ModuleThe first TS repeat is very similar in all three novel proteases and very similar to the first TS repeat of other ADAM-TSs (Fig. 3 b). It contains the same number of residues (52) in all three novel gene products (Fig. 3 b).The Cysteine-rich DomainThis TS domain is followed by a conserved cysteine-rich sequence termed the cysteine-rich domain (to distinguish it from the cysteine-free spacer domain). It contains 10 conserved cysteines in each case and demonstrates high sequence homology with the cysteine-rich domain of other ADAM-TS proteins.The Spacer DomainThis domain is of variable length, in all ADAM-TSs, and lacks the sequence landmarks so characteristic of all the other domains. It shows the least homology of all the domains.ADAMTS7 has the longest of all the spacer domains (221 amino acids), while ADAM-TS6 has the shortest of all spacer domains (127 amino acids). The spacer domain of ADAM-TS5 is intermediate in length (158 amino acids).A COOH-terminal TS ModuleThe sequence of the second TS module is more variant between the members of the ADAM-TS family than the first TS module, despite the conservation of the number and spacing of cysteine residues. The second TS module of ADAM-TS7 is followed by a short sequence containing two cysteine residues.Overall, the predicted mature forms of these proteases show 20–40% similarity to each other and to ADAM-TS1–4, although this may be considerably higher or lower for individual domains as described above. The dendrogram in Fig. 3 c indicates specific relationships between individual ADAM-TS members. The predicted molecular weights (Mr) of the full-length gene products are 108,633 (mADAM-TS5), 97,115 (hADAM-TS6), and 116,607 (hADAM-TS7). The actual Mr will almost certainly be different due to processing of these proenzymes to the mature forms and post-translational modifications. All the primary sequences predict the possibility of N-linked glycosylation at a number of potential sites. ADAM-TS5 and ADAM-TS7 contain three and two potential N-glycosylation sites, respectively, in the mature protease, of which two are in similar positions; one of these is in a constant position just upstream of the start of the spacer domain and the other lies within the spacer domain (Fig. 2, a and c). ADAM-TS5 possesses an additional site near the start of the disintegrin domain (Fig. 2 a). In ADAM-TS5, the N-linked glycosylation sites are conserved in both mouse and human sequences. ADAM-TS6 has four potential N-linked glycosylation sites within the prodomain and two others in the mature protease. These are at different positions relative to the sites in ADAM-TS5 and ADAM-TS7 (Fig. 2, a–c).Analyses of Expression of ADAM-TS5, ADAM-TS6, and ADAM-TS7 GenesNorthern AnalysisAdamts5 was specifically expressed in the 7-day mouse embryo (the peri-implantation period; hence we have given this gene the trivial name implantin) and at low or undetectable levels thereafter (Fig.4 a). Northern analysis showed undetectable expression of Adamts6 during mouse embryo development (not shown). Adamts7 was expressed at low levels throughout mouse development (Fig. 4 a). In adult human tissues examined with human cDNA probes, ADAMTS5 and ADAMTS6 mRNA were expressed at low levels in placenta, but were barely detectable in a number of other tissues examined (Fig.4 b). In contrast, ADAMTS7 mRNA was found in all tissues examined (Fig. 4 b).Figure 4Northern analysis of expression of ADAM-TS5, ADAM-TS6, and ADAM-TS7. RNA kilobase markers are shown at the left of each autoradiogram, and tissue origin is indicated above each lane . a, mouse embryo Northern blots;b , human multiple adult tissue Northern blots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The sizes of the mRNA species recognized varied between the three genes. ADAMTS5 mRNA was approximately 10 kbp in size in human tissue. The most prominent Adamts5 species was estimated at 7.5 kbp together with additional bands at 10 and 4.5 kbp (Fig. 4, a and b). The lone mRNA species detected by ADAMTS6 probe was approximately 8.5 kbp, whereas the most common mRNA species detected by ADAMTS7 probe was 5 kbp in size with an additional species seen at 7 kbp in skeletal muscle (Fig. 4 b). The ADAMTS6 probe hybridized with mRNA species of 4.5 and 3.0 kbp in mouse embryos (Fig.4 a), and a smaller band of 2.0 kbp was detected in gestational day 17 embryo mRNA (Fig. 4 a).In Situ HybridizationSpecific hybridization of the antisense Adamts5 probe to sections of 8.5-day-old mouse embryos was obtained, whereas no hybridization or low level background staining was noted with the control sense probe (Fig.5 a). Staining was uniform throughout the 8.5-day-old embryos (Fig. 5 b); we could not distinguish between the staining of various developmental components at this early stage. In addition, there was labeling of mRNA in trophoblastic cells lining the uterine cavity (Fig. 5, a and b) as well as in the developing placenta (Fig. 5, c and d). The decidual reaction (primarily cells in uterine glands) within the uterus also showed up-regulation of Adamts5 mRNA relative to the negative controls (Fig. 5, a and b). In sections from 10.5-day-old embryos, labeling was widespread but less intense compared with the 8.5-day-old embryo. Labeled cells were seen in mesenchyme and somites as well as in the neural tube and developing hindgut (Fig. 5 d).Figure 5Expression of Adamts5 mRNA in the 8.5-day-old (a–c) and 10.5-day-old (d) embryo by in situ hybridization. Magenta staining represents the hybridization to mRNA; nuclei are counterstained with methyl green.a , sense probe hybridized to uterus (asterisk) and embryo (E) (×100 magnification);b, antisense probe hybridized to embryo (E) and uterus (asterisk) (×100 magnification). c, high power view of embryo. Intense labeling is seen throughout the embryo and in the trophoblast (arrowhead) lining the uterine cavity. d, antisense probe hybridized to 10.5-day-old embryo. The section includes the caudal end of the embryo and placental bed. Staining is seen in the somite (arrow), neuroepithelium (arrowhead), and trophoblast and is less intense than at 8.5 days.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Chromosomal Mapping of Genes of the ADAM-TS FamilyHuman monochromosomal assignment was obtained using cloned cDNA probes for ADAMTS1, and ADAMTS5–7, and in addition, linkage mapping of ADAMTS3 and ADAMTS5 was obtained using PCR (TableI). For ADAMTS6 and ADAMTS7, we have yet to assign loci by linkage mapping in human or mouse genomes; the mapping of these genes is presently only available at the resolution of a single human chromosome (Table I).Linkage analysis of the mouse chromosomal loci for Adamts1, Adamts2, Adamts4, and Adamts5 was made possible by the existence of I.M.A.G.E. clones representing these genes. For Adamts1, informative Eco RV RFLVs were detected (C3H/HeJ-gld, 12.0 kbp; Mus spretus, 5.0 kbp); for Adamts2, informative Taq I RFLVs were detected (C3H/HeJ-gld, 6.4 kbp; Mus spretus, 7.0 kbp); for Adamts4, informative Bgl II RFLVs were detected (C3H/HeJ-gld, 5.8 kbp, Mus spretus, 7.0 kbp), and for Adamts5, informative Bgl I RFLVs were detected (C3H/HeJ-gld, 24.0 kbp, Mus spretus, 7.0 kbp).Haplotype analyses indicated that Adamts1 and Adamts5 co-segregated with Grik1 on mouse chromosome 16 in all 38 and 114 meiotic events examined, respectively.Adamts2 co-segregated with the Il3 locus on mouse chromosome 11 in 113/114 meiotic events examined, while Adamts4 co-segregated with the Fcgr3, Fcgr2, Mpz, Apoa2, and Fce1g loci on mouse chromosome 1 in all 114 meiotic events examined. The best gene order ± the S.D. indicated the loci as outlined in Table I.Based on linkage mapping of mouse or human genes, we were able to strongly suggest chromosomal position for the corresponding ortholog using the mouse-human homology maps. Overall, our results (Table I) demonstrate that the genes of this novel family are dispersed in the human and mouse genomes, with the exception of the Adamts1 and Adamts5 genes which are both linked to Grik1 on mouse chromosome 16. ADAMTS2 and ADAMTS6 both lie on human chromosome 5, but we do not yet know if these genes are linked.DISCUSSIONdBEST is a unique resource for identification of novel mRNAs using the BLAST programs. This, combined with the availability of the I.M.A.G.E. clones, provides a means of cloning full-length coding sequences using the inserts of I.M.A.G.E. clones as probes for RACE or library screening. The ESTs that we initially identified were small, did not encode similar peptides, and were not identical to each other, suggesting that they might represent different genes or nonoverlapping sequences from the same gene. Analysis of the monochromosomal mapping panel suggested that the human I.M.A.G.E. clones represented unique genes. In Northern blot analyses, these fragments hybridized to mRNA species of different sizes with different patterns of tissue-specific expression, which provided further evidence that they represented different genes.Because RACE provides overlapping clones in a vectorial fashion, we used this strategy for extending the sequences of these I.M.A.G.E. clones. We found that these novel genes were sufficiently represented in human fetal brain cDNA to permit cloning by RACE using this template. Since Northern analysis demonstrated an abundance of Adamts5 in the 7-day-old embryo, we used this template for RACE. The extension of ESTs led to acquisition of nonidentical, but homologous, sequences encoding similar domains, confirming that the original I.M.A.G.E. clones were derived from distinct genes whose products were related. Extension of the sequence of I.M.A.G.E. clone 569515 revealed 87% nucleotide identity and 96% amino acid identity with the partial ADAMTS5 sequence, demonstrating that this was the mouse ortholog, Adamts5. The novel sequences reported in the paper have been scanned against the data bases and all sequences with identity to, or significant relation to, the new sequence have been identified and accession numbers provided (Fig.1 d and legend).Determination of the initiation of translation requires identification of the first methionine codon in the appropriate Kozak consensus context (25Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar). Although the putative start codons identified in Adamts5 and ADAMTS7 lie within appropriate Kozak consensus sequences and are followed by typical signal peptides, the cloned nucleotide sequences do not extend far enough upstream to confirm that these are the first methionines in the ORFs we have defined. Despite many attempts to extend the sequences further using various mRNA templates, PCR conditions, and polymerases, we have not been successful.ADAM-TS Genes Are Highly RegulatedPrevious work of Kuno et al. (8Kuno 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 (433) Google Scholar) and Colige et al. (10Colige A. Li S.W. Sieron A.L. Nusgens B.V. Prockop D.J. Lapière C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2374-2379Crossref PubMed Scopus (157) Google Scholar), respectively, showed that ADAM-TS1 and ADAM-TS2 mRNA were either not detectable by Northern analysis in normal tissues or were expressed at very low levels. Colige et al. (10Colige A. Li S.W. Sieron A.L. Nusgens B.V. Prockop D.J. Lapière C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2374-2379Crossref PubMed Scopus (157) Google Scholar) used RT-PCR to detect mRNA, while Kuno et al. (8Kuno 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 (433) Google Scholar) showed a dramatic up-regulation in cachexigenic carcinoma cells and in mice stimulated with lipopolysaccaride. Our own studies demonstrate that ADAMTS5 and ADAMTS6 are expressed at low levels, primarily in the placenta. Adamts5 showed prominent expression in the 7-day-old embryo but lower expression thereafter, suggesting that this enzyme may play a role in proteolytic processing mostly during the peri-implantation period. The ADAM-TS genes (ADAMTS7 excepted) may be very highly regulated and inducible under certain circumstances only, such as has been demonstrated for ADAM-TS1 (8Kuno 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 (433) Google Scholar) and ADAM-TS5 (this study).Since all members of this family are likely to be processed by furin and are therefore likely to be constitutively activated during secretion, perhaps regulat" @default.
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• W2128487191 title "ADAM-TS5, ADAM-TS6, and ADAM-TS7, Novel Members of a New Family of Zinc Metalloproteases" @default.
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