ADAM-TS5, ADAM-TS6, and ADAM-TS7, Novel Members of aNew Family of Zinc MetalloproteasesGENERAL FEATURES AND GENOMIC DISTRIBUTION OF THE ADAM-TS FAMILY* □S

(Received for publication, April 15, 1999, and in revised form, June 14, 1999)

Tiina L. Hurskainen, Satoshi Hirohata, Michael F. Seldin‡, and Suneel S. Apte§

From the Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland,Ohio 44195 and the ‡Rowe Program in Genetics, Departments of Biological Chemistry and Medicine, University ofCalifornia, Davis, California 956161

We report the primary structure of three novel, puta-tive zinc metalloproteases designated ADAM-TS5,ADAM-TS6, and ADAM-TS7. All have a similar domainorganization, comprising a preproregion, a reprolysin-type catalytic domain, a disintegrin-like domain, athrombospondin type-1 (TS) module, a cysteine-rich do-main, a spacer domain without cysteine residues, and aCOOH-terminal TS module. These genes are differen-tially regulated during mouse embryogenesis and inadult tissues, with Adamts5 highly expressed in the peri-implantation period in embryo and trophoblast. Theseproteins are similar to four other cognate gene prod-ucts, defining a distinct family of human reprolysin-likemetalloproteases, the ADAM-TS family. The other mem-bers of the family are ADAM-TS1, an inflammation-in-duced gene, the procollagen I/II amino-propeptide proc-essing enzyme (PCINP, ADAM-TS2), and proteinspredicted by the KIAA0366 and KIAA0688 genes (ADAM-TS3 and ADAM-TS4). Individual ADAM-TS members dif-fer in the number of COOH-terminal TS modules, andsome have unique COOH-terminal domains. TheADAM-TS genes are dispersed in human and mousegenomes.

Proteolysis of extracellular matrix (ECM)1 plays a criticalrole in establishing tissue architecture during developmentand in tissue degradation in diseases such as cancer, arthritis,Alzheimer’s disease, and a variety of inflammatory conditions

(1–3). The proteolytic enzymes responsible include members ofdiverse protease families and they may work in concert or incascades to degrade or process molecules. Two groups of zincmetalloproteases in particular, ADAMs (a disintegrin and met-alloprotease) (2–4) and MMPs (matrix metalloproteinases) (1),appear broadly relevant to extracellular proteolysis. Thesefamilies include a large number of enzymes (over 20 geneproducts each) with demonstrated cleavage activity for matrixmolecules as well as nonmatrix, bioactive molecules such astumor necrosis factor-a (reviewed in Ref. 3). In other instancesof extracellular proteolysis, such as cleavage of aggrecan at theGlu373-Ala374 peptide bond (5) or the shedding of L-selectinfrom leukocytes (6), the responsible proteases have not yet beenreported. Such activities may eventually be attributed to cog-nate proteases but they may well be due to one or more hithertounknown enzymes. For these reasons, it is important to definethe full repertoire of enzymes possessed by cells, their regula-tion, and their substrate preferences.

ADAMs, also referred to as MDC (metalloprotease-disinte-grins with cysteine-rich domains, 2) have catalytic domainswith zinc-binding signatures and disintegrin domains that arevery similar to the snake venom metalloproteinases (reviewedin Ref. 7); together, the ADAMs and snake venom metallopro-teinases are referred to as reprolysins (7). Most ADAM mem-bers are quite similar in domain organization (2, 4, 7), bearingfrom amino to carboxyl termini, a signal peptide, a proregion, azinc-metalloprotease catalytic domain with the typical reproly-sin signature HEX1X2HX3X1GX1XHD (X is typically: a hydro-phobic residue (superscript 1), glycine or a hydrophobic residue(superscript 2), asparagine (superscript 3)), a disintegrin do-main, a cysteine-rich domain, an epidermal growth factor-likedomain, and in many cases a membrane-spanning region and acytoplasmic domain with signaling potential. A recently de-scribed murine gene encoded a secreted protein that differedsubstantially from the prototypic ADAM structure and wasdesignated ADAM-TS12 (8). ADAM-TS1 lacks the epidermalgrowth factor-like repeat, does not have a canonical disintegrinsequence, and possesses three modules with similar throm-bospondin type-1 (TS) repeats (8). This unique structure, theexistence of three other similar gene products (ADAM-TS2–4)in the public domain, and our discovery of three novel, relatedgene products led us to recognize and to describe here theessential features of the ADAM-TS family.

The existence of procollagen I/II amino-propeptide process-

* This work was supported by the Cleveland Clinic Foundation (to S.Apte), National Institutes of Health Grant HGO0734 (to M. F. S.), andThe Foundation Fighting Blindness and Academy of Finland (toT. L. H.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains the alignments of the ADAM-TS1–7 sequences.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF140673(Adamts5), AF141293 (ADAMTS5), AF140674 (ADAMTS6), andAF140675 (ADAMTS7).

§ To whom correspondence should be addressed: Dept. of BiomedicalEngineering, Lerner Research Institute, Cleveland Clinic Foundation(ND20), 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-3278;Fax: 216-445-4383; E-mail: aptes@bme.ri.ccf.org.

1 The abbreviations used are: ECM, extracellular matrix; ADAM-TS,a disintegrin-like and metalloprotease domain with thrombospondintype I motifs; MMP, matrix metalloproteinase; PCINP, procollagen I/IIamino-propeptide processing enzyme; EDS-VIIC, Ehlers-Danlos syn-drome type VIIC; RACE, rapid amplification of cDNA ends; PCR, po-lymerase chain reaction; kbp, kilobase pairs; bp, base pairs; cM, centi-morgan; RFLV, restriction fragment length variants; EST, expressedsequence tag; I.M.A.G.E., integrated mapping of genomes and theirexpression; ORF, open reading frame.

2 Nomenclature: gene nomenclature has been assigned in agreementwith the Human Gene Nomenclature Committee. ADAMTS5, AD-AMTS6, and ADAMTS7 etc. are human genes; Adamts5, Adamts6, andAdamts7, etc. are the mouse orthologs. The protein products of therespective genes are designated as ADAM-TS5, ADAM-TS6, andADAM-TS7.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 36, Issue of September 3, pp. 25555–25563, 1999© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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ing enzyme (PCINP) has been known for decades (9), and therecent cDNA cloning of bovine PCINP (10) demonstrated sim-ilarities of this enzyme with ADAM-TS1. The gene is nowdesignated ADAMTS2 in keeping with nomenclature recom-mended by the Human Gene Nomenclature Committee. Lack ofADAM-TS2 is known to cause dermatosparaxis in cattle (11) orEhlers-Danlos syndrome type VIIC (EDS-VIIC) in humans (12,13). EDS-VIIC is a recessively inherited disorder, characterizedclinically by severe skin fragility and biochemically by thepresence in skin of procollagen incompletely processed at theamino terminus. The precise mutations in ADAMTS2 have notyet been reported in the literature. ADAM-TS3 and ADAM-TS4designate the proteins predicted by mRNAs transcribed fromthe KIAA0366 (14) and KIAA0688 (15) genes, respectively.These genes have been deposited in GenBankTM by the KazusaDNA 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 oftheir predicted protein products to essential structural featuresof the ADAM-TS family; 3) analysis of expression of our novelgenes in various tissues; and 4) the distribution of theADAM-TS genes in human and mouse genomes.

EXPERIMENTAL PROCEDURES

cDNA Cloning and Sequence Analysis—Using the BLAST (basic localalignment search tool) programs (16), we scanned the data base ofexpressed sequence tags (dBEST, Ref. 17) using the protein sequence ofa Caenorhabditis elegans encoded ADAM-TS3 as well as ADAM-TS 1–4and identified similarities in several human and mouse ESTs. TheseESTs were derived from clones held by the I.M.A.G.E. consortium(integrated mapping of genomes and their expression): EST AA288689(clone 569515) and EST W72552 (clone 345484) for mouse and humanADAM-TS5, respectively, EST AA400393 (clone 742630) for ADAM-TS6, and EST N48032 (clone 272098) for ADAM-TS7. Translation ofthese ESTs predicted peptides with similarity, but not identity to dis-crete regions of cognate ADAM-TS members or of snake venom disin-tegrins but with no identity in GenBankTM or other protein and nucle-otide data bases. The I.M.A.G.E. clones were purchased (ResearchGenetics, Huntsville, AL) and their inserts sequenced in their entirety.

Using oligonucleotide primers based on the sequences at the ends ofthe I.M.A.G.E. clone inserts and human fetal brain or 7-day-old mouseembryo cDNA (Marathon cDNA, CLONTECH) as template, we per-formed RACE (rapid amplification of cDNA ends, Ref. 18) by PCR at 59or 39 ends or both as required. Primers were designed with calculatedTm . 72 °C, and RACE was performed with nested primers for eachamplification. For PCR, we used Advantage PCR reagents (CLON-TECH); the polymerase mix contained Taq polymerase as well as aproofreading polymerase to minimize PCR errors. We employed “hot-start” PCR for optimal efficiency using the following “touch-down” cycleconditions: 95 °C for 1 min followed by 5 cycles of 95 °C for 0.5 min,72 °C for 5 min; then 5 cycles of 95 °C for 0.5 min, 70 °C for 5 min, and20 cycles of 95 °C for 0.5 min; and finally, 68 °C for 5 min. The PCRproducts were analyzed by Southern blotting, initially using[a-32P]dCTP-labeled inserts of the I.M.A.G.E clones. For subsequentrounds of RACE, the inserts of clones obtained in the previous roundwere used as probes.

Hybridizing bands were ligated into pGEM-T Easy (Promega, Mad-ison, WI), and individual clones were selected by another round ofSouthern analysis. Automated nucleotide sequencing of both strands ofeach clone was done at the Molecular Biotechnology Core of the LernerResearch Institute, Cleveland Clinic Foundation, and nucleotide se-quence data were analyzed using software from DNAStar Inc. By inte-gration of the overlapping sequences thus obtained, we derived contig-uous nucleotide sequences and determined the complete primarystructure of mouse ADAM-TS5, human ADAM-TS6, and humanADAM-TS7 and the partial sequence of human ADAM-TS5.

Northern Analysis and in Situ Hybridization—Mouse embryo North-ern blots and multiple tissue Northern blots from human and mousetissues (CLONTECH) were hybridized to the [a-32P]dCTP-labeled in-

serts of I.M.A.G.E. clones as per the manufacturer’s recommendationsfollowed by autoradiographic exposure for 3–7 days.

In situ hybridization was done with cryosections of mouse embryos ofgestational age 8.5 and 10.5 days. Embryos, including the surroundinguterus, were fixed overnight in 4% paraformaldehdye. Sense and anti-sense probes continuously labeled with digoxigenin-UTP (Roche Molec-ular Biochemicals) were transcribed with T7 and T3 RNA polymerases,respectively, using as template a 630-bp EcoRI-SacI fragment from theAdamts5 clone 569515 (Fig. 1a) cloned into pBluescript SK1 (Strat-agene, La Jolla, CA). In situ hybridization was done essentially asdescribed previously (19) except that sections were predigested withproteinase K (Roche Molecular Biochemicals) at a lower concentration(1–5 mg/ml) than previously reported (19). Bound, digoxigenin-labeledprobe was detected using an alkaline phosphatase tagged anti-digoxi-genin antibody (Roche Molecular Biochemicals), and nuclei were coun-terstained with methyl green.

Human Chromosomal Mapping—One or each of two methods wereused for experimental determination of human gene loci of theADAM-TS family. The specific method(s) used for mapping of a partic-ular ADAM-TS gene are noted in Table I. Chromosomal assignmentwas provided by Southern analysis of a HindIII-digested monochromo-somal human-rodent radiation hybrid DNA panel (NIGMS monochro-mosomal human/rodent somatic cell hybrid mapping panel 2, CoriellCell Repositories, Camden, NJ). I.M.A.G.E. clones for ADAMTS2 (clone399065, GenBankTM accession number AA732989), ADAMTS5, AD-AMTS6, and ADAMTS7 were used as [32P]dCTP-labeled probes forSouthern analyses of the panel as described previously (20). Specificsignals in DNA from the somatic cell hybrids, which were of identicalsize to those found in control human genomic DNA (but not in mouse orhamster DNA), indicated the gene locus at a single chromosomeresolution.

For higher resolution of the ADAMTS3 and ADAMTS5 loci, weperformed linkage analyses using DNA from the Stanford Human Ge-nome Center G3 human radiation hybrid panel (Research Genetics,Huntsville, AL) for gene-specific PCR. Using oligonucleotide primersets specific for each gene (for ADAMTS3; forward primer 59-TCATC-CAGTGAGAAGTTGT-39; reverse primer, 59-CGACATTGCTCATC-CATAG-39: for ADAMTS5; forward primer 59-ACTCTGTCACTAGT-CATGGC-39; reverse primer, 59-ACATCTTATTAAAACAGCAGC-39)and stringent PCR conditions (for ADAMTS3: 95 °C 3 30 s, 55 °C 330 s, 68 °C 3 30 s for 30 cycles: for ADAMTS5 95 °C 3 30 s, 58 °C 3 30 s,68 °C 3 30 s for 30 cycles), we obtained specific amplicons in humancontrol DNA, which were not seen in DNA from the Chinese hamstersomatic cell recipient. The retention or nonretention of the band specificto the human gene was determined in the 83 hybrid DNAs that com-prise this panel. These results were compared with previously definedmarkers to obtain the linkage data together with the lod score forlinkage (Table I).

Mouse Chromosomal Mapping—In order to determine the chromo-somal location of the Adamts1, Adamts2, Adamts4, and Adamts5 genes,we analyzed a panel of DNA samples from an interspecific cross thathas been characterized for over 1200 genetic markers throughout thegenome (21). The genetic markers included in this map span between 50and 80 centimorgans (cM) on each mouse autosome and the X chromo-some. Initially, DNA from the two parental mice (C3H/HeJ-gld and(C3H/HeJ-gld 3 Mus spretus) F1) were digested with various restric-tion endonucleases and hybridized with the cDNA probes (I.M.A.G.E.clones (GenBankTM accession numbers in parentheses) 875441(AA475356), 1246561 (AA832579), 403051 (W81870), and 569515(AA288689) for Adamts1, Adamts2, Adamts4, and Adamts5, respec-tively) to determine restriction fragment length variants (RFLVs) forhaplotype analyses. Gene linkage was determined by segregation anal-ysis (22). Gene order was determined by analyzing all haplotypes andminimizing crossover frequency between all genes that were deter-mined to be within a linkage group. This method resulted in determi-nation of the most likely gene order (23). Mouse-human homology mapsdefining conserved linkage groups were used to predict the location oforthologous loci given the mapping of either the human or mouse geneby linkage (24).

RESULTS

Cloning of Novel ADAM-TS cDNAs

Using the BLAST algorithm to scan dBEST for novel ESTsthat were homologous to cognate ADAM-TS genes, we identi-fied a number of nonoverlapping sequences deposited indBEST. Full-length sequencing of the inserts of these clones3 S. S. Apte, unpublished data.

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confirmed that they were not identical to any other gene se-quences deposited in GenBankTM. Using nested oligonucleotideprimers based on the 59 and 39 ends of the sequences of theI.M.A.G.E. clones, we did successive rounds of RACE to extend

the sequences as shown in Fig. 1 (a–c). RACE primers weregenerated 50–200 bp from the ends of the sequences so that thecontiguity of RACE clones with I.M.A.G.E. clones could beclearly established. The predicted domain organization relative

FIG. 1. Cloning strategy used for isolation of mouse and human ADAM-TS5 (a), human ADAM-TS6 (b), and human ADAM-TS7 (c).The domain organization of each relative to the cDNA clones (thin line) and the extent of overlap between clones is shown. The original I.M.A.G.E.clones used are underlined. Intronic regions of incompletely spliced transcripts are shown by the angled dotted lines. DNA scale marker (in basepairs) and amino acid scale marker are at upper right. Location of the probe used for in situ hybridization (ISH) is shown in a. d, domainorganization of ADAM-TS1–4 based on the primary structure predicted from cDNA sequences. Sequences of ADAM-TS1 (mouse, GenBankTM

accession number D67076) and ADAM-TS2 (bovine, GenBankTM accession number X96389; human, GenBankTM accession number AJ003125) havebeen published, and those of ADAM-TS3 (human KIAA0366 gene, GenBankTM accession number AB002364) and ADAM-TS4 (human KIAA0688gene, GenBankTM accession number AB014588) have been deposited in the data bases. The cDNA sequence of ADAM-TS3 does not extend to thestart codon.

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to the cloned cDNA and the relationship of the products ofthese novel genes to the four cognate ADAM-TS members areshown in Fig. 1.

Cloning of Adamts5 and ADAMTS5—A single round of 59and 39 RACE sufficed for cloning of the entire coding sequenceof Adamts5 and part of the catalytic zinc binding site throughto the stop codon of ADAMTS5. The ADAMTS5 clone345484-52 contained an incompletely processed mRNA (Fig.1a) revealing a splice junction splitting the zinc binding se-quence in the ADAM-TS5 cDNA. Although the complete pri-mary structure of ADAM-TS5 could be deduced from the mousecDNA the corresponding human cDNA sequence has not yetbeen completely cloned. At its 59 end, clone TH5 contains amethionine codon (ATG) within a consensus Kozak sequencefor initiation of translation (25), but we have been unable toextend the 59 sequence further to see if this is the first methi-onine in the predicted open reading frame (ORF). The predictedamino acid sequences of Adamts5 and ADAMTS5 are shown inalignment in Fig. 2a.

Cloning of ADAMTS6—The I.M.A.G.E. clone 742630 con-tained an ORF flanked by consensus splice sequences, indicat-ing the presence of introns (Fig. 1b). Two successive rounds ofRACE at the 59 end and a single round of RACE at the 39 endprovided the complete coding sequence of ADAM-TS6. Theputative ATG codon is within a Kozak consensus sequence andencodes the first methionine within the ORF.

Cloning of ADAMTS7—The I.M.A.G.E. clone 272098 en-coded a putative preproregion and was extended in the 39-direction by two successive rounds of RACE (Fig. 1c). Attemptsto extend the sequence at its 59 end have not been successful.However, we identified a typical signal peptide sequence down-stream of the first methionine in the translated ORF; thismethionine codon lay within a satisfactory Kozak consensus fortranslation initiation, and we believe it likely that it encodesthe start codon.

Comparison of the Predicted Structure of ADAM-TS5,ADAM-TS6, and ADAM-TS7

Fig. 2 (a–c) displays the deduced primary sequence of eachprotein. An alignment of these sequences (including ADAM-TS1–4) may be viewed as supplemental data on-line.

The three novel conceptual gene products described hereshare a common domain organization. From amino to carboxyltermini, we describe them as follows.

A Preproregion—A typical signal sequence of variable lengthis followed by a putative proregion of variable length but dem-onstrating short stretches of sequence similarity (see align-ment on-line). Three cysteine residues are predicted withineach novel prodomain. The COOH-terminal most of these lieswithin a sequence context similar to the cysteine “switch” of theMMPs (26) (Fig. 2, a–c). All three novel genes predict consen-sus cleavage signals for furin, three in the case of ADAM-TS5and one each in the case of ADAM-TS6 and ADAM-TS7. Themost carboxyl-terminal furin cleavage site in ADAM-TS5 likelypredicts the processing site for generation of the mature pro-tease. The amino terminus of the mature proteins is predictedto start at the residue immediately following the cleavage site(Fig. 2, a–c).

A Catalytic Domain—The catalytic domains are very similarto each other and contain eight cysteine residues and a typicalreprolysin-type zinc binding signature (Fig. 2, a–c, and Fig.3a). Five cysteine residues are upstream of the zinc bindingsequence, while three residues are downstream, an arrange-ment that is shared with other ADAM-TS members. Like allMMPs and reprolysins, the zinc binding signature is followedin all ADAM-TS proteins by a methionine residue within a

conserved sequence context (Fig. 3a). We designate this asbeing the methionine of the “Met-turn” a structural landmarkpresent in all the MMPs and ADAMs; the Met-turn is a tightturn arranged as a right handed screw in the adamalysin andMMP polypeptides COOH-terminal to the third zinc-bindinghistidine (32). The methionine of the Met-turn is at a similar,but not constant, interval from the ADAM-TS zinc-bindingsignature, and in all the ADAM-TS members, a constant cys-teine residue is present in that interval.

A Disintegrin-like Domain—The catalytic domain is followedby a domain of 60–90 residues with 35–45% similarity to snakevenom disintegrins, but without the canonical cysteine ar-rangement seen in the latter. We term this the disintegrin-likedomain; 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 Module—The first TS repeat is very similar in all threenovel proteases and very similar to the first TS repeat of otherADAM-TSs (Fig. 3b). It contains the same number of residues(52) in all three novel gene products (Fig. 3b).

The Cysteine-rich Domain—This TS domain is followed by aconserved cysteine-rich sequence termed the cysteine-rich do-main (to distinguish it from the cysteine-free spacer domain). Itcontains 10 conserved cysteines in each case and demonstrateshigh sequence homology with the cysteine-rich domain of otherADAM-TS proteins.

The Spacer Domain—This domain is of variable length, in allADAM-TSs, and lacks the sequence landmarks so characteris-tic of all the other domains. It shows the least homology of allthe domains. ADAMTS7 has the longest of all the spacer do-mains (221 amino acids), while ADAM-TS6 has the shortest ofall spacer domains (127 amino acids). The spacer domain ofADAM-TS5 is intermediate in length (158 amino acids).

A COOH-terminal TS Module—The sequence of the secondTS module is more variant between the members of theADAM-TS family than the first TS module, despite the conser-vation of the number and spacing of cysteine residues. Thesecond TS module of ADAM-TS7 is followed by a short se-quence containing two cysteine residues.

Overall, the predicted mature forms of these proteases show20–40% similarity to each other and to ADAM-TS1–4, al-though this may be considerably higher or lower for individualdomains as described above. The dendrogram in Fig. 3c indi-cates specific relationships between individual ADAM-TSmembers. 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 willalmost certainly be different due to processing of these proen-zymes to the mature forms and post-translational modifica-tions. All the primary sequences predict the possibility of N-linked glycosylation at a number of potential sites. ADAM-TS5and ADAM-TS7 contain three and two potential N-glycosyla-tion sites, respectively, in the mature protease, of which twoare in similar positions; one of these is in a constant positionjust upstream of the start of the spacer domain and the otherlies within the spacer domain (Fig. 2, a and c). ADAM-TS5possesses an additional site near the start of the disintegrindomain (Fig. 2a). In ADAM-TS5, the N-linked glycosylationsites are conserved in both mouse and human sequences.ADAM-TS6 has four potential N-linked glycosylation siteswithin 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).

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FIG. 2. Predicted amino acid se-quences of ADAM-TS5 (a) (alignmentshows mouse sequence above, partialhuman sequence below), ADAM-TS6(b), and ADAM-TS7 (c). The active sitesequences and proposed Met-turn are en-closed in boxes. Potential furin cleavagesite(s) are indicated by arrows. Throm-bospondin type-1 modules are underlined.Potential sites for N-linked glycosylationare overlined. Cysteine residues withinthe context of an MMP-like “cysteineswitch” are indicated by the solid circles.Other cysteine residues are indicated byasterisks. The preproregion extends untilthe furin cleavage site, and the catalyticdomain extends from the furin cleavagesite to the disintegrin-like sequence (Dis).The start of the spacer domain is indi-cated; the region between the NH2-termi-nal TS domain and the spacer domain isthe cysteine-rich domain. The single let-ter amino acid code is used.

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Analyses of Expression of ADAM-TS5, ADAM-TS6, andADAM-TS7 Genes

Northern Analysis—Adamts5 was specifically expressed inthe 7-day mouse embryo (the peri-implantation period; hencewe have given this gene the trivial name implantin) and at lowor undetectable levels thereafter (Fig. 4a). Northern analysisshowed undetectable expression of Adamts6 during mouse em-bryo development (not shown). Adamts7 was expressed at lowlevels throughout mouse development (Fig. 4a). In adult hu-man tissues examined with human cDNA probes, ADAMTS5and ADAMTS6 mRNA were expressed at low levels in pla-centa, but were barely detectable in a number of other tissuesexamined (Fig. 4b). In contrast, ADAMTS7 mRNA was found inall tissues examined (Fig. 4b).

The sizes of the mRNA species recognized varied between thethree genes. ADAMTS5 mRNA was approximately 10 kbp insize in human tissue. The most prominent Adamts5 specieswas estimated at 7.5 kbp together with additional bands at 10and 4.5 kbp (Fig. 4, a and b). The lone mRNA species detectedby ADAMTS6 probe was approximately 8.5 kbp, whereas themost common mRNA species detected by ADAMTS7 probe was5 kbp in size with an additional species seen at 7 kbp in skeletalmuscle (Fig. 4b). The ADAMTS6 probe hybridized with mRNAspecies of 4.5 and 3.0 kbp in mouse embryos (Fig. 4a), and a

smaller band of 2.0 kbp was detected in gestational day 17embryo mRNA (Fig. 4a).

In Situ Hybridization—Specific hybridization of the anti-sense Adamts5 probe to sections of 8.5-day-old mouse embryoswas obtained, whereas no hybridization or low level back-ground staining was noted with the control sense probe (Fig.5a). Staining was uniform throughout the 8.5-day-old embryos(Fig. 5b); we could not distinguish between the staining ofvarious developmental components at this early stage. In ad-dition, there was labeling of mRNA in trophoblastic cells liningthe uterine cavity (Fig. 5, a and b) as well as in the developingplacenta (Fig. 5, c and d). The decidual reaction (primarily cellsin uterine glands) within the uterus also showed up-regulationof Adamts5 mRNA relative to the negative controls (Fig. 5, aand b). In sections from 10.5-day-old embryos, labeling waswidespread but less intense compared with the 8.5-day-oldembryo. Labeled cells were seen in mesenchyme and somites aswell as in the neural tube and developing hindgut (Fig. 5d).

Chromosomal Mapping of Genes of the ADAM-TS Family

Human monochromosomal assignment was obtained usingcloned cDNA probes for ADAMTS1, and ADAMTS5–7, and inaddition, linkage mapping of ADAMTS3 and ADAMTS5 wasobtained using PCR (Table I). For ADAMTS6 and ADAMTS7,we have yet to assign loci by linkage mapping in human ormouse genomes; the mapping of these genes is presently onlyavailable at the resolution of a single human chromosome(Table I).

Linkage analysis of the mouse chromosomal loci for Ad-amts1, Adamts2, Adamts4, and Adamts5 was made possible bythe existence of I.M.A.G.E. clones representing these genes.For Adamts1, informative EcoRV RFLVs were detected (C3H/HeJ-gld, 12.0 kbp; Mus spretus, 5.0 kbp); for Adamts2, inform-ative TaqI RFLVs were detected (C3H/HeJ-gld, 6.4 kbp; Musspretus, 7.0 kbp); for Adamts4, informative BglII RFLVs weredetected (C3H/HeJ-gld, 5.8 kbp, Mus spretus, 7.0 kbp), and forAdamts5, informative BglI RFLVs were detected (C3H/HeJ-gld, 24.0 kbp, Mus spretus, 7.0 kbp).

Haplotype analyses indicated that Adamts1 and Adamts5co-segregated with Grik1 on mouse chromosome 16 in all 38and 114 meiotic events examined, respectively. Adamts2 co-segregated with the Il3 locus on mouse chromosome 11 in113/114 meiotic events examined, while Adamts4 co-segre-gated with the Fcgr3, Fcgr2, Mpz, Apoa2, and Fce1g loci onmouse chromosome 1 in all 114 meiotic events examined. Thebest gene order 6 the S.D. indicated the loci as outlined inTable I.

Based on linkage mapping of mouse or human genes, wewere able to strongly suggest chromosomal position for thecorresponding ortholog using the mouse-human homologymaps. Overall, our results (Table I) demonstrate that the genesof this novel family are dispersed in the human and mousegenomes, with the exception of the Adamts1 and Adamts5genes 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.

DISCUSSION

dBEST is a unique resource for identification of novelmRNAs using the BLAST programs. This, combined with theavailability of the I.M.A.G.E. clones, provides a means of clon-ing full-length coding sequences using the inserts of I.M.A.G.E.clones as probes for RACE or library screening. The ESTs thatwe initially identified were small, did not encode similar pep-tides, and were not identical to each other, suggesting that theymight represent different genes or nonoverlapping sequencesfrom the same gene. Analysis of the monochromosomal map-

FIG. 3. a, sequence alignment of the catalytic zinc binding sites andMet-turn of two snake venom metalloproteases, four ADAMs, and theseven cognate members of the ADAM-TS family. The asterisk indicatesa glycine residue which is replaced by asparagine in ADAM-TS1 andADAM-TS4, and the solid circle indicates the methionine residue of theMet-turn. b, the first thrombospondin type-1 module of ADAMTS1–7.Residues identical to those in ADAM-TS1 are boxed. Residues indicat-ing a potential linear heparin-binding sequence are italicized. c, Den-drogram illustrating the phyllogenetic relationships between the indi-vidual members of the ADAM-TS family prepared by MegAlign.

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ping panel suggested that the human I.M.A.G.E. clones repre-sented unique genes. In Northern blot analyses, these frag-ments hybridized to mRNA species of different sizes withdifferent patterns of tissue-specific expression, which providedfurther evidence that they represented different genes.

Because RACE provides overlapping clones in a vectorialfashion, we used this strategy for extending the sequences ofthese I.M.A.G.E. clones. We found that these novel genes weresufficiently represented in human fetal brain cDNA to permitcloning by RACE using this template. Since Northern analysisdemonstrated an abundance of Adamts5 in the 7-day-old em-bryo, we used this template for RACE. The extension of ESTsled to acquisition of nonidentical, but homologous, sequencesencoding similar domains, confirming that the original I.M.-A.G.E. clones were derived from distinct genes whose productswere related. Extension of the sequence of I.M.A.G.E. clone569515 revealed 87% nucleotide identity and 96% amino acididentity with the partial ADAMTS5 sequence, demonstratingthat this was the mouse ortholog, Adamts5. The novel se-quences reported in the paper have been scanned against thedata bases and all sequences with identity to, or significantrelation to, the new sequence have been identified and acces-sion numbers provided (Fig. 1d and legend).

Determination of the initiation of translation requires iden-tification of the first methionine codon in the appropriateKozak consensus context (25). Although the putative startcodons identified in Adamts5 and ADAMTS7 lie within appro-priate Kozak consensus sequences and are followed by typical

signal peptides, the cloned nucleotide sequences do not extendfar enough upstream to confirm that these are the first methi-onines in the ORFs we have defined. Despite many attempts toextend the sequences further using various mRNA templates,PCR conditions, and polymerases, we have not been successful.

ADAM-TS Genes Are Highly Regulated—Previous work ofKuno et al. (8) and Colige et al. (10), respectively, showed thatADAM-TS1 and ADAM-TS2 mRNA were either not detectableby Northern analysis in normal tissues or were expressed atvery low levels. Colige et al. (10) used RT-PCR to detect mRNA,while Kuno et al. (8) showed a dramatic up-regulation in ca-chexigenic carcinoma cells and in mice stimulated with lipopo-lysaccaride. Our own studies demonstrate that ADAMTS5 andADAMTS6 are expressed at low levels, primarily in the pla-centa. Adamts5 showed prominent expression in the 7-day-oldembryo but lower expression thereafter, suggesting that thisenzyme may play a role in proteolytic processing mostly duringthe peri-implantation period. The ADAM-TS genes (ADAMTS7excepted) may be very highly regulated and inducible undercertain circumstances only, such as has been demonstrated forADAM-TS1 (8) and ADAM-TS5 (this study).

Since all members of this family are likely to be processed byfurin and are therefore likely to be constitutively activatedduring secretion, perhaps regulation at the transcriptionallevel may be required to control their activity. In this context,nothing is known of the functionality of the putative cysteine-switch in the ADAM-TS family. It is also not known whetherinhibitors of the MMPs and ADAMs, such as TIMP-3 (3, 27),will act as effective natural regulators of these enzymes orwhether other, more specific inhibitors exist.

Genomic Distribution of ADAM-TS Genes—We determinedthe ADAM-TS gene loci to facilitate exploration of their possi-ble role in genetic diseases. Our studies demonstrate that un-like many of the MMPs (28) the ADAM-TS genes (with theexception of ADAMTS1 and ADAMTS5) are not clusteredwithin the genome. Linkage mapping in the human or mousegenomes is a powerful tool for genetic studies, and as well, itpermits assignment of a locus in the other species using hu-man-mouse linkage homology maps. We have assigned linkagein human or mouse genomes for five out of the seven genes now

FIG. 4. Northern analysis of expression of ADAM-TS5, ADAM-TS6, and ADAM-TS7. RNA kilobase markers are shown at the left ofeach autoradiogram, and tissue origin is indicated above each lane. a,mouse embryo Northern blots; b, human multiple adult tissue Northernblots.

FIG. 5. Expression of Adamts5 mRNA in the 8.5-day-old (a–c)and 10.5-day-old (d) embryo by in situ hybridization. Magentastaining represents the hybridization to mRNA; nuclei are counter-stained with methyl green. a, sense probe hybridized to uterus (aster-isk) and embryo (E) (3100 magnification); b, antisense probe hybridizedto embryo (E) and uterus (asterisk) (3100 magnification). c, high powerview of embryo. Intense labeling is seen throughout the embryo and inthe trophoblast (arrowhead) lining the uterine cavity. d, antisenseprobe hybridized to 10.5-day-old embryo. The section includes the cau-dal end of the embryo and placental bed. Staining is seen in the somite(arrow), neuroepithelium (arrowhead), and trophoblast and is less in-tense than at 8.5 days.

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known in this family. The Online Mendelian Inheritance inMan (OMIM) data base shows that a number of human dis-eases for which the causative mutations have not been identi-fied are mapped to the vicinity of the ADAM-TS loci. Dentino-genesis imperfecta and amelogenesis imperfecta map to 4q21in the vicinity of ADAMTS3. Usher syndrome-2A (autosomalrecessive) maps to 21q21-q22, a familial platelet disorder withassociated myeloid malignancy to 21q22.1-q22, in the vicinityof ADAMTS1 and ADAMTS5. Dominant nonsyndromic senso-rineural deafness seven maps to 1q21-q23, limb girdle muscu-lar dystrophy-1B to 1q11-q21, and familial partial lipodystro-phy to 1q21-q24, the region of ADAMTS4. Mapping ofADAMTS2 to 5q23-q24 indicates the locus for the EDS-VIICmutations.

Domain Organization and Evolutionary Relationships in theADAM-TS Family—The novel ADAM-TS proteases describedhere show all the hallmarks of the ADAM-TS family (Fig. 1). Arecent review described the similarities and phyllogenetic re-lationship between the first four members of the family (29).Our results (Fig. 3c) indicate that distinct and evolutionarilyrelated subsets may exist within the ADAM-TS family. ADAM-TS3 and ADAM-TS4 are not only similar in organization andlength, but are almost identical within the catalytic domain,particularly in the vicinity of the zinc-binding consensus se-quence. This remarkable degree of identity, however, does notextend to other domains or to the unique COOH-terminal do-mains (Fig. 1d), which are totally unrelated to each other or toany other protein in the data base. The three new members wedescribe here are all very similar in domain organization, but itis also clear from the dendrogram that they do not form adistinct subset of proteases. In fact, each of the novel proteaseswe have described belongs to a distinct limb of the dendrogram(Fig. 3c). ADAM-TS1 and ADAM-TS4 have three and one TSdomains, respectively, yet their zinc-binding signatures arevery similar, and they are closely related phyllogenetically inthe ADAM-TS dendrogram, with some proximity to ADAM-TS5(Fig. 3c). In ADAM-TS1 and ADAM-TS4, but not in ADAM-TS5, a conserved glycine residue (indicated by an asterisk inFig. 3a) within the zinc binding signature, is altered to anasparagine.

The NH2-terminal most (first) TS domain shows strikingconservation within the family (Fig. 3b). All cysteine residues,a pentapeptide, CSR(T/S)C, a number of glycine residues, andtryptophans at TS domain positions 1 and 4 are conserved. Thepentapeptide CSR(T/S)C in ADAM-TS members is consistentlydifferent from the corresponding sequence in thrombospondin(CSVTC), which is known to bind to CD36 (reviewed in Ref. 30).A potential linear heparin binding motif (BBXB, B is a basic

amino acid, X denotes any amino acid) found in thrombospon-dins (30) is present in the sequence of ADAM-TS1, ADAM-TS4,ADAM-TS6, and ADAM-TS7 (Fig. 3b). Cysteine residues andtheir spacing are highly conserved in all members within thepro, catalytic, disintegrin, and cysteine-rich domains (Fig. 2,a–c, and the alignment accessible on-line).

Assuming that the even numbers of cysteines in each of thedomains of the mature ADAM-TS proteases participate in in-trachain disulfide bond formation, one can begin to visualizethe structure of ADAM-TS proteins. A catalytic domain foldedlike the metzincins (32) is likely to be followed by independ-ently folded domains in a modular fashion, with the spacerdomain providing the most variability in sequence, length, andflexibility. It will be interesting to investigate whether inter-domain interactions are essential for structure and function.

Function of the ADAM-TS Family—ADAM-TS1 transientlyexpressed in COS cells is detected within the cell substratum,but not in culture medium, indicating that it is a component ofECM (31). It interacts with ECM components through the TSand spacer domains (31). Since heparin displaces ADAM-TS1from ECM, it is possible that heparin or heparan-sulfate pro-teoglycans mediate the ECM interaction (31). The substrates ofADAM-TS1 are presently unknown. ADAM-TS1 is thought toplay a role in tumor cachexia and inflammation (8).

ADAM-TS2 cleaves native triple-helical, but not denaturedprocollagen I, and has an affinity for collagen XIV (33). Adistinct NH2-terminal processing event, mediated by an en-zyme other than ADAM-TS2, occurs in procollagen III (34). Inview of the high degree of similarity of the catalytic domains ofADAM-TS2 and ADAM-TS3, but not of their COOH-terminalhalves we speculate that these two members may be closelyrelated functionally. Perhaps ADAM-TS3 may also represent acollagen processing enzyme, with a different specificity, such asfor procollagen III. It is also possible that ADAM-TS3 may be asecond procollagen I and/or procollagen II amino propeptidase.The severity of the skin phenotype in EDS-VIIC relative to thatin bone and cartilage may suggest the existence of additionalprocollagen I/II amino-processing enzymes. The functions ofADAMTS5, ADAMTS6, and ADAMTS7 are unknown at thepresent time.

Acknowledgments—We thank Vincent Hascall and Katy Georgiadisfor critical reading of the manuscript, James Lang for photography, andDr. Hester Wain (Human Genome Nomenclature Editor) for assistancewith nomenclature.

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TABLE IMapping of ADAM-TS loci in human and mouse genomes

Human locus Mouse locus

ADAM-TS1 21q21-q22a 16 (C3–C5)b

16 (58 cM)c

ADAM-TS2 (PCINP) 5q23-q24a,d 11 (27 cM)c

ADAM-TS3 (KIAA0366) 4q21 (SHGC4–18; 9.67)d,e NAf

ADAM-TS4 (KIAA0688) 1q21-q23a,d 1 (92.5 cM)c

ADAM-TS5 (implantin) 21q21-q22 (SHGC-3322; 5.98)d,e 16 (58 cM)c

ADAM-TS6 5d NAADAM-TS7 15d NA

a Predicted human locus based on mouse locus mapping data and mouse-human chromosomal homology maps.b This locus was reported using in situ hybridization (35).c Mapping of mouse locus using the Seldin interspecific cross (consensus position in parentheses). The following gene orders were defined:

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d Mapping of human locus by Southern analysis of monochromosomal human-rodent somatic cell hybrid panel.e Mapping of human locus by PCR analysis of Stanford G3 human-hamster radiation hybrid panel (linked marker; lod score, in parentheses).f NA 5 not available.

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M G D V Q - R A A R S - - - - - - R G S L S A H M L - - - - - - - - - - L L L L 1 mADAM-TS1

M D P P A G A A R R L L C - - - - - - - - P A L L L - - - - - - - L L L L L P P 1 hADAM-TS2

S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L W L I A A 1 hADAM-TS3

M S Q T G S H P G R G L A G R W L W G A Q P C L L L P I V P L S W L V W L L L L 1 hADAM-TS4

M - - - - - - - - - - - - - R L E W A S L - - - - - - - - - - - - L L L L L L L 1 mADAM-TS5

M - - - - - - - - - - - - - - - - - - - - - - - - - - E I L W K T L T W I L S L 1 hADAM-TS6

M - - P G G P S P R S - - - - - - - - - - - - - - - P A P L L R P L L L L L C A 1 hADAM-TS7

A S I T M L L C A R G A H G R P T E E D E E L V L - - - P S L E R A P G H - - D 24 mADAM-TS1

P L L P P P P P P A N A R L A A A A D P P G G P L G H G A E R I L A V P V R T D 26 hADAM-TS2

A L V E V R T S A D G Q A G N E E M V Q I D L P I K R Y R E Y E L V T P V S T N 8 hADAM-TS3

L L A S L L P S A R - - L A S P L P R E E E I V F - - - P E K L N G S V L P G S 41 hADAM-TS4

S A S C L S L A A D S P A A A P A Q D K T R Q P Q - - - A A A A A A E P D Q P Q 16 mADAM-TS5

I M A S S E F H S D H R L S Y S S Q E E F L T Y L - - - E H Y Q L T I P I R V D 15 hADAM-TS6

L A P G A P G P A P G R A T E G R A A - - - - - - - - - - - L D I V H P V R V D 24 hADAM-TS7

S T T T R L - - R L D A F G Q Q - - - - - - - L H L K L Q P - - - - - - D - - - 59 mADAM-TS1

A Q G R L V S H V V S A A T S R A G V R A R R A A P V R T P S F P G G N E E E P 66 hADAM-TS2

L E G R Y L S H T L S A S H K K R S A R D V S S N P - - - - - - - - - - - - - - 48 hADAM-TS3

G T P A R L L C R L Q A F G E T - - - - - - - L L L E L E Q - - - - - - D - - - 76 hADAM-TS4

G E E T R E R G H L Q P L A G Q R - - R S G G L V H N I D Q L Y S G G G K - - - 53 mADAM-TS5

Q N G A F L S F T V K N D K H S R - - R R R S M D P I D P Q - - Q A V S K - - - 52 hADAM-TS6

A G G S F L S Y E L W P R A L R K - - R D V S V R R D A P A - - - - - - - - - - 53 hADAM-TS7

- - - S G F L A P G F T L Q T V - - - - - G R - - - S P G S E A Q - - - - - - - 81 mADAM-TS1

G S H L F Y N V T V F G R D L H L R L R P N A R L V A P G A T M E W Q G - - - - 106 hADAM-TS2

- E Q L F F N I T A F G K D F H L R L K P N T Q L V A P G A V V E W H E T S L V 74 hADAM-TS3

- - - S G V Q V E G L T V Q Y L - - - - - G Q - - - A P - - - - E - - - - - - - 100 hADAM-TS4

- - - V G Y L V Y A G G R R F L L D L E R D D - - - T V G A A G S - - - - - - - 88 mADAM-TS5

- - - L F F K L S A Y G K H F H L N L T L N T D F V S K H F T V E - - - - - - - 85 hADAM-TS6

- - - - F Y E L Q Y R G R E L R F N L T A N Q H L L A P G F V S E - - - - - - - 81 hADAM-TS7

- - - - - - - - - - - H L D P T G - D L A - - - - - - - H C F Y S G T V N G D P 103 mADAM-TS1

- - - - - - - - - - - E K G T T - - - - - R V E P L L G S C L Y V G D V A G L A 142 hADAM-TS2

P G N I T D P I N N H Q P G S A T Y R I R K T E P L Q T N C A Y V G D I V D I P 113 hADAM-TS3

- - - - - - - - - - - L L G - - G - A E P - - - - - - - G T Y L T G T I N G D P 118 hADAM-TS4

- - - - - - - - - - - I V T A G G - G L S A S S G H R G H C F Y R G T V D G S P 115 mADAM-TS5

- - - - - - - - - - - Y W G K D G P Q W K H - - D F L D N C H Y T G Y L Q D Q R 115 hADAM-TS6

- - - - - - - - - - - T R R R G G L G R A H I R A H T P A C H L L G E V Q D P E 110 hADAM-TS7

- - G S A A A L S L C E G - V R G A F Y L Q G E E F F I Q P A P G V A T E R L A 124 mADAM-TS1

E A S S V A - L S N C D G - L A G L I R M E E E E F F I E P L E - - - - - - - K 166 hADAM-TS2

- G T S V A - I S N C D G - L A G M I K S D N E E Y F I E P L E - - - - - - - R 153 hADAM-TS3

- - E S V A S L H W D G G A L L G V L Q Y R G A E L H L Q P - - - - - - - - L E 137 hADAM-TS4

- - R S L A V F D L C G G - L D G F F A V K H A R Y T L K P - - - L L R G S W A 143 mADAM-TS5

S T T K V A - L S N C V G - L H G V I A T E D E E Y F I E P - - - - L K N T T E 142 hADAM-TS6

L E G G L A A I S A C D G - L K G V F Q L S N E D Y F I E P - - - - L D S A P A 139 hADAM-TS7

P A V P E E E S S A R P Q F H I L R R R R - - - R - G S G - - - - G A K C G V M 161 mADAM-TS1

G L A A Q E A E Q G R V H V V Y R R P P T S - P P L G G P Q A L D - T G A S L D 197 hADAM-TS2

G K Q M E E - E K G R I H V V Y K R S A V E Q A P I D M S K D F H Y R E S D L E 183 hADAM-TS3

G G T P N S A G G - - P G A H I L R R K S - - - P - A S G Q - - - G P M C N V K 167 hADAM-TS4

E Y E R I Y G D G S S R I L H V Y N R E G F S F E - A L P P - - - R A S C E T P 177 mADAM-TS5

D S K H F S Y E N G H P H V I Y K K S A - - - L Q - Q R - H L Y D H S H C G V S 176 hADAM-TS6

R P G H - - - - - A Q P H V V Y K R Q A P E R L A - Q R G D S S A P S T C G V Q 174 hADAM-TS7

D D E T L P T S D S R P E S Q N T R N Q W P V R D P T P Q D A G K P S G P G S I 193 mADAM-TS1

S L D S L S R A L G V L E E H A N S S - - - - - - - - - - - - - - - - - - - - - 235 hADAM-TS2

G L D D L G T V Y G N I H Q Q L N E T - - - - - - - - - - - - - - - - - - - - - 222 hADAM-TS3

- - A P L G S P S P R P R - - - - - - - - - - - - - - - - - - - - - - - - - - - 198 hADAM-TS4

A S P S G P Q E S P S V H S R S R R R S A L A P Q L L D H S A F S P S G N A G P 213 mADAM-TS5

D F T R - - S G K P W W L N D T S T V S Y S L P I N N T H I - - - - - - - - - - 211 hADAM-TS6

V Y P E L E S R R E R W E Q R Q - - - - - - - Q W R R P R L - - - - - - - - - - 208 hADAM-TS7

- - - - - R K K R F V S S P R Y - V E T M L V A D Q S M A D F H G S - G L K H Y 233 mADAM-TS1

- - - R R R A R R H A A D D D Y N I E V L L G V D D S V V Q F H G K E H V Q K Y 254 hADAM-TS2

- - - M R R - R R H A G E N D Y N I E V L L G V D D S V V R F H G K E H V Q N Y 241 hADAM-TS3

- - - - - R A K R F A S L S R F - V E T L V V A D D K M A A F H G A - G L K R Y 209 hADAM-TS4

Q T W W R R R R R S I S R A R Q - V E L L L V A D S S M A R M Y G R - G L Q H Y 253 mADAM-TS5

- - - H H R Q K R S V S I E R F - V E T L V V A D K M M V G Y H G R K D I E H Y 239 hADAM-TS6

- - - R R L H Q R S V S K E K W - V E T L V V A D A K M V E Y H G Q P Q V E S Y 231 hADAM-TS7

L L T L F S V A A R F Y K H P S I R N S I S L V V V K I L V I Y E E Q K G P E V 266 mADAM-TS1

L L T L M N I V N E I Y H D E S L G A H I N V V L V R I I L L S Y G K S M S L I 291 hADAM-TS2

L L T L M N I V N E I Y H D E S L G V H I N V V L V R M I M L G Y A K S I S L I 277 hADAM-TS3

L L T V M A A A A K A F K H P S I R N P V S L V V T R L V I L G S G E E G P Q V 242 hADAM-TS4

L L T L A S I A N R L Y S H A S I E N H I R L A V V K V V V L T D K D T S L E V 291 mADAM-TS5

I L S V M N I V A K L Y R D S S L G N V V N I I V A R L I V L T E D Q P N L E I 275 hADAM-TS6

V L T I M N M V A G L F H D P S I G N P I H I T I V R L V L L E D E E E D L K I 267 hADAM-TS7

T S - N A A L T L R N F C S W Q K - - - - - - - Q H N S P S D R D P E H Y D T A 306 mADAM-TS1

E I G N P S Q S L E N V C R W A Y - - - - - - - L Q Q K P D T G H D E Y H D H A 331 hADAM-TS2

E R G N P S R S L E N V C R W A S - - - - - - - Q Q Q R S D L N H S E H H D H A 317 hADAM-TS3

G P - S A A Q T L R S F C A W Q R - - - - - - - G L N T P E D S D P D H F D T A 282 hADAM-TS4

S K - N A A T T L K N F C K W Q H - - - - - - - Q H N Q L G D D H E E H Y D A A 331 mADAM-TS5

N H - H A D K S L D S F C K W Q K S I L S H Q S D G N T I P E N G I A H H D N A 315 hADAM-TS6

T H - H A D N T L K S F C K W Q K S I - N M K G D A H P L - - - - - - H H D T A 307 hADAM-TS7

I L F T R Q D L C G - - S H T C D T L G M A D V G T V C D P S R S C S V I E D D 338 mADAM-TS1

I F L T R Q D F - - - - - G P S G M Q G Y A P V T G M C H P V R S C T L N H E D 364 hADAM-TS2

I F L T R Q D F - - - - - G P A G M Q G Y A P V T G M C H P V R S C T L N H E D 350 hADAM-TS3

I L F T R Q D L C G - - V S T C D T L G M A D V G T V C D P A R S C A I V E D D 314 hADAM-TS4

I L F T R E D L C G - - H H S C D T L G M A D V G T I C S P E R S C A V I E D D 363 mADAM-TS5

V L I T R Y D I C T Y K N K P C G T L G L A S V A G M C E P E R S C S I N E D I 354 hADAM-TS6

I L L T R K D L C A A M N R P C E T L G L S H V A G M C Q P H R S C S I N E D T 339 hADAM-TS7

G L Q A A F T T A H E L G H V F N M P H D D A - K H C A S L N G - V S G D S H L 376 mADAM-TS1

G F S S A F V V A H E T G H V L G M E H D G Q G N R C - - - - G D E V R L G S I 399 hADAM-TS2

G F S S A F V V A H E T G H V L G M E H D G Q G N R C - - - - G D E T A M G S V 385 hADAM-TS3

G L Q S A F T A A H E L G H V F N M L H D N S - K P C I S L N G P L S T S R H V 352 hADAM-TS4

G L H A A F T V A H E I G H L L G L S H D D S - K F C E E N F G T - T E D K R L 401 mADAM-TS5

G L G S A F T I A H E I V H N F G M N H D G I G N S C - - - - - - - G R K - - V 394 hADAM-TS6

G L P L A F T V A H E L G H S F G I Q H D G S G N D C E - - - - P V G K R P F I 379 hADAM-TS7

M A S M L S S L D H S Q P W S P C S A Y M V T S F L D N G H G E C L M D K P Q N 414 mADAM-TS1

M A P L V Q A A F H R F H W S R C S Q Q E L S R Y L H S - - Y D C L L D D P F A 435 hADAM-TS2

M A P L V Q A A F H R Y H W S R C S G Q E L K R Y I H S - - Y D C L L D D P F D 421 hADAM-TS3

M A P V M A H V D P E E P W S P C S A R F I T D F L D N G Y G H C L L D K P E A 391 hADAM-TS4

M S S I L T S I D A S K P W S K C T S A T I T E F L D D G H G N C L L D L P R K 439 mADAM-TS5

M K Q Q N - Y G S S H Y - - - - C - - E Y Q S F F L - - - - - V C L - - - - - - 425 hADAM-TS6

M S P Q L L Y D A A P L T W S R C S R Q Y I T R F L D R G W G L C L D D P P A K 415 hADAM-TS7

P I K - L P S D L P G T L Y D A N R Q C Q F T F G E E S K H C P D A A S - - T C 454 mADAM-TS1

H D W P A L P Q L P G L H Y S M N E Q C R F D F G L G Y M M C T A F R T F D P C 473 hADAM-TS2

H D W P K L P E L P G I N Y S M D E Q C R F D F G V G Y K M C T A F R T F D P C 459 hADAM-TS3

P L H - L P V T F P G K D Y D A D R Q C Q L T F G P D S R H C P Q L P P - - P C 431 hADAM-TS4

Q I L - G P E E L P G Q T Y D A T Q Q C N L T F G P E Y S V C P G M D - - - V C 479 mADAM-TS5

- - - - - - - - - - - - - - - - - - Q S R L H H Q L F R - - - - - - - E - - V C 447 hADAM-TS6

D I I D F P S V P P G V L Y D V S H Q C R L Q Y G A Y S A F C E D M D N - - V C 455 hADAM-TS7

T T L W C T G T S G G L L V C Q T K H F P W A D G T S C G E G K - - - - W C V S 491 mADAM-TS1

K Q L W C S - H P D N P Y F C K T K K G P P L D G T M C A P G K - - - - H C F K 513 hADAM-TS2

K Q L W C S - H P D N P Y F C K T K K G P P L D G T E C A A G K - - - - W C Y K 499 hADAM-TS3

A A L W C S G H L N G H A M C Q T K H S P W A D G T P C G P A Q - - - - A C M G 468 hADAM-TS4

A R L W C A V V R Q G Q M V C L T K K L P A V E G T P C G K G R - - - - V C L Q 515 mADAM-TS5

R E L W C L S K S N - - - R C V T N S I P A A E G T L C Q T G N I E K G W C Y Q 460 hADAM-TS6

H T L W C - S V G T - - - T C H S K L D A A V D G T R C G - - - - E N K W C L S 493 hADAM-TS7

G K C V N K T D M K H F A T P V H G S W G P W G P W G D C S R T C G G G V Q Y T 527 mADAM-TS1

G H C I W L T P D I - - - L K R D G S W G A W S P F G S C S R T C G T G V K F R 548 hADAM-TS2

G H C M W K N A N Q - - - Q K Q D G N W G S W T K F G S C S R T C G T G V R F R 534 hADAM-TS3

G R C L H M D Q L Q D F N I P Q A G G W G P W G P W G D C S R T C G G G V Q F S 504 hADAM-TS4

G K C V D K T K K K Y Y S T S S H G N W G S W G P W G Q C S R S C G G G V Q F A 551 mADAM-TS5

G D C V P F G - - - T W P Q S I D G G W G P W S L W G E C S R T C G G G V S S S 497 hADAM-TS6

G E C V P V G - - - F R P E A V D G G W S G W S A W S I C S R S C G M G V Q S A 525 hADAM-TS7

M R E C D N P V P K N G G K Y C E G K R V R Y R S C N I E D C P D N N G K T F R 567 mADAM-TS1

T R Q C D N P H P A N G G R T C S G L A Y D F Q L C S R Q D C P - D S L A D F R 585 hADAM-TS2

T R Q C N N P M P I N G G Q D C P G V N F E Y Q L C N T E E C Q - K H F E D F R 571 hADAM-TS3

S R D C T R P V P R N G G K Y C E G R R T R F R S C N T E D C P T G S A L T F R 544 hADAM-TS4

Y R H C N N P A P R N S G R Y C T G K K A I Y R S C S V T P C P P - N G K S F R 591 mADAM-TS5

L R H C D S P A P S G G G K Y C L G E R K R Y R S C N T D P C P L G S - R D F R 534 hADAM-TS6

E R Q C T Q P T P K Y K G R Y C V G E R K R F R L C N L Q A C P A G R - P S F R 562 hADAM-TS7

E E Q C E A H N E F S K A S F G N E P T - V E W T P K Y A G V S P K D R C K L T 607 mADAM-TS1

E E Q C R Q W D L Y - - - - F E H G D A Q H H W L P - H E H R D A K E R C H L Y 624 hADAM-TS2

A Q Q C Q Q R N S H - - - - F E Y Q N T K H H W L P - Y E H P D P K K R C H L Y 610 hADAM-TS3

E E Q C A A Y N H - R T D L F K S F P G P M D W V P R Y T G V A P Q D Q C K L T 584 hADAM-TS4

H E Q C E A K N G Y Q S D A - K G V K T F V E W V P K Y A G V L P A D V C K L T 630 mADAM-TS5

E K Q C A - - - D F D N M P F R G - - K Y Y N W K P - Y T G G G V K - P C A L N 573 hADAM-TS6

H V Q C S - - - H F D A M L Y K G - - Q L H T W V P - - V V N D V N - P C E L H 601 hADAM-TS7

C E A K G I G Y F F V L Q P K - V V D G T P C S P - - D S T S V C V Q G Q C V K 646 mADAM-TS1

C E S R E T G E - V V S M K R M V H D G T R C S - Y K D A F S L C V R G D C R K 659 hADAM-TS2

C Q S K E T G D - V A Y M K Q L V H D G T H C S - Y K D P Y S I C V R G E C V K 645 hADAM-TS3

C Q A R A L G Y Y Y V L E P R - V V D G T P C S P - - D S S S V C V Q G R C I H 623 hADAM-TS4

C R A K G T G Y Y V V F S P K - V T D G T E C R P - - Y S N S V C V R G R C V R 669 mADAM-TS5

C L A E G Y N F Y T E R A P - A V I D G T Q C N A - - D S L D I C I N G E C K H 606 hADAM-TS6

C R P A N - E Y F A K K L R D A V V D G T P C Y Q V R A S R D L C I N G I C K N 633 hADAM-TS7

A G C D R I I D S K K K F D K C G V C G G N G S T C K K M S G I V T S T - - R P 683 mADAM-TS1

V G C D G V I G S S K Q E D K C G V C G G D N S H C K V V K G T F T R S P K K H 697 hADAM-TS2

V G C D K E I G S N K V E D K C G V C G G D N S H C R T V K G T F T R T P R K L 683 hADAM-TS3

A G C D R I I G S K K K F D K C M V C G G D G S G C S K Q S G S F R K F - - R Y 660 hADAM-TS4

T G C D G I I G S K L Q Y D K C G V C G G D N S S C T K I I G T F N K K - - S K 706 mADAM-TS5

V G C D N I L G S D A R E D R C R V C G G G G S T C D A I E G F F N D S L P R G 643 hADAM-TS6

V G C D F E I D S G A M E D R C G V C H G N G S T C H T V S G T F E E A E G L G 672 hADAM-TS7

G Y H D I V T I P A G A T N I E V K H R N Q R G S R N N G S F L A I R A A D - G 721 mADAM-TS1

G Y I K M F E I P A G A R H L L I Q E V D A T S H H - - - - - L A V K N L E T G 737 hADAM-TS2

G Y L K M F D I P P G A R H V L I Q E D E A S P H I - - - - - L A I K N Q A T G 723 hADAM-TS3

G Y N N V V T I P A G A T H I L V R Q Q G N P G H R - - S I Y L A L K L P D - G 698 hADAM-TS4

G Y T D V V R I P E G A T H I K V R Q F K A K D Q T R F P A Y L A L K K K T - G 744 mADAM-TS5

G Y M E V V Q I P R G S V H I E V R E V A M S K N - - - - - Y I A L K S E G - D 683 hADAM-TS6

- Y V D V G L I P A G A R E I R I Q E V A E A A N - - - - - F L A L R S E D P E 712 hADAM-TS7

T Y I L N G N F T L S T L E Q D L T Y K G T V - L R Y S - - - - - - - - - - - - 760 mADAM-TS1

K F I L N E E N D V D A S S K T F I A M G - V E W E Y R D E D G R E T L Q T M G 772 hADAM-TS2

H Y I L N G K G E - E A K S R T F I D L G - V E W D Y N I E D D I E S L H T D G 758 hADAM-TS3

S Y A L N G E Y T L M P S P T D V V L P G A V S L R Y S - - - - - - - - - - - - 735 hADAM-TS4

E Y L I N G K Y M I S T S E T I I D I N G T V - M N Y S - - - - - - - - - - - - 783 mADAM-TS5

D Y Y I N G A W T I - D W P R K F D V A G T - A F H Y K R - - - - - - - - - - - 717 hADAM-TS6

K Y F L N G G W T I - Q W N G D Y Q V A G T - T F T Y A R R G N W E N L T S P G 746 hADAM-TS7

- - - - - - - - - - - - - - - - - - - - - G S S A A L E R I R S - - - - - - - - 787 mADAM-TS1

P L H G T I T V L V I P - - - V G - D T R V S L T Y K Y M I H E D S L - N - V D 811 hADAM-TS2

P L H D P V I V L I I P - - - Q E N D T R S S L T Y K Y I I H E D S V P T - I N 796 hADAM-TS3

- - - - - - - - - - - - - - - - - - - - - G A T A A S E T L S G - - - - - - - - 763 hADAM-TS4

- - - - - - - - - - - - - - - - - - - - - G W S H R D D F L H G - - - - - - - - 810 mADAM-TS5

P T D E P E S L E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 744 hADAM-TS6

P T K E P V W I Q V P A S R G P G G G S R G G V P R P S T L H G R S R P G G V S 784 hADAM-TS7

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 798 mADAM-TS1

D N N V L E E D S V V Y E W A L K K W S P C S K P C G G G S Q F T K Y G C R R R 845 hADAM-TS2

S N N V I Q E E L D T F E W A L K S W S Q V S K P C G G G F Q Y T K Y G C R R K 832 hADAM-TS3

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 774 hADAM-TS4

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 821 mADAM-TS5

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 753 hADAM-TS6

P G S V T E P G S E P - - - - - - - - G P P A A A S T S V S P S L K - - - W P N 824 hADAM-TS7

- - - - - - - - - - - - - - - - F S P L K - - - - - - - - - E P L T I - - - - - 798 mADAM-TS1

L D H K M V H R G F C A A L S K P K A I R R A C N P Q E C S Q P V W V T G E W E 885 hADAM-TS2

S D N K M V H R S F C E A N K K P K P I R R M C N I Q E C T H P L W V A E E W E 872 hADAM-TS3

- - - - - - - - - - - - - - - - H G P L A - - - - - - - - - Q P L T L - - - - - 774 hADAM-TS4

- - - - - - - - - - - - - - M G Y S A T K - - - - - - - - - E I L I V - - - - - 821 mADAM-TS5

- - - - - - - - - - - - - - - A L G P T S - - - - - - - - - E N L I V M - - - - 753 hADAM-TS6

L V A A V H R G G W G Q A P L G L G G W R - - - - - - - - - R H L V L M G P R L 853 hADAM-TS7

- - - Q V L M V G H A L R P K I K F T Y F M K K K T E - - - - - - - - - - - - S 808 mADAM-TS1

P C S Q T C G R T G M Q V R S V R C I Q P L H D N T T R S V H A K H C N D A R P 925 hADAM-TS2

H C T K T C G S S G Y Q L R T V R C L Q P L L D G T N R S V H S K Y C M G D R P 912 hADAM-TS3

- - - Q V L V A G N P Q D T R L R Y S F F V P R P T P - - - - - - - - - - - - S 784 hADAM-TS4

- - - Q I L A T D P T K A L G V R Y S F F V P K K T T Q K V N S V I S H G S N K 833 mADAM-TS5

- - - - V L L Q E Q - - N L G I R Y K F N V P - - - - - - - - - I T R T G S G D 765 hADAM-TS6

P - T Q L L F Q E S - - N P G V H Y E Y T - - - - - - - - - - - I H R E A G G H 884 hADAM-TS7

F N A I P T - - - - - F S E W V I E E W G E C S K T C G S G - - W Q R R V V Q C 833 mADAM-TS1

E S R R A C S R E L C P G R W R A G P W S Q C S V T C G N G T - - Q E R P V P C 965 hADAM-TS2

E S R R P C N R V P C P A Q W K T G P W S E C S V T C G E G T - - E V R Q V L C 952 hADAM-TS3

- T P R P T - - - - - P Q D W L - - - - - - - - - - - - - - - - - H R R - - - - 809 hADAM-TS4

V G P H S T - - - - - Q L Q W V T G P W L A C S R T C D T G - - W H T R T V Q C 870 mADAM-TS5

N E V G - - - - - - - - F T W N H Q P W S E C S A T C A G G K M P T R Q P - - - 790 hADAM-TS6

D E V P P P - - - - - V F S W H Y G P W T K C T V T C G R G E K W G R H S P T C 910 hADAM-TS7

R D I N G H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P A 866 mADAM-TS1

R T A D D S F G I C Q E E R P E T A R T C R L G P C P R N I S D P S K K S Y V V 1003 hADAM-TS2

R A G D H - - - - C D G E K P E S V R A C Q L P P C - - - - - - - - - - - - - - 990 hADAM-TS3

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 822 hADAM-TS4

Q D G N R K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L A 903 mADAM-TS5

- - - - T Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R A 819 hADAM-TS6

R G L V S G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q G 945 hADAM-TS7

S - - - - - - E C A K E V K P A S T R P C A D L P - - - - - - - - - - - - - C P 874 mADAM-TS1

Q W L S R P D P D S P I R K I S S K G H C Q G D K S I - F C R M E V L S R Y C S 1043 hADAM-TS2

- - - - - - - - - - - - - - - - N D E P C L G D K S I - F C Q M E V L A R Y C S 1012 hADAM-TS3

- - - - - - - - - A Q I L E I L R R R P - - - W A - - - - - - - - - - - - - G R 822 hADAM-TS4

K - - - - - - G C L L S Q R P S A F K Q C L L K K - - - - - - - - - - - - - C 911 mADAM-TS5

R W - - - - - - - - R T K H I L S Y A L C L L K K L I G N I S C R F A S S - C N 823 hADAM-TS6

H W L - - - Q L P A H C W A T T G L E V C F S E P Q F S I C E M R L A I A L C P 953 hADAM-TS7

H 895 mADAM-TS1

I P G Y N K L S C K S C N - - - - - - - - - - L Y N N L T N V E G R I E P P P G 1082 hADAM-TS2

I P G Y N K L C C E S C S K R S S T L P P P Y L L E A A E T H D D V I S N P S D 1035 hADAM-TS3

K 837 hADAM-TS4

930 mADAM-TS5

L A K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 854 hADAM-TS6

R P A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 990 hADAM-TS7

895 mADAM-TS1

K H N D I D V - - - - - - F M P T L P V P T V A M E V R P S P S T P L E - V P L 1112 hADAM-TS2

L P R S L V M P T S L V P Y H S E T P A K K M S L S S I S S V G G P N A Y A A F 1075 hADAM-TS3

837 hADAM-TS4

930 mADAM-TS5

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 857 hADAM-TS6

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 993 hADAM-TS7

895 mADAM-TS1

N A S S T N A T E D H P E T N A V D E P Y K I H G L - - - - - - - - E D E V Q P 1145 hADAM-TS2

R P N S K P D G A N L R Q R S A Q Q A G S K T V R L V T V P S S P P T K R V H L 1115 hADAM-TS3

837 hADAM-TS4

930 mADAM-TS5

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 857 hADAM-TS6

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 993 hADAM-TS7

895 mADAM-TS1

P N L I P R R P S P Y - - - - - - - - - - E K T R N Q R I Q E L I D E M R K K E 1177 hADAM-TS2

S S A S Q M A A A S F F A A S D S I G A S S Q A R T S K K D G K I I D N R R P T 1155 hADAM-TS3

837 hADAM-TS4

930 mADAM-TS5

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 857 hADAM-TS6

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 993 hADAM-TS7

895 mADAM-TS1

M L G K F 1207 hADAM-TS2

R S S T L E R 1195 hADAM-TS3

837 hADAM-TS4

930 mADAM-TS5

- - E T L L 857 hADAM-TS6

- - G R V H G 993 hADAM-TS7

Tiina L. Hurskainen, Satoshi Hirohata, Michael F. Seldin and Suneel S. ApteTHE ADAM-TS FAMILY

Metalloproteases: GENERAL FEATURES AND GENOMIC DISTRIBUTION OF ADAM-TS5, ADAM-TS6, and ADAM-TS7, Novel Members of a New Family of Zinc

doi: 10.1074/jbc.274.36.255551999, 274:25555-25563.J. Biol. Chem. 

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