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* This work was supported by grants from Comisión Interministerial de Ciencia y Tecnologı́a-Spain (SAF00-0217); and European Union (QLG1-CT-2000-01131). The Instituto Universitario de Oncologı́a is supported by Obra Social Cajastur-Asturias.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s)AJ512753, AJ515153, and AJ515154 ‡ Recipients of research fellowships or contracts from Ministerio de Ciencia y Tecnologı́a, Spain.
We have cloned a mouse brain cDNA encoding a new protein of the ADAMTS family (adisintegrinand metalloproteinase domain, withthrombospondin type-1 repeats), which has been called ADAMTS-20. This protein shows a domain organization similar to that described for other ADAMTSs including signal sequence, propeptide, metalloproteinase domain, disintegrin domain, central TS-1 motif, cysteine-rich region, and C-terminal TS module. However, this last module is more complex than that of other ADAMTSs, being composed of a total of 14 repeats. The structural complexity of ADAMTS-20 is further increased by the presence of an additional domain 200 residues long and located immediately adjacent to the TS module. This domain has been tentatively called GON domain and can also be recognized in some ADAMTSs such as gon-1 from Caenorhabditis elegans and human and mouse ADAMTS-9. The presence of this domain is a hallmark of a novel subfamily of structurally and evolutionarily related ADAMTSs, called GON-ADAMTSs. Expression analysis demonstrated thatADAMTS-20 transcripts can be detected at low levels in several human and mouse tissues, especially in testis. This gene is also overexpressed in some human malignant tumors, including brain, colon, and breast carcinomas. Western blot analysis using polyclonal antibodies raised against recombinant ADAMTS-20 produced inEscherichia coli showed the presence of a 70-kDa band in mouse brain and testis extracts. This recombinant ADAMTS-20 hydrolyzed a synthetic peptide used for assaying matrix metalloproteinases. These data suggest that this novel enzyme may play a role in the tissue remodeling process occurring in both normal and pathological conditions.
a disintegrin and metalloproteinase domain, with thrombospondin type-1 modules
l-dinitrophenyl-diamino propionic acid
rapid amplification of cDNA ends
The ADAMTSs1(adisintegrin andmetalloproteinase domain, withthrombospondin type-1 modules) are a growing family of zinc-dependent metalloproteinases that play important roles in a variety of normal and pathological conditions (
). These enzymes show a complex domain organization including signal sequence, propeptide, metalloproteinase domain, disintegrin-like domain, central TS-1 motif, cysteine-rich region, and a variable number of TS-like repeats at the C-terminal region. To date, 18 genetically different ADAMTSs have been identified in human tissues (
). Structural characterization of these enzymes has demonstrated that ADAMTSs are distinct from ADAMs, a related family of metalloproteinases that exhibit a similar domain organization. However, both proteinase families differ in some important aspects. Thus, ADAMs lack TS-1 repeats but contain a transmembrane domain that mediates their anchorage to the plasma membrane and a cytoplasmic tail that can participate in signal transduction events (
). By contrast, ADAMTSs display an organization of TS repeats of variable complexity and are secreted proteins devoid of transmembrane and cytoplasmic domains in their C-terminal region. The complexity of studies on ADAMTSs has further increased after the finding of a family of proteins that resemble ADAMTSs and that have been called ADAMTSLs (ADAMTS-like) or punctins (
). These proteins lack the metalloproteinase and disintegrin-like domains of ADAMTSs but contain all the remaining ADAMTS domains, including several TS-1 repeats. ADAMTSLs have been proposed to participate in the endogenous regulation of ADAMTS activity (
). Disruption of the mouse adamts-1 gene results in decreased growth, renal abnormalities, partial obstruction in the ureteropelvic junction, and alterations in adipose tissue and adrenal medullary architecture (
), a critical aspect in the invasive capacity of these tumors. ADAMTS-13 is a von Willebrand factor-cleaving protease, and mutations in the gene encoding this enzyme cause thrombotic thrombocytopenic purpura, a life-threatening disease mainly characterized by hemolytic anemia, microvascular thrombosis, low platelet count, renal failure, and neurological dysfunctions (
), and considering the growing relevance of ADAMTSs in normal and pathological processes, we have examined the possibility that additional yet uncharacterized family members could be present in the genome of these organisms. In this work, we report the identification of a novel ADAMTS that has been called ADAMTS-20. We also report the structural characterization of both human and mouse enzymes with the finding of a novel domain present in ADAMTS-20, as well as in a long isoform of ADAMTS-9, and in ADAMTSs from other organisms includingCaenorhabditis elegans, Drosophila melanogaster,Anopheles gambiae, and Fugu rubripes. Finally, we report the tissue distribution of ADAMTS-20 and perform a preliminary analysis of its enzymatic activity.
In this work, we describe the identification and characterization of a novel member of the ADAMTS family of secreted metalloproteinases with disintegrin and thrombospondin domains. The approach to identify ADAMTS-20 involved a search of human and mouse genome databases followed by a combination of RT-PCR amplifications of cDNA libraries and successive 5′- and 3′-RACE experiments to extend the originally amplified cDNA fragments. This strategy allowed us to isolate a full-length cDNA for mouse ADAMTS-20 and to deduce the complete sequence of its human orthologue. Both proteins exhibit an identical domain organization that is similar to that of previously described ADAMTSs. Thus, they harbor signal sequence, propeptide, metalloproteinase-, disintegrin-, central TS-, and cysteine rich-domains and a complex C-terminal TS-like module. Likewise, mouse and human ADAMTS-20 show several conserved residues and motifs characteristic of each of these domains, including a proprotein convertase activation sequence at the end of the propeptide, a zinc-binding site with the reprolysin signature in the catalytic domain, and conserved patterns of cysteine arrangements in the disintegrin and cysteine-rich regions. However, ADAMTS-20 also exhibits some characteristic features that allow us to distinguish this enzyme from other family members as well as to define a novel subfamily of ADAMTSs. Thus, it contains an unusually complex organization of TS repeats at the C-terminal module, being composed of a total of 14 repeats, the highest number among all equivalent modules present in vertebrate ADAMTSs identified to date. Interestingly, we have previously reported that although ADAMTS-9 has been described to possess three TS repeats at the C-terminal module (
). The second distinctive feature of ADAMTS-20 is the presence of an additional domain in its C-terminal region, immediately adjacent to the TS-terminal module. This structural motif, which we have designated as GON domain, is characterized by the presence of several conserved cysteine residues and can be recognized in gon-1 from C. elegans, in the large isoforms of human and mouse ADAMTS-9, and in proteins predicted from sequence analysis of the genomes of D. melanogaster,A. gambiae, and F. rubripes. On the basis of these structural features, we propose that all these proteins define a novel subset of ADAMTSs that could be known as GON-ADAMTSs.
The dendrogram shown in Fig. 5 confirms the structural relationships among these ADAMTS family members and allows the classification of the family of human ADAMTSs into seven subfamilies of closely related members. The first of these subfamilies should be that of hyalectanases (
). ADAMTS-9 and ADAMTS-20 should conform to the subfamily of GON-ADAMTSs. ADAMTS-13, with unique properties among all described ADAMTSs, should be the only representative of von Willebrand factor-cleaving proteases (
). Finally, ADAMTS-6, -7, -10, and -12; ADAMTS-16 and -18; and ADAMTS-17 and -19 form groups of structurally related family members that might also be indicative of putative common enzymatic and functional properties. Further studies aimed at identifying the substrates targeted by the proteases belonging to these three last ADAMTS subfamilies will be necessary to confirm that the structural similarities here defined are also supported by functional relationships between them. In this regard, the structural similarities between vertebrate and invertebrate members of the GON-ADAMTS subfamily of ADAMTSs may also allow us to speculate about putative functional roles for ADAMTS-20. To date, no physiological role has been ascribed to ADAMTS-9 nor to those related proteins identified in Drosophila, Anopheles, orFugu; however, gon-1 is an active metalloproteinase essential for controlling morphogenesis in C. elegans (
). Thus, mutagenesis studies have suggested that this ADAMTS permits and directs expansion of the gonad by remodeling the extracellular matrix and basement membrane. Interestingly, the region containing TS-1 repeats is critical for gon-1 activity because some mutations inactivating this gene are located in regions encoding these repeats (
). Since it has been suggested that similar activities may control organ morphogenesis throughout the animal kingdom, it is tempting to speculate that ADAMTS-20 may play similar roles in vertebrates to those played by gon-1 in nematodes.
As a previous step to evaluate this hypothetical function of ADAMTS-20 as an extracellular matrix remodeling enzyme, we have performed a preliminary analysis of the catalytic properties of a recombinant form of this protease produced in bacterial cells. Interestingly, ADAMTS-20 is able to hydrolyze a synthetic peptide used for analysis of vertebrate MMPs, and this hydrolyzing activity is abolished by inhibitors of metalloproteinases, demonstrating that the identified protein is an active member of this class of proteolytic enzymes. To our knowledge, this is the first report showing that a member of the ADAMTS family is able to degrade peptides such as QF-35 commonly used to assay the activity of MMPs, thus confirming the connections between both proteolytic systems (reviewed in Ref.
). Nevertheless, it is remarkable that kinetic analysis has revealed that the catalytic efficiency of ADAMTS-20 against QF-35 is much lower than that of most MMPs with the ability to hydrolyze this peptide, suggesting the occurrence of important differences in the active site of both types of metalloproteinases. Consequently, studies of substrate specificity and resolution of the three-dimensional structure of the ADAMTS-20 catalytic domain will be required to clarify the similarities and differences of this novel enzyme with members of the MMP family.
Previous studies have shown that ADAMTSs may be of relevance in tumor processes (
). Therefore, in this work, we have also explored the potential significance of ADAMTS-20 in human cancer through analysis of its expression pattern in a panel of malignant tumors. These studies have shown that ADAMTS-20 is overexpressed in several brain, colon, and breast carcinomas when compared with the paired adjacent normal tissues, suggesting that this protease could play some role in the progression of these tumors. Also, in this regard, it is interesting that the region containing the human ADAMTS-20 gene (12q12) has been found to be a recurrent site of translocations and other alterations in human malignancies (
), whose responsible gene remains to be characterized. It will be interesting to examine the possibility that ADAMTS-20 could be a target of some of these genetic abnormalities, as already demonstrated for other ADAMTS family members linked to relevant genetic diseases (
). To this purpose, as well as to clarify the role of ADAMTS-20 in physiological processes, it will be very helpful to create a mouse deficient in this protease. This work is currently in progress in our laboratory and has been facilitated by the availability of cDNA and genomic clones for mouse ADAMTS-20 generated in the present study.
In conclusion, we have cloned and characterized ADAMTS-20, a protease that, according to our exhaustive analysis of both mouse and human genomes, represents the only member of the ADAMTS family that remained to be identified in these organisms. ADAMTS-20 is an active protease with a profile of activity and sensitivity to inhibitors characteristic of metalloproteinases. However, it also exhibits a series of structural peculiarities including the presence of the newly identified GON domain, which has allowed us to define the occurrence of a novel subfamily of ADAMTSs: the GON-ADAMTSs. This structural analysis, together with that performed with other family members, has also prompted us to propose that ADAMTSs can be organized into seven different subfamilies. Hopefully, this classification may facilitate future studies aimed at exploring the multiple roles that this large and complex family of proteases may play in processes involving cell migration, tissue remodeling, and changes in cell adhesion.
We thank Drs. J. P. Freije, X. S. Puente, and G. Velasco for helpful comments and support and C. Garabaya for excellent technical assistance.