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Collagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues. A critical step in collagen fibril formation is the proteolytic removal of N- and C-terminal propeptides from procollagens by metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein 1)/Tolloid-like families, respectively. BMP1 also cleaves and activates the lysyl oxidase (LOX) precursor, the enzyme catalyzing the initial step in the formation of covalent collagen cross-links, an essential process for fibril stabilization. In this study, using murine skin fibroblasts and HEK293 cells, along with immunoprecipitation, LOX enzymatic activity, solid-phase binding assays, and proteomics analyses, we report that the LOX precursor is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 between Asp-218 and Tyr-219, 50 amino acids downstream of the BMP1 cleavage site. We noted that the LOX sequence between the BMP1- and ADAMTS-processing sites contains several conserved tyrosine residues, of which some are post-translationally modified by tyrosine O-sulfation and contribute to binding to collagen. Taken together, these findings unravel an additional level of regulation in the formation of collagen fibrils. They point to a mechanism that controls the binding of LOX to collagen and is based on differential BMP1- and ADAMTS2/14-mediated cleavage of a tyrosine-sulfated domain.

Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation. This article has an associated First Person interview with the first author of the paper., Summary: Combined application of polarisation-resolved second-harmonic generation microscopy and Raman spectroscopy identified unique, sexually dimorphic extracellular matrix signatures linking pathological matrix disorganisation with composition.

ADAMTS proteins are a superfamily of 26 secreted molecules comprising two related, but distinct families. ADAMTS proteases are zinc metalloendopeptidases, most of whose substrates are extracellular matrix (ECM) components, whereas ADAMTS-like proteins lack a metalloprotease domain, reside in the ECM and have regulatory roles vis-à-vis ECM assembly and/or ADAMTS activity. Evolutionary conservation and expansion of ADAMTS proteins in mammals is suggestive of crucial embryologic or physiological roles in humans. Indeed, Mendelian disorders or birth defects resulting from naturally occurring ADAMTS2, ADAMTS3, ADAMTS10, ADAMTS13, ADAMTS17, ADAMTS20, ADAMTSL2 and ADAMTSL4 mutations as well as numerous phenotypes identified in genetically engineered mice have revealed ADAMTS participation in major biological pathways. Important roles have been identified in a few acquired conditions. ADAMTS5 is unequivocally implicated in pathogenesis of osteoarthritis via degradation of aggrecan, a major structural proteoglycan in cartilage. ADAMTS7 is strongly associated with coronary artery disease and promotes atherosclerosis. Autoantibodies to ADAMTS13 lead to a platelet coagulopathy, thrombotic thrombocytopenic purpura, which is similar to that resulting from ADAMTS13 mutations. ADAMTS proteins have numerous potential connections to other human disorders that were identified by genome-wide association studies. Here, we review inherited and acquired human disorders in which ADAMTS proteins participate, and discuss progress and prospects in therapeutics.

Since its first description, ADAMTS14 has been considered as an aminoprocollagen peptidase based on its high similarity with ADAMTS3 and ADAMTS2. As its importance for procollagen processing was never experimentally demonstrated in vivo , we generated Adamts14 -deficient mice. They are healthy, fertile and display normal aminoprocollagen processing. They were further crossed with Adamts2 -deficient mice to evaluate potential functional redundancies between these two highly related enzymes. Initial characterizations made on young Adamts2-Adamts14 -deficient animals showed the same phenotype as that of Adamts2 -deficient mice, with no further reduction of procollagen processing and no significant aggravation of the structural alterations of collagen fibrils. However, when evaluated at older age, Adamts2-Adamts14 -deficient mice surprisingly displayed epidermal lesions, appearing in 2 month-old males and later in some females, and then worsening rapidly. Immunohistological evaluations of skin sections around the lesions revealed thickening of the epidermis, hypercellularity in the dermis and extensive infiltration by immune cells. Additional investigations, performed on young mice before the formation of the initial lesions, revealed that the primary cause of the phenotype was not related to alterations of the epidermal barrier but was rather the result of an abnormal activation and differentiation of T lymphocytes towards a Th1 profile. However, the primary molecular defect probably does not reside in the immune system itself since irradiated Adamts2-Adamts14 -deficient mice grafted with WT immune cells still developed lesions. While originally created to better characterize the common and specific functions of ADAMTS2 and ADAMTS14 in extracellular matrix and connective tissues homeostasis, the Adamts2-Adamts14 -deficient mice revealed an unexpected but significant role of ADAMTS in the regulation of immune system, possibly through a cross-talk involving mesenchymal cells and the TGFβ pathways.

Summary An eight‐week old Doberman Pinscher was diagnosed with Ehlers Danlos syndrome based on the dog's hyper‐mobile carpal, tarsal and stifle joints and abnormal skin. The skin was loose and hyper‐elastic with several wounds and large atrophic scars. The dog was euthanized after a severe degloving injury from minimal trauma. A whole‐genome sequence, generated with DNA from the dog's blood, contained a rare, homozygous C‐to‐T transition at position 2408978 on chromosome 11. This transition is predicted to alter the ADAMTS2 transcript (ADAMTS2:c.769C>T) and encode a nonsense mutation (p.Arg257Ter). Biallelic ADAMTS2 mutations have caused a type of Ehlers Danlos syndrome known as dermatosparaxis in other species.

Cartilage degeneration is believed to be the primary event in the development of osteoarthritis (OA). On the other hand, meniscal degeneration is observed with high prevalence, and some researchers have pointed out that pathological changes in menisci precede that of cartilage. The purpose of the present study is to investigate comprehensive gene expression pattern of cartilage and menisci in the initial phase of surgically induced OA and to compare them. Secondary OA was surgically induced in 10-week-old male Wistar rats by anterior cruciate ligament transection (ACLT). Articular cartilage and menisci were separately dissected from six ACLT- and six sham-operated rats. Each specimen was analyzed by microarray, histological, and immunohistochemical analysis 3 weeks after surgery. Of the 36,685 transcripts detectable by microarray, the number of upregulated transcripts in ACLT menisci was >2.5-fold compared with that in ACLT menisci in any given threshold. Cluster analysis using the Database for Annotation Visualization and Integrated Discovery (DAVID) showed genes related to OA, such as response to stimulus, angiogenesis, and apoptosis, which were predominantly found in menisci in ACLT rats. Representative proteases including Adamts2, 4, Mmp2, 12, 13, 14, 16, extracellular matrix genes including versican (Vcan), lumican (Lum), syndecan1 (Sdc1), and Prostaglandin endoperoxide synthase2 (Ptgs2) were up-regulated in menisci, but were not up-regulated in cartilage. Our results indicated that the molecular changes that occurred in menisci preceded those occurred in cartilage in the very early phase of surgically induced OA models.

ADAMTS2 (A Disintegrin and Metalloproteinase With Thrombospondin Motifs 2) is recognized as a metalloproteinase that promotes the cleavage of amino propeptides of types I, II, III, and V procollagens. However, the role of ADAMTS2 in the heart has not yet been defined. Herein, we observed the upregulated expression of ADAMTS2 in failing human hearts and hypertrophic murine hearts. Mice lacking ADAMTS2 display exacerbated cardiac hypertrophy on pressure overload–induced hypertrophic response, whereas mice with cardiac-specific overexpression of ADAMTS2 display alleviation of this detrimental phenotype. Consistent with these results, in vitro loss or gain of function experiments in neonatal rat cardiomyocytes confirmed that ADAMTS2 negatively regulates cardiomyocyte hypertrophy in response to Ang II. Mechanistically, blockage of the PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B)–dependent signaling pathway with specific inhibitors both in vivo and in vitro could rescue the aggravated hypertrophic response to the loss of ADAMTS2. Collectively, we propose that ADAMTS2 regulates the hypertrophic response through inhibiting the activation of the PI3K/AKT-dependent signaling pathway. Because ADAMTS2 is an extracellular protein, it could be effectively manipulated using pharmacological means to modulate cardiac hypertrophy.

The only documented activity of a subclass of ADAMTS proteases comprising ADAMTS2, 3 and 14 is the cleavage of the aminopropeptide of fibrillar procollagens. A limited number of in vitro studies suggested that ADAMTS3 is mainly responsible for procollagen II processing in cartilage. Here, we created an ADAMTS3 knockout mouse (Adamts3−/−) model to determine in vivo the actual functions of ADAMTS3. Heterozygous Adamts3+/− mice were viable and fertile, but their intercrosses demonstrated lethality of Adamts3−/− embryos after 15 days of gestation. Procollagens I, II and III processing was unaffected in these embryos. However, a massive lymphedema caused by the lack of lymphatics development, an abnormal blood vessel structure in the placenta and a progressive liver destruction were observed. These phenotypes are most probably linked to dysregulation of the VEGF-C pathways. This study is the first demonstration that an aminoprocollagen peptidase is crucial for developmental processes independently of its primary role in collagen biology and has physiological functions potentially involved in several human diseases related to angiogenesis and lymphangiogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s10456-015-9488-z) contains supplementary material, which is available to authorized users.

Mutations in ADAMTS2, a procollagen amino-propeptidase, cause severe skin fragility, designated as dermatosparaxis in animals, and a subtype of the Ehlers-Danlos syndrome (dermatosparactic type or VIIC) in humans. Not all collagen-rich tissues are affected to the same degree, which suggests compensation by the ADAMTS2 homologs ADAMTS3 and ADAMTS14. In situ hybridization of Adamts2, Adamts3 and Adamts14 , and of the genes encoding the major fibrillar collagens, Col1a1, Col2a1 and Col3a1 , during mouse embryogenesis, demonstrated distinct tissue-specific, overlapping expression patterns of the protease and substrate genes. Adamts3 , but not Adamts2 or Adamts14 , was co-expressed with Col2a1 in cartilage throughout development, and with Col1a1 in bone and musculotendinous tissues. ADAMTS3 induced procollagen I processing in dermatosparactic fibroblasts, suggesting a role in procollagen I processing during musculoskeletal development. Adamts2 , but not Adamts3 or Adamts14 , was co-expressed with Col3a1 in many tissues including the lungs and aorta, and Adamts2 -/- mice showed widespread defects in procollagen III processing. Adamts2 -/- mice had abnormal lungs, characterized by a decreased parenchymal density. However, the aorta and collagen fibrils in the aortic wall appeared normal. Although Adamts14 lacked developmental tissue-specific expression, it was co-expressed with Adamts2 in mature dermis, which possibly explains the presence of some processed skin procollagen in dermatosparaxis. The data show how evolutionarily related proteases with similar substrate preferences may have distinct biological roles owing to tissue-specific gene expression, and provide insights into collagen biosynthesis and the pathobiology of dermatosparaxis.

Collagen fibers are the main components of most of the extracellular matrices where they provide a structural support to cells, tissues and organs. Fibril-forming procollagens are synthetized as individual chains that associate to form homo- or hetero-trimers. They are characterized by the presence of a central triple helical domain flanked by amino and carboxy propeptides. Although there are some exceptions, these two propeptides have to be proteolytically removed to allow the almost spontaneous assembly of the trimers into collagen fibrils and fibers. While the carboxy-propeptide is mainly cleaved by proteinases from the tolloid family, the amino-propeptide is usually processed by procollagen N-proteinases: ADAMTS2, 3 and 14. This review summarizes the current knowledge concerning this subfamily of ADAMTS enzymes and discusses their potential involvement in physiopathological processes that are not directly linked to fibrillar procollagen processing.

Summary Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn ++ -binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C→T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene.

A cellular disintegrin and metalloproteinase (ADAM) is a new family of genes with structural homology to the snake venom metalloproteinases and disintegrins. We screened genes which were selectively expressed in the cachexigenic colon 26 adenocarcinoma subline in vivo. It was found that one novel cDNA clone, identified as a cachexigenic tumor selective gene, encodes a cysteine-rich protein which shows a sequence similarity to that of both the snake venom metalloproteinases and thrombospondins. We named this cDNA clone isintegrin nd etalloproteinase with hrombopondin motifs (ADAMTS-1). ADAMTS1 consists of six domains, 1) a pro- and 2) a metalloproteinase, 3) a disintegrin-like, 4) a thrombospondin (TSP) homologous domain containing TSP type I motif, 5) a spacer region, and 6) COOH-terminal TSP submotifs. Unlike other ADAMs, ADAMTS-1 does not possess a transmembrane domain and is a putative secretory protein. Therefore, ADAMTS-1 is a new type of ADAM family protein with TSP type I motifs. We demonstrated that the TSP homologous domain containing the TSP type I motif of ADAMTS-1 is functional for binding to heparin. ADAMTS-1 mRNA could be induced by stimulating colon 26 cells with an inflammatory cytokine, interleukin-1, in vitro. Moreover, intravenous administration of lipopolysaccharide in mice selectively induced ADAMTS-1 mRNA in kidney and heart. These data suggest that ADAM-TS-1 may be a gene whose expression is associated with various inflammatory processes as well as development of cancer cachexia.

Procollagen I N-proteinase (EC 3.4.24.14), the enzyme that specifically processes type I and type II procollagens to collagen, was isolated from extracts of fetal calf skin. After two chromatographic steps on concanavalin A-Sepharose and heparin-Sepharose, the semi-purified preparation was used to produce monoclonal antibodies. One reacting antibody was found to recognize not the enzyme itself but type XIV collagen on which the enzyme was bound. This binding, highly sensitive to ionic conditions (plH, salt concentrations) but not affected by non-ionic detergents, was used for affinity chromatography that strongly improved the purification procedure. The enzyme is extensively characterized: 1) it has a molecular mass of 107 kDa as determined by polyacrylamide gel electrophoresis in presence of SDS and of about 130 kDa when estimated by gel filtration on a Sephacryl-S300; 2) in standard assay (pH 7.5, 0.2 M NaCl, 35 degrees C), the activation energy for reaction with amino procollagen type I was 17,000 calories per mole. In the same conditions, Km and Vmax values were, respectively, 435 and 39 nM per hour but varied strongly with pH and salt concentration; 3) the enzyme cleaved the NH2-terminal propeptide of type I procollagen at the specific site, the Pro-Gln bond in the alpha 1 type I procollagen chain; 4) the enzyme contained a high proportion of Gly, Asx, and Glx residues but no Hyp or Hyl; 5) partial amino acid sequences obtained from internal peptides of the enzyme displayed no significant homology with known sequences. The association of procollagen I N-proteinase with a FACIT (fibril-associated collagens with interrupted triple helices) collagen as found here might be of physiological significance.

Ehlers-Danlos (ED) syndrome type VII is characterized by the accumulation of collagen precursors in connective tissues. ED VII A and B are caused by mutations in the genes of alpha 1 and alpha 2 collagen I which result in the disruption of the cleavage site of procollagen I N-proteinase. The existence of ED VII C in humans has been hypothesized on the basis of a disorder in cattle and sheep related to the absence of the enzyme. We now present evidence for the existence of this disease in humans, characterized by skin fragility, altered polymers seen as hieroglyphic pictures with electron microscopy, accumulation of p-N-alpha 1 and p-N-alpha 2 collagen type I in the dermis and absence of processing of the p-N-I polypeptides in fibroblast cultures.

ADAMTS2 belongs to the "ADAM metallopeptidase with thrombospondin type 1 motif" (ADAMTS) family. Its primary function is to process collagen type I, II, III, and V precursors into mature molecules by excising the aminopropeptide. This process allows the correct assembly of collagen molecules into fibrils and fibers, which confers to connective tissues their architectural structure and mechanical resistance. To evaluate the impact of ADAMTS2 on the pathological accumulation of extracellular matrix proteins, mainly type I and III collagens, we evaluated carbon tetrachloride-induced liver fibrosis in ADAMTS2-deficient (TS2(-/-)) and wild-type (WT) mice. A single carbon tetrachloride injection caused a similar acute liver injury in deficient and WT mice. A chronic treatment induced collagen deposition in fibrous septa that were made of thinner and irregular fibers in TS2(-/-) mice. The rate of collagen deposition was slower in TS2(-/-) mice, and at an equivalent degree of fibrosis, the resorption of fibrous septa was slightly faster. Most of the genes involved in the development and reversion of the fibrosis were similarly regulated in TS2(-/-) and WT mice. CONCLUSION: These data indicate that the extent of fibrosis is reduced in TS2(-/-) mice in comparison with their WT littermates. Inhibiting the maturation of fibrillar collagens may be a beneficial therapeutic approach to interfering with the development of fibrotic lesions.

The regulated expression of ADAMTS2 (a disintegrin and metalloproteinase with thrombospondin motifs), a secreted metalloproteinase involved in the processing of procollagen to collagen, was studied in peripheral blood mononuclear cells (PBMC). Stimulation with glucocorticoids (GC) resulted in a pronounced dose- and time-dependent increase of ADAMTS2 mRNA levels in PBMC. The increase of ADAMTS2 expression was specific for CD14++ monocytes (440-fold) and alveolar macrophages (200-fold), whereas CD3+ (T lymphocytes), phytohemagglutinin-activated CD3+ (T lymphocytes), and CD19+ (B lymphocytes) showed no significant changes in ADAMTS2 mRNA after GC treatment. Treatment of monocyte-derived macrophages (MDM) with GC also resulted in an increase of ADAMTS2 protein in the culture tissue media. Using the GC analog RU486, GC-mediated induction of ADAMTS2 mRNA was blocked, implicating that GC acts specifically via the GC-receptor. In agreement with findings in blood monocytes, cell lines of the monocytic lineage (MM6, THP-1) showed significant GC-induced significant increases in ADAMTS2 mRNA, while in epithelial cells (A549, Calu-3, Colo320, BT-20) and fibroblast (MRC-5, WI-38, and two NHDF-c cell types from adult cheek and upper arm), they showed no or little responsiveness to GC. As macrophages have important functions in immune defense and tissue homeostasis, these findings suggest that GC-mediated specific induction of ADAMTS2 in these cells may play a crucial role in the resolution of inflammation and wound repair.

International audience; Processing of fibrillar collagens is required to generate collagen monomers able to self-assemble into elongated and cylindrical collagen fibrils. ADAMTS-2 belongs to the "A disintegrin and metalloproteinase with thrombospondin type 1 motifs" (ADAMTS) family. It is responsible for most of the processing of the aminopropeptide of type I procollagen in the skin, and it also cleaves type II and type III procollagens. ADAMTS are complex secreted enzymes that are implicated in various physiological and pathological processes. Despite accumulating evidence indicating that their activity is regulated by ancillary domains, additional information is required for a better understanding of the specific function of each domain. We have generated 17 different recombinant forms of bovine ADAMTS-2 and characterized their processing, activity, and cleavage specificity. The results indicated the following: (i) activation of the ADAMTS-2 zymogen involves several cleavages, by proprotein convertases and C-terminal processing, and generates at least seven distinct processed forms; (ii) the C-terminal domain negatively regulates enzyme activity, whereas two thrombospondin type 1 repeats are enhancer regulators; (iii) the 104-kDa form displays the highest aminoprocollagen peptidase activity on procollagen type I; (iv) ADAMTS-2 processes the aminopropeptide of alpha1 type V procollagen homotrimer at the end of the variable domain; and (v) the cleaved sequence (PA) is different from the previously described sites ((P/A)Q) for ADAMTS-2, redefining its cleavage specificity. This finding and the existence of multiple processed forms of ADAMTS-2 strongly suggest that ADAMTS-2 may be involved in function(s) other than processing of fibrillar procollagen types I-III.Processing of fibrillar collagens is required to generate collagen monomers able to self-assemble into elongated and cylindrical collagen fibrils. ADAMTS-2 belongs to the "A disintegrin and metalloproteinase with thrombospondin type 1 motifs" (ADAMTS) family. It is responsible for most of the processing of the aminopropeptide of type I procollagen in the skin, and it also cleaves type II and type III procollagens. ADAMTS are complex secreted enzymes that are implicated in various physiological and pathological processes. Despite accumulating evidence indicating that their activity is regulated by ancillary domains, additional information is required for a better understanding of the specific function of each domain. We have generated 17 different recombinant forms of bovine ADAMTS-2 and characterized their processing, activity, and cleavage specificity. The results indicated the following: (i) activation of the ADAMTS-2 zymogen involves several cleavages, by proprotein convertases and C-terminal processing, and generates at least seven distinct processed forms; (ii) the C-terminal domain negatively regulates enzyme activity, whereas two thrombospondin type 1 repeats are enhancer regulators; (iii) the 104-kDa form displays the highest aminoprocollagen peptidase activity on procollagen type I; (iv) ADAMTS-2 processes the aminopropeptide of alpha1 type V procollagen homotrimer at the end of the variable domain; and (v) the cleaved sequence (PA) is different from the previously described sites ((P/A)Q) for ADAMTS-2, redefining its cleavage specificity. This finding and the existence of multiple processed forms of ADAMTS-2 strongly suggest that ADAMTS-2 may be involved in function(s) other than processing of fibrillar procollagen types I-III.

Background The ADAMTS (A Disintegrin-like and Metalloprotease with Thrombospondin motifs) proteins are a family of metalloproteases with sequence similarity to the ADAM proteases, that contain the thrombospondin type 1 sequence repeat motifs (TSRs) common to extracellular matrix proteins. ADAMTS proteins have recently gained attention with the discovery of their role in a variety of diseases, including tissue and blood disorders, cancer, osteoarthritis, Alzheimer's and the genetic syndromes Weill-Marchesani syndrome (ADAMTS10), thrombotic thrombocytopenic purpura (ADAMTS13), and Ehlers-Danlos syndrome type VIIC (ADAMTS2) in humans and belted white-spotting mutation in mice (ADAMTS20). Results Phylogenetic analysis and comparison of the exon/intron organization of vertebrate (Homo, Mus, Fugu), chordate (Ciona) and invertebrate (Drosophila and Caenorhabditis) ADAMTS homologs has elucidated the evolutionary relationships of this important gene family, which comprises 19 members in humans. Conclusions The evolutionary history of ADAMTS genes in vertebrate genomes has been marked by rampant gene duplication, including a retrotransposition that gave rise to a distinct ADAMTS subfamily (ADAMTS1, -4, -5, -8, -15) that may have distinct aggrecanase and angiogenesis functions.

Ehlers-Danlos syndrome (EDS) type VIIC, or dermatosparactic type, is a recessively inherited connective tissue disorder characterized, among other symptoms, by an extreme skin fragility resulting from mutations inactivating ADAMTS-2, an enzyme excising the aminopropeptide of procollagens type I, II, and III. All previously described mutations create premature stop codons leading to a marked reduction in the level of mRNA. In this study, we analyzed the ADAMTS2 cDNA sequences from five patients displaying clinical and/or biochemical features consistent with a diagnosis of either typical or potentially mild form of EDS type VIIC. Three different alterations were detected in the two patients with typical EDS type VIIC. The first patient was homozygous for a genomic deletion causing an in-frame skipping of exons 3-5 in the transcript. In the second patient, the allele inherited from the mother lacks exon 3, generating a premature stop codon, whereas the paternal allele has a genomic deletion resulting in an in-frame skipping of exons 14-16 at the mRNA level. Although the exons 3-5 or 14-16 encode protein domains that have not been previously recognized as crucial for ADAMTS-2 activity, the aminoprocollagen processing was strongly impaired in vitro and in vivo, providing evidence for the requirement of these domains for proper enzyme function. The three other patients with a phenotype with some resemblance to EDS type VIIC only had silent and functionally neutral variations also frequently found in a normal population.

Background Dermatosparaxis (Ehlers–Danlos syndrome in humans) is characterized by extreme fragility of the skin. It is due to the lack of mature collagen caused by a failure in the enzymatic processing of procollagen I. We investigated the condition in a commercial sheep flock. Hypothesis/Objectives Mutations in the ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2) locus, are involved in the development of dermatosparaxis in humans, cattle and the dorper sheep breed; consequently, this locus was investigated in the flock. Animals A single affected lamb, its dam, the dam of a second affected lamb and the rams in the flock were studied. Methods DNA was purified from blood, PCR primers were used to detect parts of the ADAMS2 gene and nucleotide sequencing was performed using Sanger's procedure. Skin samples were examined using standard histology procedures. Results A missense mutation was identified in the catalytic domain of ADAMTS2. The mutation is predicted to cause the substitution in the mature ADAMTS2 of a valine molecule by a methionine molecule (V15M) affecting the catalytic domain of the enzyme. Both the ‘sorting intolerant from tolerant’ (SIFT) and the PolyPhen-2 methodologies predicted a damaging effect for the mutation. Three-dimensional modelling suggested that this mutation may alter the stability of the protein folding or distort the structure, causing the protein to malfunction. Conclusions and clinical importance Detection of the mutation responsible for the pathology allowed us to remove the heterozygote ram, thus preventing additional cases in the flock.

The processing of amino- and carboxyl-propeptides of fibrillar collagens is required to generate collagen monomers that correctly assemble into fibrils. Mutations in the ADAMTS2 gene, the aminopropeptidase of procollagen I and II, result in the accumulation of non-fully processed type I procollagen, causing human Ehlers-Danlos syndrome type VIIC and animal dermatosparaxis. In this study, we show that the aminopropeptide of type I procollagen can be cleaved in vivo in absence of ADAMTS-2 activity and that this processing is performed at the cleavage site for ADAMTS-2. In an attempt to identify the enzyme responsible for this alternative aminoprocollagen peptidase activity, we have cloned the cDNA and determined the primary structure of human and mouse ADAMTS-14, a novel ADAMTS displaying striking homologies with ADAMTS-2 and -3. The structure of the human gene, which maps to 10q21.3, and the mechanisms of generation of the various transcripts are described. The existence of two sites of initiation of transcription, in two different promoter contexts, suggests that transcripts resulting from these two sites can be differently regulated. The tissue distribution of ADAMTS-14, the regulation of the gene expression by various cytokines and the activity of the recombinant enzyme are evaluated. The potential function of ADAMTS-14 as a physiological aminoprocollagen peptidase in vivo is discussed.

ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a novel family of extracellular proteases found in both mammals and invertebrates. Members of the family may be distinguished from the ADAM (a disintegrin and metalloprotease) family members based on the multiple copies of thrombospondin 1-like repeats they carry. With at least nine members in mammals alone, the ADAMTS family members are predicted by their structural domains to be extracellular matrix (ECM) proteins with a wide range of activities and functions distinct from members of the ADAM family that are largely anchored on the cell surface. ADAMTS2 is a procollagen N-proteinase, and the mutations of its gene are responsible for Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis. ADAMTS4 and ADAMTS5 are aggrecanases implicated in the degradation of cartilage aggrecan in arthritic diseases. Other members of the ADAMTS family have also been implicated in roles during embryonic development and angiogenesis. Current and future studies on this emerging group of ECM proteases may provide important insights into developmental or pathological processes involving ECM remodeling.

Procollagen N-proteinase (EC3.4.24.14) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers–Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the N-proteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I procollagen. The protein contained zinc-binding sequences of the clan MB of metallopeptidases that includes procollagen C-proteinase/BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor β or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis.

Collagen has been extracted from the skin of dermatosparaxic calves and fractionated by acrylamide-gel electrophoresis and CM-cellulose chromatography. This collagen contains in large proportion two additional types of a-chains, p-a, and p-a,. The first, p-a,, ressembles a, of a collagen extracted from the skin of a normal calf in its amino-acid composition but its molecular weight is loo/, larger. The second, p-a,, is similar to a normal a, in its amino-acid composition but its molecular weight is increased by 5O/,. The abnormal a-chains contain six to eight half cystine residues in p-a1 and one in pa,. As seen under the electron microscope on segment long spacing, the dermatosparaxic collagen is 25 nm larger than the normal collagen and the increased length results from an extension of the polypeptide chain at the N-terminal extremity of the molecules. Although the collagen is less extractable from the dermatosparaxic skin than from the normal skin and dimers and other polymers of the abnormal a-chains are observed, the dermatosparaxic collagen does not, in vitro, form fibrils of normal stability. Since an enzyme activity which can excise the N-terminal extension peptide of the dermatosparaxic collagen has been identified in extracts of normal skin but is absent in the dermatosparaxic animals, it is suggested that the dermatosparaxic collagen is a form of “procollagen)’, from which all or a part of these peptides have not been removed and that such peptides function as coordinators in the post synthesis assembly of the collagen a-chains.

A heritable connective tissue disorder of cattle, dermatosparaxis, is characterized by an extreme fragility of the skin and the presence of additional peptides at the N-terminal extremities of the collagen α chains, p-α 1 and p-α 2 . The existence of an enzyme activity is demonstrated in normal connective tissues that is capable of cleaving these additional N-terminal peptides from dermatosparaxic collagen. The activity is demonstratable with dermatosparaxic collagen in solution, as well as with reconstituted dermatosparaxic collagen fibrils polymerized in vitro . It has a pH optimum of about 7.0 and is inhibited by EDTA and mercaptoethanol. Differences in K m and V max values exist depending on the substrate utilized, i.e., p-α 1 or p-α 2 ; and the presence of additional amounts of one substrate, p-α 1 , alters the concentration requirement for the second substrate, p-α 2 . The product of the excision reaction with p-α 1 as substrate is an equimolar amount of normal α 1 monomer; the product when p-α 2 is substrate is an equimolar amount of normal α 2 monomer. The enzyme is present in normal calf skin, tendon, aorta, cartilage, and lung; it can be demonstrated in the skin of rats and humans. The enzyme activity is absent in dermatosparaxic connective tissues, thus suggesting that dermatosparaxis is caused by the absence of a normal enzyme function rather than by the production of an abnormal collagen.

© 2015 Kelwick et al.The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future.