Review

Insights on ADAMTS and ADAMTS-like from mammalian genetics

Johanne Dubail and Suneel S. Apte⁎

Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA

Correspondence to Suneel S. Apte: Department of Biomedical Engineering (ND20), Cleveland Clinic Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, USA. [email protected] http://dx.doi.org/10.1016/j.matbio.2015.03.001 Edited by R. Iozzo

Abstract

The mammalian ADAMTS superfamily comprises 19 secreted and 7 ADAMTS-like proteins, each the product of a distinct . Thus far, all appear to be relevant to function or to cell–matrix interactions. Most ADAMTS functions first emerged from analysis of spontaneous and animal mutations and genetically engineered animals. The clinical manifestations of Mendelian disorders resulting from mutations in ADAMTS2, ADAMTS10, ADAMTS13, ADAMTS17, ADAMTSL2 and ADAMTSL4 identified essential roles for each gene, but also suggested potential cooperative functions of ADAMTS proteins. These observations were extended by analysis of spontaneous animal mutations, such as in bovine ADAMTS2, canine ADAMTS10, ADAMTS17 and ADAMTSL2 and mouse ADAMTS20. These human and animal disorders are recessive and their manifestations appear to result from a loss-of-function mechanism. Genome-wide analyses have determined an association of some ADAMTS loci such as ADAMTS9 and ADAMTS7, with specific traits and acquired disorders. Analysis of genetically engineered rodent mutations, now achieved for over half the superfamily, has provided novel biological insights and animal models for the respective human genetic disorders and suggested potential candidate for related human phenotypes. Engineered mouse mutants have been interbred to generate combinatorial mutants, uncovering cooperative functions of ADAMTS proteins in morphogenesis. Specific genetic models have provided crucial insights on mechanisms of osteoarthritis (OA), a common adult-onset degenerative condition. Engineered mutants will facilitate interpretation of exome variants identified in isolated birth defects and rare genetic conditions, as well as in genome-wide screens for trait and disease associations. Mammalian forward and reverse genetics, together with genome-wide analysis, together constitute a powerful force for revealing the functions of ADAMTS proteins in physiological pathways and health disorders. Their continuing use, together with genome-editing technology and the ability to generate stem cells from mutants, presents numerous opportunities for advancing basic knowledge, human disease pathways and therapy. © 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction (ADAMTSL1-6 and papilin) in , mice and other mammals [6]. The gene name ADAMTS11 was The first A disintegrin-like and metalloprotease given in error to a gene already designated as (reprolysin type) with type 1 motif ADAMTS5 [2,7]; thus the term ADAMTS11 is no longer (ADAMTS) proteases were discovered in the late used. Phylogenetic analysis showed considerable 1990s [1–3], and the first ADAMTS-like proteins expansion of the mammalian ADAMTS family, com- (ADAMTSL) were cloned shortly after [4,5]. pared to a much smaller repertoire in Caenorhabditis ADAMTSLs resemble the non-catalytic domains of elegans, Drosophila melanogaster and Ciona intesti- ADAMTS proteases and lack a domain [6]. nalis [8] (see review by Kim and Nishiwaki in this They are not proteases, nor do they arise by alternative special issue). Mammalian ADAMTS gene expansion splicing of ADAMTS protease genes. There are 19 is postulated to have arisen primarily by gene ADAMTS genes and 7 ADAMTS-like proteins duplication, representing both sub-functionalization 0022-2836/© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Matrix Biol (2015) 44-46,24–37 Insights on ADAMTS proteases and ADAMTS-like proteins 25 and neo-functionalization of the duplicated genes [8]. post-translational modifications as a prerequisite for Sub-functionalization is suggested by the shared or cleavage that are absent in synthetic substitutes. For cooperative function of a subset of ADAMTS proteases example, ADAMTS5 proteolysis uses the chondroitin in cleavage of proteoglycans such as aggrecan and sulfate chains of versican as a crucial determinant for versican [9], and involvement of several ADAMTS proteolysis [17], and ADAMTS13 processing of von proteins in fibrillin microfibril biology [10].ADAMTS Willebrand factor (vWF) requires shear stress-me- neo-functionalization is exemplified by ADAMTS13, diated unfolding of the region containing the scissile whose major function seems to be hemostatic [11] and bond [22]. possibly related to evolution of a closed circulatory Given these challenges, it has been fortunate that system. genetics has stepped into the breach. In contrast Compared to other metalloprotease families, such to other metalloproteinases, the ADAMTS field as matrix metalloproteinases (MMPs), which have has benefited considerably from forward genetics, been studied for over half a century [12],theADAMTS i.e., resolution of the genetic basis of spontaneous field has thus existed only for a decade and a half. It is mutants, which exist for several ADAMTS genes. important to point out that ADAMTS proteases are Reverse genetics, i.e., where genetically engineered genetically distinct from the membrane-anchored A or chemically induced mutants are generated and the disintegrin and metalloprotease (ADAMs). Whereas resulting phenotype analyzed to define gene function, membrane localization endows ADAMs with a major has advanced understanding of the same genes as role in ectodomain shedding from the cell well as others of previously unknown function. That surface [13], ADAMTS proteases are secreted and ADAMTS forward genetics has revealed the basis of primarily associated with proteolytic events in extra- extremely rare inherited disorders does not diminish cellular matrix (ECM). There are distinct differences in its value, since the mutations revealed previously their respective domain structures, the hallmark of unsuspected biological pathways. As a case in point, ADAMTS proteases being a C-terminal ancillary the discovery that rare mutations in the low-density domain having a characteristic modular structure lipoprotein receptor caused hypercholesterolemia that contains one or more thrombospondin type 1 paved the way for cholesterol-lowering therapy [23]. repeats (TSRs). ADAMTS genetics has contributed new knowledge of Although there are totally 26 ADAMTS proteins to extracellular matrix assembly and degradation in be considered in defining individual and collective morphogenesis, reproductive biology and many functions of the family, and for determining their organ systems and processes as reviewed here. shared operational principles in biological networks, Other reviews in this special issue have covered the genetics has already proved to be the primary source role of ADAMTS proteases in cancer (see review by of functional insights. This was inevitable because Cal and Lopez-Otin) and cancer-related processes ADAMTS proteins are not as amenable to biochem- such as angiogenesis (see review by Rodriguez-- ical and cellular analysis as MMPs and ADAMs. For Manzaneque and Iruela-Arispe), as well as fertility example, MMPs are readily expressed, refolded and (see review by D. Russell), nervous system disor- purified in quantity from eukaryotic expression ders (see review by P. Gotschall) and procollagen systems and readily cleave peptide substrates [14]. processing (see review by Bekhouche and Colige). Analysis of ADAMs was enabled by the finding that they lacked stringent substrate sequence specificity, and mediated ectodomain shedding via a ADAMTS genomics “lawn-mower” effect enabled by their membrane anchorage [15]. Thus, ADAM activities are typically In contrast to a well-defined genomic MMP cluster determined by use of transfected constructs in which on human 11q22 [24], ADAMTS genes the target substrate's ectodomain carries an are dispersed throughout the human and mouse enzymatic reporter such as alkaline phosphatase, genomes. However, three pairs of ADAMTS genes while retaining the membrane-proximal segment of are tightly linked in regions of human-mouse synteny the ectodomain [16]. In contrast, ADAMTS protease [25], and of these, two present potential challenges for biochemistry provides many challenges, e.g., these mouse genetic engineering. These two pairs include proteases are large, have numerous disulfide bonds, evolutionarily related ADAMTS proteases, namely, the majority carry heavy and specialized glycosyla- ADAMTS1/ADAMTS5 and ADAMTS8/ADAMTS15 on tion, are poorly secreted by cells, sticky and suscep- human 21 and 11 respectively; the tible to autocatalysis, which disrupts the ancillary corresponding mouse loci are linked on mouse domain. Unlike MMPs, ADAMTS protease domains chromosomes 16 and 9 respectively. Because these expressed in isolation do not typically have specific genes have a close evolutionary relationship, there is a activity, since the ancillary domains mediate substrate possibility that they have cooperative functions. recognition [17–21]. Furthermore, unlike MMPs, most However, tight genetic linkage precludes elucidation ADAMTS proteases do not cleave short of cooperative functions by crossing knockout mice peptide substrates, and some may require substrate (such as of Adamts1 and Adamts5), because the odds 26 Insights on ADAMTS proteases and ADAMTS-like proteins

Table 1. Mendelian conditions resulting from mutations in human ADAMTS protease genes

Mendelian condition MIM number Gene name, chromosomal locus Inheritance Ehlers–Danlos syndrome (EDS), 225410 ADAMTS2, 5q35.3 [57] Autosomal recessive dermatosparaxis type or type VIIC Weill–Marchesani syndrome 1/Weill–Marchesani syndrome, 277600 ADAMTS10/19p13.2 [62] Autosomal recessive autosomal recessive/Mesodermal dysmorphodystrophy, congenital Thrombotic thrombocytopenic purpura, 274150 ADAMTS13, 9q34.2 [81] Autosomal recessive congenital/Upshaw–Schulman syndrome Weill–Marchesani-like syndrome 613195 ADAMTS17, 15q26.3 [68] Autosomal recessive Microcornea, myopic chorioretinal atrophy and 615458 ADAMTS18, 16q23.1 [82] Autosomal recessive telecanthus (MMCAT)

of recombination between closely linked loci are ADAMTS proteins appear to have a propensity for extremely poor. The third genetic linkage involves binding to the cell surface/pericellular matrix, and ADAMTS13, whose locus is linked to that of ADAMTS13, which is synthesized in the liver and by ADAMTSL2, on human chromosome 9q34 and vascular endothelium, is present in the circulation, mouse chromosome 2. These two genes are 120 where it processes vWF. During their biosynthesis, and 100 kb apart in the human and mouse genomes ADAMTS proteins undergo extensive post-transla- respectively, with CACFD1 intervening in the human tional modification, including disulfide bonding, N- and genome, plus two other intervening genes, Slc2a6 O-glycosylation, and in the case of ADAMTS7, and Tmem8c in mouse. In contrast to the first two addition of chondroitin–sulfate chains [35].Most pairs, the linkage of ADAMTSL2 and ADAMTS13 is ADAMTS proteins are modified by C-mannosylation thought to be coincidental, or to represent a very of Trp residues, and O-fucosylation of Ser/Thr distant gene duplication event. ADAMTSL2 is impli- residues in the TSRs [39–41]. The latter modification, cated in an inherited connective tissue disorder resulting in addition of either a fucose monosaccharide named geleophysic dysplasia [10,26], which has no or a fucose–glucose disaccharide, requires sequential clinical overlap with thrombotic thrombocytopenic action of two specialized glycosyltransferases, purpura resulting from ADAMTS13 deficiency. Be- POFUT2 and B3GLCT [42,43]. The fate of ADAMTS cause of the intervening genes in the syntenic regions, proteins after release from cells is poorly understood. ADAMTS13 and ADAMTSL2 genes probably do not TIMP-3 and α2-macroglobulin are known endogenous share regulatory regions. inhibitors of ADAMTS2 [44] the ADAMTS4 and ADAMTS5 [45,46], and others, but these inhibitors have not been tested against the entire Key biochemical and structural aspects of family. Some ADAMTS proteins undergo fragmenta- ADAMTS proteases relevant to tion, with potentially new functions of the derivative understanding genetic defects fragments [47], and recent work has identified a cellular clearance mechanism for ADAMTS proteases based ADAMTS proteases comprise an N-terminal on the LRP-1 receptor [48–50]. protease domain containing the catalytic activity ADAMTS proteases are implicated in fibrillar and a C-terminal ancillary domain. Biochemical and procollagen maturation (ADAMTS2, ADAMTS3), ver- 3-dimensional structural data indicate that ADAMTS sican turnover during embryogenesis (ADAMTS1, proteases have a similar catalytic mechanism as ADAMTS5, ADAMTS9, ADAMTS15, ADAMTS20) MMPs and ADAMs, employing a Zn-atom coordi- and ovulation (ADAMTS1), cartilage aggrecan de- nated by three conserved His residues [27,28].All struction in arthritis (ADAMTS4, ADAMTS5), von ADAMTS proteases, but not ADAMTSLs, have an Willebrand factor proteolysis and hemostasis N-terminal propeptide, which like that of MMPs and (ADAMTS13), and in VEGF-C activation during ADAMs, is thought to be inhibitory to catalytic lymphangiogenesis (ADAMTS3) [51]. activity and requires excision. However, ADAMTS9 and ADAMTS13 zymogens appear to be active despite retention of the propeptide, which also Insights from spontaneous animal happens to be unusually short in ADAMTS13 mutants and human Mendelian disorders [29,30]. Propeptide excision in ADAMTS proteases is mediated by proprotein convertases such as furin [30–37]. These serine proteases efficiently cleave Dermatosparaxis, a classic connective tissue after a consensus sequence Arg-Xaa-(Arg/Lys)-Arg, disorder, manifests with severe skin fragility. Indeed, with the cleavage thought to occur in the trans-Golgi, affected skin was described as having the consis- or at the cell surface [35,38]. Upon secretion, tency of wet blotting paper. It was first identified in Insights on ADAMTS proteases and ADAMTS-like proteins 27 inbred cattle, and subsequently in sheep and cats genetic findings. Tissue microfibrils are supramolec- [52–54]. Dermatosparactic skin is characterized by ular structures in extracellular matrix that are the accumulation of abnormal fibrils having assembled from large glycoproteins named fibrillins, a “hieroglyphic” ultrastructural appearance in the among which, fibrillin-1 predominates in adult dermis that is the hallmark of this disorder [53].The tissues. The potential relationship of ADAMTS10 underlying mechanism was identified a few decades with microfibrils was confirmed by the finding that ago as deficiency of the specific enzymatic activity ADAMTS10 bound to fibrillin-1 and fibrillin-2 in vitro, required for removal of the N-propeptide of procolla- and localized to fibrillin microfibrils [64]. ADAMTS10 gen I, the major collagen type in the dermis of skin [55]. lacks a perfect consensus sequence for furin Persisting pN-collagen I, the resulting incompletely processing and its zymogen is inefficiently cleaved. processed intermediary of collagen maturation, pre- Consistent with this, and despite its specific binding vents tight packing of collagen molecules by steric to fibrillin-1, ADAMTS10 cleaves fibrillin-1 poorly hindrance. Hence structurally competent fibrils are not [64]. Because ectopia lentis results from a structural formed, and as a result, the dermis is extremely weak. weakness of the fibrillin-rich structure called the Later, a human connective tissue disorder, Ehlers– zonule that centers the lens in the optic path, it is Danlos syndrome type VIIc (or dermatosparaxis type) possible that ADAMTS10 is primarily required for having similar skin fragility and collagen fibril anom- formation or stability of the zonule. Indeed, alies was identified [56] (Table 1) and both the bovine ADAMTS10 was shown to enhance microfibril and human conditions were attributed to ADAMTS2 networks in cultured fibroblasts [64], in which regard mutations in the respective species [57]. it was similar to ADAMTSL4 and ADAMTSL6 One of the persisting conundrums of this disorder [65,66], suggesting it might function as an is why bone, cartilage, tendons and arteries are not ADAMTS-like protein rather than as a protease. A as severely affected as skin, despite containing WMS-like phenotype in humans and ectopia lentis in fibrillar as a quantitatively major structural dogs result from ADAMTS17 mutations [67,68] component, and fibrillar collagen types I, II, III and V (Table 1). Affected individuals have ectopia lentis being ADAMTS2 substrates [58]. It is possible that and short stature, but not brachydactyly or congen- this could be due to compensating activities of ital cardiac anomalies [68]. Unlike ADAMTS10, closely related with similar activity, such as ADAMTS17 has a classic furin consensus site, and ADAMTS3 and ADAMTS14 [59,60]. ADAMTS3 is its zymogen is probably processed to an active highly expressed in cartilage, where collagen II is a protease. However, little is presently known about major component, as well as in embryonic bone and ADAMTS17 protein, including whether or not it binds tendon, suggesting that it could be a major procolla- to fibrillins, localizes to microfibrils or affects micro- gen processing in musculoskeletal tissues fibril biogenesis. In another example of a zonule-re- [60,61]. ADAMTS3 was recently shown to participate lated ADAMTS function, recessive isolated ectopia in proteolytic activation of the pro-angiogenic and lentis and ectopia lentis et pupillae are caused by pro-lymphangiogenic factor VEGF-C via binding to a ADAMTSL4 mutations [69–71]. A mutation com- putative co-factor, collagen- and calcium-binding monly associated with these disorders, epidermal growth factor domains 1 (CCBE1) [51]. (c767_786Del), is thought to represent an ancient The cleavage site in VEGF-C has some similarities to founder effect [71].ADAMTSL4iswidelydistribut- that in procollagens, and the presence of a C-terminal ed in various eye components as well as in domain with collagenous repeats in CCBE1 may non-ocular tissues; recent in situ hybridization data provide a basis for its interaction with ADAMTS3. (Hubmacher. D., and Apte, S.S., unpublished) show Unpublished data indicates that embryos lacking that Adamtsl4 mRNA is strongly expressed in the Adamts3 do not survive past late gestation (Dubail, J. lens equatorial epithelium whence the zonule and Colige, A., unpublished data). Presently, the roles attaches [65,72].AnADAMTSL4 mutation was of ADAMTS3 and ADAMTS14 in procollagen process- also found to cause craniosynostosis in association ing remain to be fully elucidated, and the role of with ectopia lentis, potentially related to its extrao- ADAMTS3 in lymphangiogenesis remains to be cular expression [73].LikeADAMTSL4 mRNA, validated in vivo. Procollagen N-propeptidases are ADAMTS10 and ADAMTS17 mRNAs are also reviewed extensively by Bekhouche and Colige in this widely expressed [34,74,75], suggesting that they issue. function in many organs, where they may overlap or ADAMTS10 mutations lead to Weill–Marchesani work cooperatively in the context of tissue microfi- syndrome (WMS1), with short stature, brachydac- brils. The specific phenotypes observed in each tyly, cardiac valve stenosis and ectopia lentis gene defect may therefore reflect those sites that are (dislocation of the lens) being the major clinical the least over-engineered in terms of overlapping features (Table 1) [62]. Since WMS is also caused by expression of a compensating microfibril-related dominantly inherited fibrillin-1 mutations (WMS2) ADAMTS protein. [63], a functional relationship between ADAMTS10 ADAMTSL2 mutations cause geleophysic dyspla- and tissue microfibrils was suggested by these sia, which presents with short stature, stiff skin, joint 28 Insights on ADAMTS proteases and ADAMTS-like proteins

Table 2. Spontaneous and engineered mutations in rodent ADAMTS genes

Phenotype Adamts1−/− – Growth retardation [86,132] – Urologic abnormalities possibly underlying ~ 50% post-natal lethality [86,132] – Adrenal abnormalities [86] – Reduced female fertility [86,87,89,133] – Diminished levels of synaptic proteins in the female frontal cortex [134] Adamts1− / − ; – Urologic abnormalities associated with ~95% post-natal lethality [135] Adamts4−/− − − Adamts2 / – Skin fragility [136] – Male sterility [136] − − Adamts4 / – No apparent phenotype [137] – Reduced versican cleavage during spinal cord injury [138] Adamts5−/− – Reduced cartilage loss in osteoarthritis (OA) models [139–141] – Low penetrance of soft tissue syndactyly [101] – Reduced sculpting of pulmonic valves; myxomatous mitral valve [95] – Reduced versican cleavage during myogenesis [142] – Impaired contraction and dermal collagen deposition in an excisional skin wound healing model [143] – Aggrecan accumulation and impaired tendon function [144] Adamts4− / − ; – Diminished severity of lesions in an induced OA model similar to Adamts5−/− [145] Adamts5−/− Adamts7 −/− – No reported developmental anomaly. Altered response to vascular injuries [129,130] − − Adamts9 / – Lethal at 7.5 days of gestation [100] − Adamts9+/ – Cardiac and aortic anomalies [98] – Ocular anterior segment dysgenesis (Dubail, S., and Apte, S.S., unpublished) and spontaneous corneal neovascularization [99] Adamts9+/− ; – Extensive white spotting [85] Adamts20bt/bt – Soft tissue syndactyly (complete penetrance) [101] – Cleft secondary palate (complete penetrance) [100] − Adamts9+/ ; – Delayed palate closure [100] Adamts20bt/+ − Adamts9+/ ; – High penetrance of soft tissue syndactyly [101] Adamts5+/− Adamts9fl/fl; – Soft tissue syndactyly (complete penetrance) [97] Prx-Cre Adamts9fl/fl; – Soft tissue syndactyly (complete penetrance; greater severity than Adamts9fl/fl;Prx-Cre) [97] Prx-Cre; − − Adamts5 / Adamts12−/− – Increased tumor growth and angiogenesis [146] – Increased inflammation and delayed recovery in an induced colitis model, endotoxic sepsis and pancreatitis [147] – Heightened bronchial inflammation and hyperresponsiveness in an allergic airways disease model [148] − − Adamts13 / – Thrombocytopenia in CASA/Rb mouse strain with elevated plasma vWF [149] – TTP-like syndrome after shigatoxin challenge [149] – Larger infarct after transient occlusion of left coronary artery [150] Adamts16−/− (rat) – Lower systolic blood pressure [103] – Cryptorchidism and infertility in males [102] Adams18−/− – Cataract and reduced body fat (http://www.mousephenotype.org/data/genes/MGI:2442600#section-associations) Adamts20bt/bt – White spotting of the ventral and dorsal lumbar region [85] – Low penetrance of soft tissue syndactyly [101] – Delayed palate closure [100] Adamts20bt/bt ; – Soft tissue syndactyly (complete penetrance) [101] Adamts5−/−

contractures, liver, tracheo-pulmonary and cardiac to be microfibril-related, because ADAMTSL2 anomalies, leading frequently to juvenile death [26]. bound both to fibrillin-1 and to one of its interacting Interestingly, an ADAMTSL2 point mutation, partners, latent TGFβ-binding protein 1 [26,77]. Arg221Cys, which was found in geleophysic dyspla- Intriguingly, Adamtsl2 null mice die shortly after sia and leads to anomalous disulfide bonded dimers, birth with severe lung anomalies associated with a causes Musladin–Lueke syndrome (MLS) in beagle striking increase in fibrillin-2 containing microfibrils dogs via a founder effect [76]. Joint contractures, in the bronchial wall (Hubmacher and Apte, manu- small stature and stiff skin are prominent clinical script submitted). features in affected beagles, but in contrast to These interspecies differences in the outcome of geleophysic dysplasia, pulmonary or cardiac abnor- ADAMTSL2 mutations are not an isolated example, malities are absent, and the affected dogs appear to since Adamts10 inactivation in mice did not precisely have a normal lifespan. These phenotypes are likely phenocopy WMS1; although Adamts10 null mice are Insights on ADAMTS proteases and ADAMTS-like proteins 29 slightly smaller in size, they lack specific skeletal and ADAMTS4 and ADAMTS5 are closely related cardiac anomalies and did not develop ectopia lentis enzymes termed -1 and -2 respective- (Wang. L.W., Kutz, W.E, Apte S.S., manuscript in ly, because of their implication in proteolytic destruc- preparation). The interspecies differences may tion of aggrecan in osteoarthritic joint cartilage [92]. reflect the lack of fibrillin-3 in mice, the different Proteolysis of aggrecan is a major pathogenic expression patterns of the ADAMTS proteins in the mechanism of arthritis, since it exposes other three species, and the recent observation that the cartilage components, e.g., small leucine-rich pro- mouse zonule contains both FBN1 and FBN2, teoglycans and collagens to destruction by MMPs whereas the human zonule contains FBN1 [78,79]. and other proteases. Based on the resistance of ADAMTS13 is the most extensively studied of all Adamts4−/−;Adamts5−/− mice to surgical or inflam- ADAMTS proteases but is not directly related to mation induced joint cartilage loss, the aggreca- extracellular matrix. It cleaves ultra-large vWF multi- nases became major drug targets in arthritis. An mers, which are prothrombogenic because they attractive aspect of their biology was the apparent favor platelet adhesion, into an optimal size required lack of critical physiological roles, suggesting that for normal hemostasis [22,80]. Recessive these proteases were excellent drug targets. Ac- ADAMTS13 mutations [81], or inhibitory ADAMTS13 cordingly, a number of small-molecule autoantibodies lead to abnormal platelet aggregation inhibitors and function-blocking antibodies were and widespread occlusion of microvessels by generated and investigated preclinically [92,93]. platelet thrombi, a condition termed thrombotic However, based on its expression pattern obtained thrombocytopenic purpura. Recent reviews provided from an intragenic lacZ reporter in mice, ADAMTS5 an extensive overview of this disorder [22,80]. is of potential significance to several organ systems ADAMTS18 mutations were described in a syn- [94]. For example, it is strongly expressed in cardiac drome comprising microcornea, myopic chorioretinal outflow tract endocardial cushions, where it is atrophy and telecanthus (Table 1) [82,83]. Little is required for versican proteolysis during the formation presently known about this protease. of valve leaflets, and is implicated in TGFβ signaling An Adamts20 mutant (belted, Adamts20bt) [95,96]. Indeed, Adamts5-deficient mice had re- (Table 2) occurred spontaneously in a number of duced sculpting of pulmonic valves during embryo- inbred mouse colonies, and is a white spotting genesis, and myxomatous mitral valves in adult phenotype affecting the mid-torso [84,85]. Adamts20- hearts [95]. Adamts4−/− mice are apparently nor- bt affects proper colonization of hair follicles in that mal, and neither Adamts4 null nor Adamts5 null region by neural crest derived melanoblasts. Hair mice, nor mice lacking both these genes have any follicles, but not the intervening skin, are the exclusive anomalies of cartilage turnover, endochondral ossi- domain of melanoblasts in mouse skin, and Adamts20 fication, or skeletal function. Thus, a physiological mRNA is expressed by dermal cells around melano- aggrecanase, if one exists at all, remains unidenti- blasts, but not by melanoblasts [84]. ADAMTS20 is fied for now. not required for neural crest cell migration per se, but ADAMTS9 distinguishes itself in several ways. It, acts noncell-autonomously in ensuring the prolifera- along with ADAMTS20, is the largest and most tion and survival of melanoblasts once they reach the conserved of all metalloproteases, with a clearly hair follicles [85]. recognizable nematode ortholog. It contains a C-terminal domain found only in C. elegans Gon-1 and ADAMTS20. It is also by far the physiologically Insights obtained from targeted gene most crucial metalloprotease to be uncovered, with a inactivation in mice and rats startling number of essential functions spanning early, mid and late embryogenesis, and extending into Genetic engineering of mice, and uncommonly of juvenile development and the adult. ADAMTS9 is an rats, has provided null alleles, intragenic reporters absolute requirement for early mouse development, for understanding gene expression, a means of since embryos lacking this protease die during conditional mutagenesis, and null cell lines derived gastrulation [97]. ADAMTS9 haploinsufficiency from mutant animals. ADAMTS1 deficiency in mice leads to cardiac and aortic defects, and to a highly leads to a high incidence of perinatal lethality, penetrant ocular anterior segment dysgenesis together with a high frequency of genitourinary [98,99]. In combination with the Adamts20bt homo- anomalies such as hydronephrosis [86,87]. Surviv- zygous mutant, Adamts9 haploinsufficiency leads to ing female Adamts1 null mice are infertile because death at birth from cleft palate [100], and these mice ADAMTS1 is required for versican proteolysis during have much greater white spotting than Adamts20bt/ maturation and rupture of the ovarian follicle [88–90]. bt [85]. As further evidence of of ADAMTS1 participates in versican proteolysis dur- ADAMTS proteases, mice with combined Adamts5 ing myocardial compaction, a process in which and Adamts20 deficiency have a higher incidence of cardiac myocytes form a functional myocardium soft tissue syndactyly, which results from failure of during embryogenesis [91]. interdigital web regression in the embryo, than 30 Insights on ADAMTS proteases and ADAMTS-like proteins single mutants [101]. Interdigital webs are present regions of an ADAMTS gene locus does not imply not only in aquatic birds and bats, but also during that it is necessarily in a regulatory region of that embryogenesis in humans, mice, and other mam- gene, since the SNP may actually affect regulation of mals, where they participate in development of another locus further away, e.g., if it lies within an digits. They regress by rapid sculpting after digit enhancer. Thus, SNPs do not directly implicate the formation is complete, i.e., by massive apoptosis ADAMTS protease in that disease unless additional coupled with ECM proteolysis. ADAMTS proteolysis conditions are met, for which there are currently few of versican in the interdigit matrix is required for examples. One such SNP for which this burden of apoptosis of interdigit mesenchymal cells, suggesting proof has been partially met leads to a Ser214Pro that these ADAMTS proteases couple matrix proteol- substitution in the ADAMTS7 propeptide and was ysis to cell death during web regression [101]. A similar associated with coronary artery disease [118]. role for Adamts9 in web regression was elucidated first Biochemical analysis following expression of the in combination with Adamts5 or Adamts20 and more Ser and Pro variants suggested that the Pro variant recently, by its limb-specific conditional inactivation quantitatively impaired ADAMTS7 propeptide [97,101]. These studies demonstrate the utility of excision by furin [119], which is thought to be a combining deficiency in two or more ADAMTS proteins prerequisite for proteolytic activity. Thus, the Pro sharing similar activity to elucidate cooperative and/or variant potentially has lower activity and individuals compensatory functions. with the Pro/Pro ADAMTS7 protein are predicted to Recently, rats with a targeted mutation of have reduced protease activity compared to those Adamts16 identified its potential role in regulation with Ser/Ser variants [119]. However, a definitive of blood pressure and male fertility [102,103] and mechanistic link via an effector substrate, as well as other work has suggested a connection between the cell/tissue context in which ADAMTS7 acts to Adamts16 and renal development [104]. modify coronary artery disease remains elusive for now. ADAMTS genetic variation and human disease Genetics and rigorous evaluation of ADAMTS substrate significance Epigenetic modification of ADAMTS proteins is associated with several cancers and is the subject of The genetics and biology of thrombotic thrombocy- another review by Cal and Lopez-Otin in this special topenic purpura suggests an exclusive protease-sub- issue. Recent genome-wide association studies and strate relationship between ADAMTS13 and vWF, no transcriptome analysis have connected ADAMTS loci other ADAMTS13 substrates are currently known. For with several common traits and disorders. ADAMTS9 other family members known to have multiple has been associated with multiple anthropometric substrates, questions about the substrate's biological traits, such as adiposity, age at menarche, adaptation significance arise, since the substrates could very well to high altitude, and disorders such as obesity, type 2 be cleaved in vitro, but not in vivo, e.g., because the diabetes, age-related macular degeneration (AMD), enzyme and putative substrate may never normally and arrest of cervical dilatation during labor [105–112]. encounter one another. For example, numerous The association with AMD was especially striking substrates have been identified for the prototypic because another locus implicated in this condition ADAMTS protease, ADAMTS1, including chondroitin encodes B3GLCT, the glucosyltransferase that mod- sulfate proteoglycans such as aggrecan and versican, ifies TSRs, which are most numerous in ADAMTS9 collagen I, nidogen-1 and nidogen-2, the matricellular [42,113]. Genome-wide transcriptome analysis also proteins thrombospondin-1 and -2, and the cell-an- identified reduction of ADAMTS9 mRNA in bicuspid chored EGFR ligands HB-EGF and amphiregulin aortic valve disease [114]. GWAS of pediatric stroke [120–126]. Of these, however, a significant biological identified association with ADAMTS2, ADAMTS7, impact, primarily using mouse genetic models has ADAMTS12 and ADAMTS13 [115]. ADAMTSL3 was hitherto been established mostly for proteolysis of identified as one of several loci linked to human height versican [89–91], which is also cleaved by ADAMTS4, [116] and as a candidate locus in schizophrenia [117]. ADAMTS5, ADAMTS9, ADAMTS15 and ADAMTS20 In contrast to causative gene mutations in [33,36,85,94,95,97,100,126–128]. Mendelian disorders, GWAS associations remain purely suggestive until functionally validated be- cause the majority of single nucleotide polymor- Mammalian genetics resources and phisms (SNPs) map only to the vicinity of a gene their applications locus, i.e., with few exceptions, they are not within the exons of the gene, and usually do not introduce Many of the engineered and spontaneous mouse amino acid changes in the proteins. Furthermore, genetic models described here are available to users location of the SNP in the intergenic or intronic from repositories such as the Jackson Laboratories Insights on ADAMTS proteases and ADAMTS-like proteins 31

Fig. 1. Schematic showing how Mendelian disorders and engineered rodent ADAMTS mutants, together with genome editing and induced pluripotent stem cells (IPSC), can be integrated into a discovery pipeline for substrates, biological pathway analysis, validation of genome-wide association studies (GWAS) or transcriptome surveys, and drug discovery.

(www.jax.org), or Mutant Mouse Regional Resource Knowledge based resources in genetics keep Centers (https://www.mmrrc.org). The NIH knockout pace with experimental developments. Information mouse project (KOMP; www.komp.org), a member of on human Mendelian disorders and individual the International Knockout Mouse Consortium (IKMC) mouse genes is updated regularly online and is (https://www.mousephenotype.org) is completing readily accessible at the respective websites, mutagenesis of all ADAMTS genes, with several http://www.omim.org and http://www.informatics. mouse mutants already available. The targeting jax.org. Initiatives to comprehensively establish vectors used by IKMC are versatile, since they achieve phenotypes resulting from mouse mutagenesis a “knock-out first” mouseallele,whichcanbebredto are also well under way (http://commonfund.nih. homozygosity to achieve a null phenotype and includes gov/KOMP2/index). Each of the above resources an intragenic reporter such as lacZ for easy readout of can be searched using the ADAMTS gene gene expression patterns. These alleles can then be symbol. rendered suitable for conditional mutagenesis if de- sired. Another mouse genetic resource that is well established, but has not been widely tapped, and Future directions and opportunities carries ADAMTS mutants, is the International Gene Trap Consortium (www.genetrap.org), which provides ADAMTS proteases are presently not as widely trapped mouse ES cell clones for a nominal fee. Gene known and appreciated as MMPs and ADAMs in the trap mutants result from random insertions, and thus ECM and protease communities. Their immense could potentially generate useful, diverse mutations to biological significance revealed by mammalian further understanding of mammalian biology. For genetics shows that they are deserving of consider- example, our group has recently characterized an able attention. This review has attempted to show Adamts9 gene trap allele, in which this normally that when mammalian genetics is integrated into secreted molecule is convertedintoaconstitutively experimental biology and biochemistry (Fig. 1), a membrane-anchored protease (Nandadasa et al., comprehensive, rigorous evaluation of ADAMTS submitted). function will emerge. 32 Insights on ADAMTS proteases and ADAMTS-like proteins

As the schematic in Fig. 1 illustrates, there are Foundation, and by the Knights Templar Eye multidirectional information flows between human Foundation. genetics, genetically engineered models, and in vitro experimental systems that will enrich our under- standing of ADAMTS proteases and their role in extracellular matrix. The availability of experimental Received 18 February 2015; models for common acquired diseases, such as Received in revised form 3 March 2015; diabetes, fibrosis, and atherosclerosis, in mice, Accepted 4 March 2015 means that individual ADAMTS genes implicated Available online 11 March 2015 by GWAS can be tested using knockout mice. For example, recent work used mice with inactivated Keywords: Adamts7 alleles to explore its role in vascular ADAMTS; disease [129,130]. Monoclonal and polyclonal anti- ADAMTS-like; sera can be produced in viable ADAMTS knockout ; mice and rats, since they are more likely to mount an Extracellular matrix; immune response to injected antigens derived from Mouse; the protein product of the inactivated gene. Knockout; The future holds the promise of using new gene Forward genetics; editing technologies such as CRISPR/Cas9 to Reverse genetics; generate mutants at will in any experimental Procollagen; organism. One of the benefits of this technology is Aggrecanase the ability to edit genes in cultured cells, eliminating or minimizing the use of mice to generate null cells. A potential application of this approach relevant to the discovery process is to mutate proteases in cell lines References for high throughput proteomics comparing wild type and protease deficient cells (substrate discovery). [1] Colige A, Li SW, Sieron AL, Nusgens BV, Prockop DJ, Another is the testing of ADAMTS substrate deter- Lapiere CM. cDNA cloning and expression of bovine minants by mutating the cleavage sites or altering procollagen I N-proteinase: a new member of the super- post-translational modifications for a complete family of zinc-metalloproteinases with binding sites for cells understanding of proteolytic mechanisms. For and other matrix components. Proc Natl Acad Sci U S A example, mice with a knock-in mutation of the 1997;94:2374–9. aggrecanase cleavage site that abrogated ADAMTS [2] Hurskainen TL, Hirohata S, Seldin MF, Apte SS. ADAM- processing of aggrecan confirmed that proteolysis at TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and this site was essential for cartilage loss in arthritis genomic distribution of the ADAM-TS family. J Biol Chem [131]. This remains the most rigorous test for 1999;274:25555–63. identifying definitive, biologically meaningful sub- [3] Kuno K, Kanada N, Nakashima E, Fujiki F, Ichimura F, strates, and will now become much more accessible, Matsushima K. Molecular cloning of a gene encoding a new rapid and inexpensive. The widespread utilization of type of metalloproteinase-disintegrin family protein with induced pluripotent stem cell technology (IPSC) thrombospondin motifs as an inflammation associated means that cells obtained from individuals or gene. J Biol Chem 1997;272:556–62. animals with rare Mendelian disorders or engineered [4] Hirohata S, Wang LW, Miyagi M, Yan L, Seldin MF, Keene DR, rodents can be expanded and manipulated with et al. Punctin, a novel ADAMTS-like molecule (ADAMTSL-1) in ease in culture to understand the underlying extracellular matrix. J Biol Chem 2002;22:22. [5] Kramerova IA, Kawaguchi N, Fessler LI, Nelson RE, Chen biological pathways. Taken together with in vivo Y, Kramerov AA, et al. Papilin in development; a pericellular studies that analyze tissues and cells in their natural protein with a homology to the ADAMTS metalloprotein- context in genetically engineered mice, these new ases. Development 2000;127:5475–85. technologies are likely to advance the ADAMTS field [6] Apte SS. A disintegrin-like and metalloprotease (reproly- rapidly. sin-type) with thrombospondin type 1 motif (ADAMTS) superfamily: functions and mechanisms. J Biol Chem 2009;284:31493–7. [7] Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR, et al. Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J Biol Chem 1999;274: Acknowledgments 23443–50. Work in the authors' laboratory was supported by the [8] Huxley-Jones J, Apte SS, Robertson DL, Boot-Handford National Institutes of Health (NIH) through awards RP. The characterisation of six ADAMTS proteases in the basal chordate Ciona intestinalis provides new insights into EY021151, EY024943, Program of Excellence in the vertebrate ADAMTS family. Int J Biochem Cell Biol Glycoscience award HL107147, by Sabrina's 2005;37:1838–45. Insights on ADAMTS proteases and ADAMTS-like proteins 33

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