ADAMTS proteases are complex secreted enzymes containing a prometalloprotease domain of the reprolysin type attached to an ancillary domain with a highly conserved structure that includes at least ...one thrombospondin type 1 repeat. Known functions of ADAMTS proteases include processing of procollagens and von Willebrand factor as well as catabolism of aggrecan, versican and brevican. They have been demonstrated to have important roles in connective tissue organization, coagulation, inflammation, arthritis, angiogenesis and cell migration. ADAMTS can be grouped into distinct clades within which there is conservation of modular organization, protein sequence, gene structure and possibly, of substrate preference. ADAMTS proteases are synthesized as zymogens, with constitutive proprotein convertase removal of the propeptide occurring prior to secretion. Their enzymatic specificity is heavily influenced by their ancillary domain, which plays a critical role in directing these enzymes to their substrates, the cell surface and the extracellular matrix.
ADAMTS proteases mediate biosynthesis and breakdown of secreted extracellular matrix (ECM) molecules in numerous physiological and disease processes. In addition to their catalytic domains, ADAMTS ...proteases contain ancillary domains, which mediate substrate recognition and ECM binding and confer distinctive properties and roles to individual ADAMTS proteases. Although alternative splicing can greatly expand the structural and functional diversity of ADAMTS proteases, it has been infrequently reported and functional consequences have been rarely investigated. Here, we characterize the structural and functional impact of alternative splicing of ADAMTS17, mutations in which cause Weill‐Marchesani syndrome 4. Two novel ADAMTS17 splice variants, ADAMTS17A and ADAMTS17B, were investigated by structural modeling, mass spectrometry, and biochemical approaches. Our results identify a novel disulfide‐bridged insertion in the ADAMTS17A spacer that originates from inclusion of a novel exon. This insertion results in differential autoproteolysis of ADAMTS17, and thus, predicts altered proteolytic activity against other substrates. The second variant, ADAMTS17B, results from an in‐frame exon deletion and prevents ADAMTS17B secretion. Thus, alternative splicing of the ADAMTS spacer significantly regulates the physiologically relevant proteolytic activity of ADAMTS17, either by altering proteolytic specificity (ADAMTS17A) or by altering cellular localization (ADAMTS17B).
Although hundreds of cytosolic or transmembrane molecules form the primary cilium, few secreted molecules are known to contribute to ciliogenesis. Here, homologous secreted metalloproteases ADAMTS9 ...and ADAMTS20 are identified as ciliogenesis regulators that act intracellularly. Secreted and furin-processed ADAMTS9 bound heparan sulfate and was internalized by LRP1, LRP2 and clathrin-mediated endocytosis to be gathered in Rab11 vesicles with a unique periciliary localization defined by super-resolution microscopy. CRISPR-Cas9 inactivation of ADAMTS9 impaired ciliogenesis in RPE-1 cells, which was restored by catalytically active ADAMTS9 or ADAMTS20 acting in trans, but not by their proteolytically inactive mutants. Their mutagenesis in mice impaired neural and yolk sac ciliogenesis, leading to morphogenetic anomalies resulting from impaired hedgehog signaling, which is transduced by primary cilia. In addition to their cognate extracellular proteolytic activity, ADAMTS9 and ADAMTS20 thus have an additional proteolytic role intracellularly, revealing an unexpected regulatory dimension in ciliogenesis.
•Versican- and hyaluronan-deficient mouse mutants fail to form yolk sac vasculature and blood.•Mouse embryonic stem cells with CRISPR-Cas9 Vcan mutation have impaired angiogenesis and ...hematopoiesis.•The versican-hyaluronan complex is strongly associated with emerging Flk1+ hematoendothelial precursors.•Versican and hyaluronan are each destabilized by the absence of the other.•The versican chondroitin sulfate chains sequester vasculogenic factors VEGF and Indian Hedgehog.
Little is known about extracellular matrix (ECM) contributions to formation of the earliest cell lineages in the embryo. Here, we show that the proteoglycan versican and glycosaminoglycan hyaluronan are associated with emerging Flk1+ hematoendothelial progenitors at gastrulation. The mouse versican mutant Vcanhdf lacks yolk sac vasculature, with attenuated yolk sac hematopoiesis. CRISPR/Cas9-mediated Vcan inactivation in mouse embryonic stem cells reduced vascular endothelial and hematopoietic differentiation within embryoid bodies, which generated fewer blood colonies, and had an impaired angiogenic response to VEGF165. Hyaluronan was severely depleted in Vcanhdf embryos, with corresponding upregulation of the hyaluronan-depolymerase TMEM2. Conversely, hyaluronan-deficient mouse embryos also had vasculogenic suppression but with increased versican proteolysis. VEGF165 and Indian hedgehog, crucial vasculogenic factors, utilized the versican-hyaluronan matrix, specifically versican chondroitin sulfate chains, for binding. Versican-hyaluronan ECM is thus an obligate requirement for vasculogenesis and primitive hematopoiesis, providing a vasculogenic factor-enriching microniche for Flk1+ progenitors from their origin at gastrulation.
ADAMTSL2 mutations cause an autosomal recessive connective tissue disorder, geleophysic dysplasia 1 (GPHYSD1), which is characterized by short stature, small hands and feet, and cardiac defects. ...ADAMTSL2 is a matricellular protein previously shown to interact with latent transforming growth factor-β binding protein 1 and influence assembly of fibrillin 1 microfibrils. ADAMTSL2 contains seven thrombospondin type-1 repeats (TSRs), six of which contain the consensus sequence for O-fucosylation by protein O-fucosyltransferase 2 (POFUT2). O-fucose–modified TSRs are subsequently elongated to a glucose β1-3-fucose (GlcFuc) disaccharide by β1,3-glucosyltransferase (B3GLCT). B3GLCT mutations cause Peters Plus Syndrome (PTRPLS), which is characterized by skeletal defects similar to GPHYSD1. Several ADAMTSL2 TSRs also have consensus sequences for C-mannosylation. Six reported GPHYSD1 mutations occur within the TSRs and two lie near O-fucosylation sites. To investigate the effects of TSR glycosylation on ADAMTSL2 function, we used MS to identify glycan modifications at predicted consensus sequences on mouse ADAMTSL2. We found that most TSRs were modified with the GlcFuc disaccharide at high stoichiometry at O-fucosylation sites and variable mannose stoichiometry at C-mannosylation sites. Loss of ADAMTSL2 secretion in POFUT2−/− but not in B3GLCT−/− cells suggested that impaired ADAMTSL2 secretion is not responsible for skeletal defects in PTRPLS patients. In contrast, secretion was significantly reduced for ADAMTSL2 carrying GPHYSD1 mutations (S641L in TSR3 and G817R in TSR6), and S641L eliminated O-fucosylation of TSR3. These results provide evidence that abnormalities in GPHYSD1 patients with this mutation are caused by loss of O-fucosylation on TSR3 and impaired ADAMTSL2 secretion.
Mutations in the secreted metalloproteinase ADAMTS10 cause recessive Weill-Marchesani syndrome (WMS), comprising ectopia lentis, short stature, brachydactyly, thick skin and cardiac valve anomalies. ...Dominant WMS caused by FBN1 mutations is clinically similar and affects fibrillin-1 microfibrils, which are a major component of the ocular zonule. ADAMTS10 was previously shown to enhance fibrillin-1 assembly in vitro. Here, Adamts10 null mice were analyzed to determine the impact of ADAMTS10 deficiency on fibrillin microfibrils in vivo. An intragenic lacZ reporter identified widespread Adamts10 expression in the eye, musculoskeletal tissues, vasculature, skin and lung. Adamts10−/− mice had reduced viability on the C57BL/6 background, and although surviving mice were slightly smaller and had stiff skin, they lacked brachydactyly and cardiovascular defects. Ectopia lentis was not observed in Adamts10−/− mice, similar to Fbn1−/− mice, most likely because the mouse zonule contains fibrillin-2 in addition to fibrillin-1. Unexpectedly, in contrast to wild-type eyes, Adamts10−/− zonule fibers were thicker and immunostained strongly with fibrillin-2 antibodies into adulthood, whereas fibrillin-1 staining was reduced. Furthermore, fibrillin-2 staining of hyaloid vasculature remnants persisted post-natally in Adamts10−/− eyes. ADAMTS10 was found to cleave fibrillin-2, providing an explanation for persistence of fibrillin-2 at these sites. Thus, analysis of Adamts10−/− mice led to identification of fibrillin-2 as a novel ADAMTS10 substrate and defined a proteolytic mechanism for clearance of ocular fibrillin-2 at the end of the juvenile period.
•Mutations affecting the metalloproteinase ADAMTS10 and fibrillin-1 cause Weill-Marchesani syndrome.•An Adamts10 null-reporter allele is partly lethal in the C57BL/6 strain and shows widespread Adamts10 mRNA expression.•Adamts10 null mouse eyes show fibrillin-2 accumulation in the zonule and vitreous.•Fibrillin-2 is cleaved by ADAMTS10 and is identified as a novel ADAMTS10 substrate.
As a result of an aging population, musculoskeletal disease is a growing source of health and economic burden in the United States. In 2019, musculoskeletal conditions affected approximately 127.4 ...million people (more than a third of the U.S. population); they were the top driver of health-care spending in 2016, with an estimated direct annual cost of $380.9 billion. While musculoskeletal conditions represent a substantial and growing burden in terms of prevalence, disability, and health-care costs, National Institutes of Health (NIH) research funding has remained disproportionately allocated to other disease conditions. Therefore, our purpose was to provide an assessment of the current burden of musculoskeletal disease in terms of prevalence, disability, and health-care costs, and compare the changing burden of disease to trends in NIH funding.
Estrogens and progesterone control breast development and carcinogenesis via their cognate receptors expressed in a subset of luminal cells in the mammary epithelium. How they control the ...extracellular matrix, important to breast physiology and tumorigenesis, remains unclear. Here we report that both hormones induce the secreted protease Adamts18 in myoepithelial cells by controlling Wnt4 expression with consequent paracrine canonical Wnt signaling activation. Adamts18 is required for stem cell activation, has multiple binding partners in the basement membrane and interacts genetically with the basal membrane-specific proteoglycan, Col18a1, pointing to the basement membrane as part of the stem cell niche. In vitro, ADAMTS18 cleaves fibronectin; in vivo, Adamts18 deletion causes increased collagen deposition during puberty, which results in impaired Hippo signaling and reduced Fgfr2 expression both of which control stem cell function. Thus, Adamts18 links luminal hormone receptor signaling to basement membrane remodeling and stem cell activation.
Secreted metalloproteases have diverse roles in the formation, remodeling, and the destruction of extracellular matrix. Recessive mutations in the secreted metalloprotease ADAMTS17 cause ectopia ...lentis and short stature in humans with Weill-Marchesani-like syndrome and primary open angle glaucoma and ectopia lentis in dogs. Little is known about this protease or its connection to fibrillin microfibrils, whose major component, fibrillin-1, is genetically associated with ectopia lentis and alterations in height. Fibrillin microfibrils form the ocular zonule and are present in the drainage apparatus of the eye. We show that recombinant ADAMTS17 has unique characteristics and an unusual life cycle. It undergoes rapid autocatalytic processing in trans after its secretion from cells. Secretion of ADAMTS17 requires O-fucosylation and its autocatalytic activity does not depend on propeptide processing by furin. ADAMTS17 binds recombinant fibrillin-2 but not fibrillin-1 and does not cleave either. It colocalizes to fibrillin-1 containing microfibrils in cultured fibroblasts and suppresses fibrillin-2 (FBN2) incorporation in microfibrils, in part by transcriptional downregulation of Fbn2 mRNA expression. RNA in situ hybridization detected Adamts17 expression in specific structures in the eye, skeleton and other organs, where it may regulate the fibrillin isoform composition of microfibrils.
The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 ...was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.
Display omitted
•ADAMTS9-deficient RPE-1 cells have impaired attachment to a glass substrate.•ADAMTS9 and ADAMTS20 release the MT1-MMP catalytic domain from the cell surface.•Increased cell surface MT1-MMP increases pro-MMP2 activation and collagenolysis.•MT1-MMP knockdown restores attachment of ADAMTS9-deficient RPE-1 cells.
ADAMTS9 and ADAMTS20 are homologous secreted proteases implicated in extracellular matrix proteolysis and ciliogenesis, but few relevant substrates of these proteases are known. Quantitative terminomics comparison of RPE-1 cells with inactivation of ADAMTS9 and parental RPE-1 cells identified transmembrane protease MT1-MMP (MMP14) as a novel ADAMTS9 substrate. Enhanced cell surface MT1-MMP activity in ADAMTS9 gene–edited cells contributes to their adhesion defect but not lack of cilia. One physiological function of ADAMTS9/20 may be to dampen cell surface MT1-MMP activity.
PDF
