ADAMTS9 Cleaves Fibronectin 1

JBC Papers in Press. Published on May 13, 2019 as Manuscript RA118.006479 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.006479 ADAMTS9 cleaves fibronectin A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif 9 (ADAMTS9) regulates fibronectin fibrillogenesis and turnover Lauren W. Wang1, Sumeda Nandadasa1, Douglas S. Annis2, Joanne Dubail1*, Deane F. Mosher2, Belinda B. Willard3 and Suneel S. Apte1 From the 1Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195, 2Departments of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI 53706, 3Proteomics and Metabolomics Core, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195. *Present address: Department of Genetics, INSERM UMR 1163, Université Paris Descartes- Sorbonne Paris Cité, Institut Imagine, AP-HP, Hôpital Necker Enfants Malades, 75015 Paris, France Running title: Fibronectin proteolysis by ADAMTS9 To whom correspondence should be addressed: Suneel S. Apte, MBBS, DPhil, Department of Biomedical Engineering-ND20, Cleveland Clinic Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195. Tel: 216 445 3278; fax: 216 444 9198; email: [email protected] Downloaded from Keywords: metalloprotease, fibronectin, extracellular matrix (ECM), proteolysis, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS), proteolytic enzyme, mass spectrometry, primary cilium, fibrillogenesis _____________________________________________________________________________________ http://www.jbc.org/ ABSTRACT not a catalytically inactive variant, disrupted The secreted metalloprotease ADAMTS9 fibronectin fibril networks formed by fibroblasts in has dual roles in extracellular matrix (ECM) vitro, and ADAMTS9-deficient RPE1 cells by guest on May 13, 2019 turnover and biogenesis of the primary cilium assembled a robust fibronectin fibril network, during mouse embryogenesis. Its gene locus is unlike wild type cells. Targeted LC–MS analysis associated with several human traits and disorders, of fibronectin digested by ADAMTS9-expressing but ADAMTS9 has few known interacting cells identified a semi-tryptic peptide arising from partners or confirmed substrates. Here, using a cleavage at Gly-2196–Leu-2197. We noted that yeast two-hybrid screen for proteins interacting this scissile bond is in the linker between with its C-terminal Gon1 domain, we identified fibronectin modules III17 and I10, a region three putative ADAMTS9-binding regions in the targeted also by other proteases. These findings, ECM glycoprotein fibronectin. Using solid-phase along with stronger fibronectin staining previously binding assays and surface plasmon resonance observed in Adamts9 mutant embryos, suggest that experiments with purified proteins, we ADAMTS9 contributes to fibronectin turnover demonstrate that ADAMTS9 and fibronectin during ECM remodeling. interact. ADAMTS9 constructs, including those ________________________________________ lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lacking fibrillin-1, Extracellular matrix (ECM) is a complex which co-localizes with fibronectin and binds network of proteins, glycoproteins, proteoglycans several ADAMTSs. We observed no fibrillar and glycosaminoglycans that ensures tissue ADAMTS9 staining after blockade of fibroblast structural integrity. It also provides positional fibronectin fibrillogenesis with a peptide based on information and mechanical signals to cells and the functional upstream domain of a sequesters growth factors and cytokines (1, 2). Staphylococcus aureus adhesin. These findings Since nearly all signals perceived by cells are indicate that ADAMTS9 binds fibronectin dimers potentially modifiable by the ECM, a highly and fibrils directly through multiple sites in both regulated ECM composition and organization are molecules. Proteolytically active ADAMTS9, but necessary for correct cell and organ function. 1 ADAMTS9 cleaves fibronectin ECM is continuously remodeled by coordinated disorders of complex etiology, including type 2 biosynthesis and proteolysis of its components (3). diabetes mellitus, obesity and age-related macular Among several enzymes participating in ECM degeneration (22-26). ADAMTS9 was identified as proteolysis are ADAMTS proteases, a family of 19 a tumor suppressor gene in gastric, secreted zinc proteases with diverse and essential nasopharyngeal and esophageal squamous cell roles in mammalian development and homeostatic carcinoma, where it may act via suppression of processes [reviewed in (4-6)]. Naturally occurring angiogenesis (27-29). Despite this considerable mutations affecting ADAMTS proteases cause a biological and disease relevance, the only known variety of Mendelian disorders in humans and ADAMTS9 substrates to date are the ECM other species (4) and ADAMTS proteolytic proteoglycans aggrecan and versican (11). activity may drive the pathology of acquired Previous work has demonstrated that disorders such as osteoarthritis and coronary artery ADAMTS9 enters the secretory pathway, where it disease (7, 8). undergoes obligate glycosylation, binds therein to ADAMTS proteases comprise an N- several chaperones, and arrives at the cell surface, terminal pro-protease domain upstream of a series where its propeptide is cleaved by furin (11, 13, of canonical modules constituting an ancillary 14, 30-32). Recent work shows that furin- domain, whose hallmark is the presence of one or processed, catalytically active ADAMTS9 is several thrombospondin type 1 repeats (TSRs) (9). endocytosed and trafficked to Rab11 vesicles that Downloaded from ADAMTS9 and its homolog ADAMTS20 each form a girdle around the basal body of the primary contain 15 TSRs and a unique C-terminal module cilium (33). There, through an as yet, unknown named Gon1 after their C.elegans ortholog Gon-1 mechanism, ADAMTS9 mediates ciliary growth. (10, 11). They have high domain and sequence Here, adding to the growing understanding of this homology with Gon-1, which is necessary for important protease, we report identification of the http://www.jbc.org/ gonadal morphogenesis (10) as well as with the D. ECM and circulating glycoprotein fibronectin as a melanogaster metalloprotease ADAMTS-A, novel ADAMTS9 binding partner and show that which is essential for tracheal development (12). its assembly and abundance are reduced in the Thus, these proteases are evolutionarily conserved presence of ADAMTS9. These findings are by guest on May 13, 2019 and likely to have significant functions in consistent with stronger fibronectin staining mammals. Indeed, genetically engineered mouse previously observed in mouse embryos mutants has shown that ADAMTS9 is homozygous for the loss-of-function Adamts9Gt indispensable for several aspects of mammalian mutation (14, 19). embryogenesis. Adamts9 null mouse embryos fail to undergo gastrulation, a crucial early Results morphogenetic event during which the definitive Fibronectin is a novel binding binding partner of germ layers are formed, and die by 7 days of the ADAMTS9 Gon1 domain gestation (13). This mouse allele, and others, A yeast two-hybrid screen of a human including a hypomorphic Adamts9 allele placenta library of Gal4 fusion proteins was (Adamts9Gt), chemically-induced point mutations, undertaken using the human ADAMTS9 Gon1 and conditional Adamts9 deletion have identified module (residues 1733-1935, SwissProt accession crucial functions in craniofacial, neural, vascular, no. Q9P2N4) (Fig. 1A) as the “bait”, fused in- cardiac, eye, pigment and limb development (14- frame and downstream of the Gal4 DNA-binding 19). In adult mice, ADAMTS9 has a role in domain. In addition to several other secreted or parturition via regulation of ECM control of cell-surface proteins, which could be putative uterine smooth muscle cell differentiation (20). binding partners, eight independent ADAMTS9 ADAMTS9 and ADAMTS20 were recently found Gon1 interacting cDNA clones, each encoding to be essential for biogenesis of the primary part of fibronectin (Fig. 1B) were identified in- cilium, providing an unexpected role for these frame with the Gal4 DNA-activating domain. The secreted proteases in formation of a cellular selected interacting domains (SIDs), of organelle (6, 21). Genome-wide analysis has fibronectin, which are minimum interacting linked the ADAMTS9 locus to diverse genetic regions determined by the overlap between these traits, disease susceptibility markers and common clones (34), are shown in Fig. 1B. The 3 SIDs, 2 ADAMTS9 cleaves fibronectin SID1, SID2 and SID3, spanned three non- as identified in the yeast-two-hybrid screen was overlapping regions of fibronectin comprising replicated using proteins purified after secretion both type I and type III repeats (Fig. 1B). from eukaryotic cells, and the TSR9-15 region of Yeast two-hybrid interactions occur in a ADAMTS9 was also found to contain one or more reducing environment between proteins that are binding sites for fibronectin. synthesized without post-translational modifications typical for secreted mammalian ADAMTS9 constructs bind fibronectin fibrils proteins. Such modifications include introduction Many cell types that secrete fibronectin or are of disulfide bonds and N-glycosylation. Two provided exogenous fibronectin assemble it into disulfide bonds stabilize fibronectin type I fibrils, starting within a few hours of attachment if modules (35). The human ADAMTS9 Gon1 plated at sufficiently high density (36). Fibronectin

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