The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9+/- mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9+/- mice developed spontaneous corneal neovascularization. beta-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9+/- mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor165, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor165. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1.
Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network.Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furindeficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cellsurface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.Zinc metalloendopeptidases, like most proteases, are synthesized as zymogens, and the N-terminal propeptide is usually excised. Propeptide excision usually leads to enzymatic activation, an important regulatory event, and it can occur intracellularly, at the cell surface, or extracellularly through a variety of proteolytic mechanisms. In one such mechanism, the propeptide is proteolytically excised by serine proteases of the mammalian subtilisin-like proprotein convertase (PC) 4 family (1-5). This mechanism is used by some MMPs (6, 7), many ADAMs (8 -10), and all ADAMTS proteases studied thus far (11,12). In these proteases, removal of the propeptide appears to be mediated by the most widely distributed PC, furin, and occurs within the constitutive secretory pathway, specifically in the trans-Golgi network (TGN) (7-9, 13, 14).Furin is the best studied of the seven PCs implicated in proprotein processing within the constitutive secretory pathway, and it is present in virtually all cells (1, 15). It is a type I transmembrane protein that is itself synthesized as a zymogen that undergoes autocatalytic intramolecular activation (16). Furin cleaves on the carboxyl side of a consensus recognition site that is rich in basic residues (e.g. Arg-Xaa-Arg/Lys-Arg2) (2,4,5,17). Most furin resides in the TGN, but some is present at the plasma membrane and shuttles between the cell surface and the . Furin is also shed from cells and may be functional in the extracellular space (21). Microbial toxins such as the anthrax protective antigen and diphtheria toxin are processed by cell-surface furin, with important implications for their toxicity (20,22). However, the physiological role of cell-surface or secreted furin in processing endogenous cellular products has remained elusive.The ADAMTS proteases are a family of 19 secreted enzymes, of which some have critical physiological functions and have been implicated in inherited human disorders, namely Ehlers-Danlos syndrome type VIIC (ADAMTS2), Weill-Marchesani syndrom...
ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg 74 2 and Arg 287 2) the ADAMTS9 propeptide was also furin-processed at Arg 209 . Substitution of Ala for Arg 74 , Arg 209 , and Arg 287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface. ADAMTS4 proteases have critical roles in many biological processes and in inherited and acquired human disorders (1-5). The 19 enzymes of this family share a conserved organization comprising an N-terminal metalloprotease domain and a C-terminal ancillary domain, which contains the thrombospondin type-1 repeats that are the hallmark of the family (6). ADAMTS9 is the largest enzyme of the family, containing 15 thrombospondin type-1 repeats, and its mRNA is widely expressed during embryonic development and in adult tissues (7-9). In previously published work, we showed that when expressed in COS-1 or HEK293F cells, ADAMTS9 is located at the cell surface or within the pericellular matrix (8), suggesting that, despite the lack of a membrane anchor, it could be considered as an operational cell-surface protease. ADAMTS9 can cleave the large aggregating proteoglycans aggrecan and versican (8), suggesting a role in turnover of extracellular matrix. Thus, like its Caenorhabditis elegans ortholog, Gon-1, which is required for cell migration during gonadal morphogenesis (10), it is possible that ADAMTS9 participates in extracellular proteolysi...
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