OBJECTIVE: As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988-90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800-8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a(2)M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a(2)M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP. METHODS: An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1-beta (IL-1beta) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a(2)M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a(2)M were also analyzed. RESULTS: The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-alpha- or IL-1beta-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-alpha- or IL-1beta-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a(2)M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a(2)M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. CONCLUSION: Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, a(2)M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a(2)M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.
Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteolytic enzymes are implicated in a variety of physiological processes, such as collagen maturation, organogenesis, angiogenesis, reproduction and inflammation. Moreover, deficiency or overexpression of certain ADAMTS proteins is directly involved in serious human diseases, including cancer. However, the functional roles of other family members, such as ADAMTS12, remain unknown. Here, by using different in vitro and in vivo approaches, we have evaluated the possible role of ADAMTS12 in the development and progression of cancer. First, we show that expression of ADAMTS12 in Madin-Darby canine kidney (MDCK) cells prevents the tumorigenic effects of hepatocyte growth factor (HGF) by blocking the activation of the Ras-MAPK signalling pathway and that this regulation involves the thrombospondin domains of the metalloproteinase. We also show that addition of recombinant human ADAMTS12 to bovine aortic endothelial cells (BAE-1 cells) abolishes their ability to form tubules upon stimulation with vascular endothelial growth factor (VEGF). Additionally, tumours induced in immunodeficient SCID mice injected with A549 cells overexpressing ADAMTS12 show a remarkable growth deficiency in comparison with tumours formed in animals injected with parental A549 cells. Overall, our data suggest that ADAMTS12 confers tumour-protective functions upon cells that produce this proteolytic enzyme.
Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.
Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.
OBJECTIVE: As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988-90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800-8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a(2)M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a(2)M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP. METHODS: An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1-beta (IL-1beta) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a(2)M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a(2)M were also analyzed. RESULTS: The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-alpha- or IL-1beta-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-alpha- or IL-1beta-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a(2)M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a(2)M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. CONCLUSION: Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, a(2)M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a(2)M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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