Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.
(9/1953)
We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis. (+info)
ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix.
(10/1953)
Cellular disintegrin and metalloproteinases (ADAMs) are a family of genes with a sequence similar to the snake venom metalloproteinases and disintegrins. ADAMTS-1 is a unique ADAM family protein with respect to the presence of thrombospondin type I motifs and the capacity to bind to the extracellular matrix. Because ADAMTS-1 has a potential zinc-binding motif in the metalloproteinase domain, we examined in this study whether ADAMTS-1 is an active metalloproteinase by means of the proteinase trapping mechanism of alpha2-macroglobulin. We found that the soluble type of ADAMTS-1 protein is able to form a covalent-binding complex with alpha2-macroglobulin. Furthermore, the point mutation within the zinc-binding motif of ADAMTS-1 protein eliminates its capacity to bind to alpha2-macroglobulin. These data demonstrate that the metalloproteinase domain of ADAMTS-1 is catalytically active. In addition, we showed that the removal of the pro-domain from the ADAMTS-1 precursor is impaired in the furin-deficient cell line, LoVo, and that the processing ability of the cells is restored by the co-expression of the furin cDNA. These data provide evidence that the ADAMTS-1 precursor is processed in vivo by furin endopeptidase in the secretory pathway. Consequently, ADAMTS-1 is an active metalloprotease that is associated with the extracellular matrix. This study strongly suggests that ADAMTS-1 may play a role in the inflammatory process through its protease activity. (+info)
Tumor necrosis factor-alpha-converting enzyme and tumor necrosis factor-alpha in human dilated cardiomyopathy.
(11/1953)
BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of dilated cardiomyopathy (DCM). TNF-alpha-converting enzyme (TACE) has recently been purified and its complementary DNA cloned. The expression of TACE results in the production of a functional enzyme that has precursor TNF-alpha in the mature form. The aim of this study was to determine whether TACE is expressed with TNF-alpha in myocardium and whether levels of TACE and TNF-alpha are related to clinical severity of DCM. METHODS AND RESULTS: Endomyocardial tissues were obtained from 30 patients with DCM and 5 control subjects. TNF-alpha and TACE mRNA levels were measured by a novel real-time quantitative reverse transcriptase-polymerase chain reaction method. Expression of TNF-alpha and TACE proteins was determined by immunohistochemical analysis. TNF-alpha mRNA was expressed in DCM patients (TNF-alpha/GAPDH ratio 0.85+/-0.24) but not in control subjects. TACE mRNA expression was significantly greater in DCM patients than in control subjects (TACE/GAPDH ratio 2.52+/-0.59 vs 0.03+/-0.02, P<0.05). A positive correlation was found between TNF-alpha and TACE mRNA levels (r=0.779, P<0.001). TACE and TNF-alpha immunostaining was observed in myocytes in patients with DCM. When 2 subgroups of DCM were divided on the basis of left ventricular end-systolic diameter (LVESD) of 45 mm and left ventricular ejection fraction (LVEF) of 40%, the DCM subgroup with high LVESD (>/=45 mm) showed significantly greater expression of TACE (P=0.02) and TNF-alpha (P=0. 001) than did the low LVESD subgroup (<45 mm). In addition, the DCM subgroup with lower LVEF (<40%) showed higher expression of TACE (P=0.006) and TNF-alpha (P=0.01) than did the subgroup with high LVEF (>/=40%). CONCLUSIONS: This study has shown that increased myocardial TACE expression is associated with elevated myocardial TNF-alpha expression in both mRNA and protein levels in clinically advanced DCM. (+info)
Transcripts encoding the sperm surface protein tMDC II are non-functional in the human.
(12/1953)
Five members of the MDC (metalloproteinase-like,disintegrin-like cysteine-rich domain) family of proteins (fertilin alpha, fertilin beta, tMDC I, tMDC II and tMDC III) are expressed on the surface of macaque (Macaca fascicularis) sperm, where they have been proposed to play a role in sperm-egg binding via an interaction between their disintegrin-like domain and one or more integrins on the egg plasma membrane. Of these, two (fertilin alpha and tMDC I) have recently been shown to be non-functional in the human. Here we report the existence of multiple isoforms of human tMDC II transcripts in the human, all of which are also non-functional owing to the presence of deletions and in-frame termination codons, when compared with the macaque orthologue, a finding which is further supported by the lack of immunoreactivity on Western blots of human testis and sperm extracts probed with a macaque anti-tMDC II polyclonal antiserum. These results are discussed in the context of our proposed model for multiple proteins implicated in sperm-egg interactions. (+info)
Regulation of tumor necrosis factor-alpha and tumor necrosis factor converting enzyme in human osteoarthritis.
(13/1953)
A snake venom-like protease isolated by a differential display screen between normal and osteoarthritis (OA)-affected cartilage (designated as cSVP) has a cDNA sequence identical to tumor necrosis factor (TNF)alpha convertase enzyme (TACE) and belongs to the adamalysin group of proteases. It has unique structural properties and when expressed in baculovirus, cleaves preferentially proTNFalpha to TNFalpha. The OA-affected cartilage has upregulated mRNA for TNFalpha and TACE as compared to normal cartilage. TNFalpha and TACE regulate inflammatory mediators in OA-affected cartilage which can be inhibited by both soluble TNFalpha receptors and inhibitors of TACE. These experiments demonstrate a functional paracrine/autocrine role of TNFalpha in OA-affected cartilage that is modulated by upregulated levels of chondrocyte-derived TACE. (+info)
METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity.
(14/1953)
We have studied two related proteins that contain a repeated amino acid motif homologous to the anti-angiogenic type 1 repeats of thrombospondin-1 (TSP1). Complete sequence analysis revealed no other similarities with TSP1, but identified unique signal sequences, as well as metalloprotease and disintegrin-like domains in the NH(2) termini. We named these proteins METH-1 and METH-2 due to the novel combination of metalloprotease and thrombospondin domains. Overall amino acid sequence identity between METH-1 and METH-2 is 51. 7%, yet transcript distribution revealed non-overlapping patterns of expression in tissues and cultured cell lines. To characterize these proteins functionally, we isolated full-length cDNAs, produced recombinant protein, and generated antisera to the recombinant proteins. Both METH-1 and METH-2 represent single copy genes, which encode secreted and proteolytically processed proteins. METH proteins suppressed fibroblast growth factor-2-induced vascularization in the cornea pocket assay and inhibited vascular endothelial growth factor-induced angiogenesis in the chorioallantoic membrane assay. Suppression of vessel growth in both assays was considerably greater than that mediated by either thrombospondin-1 or endostatin on a molar basis. Consistent with an endothelial specific response, METH-1 and METH-2 were shown to inhibit endothelial cell proliferation, but not fibroblast or smooth muscle growth. We propose that METH-1 and METH-2 represent a new family of proteins with metalloprotease, disintegrin, and thrombospondin domains. The distinct distribution of each gene product suggests that each has evolved distinct regulatory mechanisms that potentially allow for fine control of activity during distinct physiological and pathological states. (+info)
Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.
(15/1953)
Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease. (+info)
ADAM-TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family.
(16/1953)
We report the primary structure of three novel, putative zinc metalloproteases designated ADAM-TS5, ADAM-TS6, and ADAM-TS7. All have a similar domain organization, comprising a preproregion, a reprolysin-type catalytic domain, a disintegrin-like domain, a thrombospondin type-1 (TS) module, a cysteine-rich domain, a spacer domain without cysteine residues, and a COOH-terminal TS module. These genes are differentially regulated during mouse embryogenesis and in adult tissues, with Adamts5 highly expressed in the peri-implantation period in embryo and trophoblast. These proteins are similar to four other cognate gene products, defining a distinct family of human reprolysin-like metalloproteases, the ADAM-TS family. The other members of the family are ADAM-TS1, an inflammation-induced gene, the procollagen I/II amino-propeptide processing enzyme (PCINP, ADAM-TS2), and proteins predicted by the KIAA0366 and KIAA0688 genes (ADAM-TS3 and ADAM-TS4). Individual ADAM-TS members differ in the number of COOH-terminal TS modules, and some have unique COOH-terminal domains. The ADAM-TS genes are dispersed in human and mouse genomes. (+info)