title: TNF-alpha convertase enzyme from human arthritis-affected cartilage: isolation of cDNA by differential display, expression of the active enzyme, and regulation of TNF-alpha.
abstract: 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 TNF-alpha convertase enzyme (TACE). TACE shows the presence of an unknown prodomain, a cysteine switch, a catalytic domain, a zinc binding region, a disintegrin region, an EGF-like domain, a transmembrane domain, and a unique cytoplasmic region. A TACE construct harboring the signal + prodomain + catalytic region (TACE-SPCdeltaDETCy), expressed in baculovirus could cleave preferentially (approximately 12-fold) the TNF-specific peptide over the matrix metalloproteases peptide in vitro. This recombinant protein also cleaved the natural substrate GST-ProTNF-alpha to TNF-alpha (17 kDa) in vitro. The mRNA for TACE, which is broadly distributed and differentially expressed in a variety of human tissues, is up-regulated in arthritis-affected cartilage, but not normal cartilage. OA-affected cartilage also expressed TNF-alpha mRNA that was not detected in normal cartilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-alpha and IL-8 in ex vivo conditions. Addition of TNF-alphaR fused to IgG Fc fragment (TNF-alphaR:Fc) in the presence or absence of soluble IL-1R (with which it acted additively) significantly attenuated the spontaneous/autocrine release of articular IL-8 in this assay. These experiments demonstrate a functional paracrine/autocrine role of TNF-alpha in OA-affected cartilage that may depend, in part, on up-regulated levels of chondrocyte-derived TACE.
authors.affiliation: Department of Rheumatology and Medicine, Hospital for Joint Diseases, New York, NY 10003, USA.
authors.forename: I R
authors.initials: IR
authors.lastname: Patel
authors.forename: M G
authors.initials: MG
authors.lastname: Attur
authors.forename: R N
authors.initials: RN
authors.lastname: Patel
authors.forename: S A
authors.initials: SA
authors.lastname: Stuchin
authors.forename: R A
authors.initials: RA
authors.lastname: Abagyan
authors.forename: S B
authors.initials: SB
authors.lastname: Abramson
authors.forename: A R
authors.initials: AR
authors.lastname: Amin
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D001172:Arthritis, Rheumatoid / Q000201:enzymology / Q000235:genetics*; D002356:Cartilage / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D017421:Sequence Analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D018076:DNA, Complementary; D014409:Tumor Necrosis Factor-alpha; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human