The Τumor Necrosis Factor-R Converting Enzyme (TACE): A Unique Metalloproteinase with Highly Defined Substrate Selectivity † Mohita J. Mohan, ‡ Theresa Seaton, §,| Justin Mitchell, § Anne Howe, § Kevin Blackburn, § William Burkhart, § Mary Moyer, § Inder Patel, §,⊥ Gregory M. Waitt, § J. David Becherer, § Marcia L. Moss, # and Marcos E. Milla* ,‡ Department of Biochemistry and Biophysics and Johnson Research Foundation, UniVersity of PennsylVania School of Medicine, Philadelphia, PennsylVania 19104, GlaxoSmithKline Inc., Research Triangle Park, North Carolina 27709, and Cognosci Inc., 2 DaVis DriVe, Research Triangle Park, North Carolina 27709 ReceiVed April 24, 2002; ReVised Manuscript ReceiVed May 30, 2002 ABSTRACT: TNFR converting enzyme (TACE) processes precursor TNFR between Ala76 and Val77, yielding a correctly processed bioactive 17 kDa protein. Genetic evidence indicates that TACE may also be involved in the shedding of other ectodomains. Here we show that native and recombinant forms of TACE efficiently processed a synthetic substrate corresponding to the TNFR cleavage site only. For all other substrates, conversion occurred only at high enzyme concentrations and prolonged reaction times. Often, cleavage under those conditions was accompanied by nonspecific reactions. We also compared TNFR cleavage by TACE to cleavage by those members of the matrix metalloproteinase (MMP) family previously implied in TNFR release. The specificity constants for TNFR cleavage by the MMPs were approximately 100-1000-fold slower relative to TACE. MMP 7 also processed precursor TNFR at the correct cleavage site but did so with a 30-fold lower specificity constant relative to TACE. In contrast, MMP 1 processed precursor TNFR between Ala74 and Gln75, in addition to between Ala76 and Val77, while MMP 9 cleaved this natural substrate solely between Ala74 and Gln75. Additionally, the MMP substrate Dnp-PChaGC(Me)HK(NMA)-NH 2 was not cleaved at all by TACE, while collagenase (ΜΜP 1), gelatinase (ΜΜP 9), stromelysin 1 (MMP 3), and matrilysin (MMP 7) all processed this substrate efficiently. All of these results indicate that TACE is unique in terms of its specificity requirements for substrate cleavage. One of the most promising targets for the treatment of inflammatory diseases is the TNFR 1 converting enzyme (TACE or ADAM 17). TACE is a zinc metalloproteinase that cleaves 26 kDa precursor TNFR to its mature, 17 kDa form (1, 2). Precursor TNFR exists as a membrane-bound form in certain cell types, while mature TNFR is secreted into the extracellular compartment (3). If the precursor form of TNFR contains a mutated cleavage site, it does not get released from cells, indicating that a processing event is required for TNFR to be secreted (4, 5). Inhibitors of TACE may therefore be therapeutically useful in the treatment of any disease state where inhibition of TNFR release has been validated as a target, such as arthritis and Crohn’s disease (6-8). A class of inhibitors known as hydroxamates have been shown to inhibit TNFR secretion from a variety of cell types (9-11). These inhibitors were previously demonstrated to inhibit a class of enzymes known as the matrix metallopro- teases (ΜΜPs). A microsomal activity that processes precur- sor TNFR in vitro was purified from membrane preparations, and the enzyme was cloned and expressed (1, 2). TACE, while inhibited by matrix metalloprotease inhibitors, belongs to the disintegrin family of metalloproteases or ADAMs (12). TACE appears to be the physiological TNFR converting enzyme since TNFR release is reduced by 90% in cells derived from TACE -/- mice (1). Surprisingly, it was later found that the shedding of other ectodomains was also blocked in these knockout cells. Those ectodomains affected so far include the amyloid precursor protein (APP; 13), L-selectin (14), TNFR receptors I and II (TNFR-I and -II; † Supported partly by National Institutes of Health Grant AR45949 to M.E.M. * To whom correspondence should be addressed. Tel: 215-898-7500. Fax: 215-573-4764. E-mail: mmilla@mail.med.upenn.edu. ‡ University of Pennsylvania School of Medicine. § GlaxoSmithKline Inc. | Current address: Dow Corning Corp., Midland MI 48686-0994. ⊥ Current address: Rheumatology Research Labs, Hospital for Joint Diseases, New York University, New York, NY 10003. # Cognosci Inc. 1 Abbreviations: TNFR, tumor necrosis factor-R; TACE, TNFR converting enzyme; mTACE, microsomal TACE; rTACE, mature ectodomain of recombinant TACE; ΜΜP, matrix metalloproteinase; ADAM, a disintegrin and metalloproteinase; GW 9471, (2R,3S)-2-(2- methyl-1-propyl)-3-N-hydroxycarbamoyl-4-phenylsulfanylbutanoic acid [(1S)-1-methylcarbamoyl-2-phenyl-1-ethyl]amide; MMP 1, human fibroblast collagenase; ΜΜP 3, stromelysin 1; ΜΜP 7, matrilysin; ΜΜP 9, 92 kDa gelatinase; Dnp, 2,4-dinitrophenyl; Nma, N-methyl- anthranilic acid; NBD, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]- hexanoic acid; DMC, 7-dimethylaminocoumarin-4-acetate; DMSO, dimethyl sulfoxide; Cha, -cyclohexylalanyl; C(Me), S-methylcysteine; Nva, norvaline; HFBA, heptafluorobutyric acid; NR, no reaction; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, poly(vinylidene difluoride); APP, amyloid precursor protein; TNFR, TNFR receptor; TGFR, transforming growth factor-R; HER4, erbB4/HER4 epidermal growth factor receptor isoform JM-a; IL-6R, interleukin-6 receptor; M-CSFR, macrophage colony-stimulating factor receptor; GHR, growth hormone receptor; PrP c , cellular prion protein; KL-1, c-kit ligand; RANKL, receptor of activated nuclear factor κ-B (NFκB) ligand; MT1-MMP, membrane-type 1 matrix metalloproteinase. 9462 Biochemistry 2002, 41, 9462-9469 10.1021/bi0260132 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/27/2002