Specific Sequence Elements Are Required for the Expression of Functional Tumor Necrosis Factor-α-converting Enzyme (TACE)
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.30563
ISSN1083-351X
AutoresMarcos E. Milla, M. Anthony Leesnitzer, Marcia L. Moss, William C. Clay, H. Luke Carter, Ann B. Miller, Jui‐Lan Su, Millard H. Lambert, Derril H. Willard, Douglas M. Sheeley, Thomas A. Kost, William Burkhart, Mary B. Moyer, Kevin Blackburn, Gregory Pahel, Justin Mitchell, Christine R. Hoffman, J. David Becherer,
Tópico(s)Research on Leishmaniasis Studies
ResumoThe tumor necrosis factor-α-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-α secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE. The tumor necrosis factor-α-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-α secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE. tumor necrosis factor-α TNFα-converting enzyme 4-aminophenylmercuric acetate a disintegrin and metalloprotease polyacrylamide gel electrophoresis high performance liquid chromatography liquid chromatography-mass spectrometry TNFα1 is a potent cytokine that is secreted by activated monocytes and macrophages in a tightly regulated manner (1Vasalli P. Annu. Rev. Immunol. 1992; 10: 411-452Crossref PubMed Scopus (1798) Google Scholar). Upon release, TNFα mediates the recruitment and activation of inflammatory cells to injured or infected tissues (2Old L.J. Science. 1985; 230: 630-632Crossref PubMed Scopus (1290) Google Scholar). Elevated levels of circulating TNFα have been demonstrated in several acute and chronic pathological states, such as lipopolysaccharide-induced septic shock, arthritis, pleurisy, Crohn's disease, and inflammatory bowel disease (3Beutler B. Cerami A. Annu. Rev. Biochem. 1988; 57: 505-518Crossref PubMed Scopus (728) Google Scholar). TNFα is synthesized as a pro, membrane-anchored form facing the lumenal/extracellular side of the secretory pathway. Our group and others have shown that proTNFα is released from cells after endoproteolytic cleavage at positions Ala76-Val77, mediated by a zinc metalloprotease sensitive to hydroxamic acid inhibitors (4McGeehan G.M. Becherer J.D. Bast Jr., R.C. Boyer C.M. Champion B. Connolly K.M. Conway J.G. Furdon P. Karp S. Kidao S. McElroy A.B. Nichols J. Pryzwansky K.M. Schoenen F. Sekut L. Truesdale A. Verghese M. Warner J. Ways J.P. Nature. 1994; 370: 558-561Crossref PubMed Scopus (544) Google Scholar, 5Mohler K.M. Sleath P.R. Fitzner J.N. Cerretti D.P. Alderson M. Kerwar S.S. Torrance D.S. Otten-Evans C. Greenstreet T. Weerawarna K. Kronheim S.R. Petersen M. Gerhart M. Kozlosky C.J. March C.J. Black R.A. Nature. 1994; 370: 218-220Crossref PubMed Scopus (571) Google Scholar, 6Gearing A.J.H. Beckett P. Christodoulou M. Churchill M. Clements J. Davidson A.H. Drummond A.H. Galloway W.A. Gilbert R. Gordon J.L. Leber T.M. Mangan M. Miller K. Nayee P. Owen K. Patel S. Thomas W. Wells G. Wood L.M. Woolley K. Nature. 1994; 370: 555-557Crossref PubMed Scopus (1104) Google Scholar). Because neutralization of TNFα activity has been demonstrated in the clinic, this enzyme constitutes a potential target for drug discovery.The TNFα-converting enzyme (TACE) was purified to homogeneity and cloned (7Moss M.L. Jin S.-L.C. Milla M.E. Burkhart W. Carter H.L. Chen W.-J. Clay W.C. Didsbury J.R. Hassler D. Hoffman C.R. Kost T.A. Lambert M.H. Leesnitzer M.A. McCauley P. McGeehan G. Mitchell J. Moyer M. Pahel G. Rocque W. Overton L.K. Schoenen F. Seaton T. Su J.-L. Warner J. Willard D. Becherer J.D. Nature. 1997; 385: 733-736Crossref PubMed Scopus (1467) Google Scholar, 8Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2675) Google Scholar). Analysis of its amino acid sequence demonstrates a multidomain protein closely resembling members of the disintegrin family of metalloproteases, also commonly referred to as ADAMs or metalloprotease and disintegrin-containing proteins (9Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). Starting at the N terminus, TACE exhibits a classical signal peptide followed by a ∼200-residue pro domain that includes a consensus cysteine switch motif (PKVCGY186), which can act as an inhibitor by ligating the zinc ion in the catalytic site (10Van Wart H.E. Birkedal-Hansen B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5581Crossref PubMed Scopus (1192) Google Scholar, 32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjumen-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The catalytic domain starts downstream from a consensus furin cleavage site (RVKRR215) and contains a canonical zinc binding site and a MYP loop involved in formation of the P1′ pocket (11Maskos K. Fernandez-Catalan C. Huber R. Bourenkov G.P. Bartunik H. Ellestad G.A. Reddy P. Wolfson M.F. Rauch C.T. Castner B.J. Davis R. Clarke H.R.G. Petersen M. Fitzner J.M. Cerretti D.P. March C.J. Paxton R.J. Black R.A. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3408-3412Crossref PubMed Scopus (361) Google Scholar). A ∼200-amino acid cysteine-rich domain follows, including a 100-amino acid disintegrin-like region. A single transmembrane domain defines the end of the catalytic domain of TACE and is followed by a ∼150-residue cytosolic tail that contains consensus sequences for binding to proteins containing Src homology 2 and Src homology 3 domains. Little is known at this point about the role these different domains play in regulating catalytic activity and physiological substrate recognition. TACE, ADAM 9, and ADAM 10 are the only members of this family for which proteolytic activity on a specific substrate has been demonstrated. TACE and Kuzbanian (an ADAM metalloprotease involved in processing of the Drosophila protein Notch and neuronal development (12Qi H. Rand M.D. Wu X. Sestan N. Wang W. Rakic P. Xu T. Artavanis-Tsakonas S. Science. 1999; 283: 91-94Crossref PubMed Scopus (374) Google Scholar)) are the only two members of this family for which the physiological substrates are known.We report here the construction, overexpression, and characterization of full-length recombinant TACE and a series of truncates nested around the predicted catalytic domain sequence using baculoviral expression vectors. A comparison is presented of the activity of the different truncates against a synthetic peptide substrate containing the cleavage site of this cytokine. Such recombinant forms of TACE will be of use in establishing relationships between the domain architecture of TACE and the function and regulation of this enzyme.DISCUSSIONOur goal is to understand the relationship among the domain architecture, function, and regulation of TACE. The studies reported here show that only certain recombinant forms of TACE can be overexpressed in insect cells in a soluble, secreted form, fully active as compared with native TACE purified from a human monocytic cell line.Insect cells expressed mature, functional full-length TACE at very low levels. Most of the protease remained in its immature form, with the pro domain intact, as judged from Western blots of cell extracts. This result is particularly perplexing because truncates Arg473(catalytic domain) and Arg651 (catalytic and cysteine-rich domains) are maturated at the predicted furin cleavage site in insect cells. Full-length TACE contains a C-terminal extension of Arg651, including a transmembrane domain and a cytoplasmic tail. We hypothesize that this cytoplasmic domain contains a negative signal that must be reversed to allow transport of TACE to the secretory compartment where activation occurs. It is intriguing that this domain contains a potential tyrosine phosphorylation site in the context of a Src homology 2 domain binding site, KKLDKQYESL705, as well as a potential Src homology 3 domain binding site, PAPQTPGR738. In fact, the tyrosine kinase domain of the epidermal growth factor receptor can convert the recombinant TACE cytoplasmic tail (produced in E. coli) to its phosphotyrosine form.2Pradines-Figueres and Raetz (24Pradines-Figueres A. Raetz C.R.H. J. Biol. Chem. 1992; 267: 23261-23268Abstract Full Text PDF PubMed Google Scholar) have shown that TNFα secretion from MonoMac 6 cells occurs only after stimulation of these cells with both bacterial lipopolysaccharide and phorbol-12-myristate-13-acetate. Although proTNFα biosynthesis is enhanced after lipopolysaccharide stimulation, release does not occur to significant levels in the absence of phorbol 12-myristate 13-acetate (13Moss M.L. Becherer J.D. Milla M.E. Pahel G. Lambert M. Andrews R. Frye S. Haffner C. Cowan D. Maloney P. Dixon E.P. Jansen M. Mitchell J.L. Leesnitzer T. Warner J. Conway J. Bickett D.M. Bird M. Priest R. Reinhard J. Lin P. Bradshaw D. Nixon J.S. Bottomley K. Metalloproteinases as Targets for Anti-inflammatory Drugs. Birkhauser Publishers, Basel1998: 187-204Google Scholar). Potentially, a phorbol 12-myristate 13-acetate-initiated phosphorylation or dephosphorylation event at the cytosolic tail of TACE precedes TACE activation, either via a conformational change that exposes the cleavage site to the processing protease(s) or by directing transport of TACE to the compartment where activation takes place. Remarkably, it has been shown that mutation of an equivalent tyrosine residue to phenylalanine in the cytoplasmic tail of furin results in failure of this enzyme to localize properly inside the cell (25Schäfer W. Stroh A. Berhöfer S. Seiler J. Vey M. Kruse M.-L. Kern H.F. Klenk H.-D. Garten W. EMBO J. 1995; 14: 2424-2435Crossref PubMed Scopus (219) Google Scholar, 26Takahashi S. Nakagawa T. Banno T. Watanabe T. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 28397-28401Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Therefore, it is possible that insect cells contain a TACE maturating activity but lack the cellular factors responsible for making TACE available to such protease. Both Arg473 and Arg651, after signal peptide cleavage, likely reach the compartment where maturation occurs via bulk flow secretion.Maturation by proteolytic cleavage is essential in order to generate active TACE. The pro domain strongly inhibits TACE, probably through complexing the catalytic zinc in the active site via an unpaired cysteine residue in the putative cysteine switch box. Our observation that the mercurial compound APMA efficiently mediates dissociation of the pro-catalytic domain complex supports this hypothesis. Additionally, peptides from the putative Cys-switch region do inhibit TACE activity (32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjumen-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar).Interestingly, APMA-mediated dissociation of the pro-catalytic domain complex occurs at concentrations well below the ones used for activating matrix metalloproteinases. At higher concentrations, APMA acts as an inhibitor of TACE. We do not know the mechanism of this inactivation effect. APMA may react with one or more cysteine residues in the catalytic domain that are essential for keeping TACE in its functional form. Release of the cleaved pro domain appears not to be a simple diffusion-driven process, because pro remained tightly associated to the catalytic domain through several purification steps and even in the presence of substantial concentrations of the chemical denaturant urea. This suggests a role for the cysteine-rich domain in freeing the catalytic domain from the pro domain as indicated by the apparent absence of complex when Arg651 was overexpressed and purified. The cysteine-rich domain may have an interacting surface with the catalytic domain that overlaps the one used by the pro domain. Given the remarkable sensitivity of TACE pro domain to proteolysis, it is likely that once it is detached from the catalytic domain, its degradation prevents reassociation with the catalytic domain. It might also be possible that the cysteine-rich domain induces changes in conformation that dramatically reduce the affinity of the pro domain for the catalytic domain. The observed differences in glycosylation sites between Arg473 and Arg651, as well as the observed differences in oligosaccharide processing, may also have a role in stabilizing/destabilizing the pro-catalytic domain complex. It is less likely, however, that the cysteine-rich domain acts as an exchange factor, accepting pro after TACE activation, because of the apparent absence of Arg651-pro complexes.The pro domain is not only an inhibitor of the catalytic domain, but also appears to have at least some of the properties observed in chaperones, as it seems to facilitate either secretion or folding or both. When expressed separately, both domains failed to be secreted and appeared to be extremely sensitive to proteolysis. It seems unlikely that this result is due to inability of the endoplasmic reticulum signal peptidase to access its cleavage site: about half of the purified R473Δpro protein did have Ala17 as its N terminus, consistent with signal peptidase processing. In addition, the R473Δpro construct contains a signal peptide-catalytic domain fusion starting well downstream (after Pro24) from the signal peptidase cleavage site. Furthermore, a similar fusion of the signal peptide to the cysteine-rich domain (Fig. 1) is efficiently secreted by insect cells, and is recognized by a monoclonal antibody specific for the native form of this domain. 3A. Miller and M. Milla, unpublished work. This dependence is not surprising: it has been previously reported for several proteases, including zinc metalloproteases, that the pro domain is essential for proper secretion (27Wetmore D.R. Hardman K.D. Biochemistry. 1996; 35: 6549-6558Crossref PubMed Scopus (46) Google Scholar, 28Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hagmann W.K. Hermes J.D. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (270) Google Scholar). Similar results have been described for other members of the ADAMs family, namely metalloprotease and disintegrin-containing proteins 9 and 15 (32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjumen-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). For at least two proteases, the α-lytic protease and subtilisin, the pro domain seems to control the kinetics of the folding reaction, supporting an intramolecular chaperone role (29Baker D. Sohl J.L. Agard D.A. Nature. 1992; 356: 263-265Crossref PubMed Scopus (283) Google Scholar, 30Wang L. Ruan B. Ruvinov S. Bryan P.N. Biochemistry. 1998; 37: 3165-3171Crossref PubMed Scopus (43) Google Scholar).How does TACE quaternary structure relate to its function? Both proTNFα and TNFα are homotrimers. As the release of proTNFα from cellular membranes requires cleaving three stems, we investigated the oligomerization state of these recombinant forms by equilibrium sedimentation analysis to address whether TACE itself is a trimer. Both Arg473 and Arg651 were monomeric at concentrations well above the ones used to demonstrate cleavage against the synthetic peptide substrate. Bode and co-workers (11Maskos K. Fernandez-Catalan C. Huber R. Bourenkov G.P. Bartunik H. Ellestad G.A. Reddy P. Wolfson M.F. Rauch C.T. Castner B.J. Davis R. Clarke H.R.G. Petersen M. Fitzner J.M. Cerretti D.P. March C.J. Paxton R.J. Black R.A. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3408-3412Crossref PubMed Scopus (361) Google Scholar) published the three-dimensional structure of the catalytic domain of TACE (31Clarke H.R. Wolfson M.F. Rauch C.T. Castner B.J. Huang C.P. Gerhart M.J. Johnson R.S. Cerretti D.P. Paxton R.J. Price V.L. Black R.A. Protein Expression Purif. 1998; 13: 104-110Crossref PubMed Scopus (17) Google Scholar). They propose a model in which the "right side" of the catalytic domain forms such interactions with the base of the proTNFα trimer. A role for the transmembrane or cytoplasmic domains in substrate recognition or oligomerization remains to be investigated.The extreme salt sensitivity of TACE is intriguing. Concentrations of NaCl that completely inhibit its activity (100 mm) do not seem to have an effect on its oligomeric state or solubility. Examination of electrostatic surface potential models of TACE (11Maskos K. Fernandez-Catalan C. Huber R. Bourenkov G.P. Bartunik H. Ellestad G.A. Reddy P. Wolfson M.F. Rauch C.T. Castner B.J. Davis R. Clarke H.R.G. Petersen M. Fitzner J.M. Cerretti D.P. March C.J. Paxton R.J. Black R.A. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3408-3412Crossref PubMed Scopus (361) Google Scholar) reveals that the relatively flat left-hand side of the active-site cleft is positively charged. Therefore, disruption of favorable electrostatic interactions with the substrate peptide by chloride ions is formally possible. The physiological meaning of this effect remains to be established.We believe that some of these truncates will be useful in studies aimed at understanding how TACE interacts with its natural substrate, proTNFα, as well as to dissect the levels of regulation of its biogenesis and activity. In turn, this should allow us to explore new avenues for targeting this key enzyme for therapeutic intervention in inflammatory diseases, cancer, and AIDS. TNFα1 is a potent cytokine that is secreted by activated monocytes and macrophages in a tightly regulated manner (1Vasalli P. Annu. Rev. Immunol. 1992; 10: 411-452Crossref PubMed Scopus (1798) Google Scholar). Upon release, TNFα mediates the recruitment and activation of inflammatory cells to injured or infected tissues (2Old L.J. Science. 1985; 230: 630-632Crossref PubMed Scopus (1290) Google Scholar). Elevated levels of circulating TNFα have been demonstrated in several acute and chronic pathological states, such as lipopolysaccharide-induced septic shock, arthritis, pleurisy, Crohn's disease, and inflammatory bowel disease (3Beutler B. Cerami A. Annu. Rev. Biochem. 1988; 57: 505-518Crossref PubMed Scopus (728) Google Scholar). TNFα is synthesized as a pro, membrane-anchored form facing the lumenal/extracellular side of the secretory pathway. Our group and others have shown that proTNFα is released from cells after endoproteolytic cleavage at positions Ala76-Val77, mediated by a zinc metalloprotease sensitive to hydroxamic acid inhibitors (4McGeehan G.M. Becherer J.D. Bast Jr., R.C. Boyer C.M. Champion B. Connolly K.M. Conway J.G. Furdon P. Karp S. Kidao S. McElroy A.B. Nichols J. Pryzwansky K.M. Schoenen F. Sekut L. Truesdale A. Verghese M. Warner J. Ways J.P. Nature. 1994; 370: 558-561Crossref PubMed Scopus (544) Google Scholar, 5Mohler K.M. Sleath P.R. Fitzner J.N. Cerretti D.P. Alderson M. Kerwar S.S. Torrance D.S. Otten-Evans C. Greenstreet T. Weerawarna K. Kronheim S.R. Petersen M. Gerhart M. Kozlosky C.J. March C.J. Black R.A. Nature. 1994; 370: 218-220Crossref PubMed Scopus (571) Google Scholar, 6Gearing A.J.H. Beckett P. Christodoulou M. Churchill M. Clements J. Davidson A.H. Drummond A.H. Galloway W.A. Gilbert R. Gordon J.L. Leber T.M. Mangan M. Miller K. Nayee P. Owen K. Patel S. Thomas W. Wells G. Wood L.M. Woolley K. Nature. 1994; 370: 555-557Crossref PubMed Scopus (1104) Google Scholar). Because neutralization of TNFα activity has been demonstrated in the clinic, this enzyme constitutes a potential target for drug discovery. The TNFα-converting enzyme (TACE) was purified to homogeneity and cloned (7Moss M.L. Jin S.-L.C. Milla M.E. Burkhart W. Carter H.L. Chen W.-J. Clay W.C. Didsbury J.R. Hassler D. Hoffman C.R. Kost T.A. Lambert M.H. Leesnitzer M.A. McCauley P. McGeehan G. Mitchell J. Moyer M. Pahel G. Rocque W. Overton L.K. Schoenen F. Seaton T. Su J.-L. Warner J. Willard D. Becherer J.D. Nature. 1997; 385: 733-736Crossref PubMed Scopus (1467) Google Scholar, 8Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2675) Google Scholar). Analysis of its amino acid sequence demonstrates a multidomain protein closely resembling members of the disintegrin family of metalloproteases, also commonly referred to as ADAMs or metalloprotease and disintegrin-containing proteins (9Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). Starting at the N terminus, TACE exhibits a classical signal peptide followed by a ∼200-residue pro domain that includes a consensus cysteine switch motif (PKVCGY186), which can act as an inhibitor by ligating the zinc ion in the catalytic site (10Van Wart H.E. Birkedal-Hansen B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5581Crossref PubMed Scopus (1192) Google Scholar, 32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjumen-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The catalytic domain starts downstream from a consensus furin cleavage site (RVKRR215) and contains a canonical zinc binding site and a MYP loop involved in formation of the P1′ pocket (11Maskos K. Fernandez-Catalan C. Huber R. Bourenkov G.P. Bartunik H. Ellestad G.A. Reddy P. Wolfson M.F. Rauch C.T. Castner B.J. Davis R. Clarke H.R.G. Petersen M. Fitzner J.M. Cerretti D.P. March C.J. Paxton R.J. Black R.A. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3408-3412Crossref PubMed Scopus (361) Google Scholar). A ∼200-amino acid cysteine-rich domain follows, including a 100-amino acid disintegrin-like region. A single transmembrane domain defines the end of the catalytic domain of TACE and is followed by a ∼150-residue cytosolic tail that contains consensus sequences for binding to proteins containing Src homology 2 and Src homology 3 domains. Little is known at this point about the role these different domains play in regulating catalytic activity and physiological substrate recognition. TACE, ADAM 9, and ADAM 10 are the only members of this family for which proteolytic activity on a specific substrate has been demonstrated. TACE and Kuzbanian (an ADAM metalloprotease involved in processing of the Drosophila protein Notch and neuronal development (12Qi H. Rand M.D. Wu X. Sestan N. Wang W. Rakic P. Xu T. Artavanis-Tsakonas S. Science. 1999; 283: 91-94Crossref PubMed Scopus (374) Google Scholar)) are the only two members of this family for which the physiological substrates are known. We report here the construction, overexpression, and characterization of full-length recombinant TACE and a series of truncates nested around the predicted catalytic domain sequence using baculoviral expression vectors. A comparison is presented of the activity of the different truncates against a synthetic peptide substrate containing the cleavage site of this cytokine. Such recombinant forms of TACE will be of use in establishing relationships between the domain architecture of TACE and the function and regulation of this enzyme. DISCUSSIONOur goal is to understand the relationship among the domain architecture, function, and regulation of TACE. The studies reported here show that only certain recombinant forms of TACE can be overexpressed in insect cells in a soluble, secreted form, fully active as compared with native TACE purified from a human monocytic cell line.Insect cells expressed mature, functional full-length TACE at very low levels. Most of the protease remained in its immature form, with the pro domain intact, as judged from Western blots of cell extracts. This result is particularly perplexing because truncates Arg473(catalytic domain) and Arg651 (catalytic and cysteine-rich domains) are maturated at the predicted furin cleavage site in insect cells. Full-length TACE contains a C-terminal extension of Arg651, including a transmembrane domain and a cytoplasmic tail. We hypothesize that this cytoplasmic domain contains a negative signal that must be reversed to allow transport of TACE to the secretory compartment where activation occurs. It is intriguing that this domain contains a potential tyrosine phosphorylation site in the context of a Src homology 2 domain binding site, KKLDKQYESL705, as well as a potential Src homology 3 domain binding site, PAPQTPGR738. In fact, the tyrosine kinase domain of the epidermal growth factor receptor can convert the recombinant TACE cytoplasmic tail (produced in E. coli) to its phosphotyrosine form.2Pradines-Figueres and Raetz (24Pradines-Figueres A. Raetz C.R.H. J. Biol. Chem. 1992; 267: 23261-23268Abstract Full Text PDF PubMed Google Scholar) have shown that TNFα secretion from MonoMac 6 cells occurs only after stimulation of these cells with both bacterial lipopolysaccharide and phorbol-12-myristate-13-acetate. Although proTNFα biosynthesis is enhanced after lipopolysaccharide stimulation, release does not occur to significant levels in the absence of phorbol 12-myristate 13-acetate (13Moss M.L. Becherer J.D. Milla M.E. Pahel G. Lambert M. Andrews R. Frye S. Haffner C. Cowan D. Maloney P. Dixon E.P. Jansen M. Mitchell J.L. Leesnitzer T. Warner J. Conway J. Bickett D.M. Bird M. Priest R. Reinhard J. Lin P. Bradshaw D. Nixon J.S. Bottomley K. Metalloproteinases as Targets for Anti-inflammatory Drugs. Birkhauser Publishers, Basel1998: 187-204Google Scholar). Potentially, a phorbol 12-myristate 13-acetate-initiated phosphorylation or dephosphorylation event at the cytosolic tail of TACE precedes TACE activation, either via a conformational change that exposes the cleavage site to the processing protease(s) or by directing transport of TACE to the compartment where activation takes place. Remarkably, it has been shown that mutation of an equivalent tyrosine residue to phenylalanine in the cytoplasmic tail of furin results in failure of this enzyme to localize properly inside the cell (25Schäfer W. Stroh A. Berhöfer S. Seiler J. Vey M. Kruse M.-L. Kern H.F. Klenk H.-D. Garten W. EMBO J. 1995; 14: 2424-2435Crossref PubMed Scopus (219) Google Scholar, 26Takahashi S. Nakagawa T. Banno T. Watanabe T. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 28397-28401Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Therefore, it is possible that insect cells contain a TACE maturating activity but lack the cellular factors responsible for making TACE available to such protease. Both Arg473 and Arg651, after signal peptide cleavage, likely reach the compartment where maturation occurs via bulk flow secretion.Maturation by proteolytic cleavage is essential in order to generate active TACE. The pro domain strongly inhibits TACE, probably through complexing the catalytic zinc in the active site via an unpaired cysteine residue in the putative cysteine switch box. Our observation that the mercurial compound APMA efficiently mediates dissociation of the pro-catalytic domain complex supports this hypothesis. Additionally, peptides from the putative Cys-switch region do inhibit TACE activity (32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjumen-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar).Interestingly, APMA-mediated dissociation of the pro-catalytic domain complex occurs at concentrations well below the ones used for activating matrix metalloproteinases. At higher concentrations, APMA acts as an inhibitor of TACE. We do not know the mechanism of this inactivation effect. APMA may react with one or more cysteine residues in the catalytic domain that are essential for keeping TACE in its functional form. Release of the cleaved pro domain appears not to be a simple diffusion-driven process, because pro remained tightly associated to the catalytic domain through several purification steps and even in the presence of substantial concentrations of the chemical denaturant urea. This suggests a role for the cysteine-rich domain in freeing the catalytic domain from the pro domain as indicated by the apparent absence of complex when Arg651 was overexpressed and purified. The cysteine-rich domain may have an interacting surface with the catalytic domain that overlaps the one used by the pro domain. Given the remarkable sensitivity of TACE pro domain to proteolysis, it is likely that once it is detached from the catalytic domain, its degradation prevents reassociation with the catalytic domain. It might also be possible that the cysteine-rich domain induces changes in conformation that dramatically reduce the affinity of the pro domain for the catalytic domain. The observed differences in glycosylation sites between Arg473 and Arg651, as well as the observed differences in oligosaccharide processing, may also have a role in stabilizing/destabilizing the pro-catalytic domain complex. It is less likely, however, that the cysteine-rich domain acts as an exchange factor, accepting pro after TACE activation, because of the apparent absence of Arg651-pro complexes.The pro domain is not only an inhibitor of the catalytic domain, but also appears to have at least some of the properties observed in chaperones, as it seems to facilitate either secretion or folding or both. When expressed separately, both domains failed to be secreted and appeared to be extremely sensitive to proteolysis. It seems unlikely that this result is due to inability of the endoplasmic reticulum signal peptidase to access its cleavage site: about half of the purified R473Δpro protein did have Ala17 as its N terminus, consistent with signal peptidase processing. In addition, the R473Δpro construct contains a signal peptide-catalytic domain fusion starting well downstream (after Pro24) from the signal peptidase cleavage site. Furthermore, a similar fusion of the signal peptide to the cysteine-rich domain (Fig. 1) is efficiently secreted by insect cells, and is recognized by a monoclonal antibody specific for the native form of this domain. 3A. Miller and M. Milla, unpublished work. This dependence is not surprising: it has been previously reported for several proteases, including zinc metalloproteases, that the pro domain is essential for proper secretion (27Wetmore D.R. Hardman K.D. Biochemistry. 1996; 35: 6549-6558Crossref PubMed Scopus (46) Google Scholar, 28Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hagmann W.K. Hermes J.D. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (270) Google Scholar). Similar results have been described for other members of the ADAMs family, namely metalloprotease and disintegrin-containing proteins 9 and 15 (32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjumen-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). For at least two proteases, the α-lytic protease and subtilisin, the pro domain seems to control the kinetics of the folding reaction, supporting an intramolecular chaperone role (29Baker D. Sohl J.L. Agard D.A. Nature. 1992; 356: 263-265Crossref PubMed Scopus (283) Google Scholar, 30Wang L. Ruan B. Ruvinov S. Bryan P.N. Biochemistry. 1998; 37: 3165-3171Crossref PubMed Scopus (43) Google Scholar).How does TACE quaternary structure relate to its function? Both proTNFα and TNFα are homotrimers. As the release of proTNFα from cellular membranes requires cleaving three stems, we investigated the oligomerization state of these recombinant forms by equilibrium sedimentation analysis to address whether TACE itself is a trimer. Both Arg473 and Arg651 were monomeric at concentrations well above the ones used to demonstrate cleavage against the synthetic peptide substrate. Bode and co-workers (11Maskos K. Fernandez-Catalan C. Huber R. Bourenkov G.P. Bartunik H. Ellestad G.A. Reddy P. Wolfson M.F. Rauch C.T. Castner B.J. Davis R. Clarke H.R.G. Petersen M. Fitzner J.M. Cerretti D.P. March C.J. Paxton R.J. Black R.A. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3408-3412Crossref PubMed Scopus (361) Google Scholar) published the three-dimensional structure of the catalytic domain of TACE (31Clarke H.R. Wolfson M.F. Rauch C.T. Castner B.J. Huang C.P. Gerhart M.J. Johnson R.S. Cerretti D.P. Paxton R.J. Price V.L. Black R.A. Protein Expression Purif. 1998; 13: 104-110Crossref PubMed Scopus (17) Google Scholar). They propose a model in which the "right side" of the catalytic domain forms such interactions with the base of the proTNFα trimer. A role for the transmembrane or cytoplasmic domains in substrate recognition or oligomerization remains to be investigated.The extreme salt sensitivity of TACE is intriguing. Concentrations of NaCl that completely inhibit its activity (100 mm) do not seem to have an effect on its oligomeric state or solubility. Examination of electrostatic surface potential models of TACE (11Maskos K. Fernandez-Catalan C. Huber R. Bourenkov G.P. Bartunik H. Ellestad G.A. Reddy P. Wolfson M.F. Rauch C.T. Castner B.J. Davis R. Clarke H.R.G. Petersen M. Fitzner J.M. Cerretti D.P. March C.J. Paxton R.J. Black R.A. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3408-3412Crossref PubMed Scopus (361) Google Scholar) reveals that the relatively flat left-hand side of the active-site cleft is positively charged. Therefore, disruption of favorable electrostatic interactions with the substrate peptide by chloride ions is formally possible. The physiological meaning of this effect remains to be established.We believe that some of these truncates will be useful in studies aimed at understanding how TACE interacts with its natural substrate, proTNFα, as well as to dissect the levels of regulation of its biogenesis and activity. In turn, this should allow us to explore new avenues for targeting this key enzyme for therapeutic intervention in inflammatory diseases, cancer, and AIDS. Our goal is to understand the relationship among the domain architecture, function, and regulation of TACE. The studies reported here show that only certain recombinant forms of TACE can be overexpressed in insect cells in a soluble, secreted form, fully active as compared with native TACE purified from a human monocytic cell line. Insect cells expressed mature, functional full-length TACE at very low levels. Most of the protease remained in its immature form, with the pro domain intact, as judged from Western blots of cell extracts. This result is particularly perplexing because truncates Arg473(catalytic domain) and Arg651 (catalytic and cysteine-rich domains) are maturated at the predicted furin cleavage site in insect cells. Full-length TACE contains a C-terminal extension of Arg651, including a transmembrane domain and a cytoplasmic tail. We hypothesize that this cytoplasmic domain contains a negative signal that must be reversed to allow transport of TACE to the secretory compartment where activation occurs. It is intriguing that this domain contains a potential tyrosine phosphorylation site in the context of a Src homology 2 domain binding site, KKLDKQYESL705, as well as a potential Src homology 3 domain binding site, PAPQTPGR738. In fact, the tyrosine kinase domain of the epidermal growth factor receptor can convert the recombinant TACE cytoplasmic tail (produced in E. coli) to its phosphotyrosine form.2 Pradines-Figueres and Raetz (24Pradines-Figueres A. Raetz C.R.H. J. Biol. Chem. 1992; 267: 23261-23268Abstract Full Text PDF PubMed Google Scholar) have shown that TNFα secretion from MonoMac 6 cells occurs only after stimulation of these cells with both bacterial lipopolysaccharide and phorbol-12-myristate-13-acetate. Although proTNFα biosynthesis is enhanced after lipopolysaccharide stimulation, release does not occur to significant levels in the absence of phorbol 12-myristate 13-acetate (13Moss M.L. Becherer J.D. Milla M.E. Pahel G. Lambert M. Andrews R. Frye S. Haffner C. Cowan D. Maloney P. Dixon E.P. Jansen M. Mitchell J.L. Leesnitzer T. Warner J. Conway J. Bickett D.M. Bird M. Priest R. Reinhard J. Lin P. Bradshaw D. Nixon J.S. Bottomley K. Metalloproteinases as Targets for Anti-inflammatory Drugs. Birkhauser Publishers, Basel1998: 187-204Google Scholar). Potentially, a phorbol 12-myristate 13-acetate-initiated phosphorylation or dephosphorylation event at the cytosolic tail of TACE precedes TACE activation, either via a conformational change that exposes the cleavage site to the processing protease(s) or by directing transport of TACE to the compartment where activation takes place. Remarkably, it has been shown that mutation of an equivalent tyrosine residue to phenylalanine in the cytoplasmic tail of furin results in failure of this enzyme to localize properly inside the cell (25Schäfer W. Stroh A. Berhöfer S. Seiler J. Vey M. Kruse M.-L. Kern H.F. Klenk H.-D. Garten W. EMBO J. 1995; 14: 2424-2435Crossref PubMed Scopus (219) Google Scholar, 26Takahashi S. Nakagawa T. Banno T. Watanabe T. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 28397-28401Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Therefore, it is possible that insect cells contain a TACE maturating activity but lack the cellular factors responsible for making TACE available to such protease. Both Arg473 and Arg651, after signal peptide cleavage, likely reach the compartment where maturation occurs via bulk flow secretion. Maturation by proteolytic cleavage is essential in order to generate active TACE. The pro domain strongly inhibits TACE, probably through complexing the catalytic zinc in the active site via an unpaired cysteine residue in the putative cysteine switch box. Our observation that the mercurial compound APMA efficiently mediates dissociation of the pro-catalytic domain complex supports this hypothesis. Additionally, peptides from the putative Cys-switch region do inhibit TACE activity (32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjumen-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Interestingly, APMA-mediated dissociation of the pro-catalytic domain complex occurs at concentrations well below the ones used for activating matrix metalloproteinases. At higher concentrations, APMA acts as an inhibitor of TACE. We do not know the mechanism of this inactivation effect. APMA may react with one or more cysteine residues in the catalytic domain that are essential for keeping TACE in its functional form. Release of the cleaved pro domain appears not to be a simple diffusion-driven process, because pro remained tightly associated to the catalytic domain through several purification steps and even in the presence of substantial concentrations of the chemical denaturant urea. This suggests a role for the cysteine-rich domain in freeing the catalytic domain from the pro domain as indicated by the apparent absence of complex when Arg651 was overexpressed and purified. The cysteine-rich domain may have an interacting surface with the catalytic domain that overlaps the one used by the pro domain. Given the remarkable sensitivity of TACE pro domain to proteolysis, it is likely that once it is detached from the catalytic domain, its degradation prevents reassociation with the catalytic domain. It might also be possible that the cysteine-rich domain induces changes in conformation that dramatically reduce the affinity of the pro domain for the catalytic domain. The observed differences in glycosylation sites between Arg473 and Arg651, as well as the observed differences in oligosaccharide processing, may also have a role in stabilizing/destabilizing the pro-catalytic domain complex. It is less likely, however, that the cysteine-rich domain acts as an exchange factor, accepting pro after TACE activation, because of the apparent absence of Arg651-pro complexes. The pro domain is not only an inhibitor of the catalytic domain, but also appears to have at least some of the properties observed in chaperones, as it seems to facilitate either secretion or folding or both. When expressed separately, both domains failed to be secreted and appeared to be extremely sensitive to proteolysis. It seems unlikely that this result is due to inability of the endoplasmic reticulum signal peptidase to access its cleavage site: about half of the purified R473Δpro protein did have Ala17 as its N terminus, consistent with signal peptidase processing. In addition, the R473Δpro construct contains a signal peptide-catalytic domain fusion starting well downstream (after Pro24) from the signal peptidase cleavage site. Furthermore, a similar fusion of the signal peptide to the cysteine-rich domain (Fig. 1) is efficiently secreted by insect cells, and is recognized by a monoclonal antibody specific for the native form of this domain. 3A. Miller and M. Milla, unpublished work. This dependence is not surprising: it has been previously reported for several proteases, including zinc metalloproteases, that the pro domain is essential for proper secretion (27Wetmore D.R. Hardman K.D. Biochemistry. 1996; 35: 6549-6558Crossref PubMed Scopus (46) Google Scholar, 28Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hagmann W.K. Hermes J.D. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (270) Google Scholar). Similar results have been described for other members of the ADAMs family, namely metalloprotease and disintegrin-containing proteins 9 and 15 (32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjumen-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). For at least two proteases, the α-lytic protease and subtilisin, the pro domain seems to control the kinetics of the folding reaction, supporting an intramolecular chaperone role (29Baker D. Sohl J.L. Agard D.A. Nature. 1992; 356: 263-265Crossref PubMed Scopus (283) Google Scholar, 30Wang L. Ruan B. Ruvinov S. Bryan P.N. Biochemistry. 1998; 37: 3165-3171Crossref PubMed Scopus (43) Google Scholar). How does TACE quaternary structure relate to its function? Both proTNFα and TNFα are homotrimers. As the release of proTNFα from cellular membranes requires cleaving three stems, we investigated the oligomerization state of these recombinant forms by equilibrium sedimentation analysis to address whether TACE itself is a trimer. Both Arg473 and Arg651 were monomeric at concentrations well above the ones used to demonstrate cleavage against the synthetic peptide substrate. Bode and co-workers (11Maskos K. Fernandez-Catalan C. Huber R. Bourenkov G.P. Bartunik H. Ellestad G.A. Reddy P. Wolfson M.F. Rauch C.T. Castner B.J. Davis R. Clarke H.R.G. Petersen M. Fitzner J.M. Cerretti D.P. March C.J. Paxton R.J. Black R.A. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3408-3412Crossref PubMed Scopus (361) Google Scholar) published the three-dimensional structure of the catalytic domain of TACE (31Clarke H.R. Wolfson M.F. Rauch C.T. Castner B.J. Huang C.P. Gerhart M.J. Johnson R.S. Cerretti D.P. Paxton R.J. Price V.L. Black R.A. Protein Expression Purif. 1998; 13: 104-110Crossref PubMed Scopus (17) Google Scholar). They propose a model in which the "right side" of the catalytic domain forms such interactions with the base of the proTNFα trimer. A role for the transmembrane or cytoplasmic domains in substrate recognition or oligomerization remains to be investigated. The extreme salt sensitivity of TACE is intriguing. Concentrations of NaCl that completely inhibit its activity (100 mm) do not seem to have an effect on its oligomeric state or solubility. Examination of electrostatic surface potential models of TACE (11Maskos K. Fernandez-Catalan C. Huber R. Bourenkov G.P. Bartunik H. Ellestad G.A. Reddy P. Wolfson M.F. Rauch C.T. Castner B.J. Davis R. Clarke H.R.G. Petersen M. Fitzner J.M. Cerretti D.P. March C.J. Paxton R.J. Black R.A. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3408-3412Crossref PubMed Scopus (361) Google Scholar) reveals that the relatively flat left-hand side of the active-site cleft is positively charged. Therefore, disruption of favorable electrostatic interactions with the substrate peptide by chloride ions is formally possible. The physiological meaning of this effect remains to be established. We believe that some of these truncates will be useful in studies aimed at understanding how TACE interacts with its natural substrate, proTNFα, as well as to dissect the levels of regulation of its biogenesis and activity. In turn, this should allow us to explore new avenues for targeting this key enzyme for therapeutic intervention in inflammatory diseases, cancer, and AIDS. We thank Dr. Robert Fuller for generously sharing a baculovirus strain encoding full-length furin. We also thank Mike Luther, Tom Consler, Fred Kull, Blaine Knight, Jerry McGeehan, Daniel Hassler, Phillip McCauley, and Perry Brignola for help, advice, or comments.
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