Catalytic Activity of Human ADAM33
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m309696200
ISSN1083-351X
AutoresJun Zou, Feng Zhu, Jianjun Liu, Wenyan Wang, Rumin Zhang, Charles G. Garlisi, Yan‐Hui Liu, Shihong Wang, Himanshu Shah, Yuntao Wan, Shelby P. Umland,
Tópico(s)Cell Adhesion Molecules Research
ResumoADAM33 (a disintegrin and metalloproteinase) is an asthma susceptibility gene recently identified through a genetic study of asthmatic families (van Eerdewegh et al. (2002) Nature 418, 426–430). In order to characterize the catalytic properties of ADAM33, the metalloproteinase domain of human ADAM33 was expressed in Drosophila S2 cells and purified. The N-terminal sequence of the purified metalloproteinase was exclusively 204EARR, indicating utilization of one of three furin recognition sites. Of many synthetic peptides tested as potential substrates, four peptides derived from β-amyloid precursor protein (APP), Kit-ligand-1 (KL-1), tumor necrosis factor-related activation-induced cytokine, and insulin B chain were cleaved by ADAM33; mutation at the catalytic site, E346A, inactivated catalytic activity. Cleavage of APP occurred at His14↓Gln15, not at the α-secretase site and was inefficient (kcat/Km (1.6 ± 0.3) × 102 m–1 s–1). Cleavage of a juxtamembrane KL-1 peptide occurred at a site used physiologically with a similar efficiency. Mutagenesis of KL-1 peptide substrate indicated that the P3, P2, P1, and P3′ residues were critical for activity. In a transfected cell-based sheddase assay, ADAM33 functioned as a negative regulator of APP shedding and mediated some constitutive shedding of KL-1, which was not regulated by phorbol 12-myristate 13-acetate activation. ADAM33 activity was sensitive to several hydroxamate inhibitors (IK682, Ki = 23 ± 7 nm) and to tissue inhibitors of metalloproteinase (TIMPs). Activity was inhibited moderately by TIMP-3 and TIMP-4 and weakly inhibited by TIMP-2 but not by TIMP-1, a profile distinct from other ADAMs. The identification of ADAM33 peptide substrates, cellular activity, and a distinct inhibitor profile provide the basis for further functional studies of ADAM33. ADAM33 (a disintegrin and metalloproteinase) is an asthma susceptibility gene recently identified through a genetic study of asthmatic families (van Eerdewegh et al. (2002) Nature 418, 426–430). In order to characterize the catalytic properties of ADAM33, the metalloproteinase domain of human ADAM33 was expressed in Drosophila S2 cells and purified. The N-terminal sequence of the purified metalloproteinase was exclusively 204EARR, indicating utilization of one of three furin recognition sites. Of many synthetic peptides tested as potential substrates, four peptides derived from β-amyloid precursor protein (APP), Kit-ligand-1 (KL-1), tumor necrosis factor-related activation-induced cytokine, and insulin B chain were cleaved by ADAM33; mutation at the catalytic site, E346A, inactivated catalytic activity. Cleavage of APP occurred at His14↓Gln15, not at the α-secretase site and was inefficient (kcat/Km (1.6 ± 0.3) × 102 m–1 s–1). Cleavage of a juxtamembrane KL-1 peptide occurred at a site used physiologically with a similar efficiency. Mutagenesis of KL-1 peptide substrate indicated that the P3, P2, P1, and P3′ residues were critical for activity. In a transfected cell-based sheddase assay, ADAM33 functioned as a negative regulator of APP shedding and mediated some constitutive shedding of KL-1, which was not regulated by phorbol 12-myristate 13-acetate activation. ADAM33 activity was sensitive to several hydroxamate inhibitors (IK682, Ki = 23 ± 7 nm) and to tissue inhibitors of metalloproteinase (TIMPs). Activity was inhibited moderately by TIMP-3 and TIMP-4 and weakly inhibited by TIMP-2 but not by TIMP-1, a profile distinct from other ADAMs. The identification of ADAM33 peptide substrates, cellular activity, and a distinct inhibitor profile provide the basis for further functional studies of ADAM33. ADAM33 (a disintegrin and metalloproteinase) was identified as an asthma susceptibility gene by a genetic linkage and polymorphism study of asthmatic families (1Van Eerdewegh P. Little R.D. Dupuis J. Del Mastro R.G. Falls K. Simon J. Torrey D. Pandit S. McKenny J. Braunschweiger K. Walsh A. Liu Z. Hayward B. Folz C. Manning S.P. Bawa A. Saracino L. Thackston M. Benchekroun Y. Capparell N. Wang M. Adair R. Feng Y. Dubois J. FitzGerald M.G. Huang H. Gibson R. Allen K.M. Pedan A. Danzig M.R. Umland S.P. Egan R.W. Cuss F.M. Rorke S. Clough J.B. Holloway J.W. Holgate S.T. Keith T.P. Nature. 2002; 418: 426-430Crossref PubMed Scopus (945) Google Scholar). Determining the role of ADAM33 in the pathophysiology of asthma will require defining its function at several levels. ADAM33 belongs to a family of type I transmembrane metalloproteinases. These integral membrane glycoproteins play important physiological roles in fertilization, myogenesis, and neurogenesis due to their participation in cell-cell interactions and proteolytic release of cell surface membrane proteins such as cytokines, growth factors, and receptors (2Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (224) Google Scholar, 3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (886) Google Scholar). The structure of the ADAM 1The abbreviations used are: ADAM, a disintegrin and metalloproteinase; MMP, matrix metalloproteinase; KL-1, Kit-ligand-1; APP, amyloid precursor protein; TRANCE, TNF-related activation-induced cytokine; TNF-α, tumor necrosis factor-α; TGF-α, transforming growth factor-α; TIMP, tissue inhibitor of metalloproteinase; PNGase, peptide: N-glycosidase F; Ni2+-NTA, nickel-nitrilotriacetic acid; HPLC, high performance liquid chromatography; WT, wild type; PMA, phorbol 12-myristate 13-acetate; MALDI-TOF, matrix-assisted laser-desorption/ionization time-of-flight; pMT, metallothionein promoter; Dabcyl, 4-{[(4-dimethylamino)phenyl]azo}benzoic acid; Edans, N-acetyl-N′-(5-sulfo-l-naphthy1)ethylenediamine.1The abbreviations used are: ADAM, a disintegrin and metalloproteinase; MMP, matrix metalloproteinase; KL-1, Kit-ligand-1; APP, amyloid precursor protein; TRANCE, TNF-related activation-induced cytokine; TNF-α, tumor necrosis factor-α; TGF-α, transforming growth factor-α; TIMP, tissue inhibitor of metalloproteinase; PNGase, peptide: N-glycosidase F; Ni2+-NTA, nickel-nitrilotriacetic acid; HPLC, high performance liquid chromatography; WT, wild type; PMA, phorbol 12-myristate 13-acetate; MALDI-TOF, matrix-assisted laser-desorption/ionization time-of-flight; pMT, metallothionein promoter; Dabcyl, 4-{[(4-dimethylamino)phenyl]azo}benzoic acid; Edans, N-acetyl-N′-(5-sulfo-l-naphthy1)ethylenediamine. proteins is conserved and characterized by multiple domains (an N-terminal secretion signal sequence, pro- and catalytic domains, disintegrin, and cysteine-rich domains), which are usually followed by an epidermal growth factor repeat, a transmembrane, and a carboxyl-terminal cytoplasmic tail (3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (886) Google Scholar). Of 34 ADAMs identified, about half of them, including ADAM33, were predicted to be active proteinases based on the presence of the HEXXHXXGXXH zinc binding motif and a glutamic acid in the catalytic domain (4Black R.A. White J.M. Curr. Opin. Cell Biol. 1998; 10: 654-659Crossref PubMed Scopus (428) Google Scholar, 5Becherer J.D. Blobel C.P. Curr. Top. Dev. Biol. 2003; 54: 101-123Crossref PubMed Google Scholar). However, only some of these ADAMs were demonstrated experimentally to possess catalytic activity, including ADAM8 (6Schlomann U. Wildeboer D. Webster A. Antropova O. Zeuschner D. Knight C.G. Docherty A.J. Lambert M. Skelton L. Jockusch H. Bartsch J.W. J. Biol. Chem. 2002; 277: 48210-48219Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 7Amour A. Knight C.G. English W.R. Webster A. Slocombe P.M. Knauper V. Docherty A.J. Becherer J.D. Blobel C.P. Murphy G. FEBS Lett. 2002; 524: 154-158Crossref PubMed Scopus (128) Google Scholar), ADAM9 (8Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjument-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), ADAM10 (9Howard L. Glynn P. Methods Enzymol. 1995; 248: 388-395Crossref PubMed Scopus (49) Google Scholar), ADAM12 (10Loechel F. Gilpin B.J. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 16993-16997Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), ADAM15 (11Fourie A.M. Coles F. Moreno V. Karlsson L. J. Biol. Chem. 2003; 278: 30469-30477Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12Lum L. Reid M.S. Blobel C.P. J. Biol. Chem. 1998; 273: 26236-26247Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), ADAM17 (13Black 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 (2689) Google Scholar, 14Moss M.L. Jin S.L. Milla M.E. Bickett D.M. 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. Becherer J.D. et al.. 1997; 385: 733-736Google Scholar), ADAM19 (15Shirakabe K. Wakatsuki S. Kurisaki T. Fujisawa-Sehara A. J. Biol. Chem. 2001; 276: 9352-9358Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 16Kang T. Park H.I. Suh Y. Zhao Y.G. Tschesche H. Sang Q.X. J. Biol. Chem. 2002; 277: 48514-48522Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 17Chesneau V. Becherer J.D. Zheng Y. Erdjument-Bromage H. Tempst P. Blobel C.P. J. Biol. Chem. 2003; 278: 22331-22340Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), ADAM28 (18Howard L. Zheng Y. Horrocks M. Maciewicz R.A. Blobel C. FEBS Lett. 2001; 498: 82-86Crossref PubMed Scopus (63) Google Scholar), and ADAM33 (19Garlisi C.G. Zou J. Devito K.E. Tian F. Zhu F.X. Liu J. Shah H. Wan Y. Billah M.M. Egan R.W. Umland S.P. Biochem. Biophys. Res. Commun. 2003; 301: 35-43Crossref PubMed Scopus (53) Google Scholar). For only a few ADAMs, a physiological substrate has been identified (3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (886) Google Scholar). ADAM17/TNF-α-converting enzyme is the best studied member of the ADAM family. In addition to shedding soluble TNF-α after cleavage of the membrane-anchored precursor (13Black 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 (2689) Google Scholar), ADAM17 also processes many other integral membrane proteins including the amyloid precursor protein (APP) (20Buxbaum, J. D., Liu, K. N., Luo, Y., Slack, J. L., Stocking, K. L., Peschon, J. J., Johnson, R. S., Castner, B. J., Cerretti, D. P., and Black, R. A. (1998) 273, 27765–27767Google Scholar), transforming growth factor-α (TGF-α), L-selectin, and TNF receptor II (21Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1358) Google Scholar) as well as IL-1 receptor and TNF receptor I (22Reddy P. Slack J.L. Davis R. Cerretti D.P. Kozlosky C.J. Blanton R.A. Shows D. Peschon J.J. Black R.A. J. Biol. Chem. 2000; 275: 14608-14614Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). ADAM10/Kuzbanian is involved in neuronal development through the cleavage of Notch, a type I transmembrane receptor that controls cell fate determination (23Schlöndorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar). The cDNAs of ADAM33 have been cloned from humans and mice and are most closely related to ADAM19, ADAM12, and Xenopus ADAM13 (1Van Eerdewegh P. Little R.D. Dupuis J. Del Mastro R.G. Falls K. Simon J. Torrey D. Pandit S. McKenny J. Braunschweiger K. Walsh A. Liu Z. Hayward B. Folz C. Manning S.P. Bawa A. Saracino L. Thackston M. Benchekroun Y. Capparell N. Wang M. Adair R. Feng Y. Dubois J. FitzGerald M.G. Huang H. Gibson R. Allen K.M. Pedan A. Danzig M.R. Umland S.P. Egan R.W. Cuss F.M. Rorke S. Clough J.B. Holloway J.W. Holgate S.T. Keith T.P. Nature. 2002; 418: 426-430Crossref PubMed Scopus (945) Google Scholar, 24Yoshinaka T. Nishii K. Yamada K. Sawada H. Nishiwaki E. Smith K. Yoshino K. Ishiguro H. Higashiyama S. Gene (Amst.). 2002; 282: 227-236Crossref PubMed Scopus (111) Google Scholar, 25Gunn T.M. Azarani A. Kim P.H. Hyman R.W. Davis R.W. Barsh G.S. BMC Genetics.http://www.biomedcentral.com/1471-2156/3/2Date: 2002Google Scholar). Overexpression of ADAM19 in L929 cells suggested that ADAM19 may participate in the shedding of β1-neuregulin, a member of the epidermal growth factor family (15Shirakabe K. Wakatsuki S. Kurisaki T. Fujisawa-Sehara A. J. Biol. Chem. 2001; 276: 9352-9358Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Also, purified ADAM19 metalloproteinase cleaved peptides corresponding to the known cleavage sites of TNF-α, TRANCE, and KL-1 (17Chesneau V. Becherer J.D. Zheng Y. Erdjument-Bromage H. Tempst P. Blobel C.P. J. Biol. Chem. 2003; 278: 22331-22340Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). ADAM12 was reported to process heparin-binding epidermal growth factor in vivo (26Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (639) Google Scholar) and to cleave insulin-like growth factor-binding protein-3 and -5 (27Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (276) Google Scholar). ADAMs are synthesized as latent proforms in the endoplasmic reticulum. This latency is the result of a complex formation via a thiol-zinc bond between a conserved cysteine in the prodomain and the essential catalytic Zn2+ in the metalloprotease domain (28Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (699) Google Scholar, 29Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Crossref PubMed Scopus (439) Google Scholar). The latent ADAMs can be activated by multiple means that dissociate the thiol-zinc bond in the complex, and this mechanism is referred as the "cysteine switch" of activation (30Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1197) Google Scholar). Many ADAMs possess a furin recognition site (RXXR) between their pro- and catalytic domains (31Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar, 32Stone A.L. Kroeger M. Sang Q.X. J. Protein Chem. 1999; 18: 447-465Crossref PubMed Scopus (134) Google Scholar). A common mechanism to activate these zymogens is through dissociation of the pro-domain following cleavage of the prodomain by furin or furin-like proteases in the trans-Golgi (3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (886) Google Scholar). ADAMs belong to the metzincin superfamily of metalloproteinases, which also includes matrix metalloproteinases (MMPs) and snake venom metalloproteinases (3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (886) Google Scholar). The activities of MMPs are regulated by tissue inhibitors of metalloproteinase (TIMPs) during tissue remodeling; to date, four mammalian TIMPs have been identified, which all inhibit the MMPs, but only TIMP-1 and TIMP-3 inhibit some ADAM family members (33Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (967) Google Scholar). For instance, TIMP-3 was found to inhibit ADAM12 (27Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (276) Google Scholar), ADAM17 (34Amour A. Slocombe P.M. Webster A. Butler M. Knight C.G. Smith B.J. Stephens P.E. Shelley C. Hutton M. Knauper V. Docherty A.J. Murphy G. FEBS Lett. 1998; 435: 39-44Crossref PubMed Scopus (542) Google Scholar), and ADAM19 (16Kang T. Park H.I. Suh Y. Zhao Y.G. Tschesche H. Sang Q.X. J. Biol. Chem. 2002; 277: 48514-48522Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), whereas TIMP-1 and TIMP-3 inhibited ADAM10 (35Amour A. Knight C.G. Webster A. Slocombe P.M. Stephens P.E. Knauper V. Docherty A.J. Murphy G. FEBS Lett. 2000; 473: 275-279Crossref PubMed Scopus (350) Google Scholar). The TIMPs are secreted proteins but may be found at the cell surface in association with membrane-bound proteins. Only TIMP-3 is located in the extracellular matrix via its C-terminal domain binding to heparan sulfate proteoglycan (36Nagase H. Brew K. Arthritis Res. 2002; 4: S51-S61Crossref PubMed Scopus (47) Google Scholar). The physiological significance of the inhibition of ADAMs by TIMPs is still unclear, but it has been suggested that characterization of the TIMP inhibition profile of the remaining ADAMs can aid in endogenous substrate identification (7Amour A. Knight C.G. English W.R. Webster A. Slocombe P.M. Knauper V. Docherty A.J. Becherer J.D. Blobel C.P. Murphy G. FEBS Lett. 2002; 524: 154-158Crossref PubMed Scopus (128) Google Scholar). In this study, we expressed and purified a soluble recombinant form of human ADAM33 catalytic protein and used it to test a number of potential candidate substrate peptides. The identified ADAM33-cleavable peptide substrates, in combination with the knowledge gained from the study of substrate mutagenesis, were used to characterize the substrate specificity and enzymatic activity of ADAM33 and its regulation by various synthetic and physiological inhibitors. Reagents—Restriction enzymes, T4 ligase, and peptide:N-glycosidase F (PNGase) were purchased from New England Biolabs (Beverly, MA). All chemicals and insulin B chain peptide were from Sigma unless indicated otherwise. Human TIMPs were purchased from R & D Systems (Minneapolis, MN). All reagents and media for Drosophila S2 cell expression including anti-His6 antibody conjugated with horseradish peroxidase were from Invitrogen unless indicated otherwise. A pcDNA3.1 plasmid (Invitrogen, San Diego, CA) containing full-length APP or full-length c-Kit ligand (KL-1) was obtained from Dr. L. Zhang (Schering Plough, Kenilworth, NJ) (37Zhang L. Song L. Parker E.M. J. Biol. Chem. 1999; 274: 8966-8972Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) or Dr. T. McClanahan (DNAX, Palo Alto, CA), respectively. Antibodies to peptides in the ADAM33 pro-domain (Pro1), catalytic domain (ASP2), or cytoplasmic domain (Cyt2) and their use are described elsewhere (19Garlisi C.G. Zou J. Devito K.E. Tian F. Zhu F.X. Liu J. Shah H. Wan Y. Billah M.M. Egan R.W. Umland S.P. Biochem. Biophys. Res. Commun. 2003; 301: 35-43Crossref PubMed Scopus (53) Google Scholar), and they were used for Western blotting. The W0 –2 antibody was isolated from a hybridoma cell obtained from Dr. Konrad Beyreuther (University of Heidelberg, Heidelberg, Germany) (38Ida N. Hartmann T. Pantel J. Schroder J. Zerfass R. Forstl H. Sandbrink R. Masters C.L. Beyreuther K. J. Biol. Chem. 1996; 271: 22908-22914Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar) and was used to detect APP by Western blotting. Human stem cell factor, also know as c-Kit ligand (KL-1), was detected in culture supernatants of transfected cells by enzyme-linked immunosorbent assay using the Quantakine kit from R & D Systems. Chromatographic columns and the ECL Western blot system were from Amersham Biosciences. Peptides from amyloid precursor protein (APP28) and myelin basic protein were purchased from Bachem (Torrance, CA). Peptide substrate for the ADAM17 assay, K(7-methoxycoumarin-4-ylacetyl)SPLAQAVRSSSRK(2,4-dinitrophenyl), was derived from TNF-α and synthesized in-house (see below). Recombinant human ADAM17 (catalytic domain) was a gift from R. Ingram (Schering Plough), and IK682, Marimastat, and Immunex compound 1 were synthesized by Medicinal Chemistry at Schering Plough Research Institute. Expression of ADAM33 in Drosophila S2 Cells—The nucleotides of ADAM33 pro- and catalytic domains (bases 91–1227, corresponding to amino acids Leu31–Pro409) were amplified by PCR using a full-length ADAM33 cDNA template (GenBank™ accession number AF466287) (1Van Eerdewegh P. Little R.D. Dupuis J. Del Mastro R.G. Falls K. Simon J. Torrey D. Pandit S. McKenny J. Braunschweiger K. Walsh A. Liu Z. Hayward B. Folz C. Manning S.P. Bawa A. Saracino L. Thackston M. Benchekroun Y. Capparell N. Wang M. Adair R. Feng Y. Dubois J. FitzGerald M.G. Huang H. Gibson R. Allen K.M. Pedan A. Danzig M.R. Umland S.P. Egan R.W. Cuss F.M. Rorke S. Clough J.B. Holloway J.W. Holgate S.T. Keith T.P. Nature. 2002; 418: 426-430Crossref PubMed Scopus (945) Google Scholar). The following PCR primers were used, which added a KpnI site to the 5′-end of the gene fragment: primer 1, 5′-TTA GAT TCA TAG G GTA CCG CTT CAA GGA CAT ATC CCT GGG CAG-3′ and nucleotides encoding a His6 tag plus a XhoI site at the 3′ end; primer 2, 5′-ATC TGA TAT CTC GAG TCA ATG ATG GTG ATG ATG ATG TCC TGA CGG GGC ATT GGA GAG GCA AGC GC-3′. To generate a mutant ADAM33 construct with a single amino acid mutation from glutamic acid to alanine (E346A), a full-length ADAM33 cDNA with an E346A mutation (19Garlisi C.G. Zou J. Devito K.E. Tian F. Zhu F.X. Liu J. Shah H. Wan Y. Billah M.M. Egan R.W. Umland S.P. Biochem. Biophys. Res. Commun. 2003; 301: 35-43Crossref PubMed Scopus (53) Google Scholar) was used as a template and amplified with the above PCR primer 1 and primer 3 (5′-A TCT GAT ATC TCG AGC CGG GGC ATT GGA GAG GCA AGC G-3′). This set of primers generated the same 5′- and 3′-ends of the ADAM33 cDNA fragment as the above, whereas its 3′-end fuses with V5-His6 tag in frame in the vector, pMT/Bip/V5-His-C (Invitrogen). The PCR-amplified cDNAs of wild type (WT) and E346A mutant were digested with KpnI and XhoI restriction enzymes and ligated into the Drosophila cell expression vector, pMT/Bip/V5-His-C. Both the WT and the E346A mutant cDNA constructs were verified by sequencing, and their amino acid sequences were identical to that encoded by GenBank™ accession number AF466287 (WT), with the exception of the E346A mutation. Each of the plasmid constructs was co-transfected with pCoHYGRO into Drosophila S2 cells according to the manufacturer's instructions, and stable transfectant cells were selected in the growth medium containing 300 μg/ml hygromycin-B. The stable transfected Drosophila S2 cell line was maintained at 23 °C in DES® medium containing 10% fetal bovine serum, 0.1% fluronic F-68, 300 μg/ml hygromycin-B. Expression of human ADAM33 protein was induced in the presence of 10 μm CdCl2 and 200 μm ZnCl2 in serum-free medium. The culture medium was harvested 6 days post-induction and cleared by centrifugation at 6000 rpm for 20 min. Purification and Characterization of Recombinant Human ADAM33—Conditioned medium was mixed with an equal volume of S-buffer A (25 mm HEPES, pH 6.8, 50 mm NaCl, and 10% glycerol) and then applied to an SP-Sepharose Fast Flow column equilibrated with S-Buffer A, using the AKTA FPLC system (Amersham Biosciences). Bound protein on the column was washed with S-buffer A and 0.1 m NaCl, followed by elution with a linear gradient of NaCl (0.1–0.5 m). Fractions containing ADAM33 were identified by SDS-PAGE, Western blotting (19Garlisi C.G. Zou J. Devito K.E. Tian F. Zhu F.X. Liu J. Shah H. Wan Y. Billah M.M. Egan R.W. Umland S.P. Biochem. Biophys. Res. Commun. 2003; 301: 35-43Crossref PubMed Scopus (53) Google Scholar), or enzyme activity assays. Fractions containing ADAM33 were combined, adjusted to 20 mm imidazole, and loaded onto a Ni2+-NTA column equilibrated with Ni2+-NTA buffer A (25 mm HEPES, pH 7.9, 20 mm imidazole, 500 mm NaCl, and 10% glycerol). The bound protein was washed with Ni2+-NTA buffer A and eluted with Ni2+-NTA buffer B (25 mm HEPES, pH 7.9, 500 mm imidazole, and 10% glycerol). Fractions containing ADAM33 were combined and concentrated by stirred ultrafiltration cell (Millipore Corp., Bedford, MA) before loading onto a Superdex-75 column equilibrated with G-buffer (25 mm HEPES, pH 7.5, 50 mm imidazole, 150 mm NaCl, and 10% glycerol). Protein was eluted with 1.5× column volumes of G-buffer. Fractions containing ADAM33 were identified by enzyme activity assays and SDS-PAGE. Protein concentration was measured by Bradford assay (Bio-Rad), and ADAM33 catalytic domain (ADAM33cat) concentration was estimated by UV absorption using a molar extinction coefficient, ∈280 = 26,780 m–1 cm–1, which was calculated by Genetics Computer Group (GCG) software (Madison, WI). The GCG software was also used to estimate the isoelectric points of recombinant ADAM33 pro- and catalytic domains (pI = 7.9 and 8.3, respectively). The N-terminal amino acid sequence of ADAM33 protein was determined as follows. The protein was separated on a 14% Tris-glycine gel (Novex, San Diego, CA), transferred to polyvinylidene fluoride membrane, and analyzed by automated Edman degradation (39Tempst P. Geromanos S. Elicone C. Erdjument-Bromage H. Methods Companion Methods Enzymol. 1994; 6: 248-261Crossref Scopus (57) Google Scholar). For mass spectrometry sequence identification, Coomassie Blue-stained protein bands were excised from SDS-PAGE gel and digested with trypsin (Promega, Madison, WI) as described (40Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7784) Google Scholar). Tryptic peptides were then subject to mass spectrometric analysis. Deglycosylation of ADAM33 by PNGase F (New England Biolabs, Beverly, MA) was done according to the manufacturer's instructions. Briefly, purified ADAM33 protein was denatured in denaturing buffer (0.5% SDS, 1% β-mercaptoethanol) for 10 min at 100 °C prior to deglycosylation with PNGase F for 2 h at 37 °C. The sample was then analyzed by SDS-PAGE. Peptide Synthesis—Peptides were synthesized in house or purchased from Anaspec (San Jose, CA). Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry was used on either an ABI model 431A Peptide Synthesizer (Applied Biosystems, Foster City, CA) or an ACT model 496 multiple synthesizer (Advanced ChemTech, Louisville, KY). The molecular masses of purified peptides were confirmed by electrospray ionization mass spectrometry. Peptide Cleavage Assay—The peptide substrate (50 μm) was incubated with or without ADAM33 (0.1–0.5 μm) in assay buffer (20 mm HEPES, pH 7.5, 0.5 m NaCl, 0.2 mg/ml bovine serum albumin) for 2 h at room temperature in the presence or absence of various inhibitors (5 mm 1,10-phenanthroline or a protease inhibitor mixture (2 μg/ml leupeptin, 0.4 μm benzamidine, 10 μg/ml soybean trypsin inhibitor, and 0.5 mm iodoacetamide)) (17Chesneau V. Becherer J.D. Zheng Y. Erdjument-Bromage H. Tempst P. Blobel C.P. J. Biol. Chem. 2003; 278: 22331-22340Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The reaction was quenched by adding 10% trifluoroacetic acid to a final concentration of 1%, and samples were analyzed on an Agilent model 1100 high performance liquid chromatograph with C8 column (4.6-mm inner diameter × 50-mm length). Solvents were as follows: A, 0.1% trifluoroacetic acid in water; B, 0.09% trifluoroacetic acid in acetonitrile. A linear gradient from 2 to 42% B was run over 7 min at 1.5 ml/min, and the eluate was monitored at 214 nm. The percentage of peptide cleavage was calculated using the peak area of the cleaved products divided by the sum of the peak areas of both the products and the remaining substrate. The cleavage sites of the peptide were identified by either electrospray or matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Kinetic Studies—Kinetic constants of peptide substrates, APP, and KL-1, were obtained by using the HPLC assay described above and analysis as reported previously (8Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjument-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, 41Park H.I. Turk B.E. Gerkema F.E. Cantley L.C. Sang Q.X. J. Biol. Chem. 2002; 277: 35168-35175Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 42Zhang R. Beyer B.M. Durkin J. Ingram R. Njoroge F.G. Windsor W.T. Malcolm B.A. Anal. Biochem. 1999; 270: 268-275Crossref PubMed Scopus (88) Google Scholar). In brief, ADAM33cat (80 nm) was incubated with peptide substrate (30–2000 μm) in assay buffer for 10–60 min at room temperature. Reactions were timed to allow less than 15% turnover of the substrate. The initial cleavage velocities of the peptides were obtained by plotting cleaved product versus reaction time. The kinetic constants, kcat, Km, and kcat/Km, were determined based on best fit of the data with the Michaelis-Menten equation (43Lehninger A.L. Biochemistry.2nd Ed. Worth Publishers, Inc., New York1975: 189-195Google Scholar). The inhibition dissociation constants (Ki) for inhibitors were determined according to the methods described (41Park H.I. Turk B.E. Gerkema F.E. Cantley L.C. Sang Q.X. J. Biol. Chem. 2002; 277: 35168-35175Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 44Kuzmic P. Elrod K.C. Cregar L.M. Sideris S. Rai R. Janc J.W. Anal. Biochem. 2000; 286: 45-50Crossref PubMed Scopus (46) Google Scholar). In brief, the HPLC assay was performed in the pre
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