The Metalloprotease Disintegrin ADAM8

ADAMs (adisintegrinand metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu330 to Gln exchange (EQ-A8) in the Zn2+ binding motif (HE330LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC50∼50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion. ADAMs (adisintegrinand metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu330 to Gln exchange (EQ-A8) in the Zn2+ binding motif (HE330LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC50∼50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion. ADAM 1The abbreviations used are: ADAM, a disintegrin and metalloprotease; DD, disintegrin/cysteine-rich domain of ADAM; dec-RVKR-CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; OPT, 1,10-ortho-phenanthroline; Suc, succinic acid; Mcp, dl-2-amino-3-(7-methoxycoumaryl)propionic acid; Dpa, l-2-amino-3-(2,4-dinitrophenyl)aminopropionic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MBP, myelin basic protein; CNS, central nervous system; BB-94, batimastat; PBS, phosphate-buffered saline; GFP, green fluorescent protein; GST, glutathione S-transferase; FCS, fetal calf serum; EndoH, endoglycosidase H; HRP, horseradish peroxidase; fw, forward; rev, reverse; MALDI, matrix-assisted laser desorption ionization; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; NHS, N-hydroxysuccinimide; PNGaseF, peptide-N-glycosidase F. 1The abbreviations used are: ADAM, a disintegrin and metalloprotease; DD, disintegrin/cysteine-rich domain of ADAM; dec-RVKR-CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; OPT, 1,10-ortho-phenanthroline; Suc, succinic acid; Mcp, dl-2-amino-3-(7-methoxycoumaryl)propionic acid; Dpa, l-2-amino-3-(2,4-dinitrophenyl)aminopropionic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MBP, myelin basic protein; CNS, central nervous system; BB-94, batimastat; PBS, phosphate-buffered saline; GFP, green fluorescent protein; GST, glutathione S-transferase; FCS, fetal calf serum; EndoH, endoglycosidase H; HRP, horseradish peroxidase; fw, forward; rev, reverse; MALDI, matrix-assisted laser desorption ionization; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; NHS, N-hydroxysuccinimide; PNGaseF, peptide-N-glycosidase F. (adisintegrinand metalloprotease domain) proteins constitute a family of transmembrane glycoproteins and serve essential physiological roles in fertilization, myogenesis, and neurogenesis. These functions are due to distinct protein domains involved in cell-cell fusion, cell-cell interaction, or proteolysis of membrane proteins, a process termed ectodomain shedding (1Peschon 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. 1997; 282: 1281-1284Google Scholar). To date, the family of ADAM proteinases comprises more than 30 members in different species (2Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Google Scholar, 3Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Google Scholar), and 24 ADAM genes were found in the mouse genome. Fourteen of the murine ADAMs contain the catalytic consensus sequence HEXXHXXGXXHD in their metalloprotease domains and are therefore predicted to be proteolytically active (4Stöcker W. Bode W. Curr. Opin. Struct. Biol. 1995; 5: 383-390Google Scholar). The cleavage of myelin basic protein (MBP) by ADAM10/MADM was the first demonstration of proteolysis by ADAMs (5Chantry A. Gregson N.A. Glynn P. J. Biol. Chem. 1989; 264: 21603-21607Google Scholar). The tumor necrosis factor-α convertase (ADAM17) was purified on the basis of its ability to cleave tumor necrosis factor-α (6Moss 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. Nature. 1997; 385: 733-736Google Scholar, 7Black 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: 729-733Google Scholar) and a number of other peptide and protein substrates in vitro (1Peschon 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. 1997; 282: 1281-1284Google Scholar,8Brou C. Logeat F. Gupta N. Bessia C. LeBail O. Doedens J.R. Cumano A. Roux P. Black R.A. Israel A. Mol. Cell. 2000; 5: 207-216Google Scholar). Proteolysis of membrane-bound surface molecules was also demonstrated for heparin-binding epidermal growth factor (9Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Google Scholar, 10Buxbaum 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. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Google Scholar, 11Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Google Scholar) and amyloid precursor protein (10,11), which are cleaved by ADAMs 9 and 10, respectively. Catalytically active ADAMs are usually activated by furin-catalyzed removal of the prodomain or by other proprotein convertases. For cleavage by furin-like convertases or prohormone convertases, such ADAMs possess a consensus sequence RX(K/R)R localized between the pro- and metalloprotease domains. This type of activation has been demonstrated for a number of ADAM proteases, among them ADAMs 9 (12Roghani 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: 3531-3540Google Scholar), 10 (13Anders A. Gilbert S. Garten W. Postina R. Fahrenholz F. FASEB J. 2001; 15: 1837-1839Google Scholar), 12 (14Loechel F. Gilpin B.J. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 16993-16997Google Scholar), 15 (15Lum L. Reid M.S. Blobel C.P. J. Biol. Chem. 1998; 273: 26236-26247Google Scholar), and 17 (16Clarke 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 Expr. Purif. 1998; 13: 104-110Google Scholar). Two members of the ADAM family in the mouse, ADAM8 (17Yoshida S. Setoguchi M. Higuchi Y. Akizuki S. Yamamoto S. Int. Immunol. 1990; 2: 585-591Google Scholar) and ADAM28 (18Howard L. Maciewicz R.A. Blobel C.P. Biochem. J. 2000; 348: 21-27Google Scholar), do not contain the consensus sequence for activation by furin-like proteases. In ADAM28, a recently cloned ADAM, a point mutation from glutamate 343 to alanine in the catalytic domain prevents prodomain removal, suggesting an autocatalytic mechanism for the protease activation (18Howard L. Maciewicz R.A. Blobel C.P. Biochem. J. 2000; 348: 21-27Google Scholar). Up to now, the mechanism of activation of ADAM8 is unknown. In matrix metalloproteases and in the snake venom metalloprotease Adamalysin II, the free sulfhydryl of the cysteine switch residue in the prodomain is thought to bind to the Zn2+ ion in the catalytic site. This interaction between the prodomain and the active site appears to inhibit the catalytic site and acts as an intramolecular chaperone, allowing the pro- and the metalloprotease domain to fold properly during transport through the secretory pathway (19Suzuki T. Yan Q. Lennarz W.J. J. Biol. Chem. 1998; 273: 10083-10086Google Scholar). In addition to their proteolytic properties, ADAMs are able to mediate cell-cell interactions via their disintegrin/cysteine-rich domains. A role in cell adhesion has been demonstrated for ADAM9, ADAM12, and ADAM15 (20Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Google Scholar, 21Zhang X.P. Kamata T. Yokoyama K. Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1998; 273: 7345-7350Google Scholar, 22Nath D. Slocombe P.M. Webster A. Stephens P.E. Docherty A.J. Murphy G. J. Cell Sci. 2000; 113: 2319-2328Google Scholar). The recombinant disintegrin/cysteine-rich domain of ADAM12 mediates cell adhesion (23Zolkiewska A. Exp. Cell Res. 1999; 252: 423-431Google Scholar). Human ADAM15 is the only metalloprotease-disintegrin containing an RGD sequence within the integrin-binding loop of the disintegrin domain (24Krätzschmar J. Lum L. Blobel C.P. J. Biol. Chem. 1996; 271: 4593-4596Google Scholar), and this has been shown to bind to the integrins αvβ3 and α5β1 (21Zhang X.P. Kamata T. Yokoyama K. Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1998; 273: 7345-7350Google Scholar, 25Nath D. Slocombe P.M. Stephens P.E. Warn A. Hutchinson G.R. Yamada K.M. Docherty A.J. Murphy G. J. Cell Sci. 1999; 112: 579-587Google Scholar). However, although necessary for integrin binding, the tripeptide alone is not sufficient to determine specificity for cell adhesion; rather, the overall loop structure within the integrin binding loop forms the determinant for integrin binding (21Zhang X.P. Kamata T. Yokoyama K. Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1998; 273: 7345-7350Google Scholar). Two ADAMs, ADAM22 and 23, lacking the conserved catalytic motif are highly expressed in the CNS. Deficiency in ADAM23 leads to an embryonic lethality (26Leighton P.A. Mitchell K.J. Goodrich L.V. Lu X. Pinson K. Scherz P. Skarnes W.C. Tessier-Lavigne M. Nature. 2001; 410: 174-179Google Scholar), whereas a knockout of ADAM22 causes ataxia (27Sagane K. Yamazaki K. Miziui Y. Tanaka I. Gene (Amst.). 1999; 236: 79-86Google Scholar), demonstrating that these ADAMs play essential roles in the CNS. ADAM8 was originally cloned as MS2 or CD156 from mouse macrophages (17Yoshida S. Setoguchi M. Higuchi Y. Akizuki S. Yamamoto S. Int. Immunol. 1990; 2: 585-591Google Scholar). It is expressed in macrophages, neurons, and oligodendrocytes and is up-regulated in the CNS following neurodegeneration and subsequent activation of glia cells, astrocytes, and microglia, suggesting that ADAM8 plays a role in neuron-glia interactions (28Schlomann U. Rathke-Hartlieb S. Yamamoto S. Jockusch H. Bartsch J.W. J. Neurosci. 2000; 20: 7964-7971Google Scholar). In rats immunized with a recombinant ADAM8 disintegrin domain, experimental autoimmune encephalomyelitis induced by myelin basic protein fragments is significantly ameliorated (29Schluesener H.J. J. Neuroimmunol. 1998; 87: 197-202Google Scholar). ADAM8 has also been implicated in the differentiation of osteoclasts, a process involving cell-cell fusion (30Choi S.J. Han J.H. Roodman G.D. J. Bone Miner. Res. 2001; 16: 814-822Google Scholar). These results suggest a role of ADAM8 in cell adhesion and cell fusion; however, the mechanisms of these activities are unknown. In the current study of ADAM8 we present the biochemical properties of ADAM8 with respect to catalytic activity, autocatalytic processing, and cell adhesion. Taq DNA polymerase (Master-Mix) was obtained from Qiagen (Hilden, Germany) and Pfupolymerase from Stratagene (Heidelberg, Germany). The furin inhibitor decanoyl-RVKR-chloromethylketone (CMK) was from Bachem (Heidelberg) and biotin-NHS from Amersham Biosciences (Lund, Sweden). Recombinant TIMPs 1 and 2, the inhibitory domain of TIMP4, and BB-94 were kindly provided by Dr. Harald Tschesche (University of Bielefeld). TIMP3 was obtained from Chemicon (Heidelberg, Germany). The catalytic domain of murine ADAM8 (amino acids 190–398) with a C-terminal histidine (HIS) tag was cloned using the Escherichia coli expression vector pRSETA. ADAM8catHIS was expressed in E. coli strain BL21 and purified from inclusion bodies using nickel-Sepharose according to manufacturer's instructions (Qiagen). After removal of urea by dialysis against PBS, the renatured protein was used to generate polyclonal rabbit antisera. Antibodies were affinity-purified by coupling the recombinant ADAM8catHIS to a HiTrap NHS column, passing 5 ml of serum over the column, washing, and eluting specific antibodies with 0.1 m glycine, pH 2.5. Eluted antibodies were neutralized with 1 m Tris-HCl, pH 8, and then dialyzed into PBS. The disintegrin domain (DD) of ADAM8 (amino acid residues 406–482) was cloned using the E. coli expression vector PGEX-2T. The resulting GST-A8DD fusion protein was expressed inE. coli strain BL21 after induction with 1 mmisopropyl-1-thio-β-d-galactopyranoside at 25 °C for 6 h. E. coli lysates were prepared and the GST fusion protein was purified on a glutathione agarose column. Peak fractions were analyzed by SDS-PAGE and dialyzed against PBS. The resulting protein was used to generate a polyclonal rabbit antisera. Antibodies were affinity-purified by coupling the recombinant GSTA8DD protein to a HiTrap NHS column, passing 5 ml of serum over the column, washing, and eluting with 0.1 m glycine, pH 2.5. Eluted antibodies were neutralized with 1 m Tris-HCl and then dialyzed into PBS. To remove anti-GST antibodies and cross-reacting antibodies, the affinity-purified antibody was passed over an additional HiTrap NHS column to which a similarly expressed and purified ADAM15 GST-DD had been coupled. The flowthrough was collected and shown to be specific for GSTA8DD compared with the ADAM15 GST-DD in an enzyme-linked immunoabsorbance assay. Affinity-purified antibodies were stored in aliquots at −20 °C. Antibody a-CTD was generated as described (28Schlomann U. Rathke-Hartlieb S. Yamamoto S. Jockusch H. Bartsch J.W. J. Neurosci. 2000; 20: 7964-7971Google Scholar) and was purified by protein A-Sepharose chromatography. 1321N1 astrocytoma cells were obtained from European Collection of Cell Cultures and were grown in Iscove's modified Dulbecco's medium (Invitrogen, Groningen, Netherlands) containing 5% FCS and 1% glutamine. COS-7 cells were grown in Dulbecco's modified Eagle's medium in the presence of 10% FCS and 1% glutamine. Transient transfections were performed with LipofectAMINE (Invitrogen) or by the calcium phosphate technique. Stable 1321N1 transfectants were selected with 400 μg/ml G418. Cells were lysed in RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10 mm 1,10-o-phenanthroline, 10 mmEGTA, and CompleteTM EDTA-free inhibitor mixture (Roche Molecular Biochemicals, Mannheim). The samples were incubated on ice for 1 h. After sonification, cell lysates were further enriched by ConA-Sepharose (Amersham Biosciences, Braunschweig, Germany). Proteins bound to ConA-Sepharose were washed 2× with 20 mmTris-HCl, pH 7.4, 0.5 m NaCl, 1 mmMgCl2, 1 mm CaCl2 and were eluted either with 100–200 μl of 2× SDS-PAGE loading buffer (for direct analysis) or with 100–200 μl of 0.2 mα-methylmannoside (Sigma, Deisenhofen, Germany) plus inhibitor mixture to recover active protease. Where indicated, samples were deglycosylated with endoglycosidase H (EndoH) or PNGaseF (New England BioLabs, Frankfurt) according to the manufacturer's instructions. For analysis of soluble ADAM8 proteins, cell supernatants from transfected cells were collected after adding serum-free medium 24 h before cell harvest. Supernatants were concentrated ∼10-fold by using YM columns (Millipore, Eschborn, Germany) and ConA-Sepharose. Samples were run on 7.5–10% SDS-PAGE and blotted onto Nylon membranes (Biodyne B, PALL, Portsmouth, UK) by electroblotting. After staining with 1% (w/v) Ponceau solution, proteins were subjected to immunoblotting by blocking with 5% milk powder overnight and antibodies were used in a 1:1000 dilution (pc antibodies) or 1:50 (monoclonal a-FLAG antibody) (31Rüdiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Google Scholar). Detection of the proteins was performed with anti-rabbit-IgG-HRP or anti-mouse-IgG-HRP (1:4000 to 1:5000, Sigma) using LumiLight Plus (Roche Molecular Biochemicals) as the chemiluminescent substrate. A full-length mouse ADAM8 cDNA (2481 bp) was amplified by PCR using Pfu polymerase with proofreading activity with the following primers: A8 fw, 5′-ATG CTT GGC CTC TGG CTG CTC AG-3′; A8 rev (FLAG sequence in italics), 5′-TCAGAT CTC CTG AGG CTT AAA CTG AGG GAA GGA CCT CTT CTG GAT GGG-3′. At the 3′-end, we used an oligonucleotide encoding 10 additional amino acids (SFPQFKPQEI, "Bi-Pro" FLAG) (29Schluesener H.J. J. Neuroimmunol. 1998; 87: 197-202Google Scholar) for immunodetection. The resulting PCR product ("A8cF") was cloned in the mammalian expression vector pTARGET (Promega, Heidelberg). Additional constructs with domain deletions or mutations in the catalytic domain were derived by PCR with A8 fw and the following primers: A8EC, 5′-TCA-FLAGsequence-ACA GTT GGG TGG TGC CCA GCC-3′; A8MP, 5′-TCA-FLAGsequence-CAC GAA CCG GTT GAC ATC TGG-3′. For introducing the point mutation glutamate to glutamine (E330Q), the amplification product of the primer pair A8fw and EQA8 (5′-CCA GGT TGT GGC CCA GCT GAT G-3′) was used as a sense primer in conjunction with the A8rev primer for amplification of full-length EQ-A8cF or primer A8MP for the soluble EQ-A8MP construct. For generation of the construct A8-ΔDD, the amplification products of the primer pair A8fw and A8ΔDD, 5′-TCT AGA CAC GAA CCG GTT GAC ATC TGG-3′, as well as sA8ΔDD, 5′-TCT AGA TGC CCA GGG GGC TAC TGC TTT-3′, and A8rev were subcloned in the pCRII vector (Invitrogen), and the fragments were ligated together by the extra XbaI site, thereby introducing two additional residues, serine and arginine. Cells grown on collagen-coated coverslips were fixed with ice-cold methanol for 5 min at room temperature. To detect ADAM8, we used polyclonal antibodies against the cytoplasmic domain (a-CTD, 1:500 in PBS) (28Schlomann U. Rathke-Hartlieb S. Yamamoto S. Jockusch H. Bartsch J.W. J. Neurosci. 2000; 20: 7964-7971Google Scholar), the disintegrin domain (a-DD, 1:200), or an antibody against the catalytic domain of ADAM8 (a-MP, 1:200) for 1 h at 37 °C or overnight at 4 °C. As secondary antibody, we used a goat anti-rabbit antibody coupled to Cy3 (Dianova, Hamburg, Germany). Fluorescent stainings were visualized using an Axiophot fluorescence microscope or by confocal laser microscopy (TCS SP2, Leica, Germany). Images were further processed with Adobe Photoshop 6.0. Transfected COS-7 cells were grown to confluency on 90-mm plates, washed with PBS at 4 °C, and incubated for 45 min with the non-membrane-permeant biotinylation reagent NHS-LC biotin (Pierce, Bonn, Germany) at room temperature. After washing with 0.1 m glycine in PBS, the cells were lysed in RIPA buffer plus protease inhibitors. The lysates were subjected to immunoprecipitation with anti-Bi-Pro antibody and protein G-Sepharose (Sigma) (32Giesemann T. Rathke-Hartlieb S. Rothkegel M. Bartsch J.W. Buchmeier S. Jockusch B.M. Jockusch H. J. Biol. Chem. 1999; 274: 37908-37914Google Scholar). After elution with 2× Laemmli buffer, the samples were applied on SDS-PAGE gels, and the ADAM8-specific bands were detected by Western blotting and staining with a-CTD (1:1000) or streptavidin-HRP (1:1000, Roche Molecular Biochemicals). The cDNA fragment encoding the A8 DC domain was generated by PCR using PlatinumTM Pfx polymerase (Invitrogen) with the following primers: DCE-A8f, 5′-GGT GGC CCT GTG TGT GGA AAC-3′; DCE-A8r, 5′-TAC ACA GTT GGG TGG TGC CCA-3′. The resulting cDNA fragment was cloned into the bacterial expression vector pTrcHis2 (Invitrogen) containing a C-terminal Myc and His6 tag. This vector was transformed into E. coli strain TOP10 (Invitrogen). Recombinant protein expression was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside for 5 h to overnight. Purification of the recombinant DCE domain was done using the XpressTM system protein purification kit (Invitrogen) according to the manufacturer's instructions for native protein preparations. COS-7 cells were transiently transfected with constructs encoding various forms of C-terminally FLAG-tagged soluble catalytic domain lacking the transmembrane domain (see Fig. 1). The cells were grown to confluency, and 24 h before harvest, growth medium was exchanged by serum-free Dulbecco's modified Eagle's medium. The supernatants were collected and concentrated by centrifugation on Amicon YM-30 (Millipore) columns. The concentrated supernatants were further purified by affinity purification with ConA-Sepharose or as Fc fusion protein by protein A-Sepharose (Sigma). The purity of soluble proteases was confirmed by SDS-PAGE, and concentrations were determined using the BCA reagent. Further supplies of the ADAM8 catalytic domain expressed as an Fc fusion were generated essentially as described previously for ADAM17 (33Amour A. Knight C.G. Webster A. Slocombe P.M. Stephens P.E. Knauper V. Docherty A.J. Murphy G. FEBS Lett. 2000; 473: 275-279Google Scholar). PCR-amplified DNA encoding the prepro-catalytic domain was inserted upstream of an enterokinase cleavage site ([V405 in ADAM8V]DDDDK−) followed by the human IgG1 heavy chain constant region, hinge, CH2, and CH3 in vector pEE12. Following stable transfection of NSO myeloma cells (31Rüdiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Google Scholar), conditioned media were incubated with protein A-Sepharose from which ADAM8 was eluted following washing and cleavage with recombinant enterokinase (33Amour A. Knight C.G. Webster A. Slocombe P.M. Stephens P.E. Knauper V. Docherty A.J. Murphy G. FEBS Lett. 2000; 473: 275-279Google Scholar). Purified A8 proteins were incubated with 1–10 μg of bovine myelin basic protein (MBP, Sigma) in Tris-HCl (pH 7.4) in the presence of 100 μm ZnCl2 and 5 mm CaCl2, 200 mm NaCl, and EDTA-free inhibitor MixtureTM (Roche Molecular Biochemicals). The digestion products were analyzed on 15% PAGE gels and stained with Coomassie Brilliant Blue. ADAM8 protease activity was measured by monitoring the cleavage of the quenched fluorescent peptide [Suc-H-(Mcp)-GSLPQKSH-K(Dpa)-R-amide] substrate. Assays were performed in a final volume of 100 μl containing 1 μg of ADAM8, 10 mm HEPES, 0.98 mm CHAPS, and 2% Me2SO at pH 7.5. After a 1-h incubation at 25 °C, the increase in fluorescence (λ excitation: 340 nm; λ emission: 405 nm) associated with peptide cleavage was measured with a fluorometer system LB-970 (Berthold Technologies, Bad Wildbad, Germany). To determine the IC50 values, test compounds were prepared at an initial concentration of 1.5 mm in 100% Me2SO, then diluted in assay buffer to 0.3 mm. Further dilutions were made in assay buffer containing 20% Me2SO, prior to diluting 10-fold into the assay, to allow testing across the range 1 nm to 30 μm. IC50 values were calculated using GraphPad Prism. Data were fitted by using non-linear regression analysis. For preparation of sequencing samples, cell lysates from ten 150-mm plates of COS-7 cells transfected with the cDNA encoding the complete A8 protein were lysed with RIPA buffer (see above). After ConA-Sepharose purification and immunoprecipitation with anti-FLAG antibody 4A6 (31Rüdiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Google Scholar), samples were run on an SDS-PAGE gel in borate buffer (50 mmH3BO3, pH 9.0, 20% ethanol, 1 mmdithiothreitol). After electrophoresis, samples were electroblotted onto polyvinylidene difluoride) membranes (Porablot, Macherey and Nagel, Düren, Germany) and stained with either Coomassie Brilliant Blue or anti-A8-CD. For N-terminal sequencing, ADAM8 bands were cut out and applied on a protein sequencer (Knauer, Germany). Usually, 5 cycles of automated Edman degradation were run to obtain sufficient sequence information. Cell adhesion was tested essentially as described previously (23Zolkiewska A. Exp. Cell Res. 1999; 252: 423-431Google Scholar). Briefly, 96-well plates were covered with the indicated amount of recombinant protein in PBS at 4 °C for 16 h. After blocking with bovine serum albumin for 1 h, 105 cells in PBS or medium (with 5% FCS) were seeded onto the plates. For blocking experiments, cells were incubated prior to seeding with a-DD (10 μg/ml) for 15 min at room temperature. After 1-h incubation at 37 °C, the wells were rinsed 3× with PBS, and the remaining cells were quantified by counting 10 randomly chosen viewing fields (100-fold magnification) or by quantification through SDS-PAGE. The 100% value of cell adhesion was obtained by allowing 105 cells to adhere completely. Transiently transfected cells were co-transfected with a GFP vector, and the amount of GFP+ cells was counted in comparison to the total number of GFP+ cells seeded into control wells. The construct A8cF (Fig. 1) contains the complete cDNA sequence of mouse ADAM8 with 826 amino acids. For immunodetection and affinity purification, A8cF contains an additional FLAG sequence (F) of 10 amino acids derived from birch pollen profilin ("BiPro-flag") (31Rüdiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Google Scholar). Upon expression of the full-length construct A8cF in COS-7 cells, three bands representing ADAM8 protein were observed in cell lysates: 1) a proform of Mr 120,000; 2) a processed form with an Mr of 90,000, which is consistent with propeptide removal; 3) a "remnant" form of ADAM8 protein with an apparent molecular weight of ∼60,000 (Fig. 1B). The remnant ADAM8 protein has been detected even in the permanent presence of protease inhibitors during the preparation of the cell lysates and was found to be abundant in all tissues and cells expressing ADAM8 investigated to date (28Schlomann U. Rathke-Hartlieb S. Yamamoto S. Jockusch H. Bartsch J.W. J. Neurosci. 2000; 20: 7964-7971Google Scholar). As expected, a-MP detected only pro-ADAM8 and the 90-kDa processed form (Fig. 1B), but not the remnant ADAM8 protein, which lacks the catalytic domain. To investigate the maturation of ADAM8, we performed Western blot analysis with an EndoH-treated sample of ADAM8 expressing COS-7 cells. Only pro-ADAM8 was sensitive to EndoH, whereas the mature and the remnant form of ADAM8 were EndoH-resistant. An identical sample cleaved with PNGaseF, which removes most or all carbohydrate moieties, showed a shift of all three ADAM8 bands, demonstratingN-glycosylation (Fig. 2A). From surface-biotinylated COS-7 cells, only the processed forms of ADAM8 with molecular masses of 90 and 60 kDa were immunoprecipitated (Fig. 2B), indicating that prodomain removal occurs intracellularly. Taken together these results suggest that the observed processing of ADAM8 occurs by prodomain removal during passage through the secretory pathway. ADAM8 contains a non-perfect consensus cleavage sequence for furin (RETR in amino acid position 193). A specific inhibitor of furin-like proteases (decanoyl-RVKR-chloromethylketone) did not to inhibit the processing of A8cF in COS-7 cells, even in high concentrations of 50 μm(Fig. 3A). This finding suggested that prodo