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Ectodomain Shedding of the Neural Recognition Molecule CHL1 by the Metalloprotease-disintegrin ADAM8 Promotes Neurite Outgrowth and Suppresses Neuronal Cell Death

2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês

10.1074/jbc.m400560200

ISSN

1083-351X

Autores

Silvia Naus, Melanie Richter, Dirk Wildeboer, Marcia L. Moss, Melitta Schachner, Jörg W. Bartsch,

Tópico(s)

Cellular Mechanics and Interactions

Resumo

The neural cell adhesion molecule "close homologue of L1," termed CHL1, has functional importance in the nervous system. CHL1 is expressed as a transmembrane protein of 185 kDa, and ectodomain shedding releases soluble fragments relevant for its physiological function. Here we describe that ADAM8, a member of the family of metalloprotease disintegrins cleaves a CHL1-Fc fusion protein in vitro at two sites corresponding to release of the extracellular domain of CHL1 in fibronectin (FN) domains II (125 kDa) and V (165 kDa), inhibited by batimastat (BB-94). Cleavage of CHL1-Fc in the 125-kDa fragment was not detectable under non-reducing conditions arguing that cleavage resulting in the 165-kDa fragment is more relevant in releasing soluble CHL1 in vivo. In cells transfected with full-length ADAM8, membrane proximal cleavage of CHL1 was similar and not stimulated by phorbol ester 12-O-tetradecanoylphorbol-13-acetate and pervanadate. No cleavage of CHL1 was observed in cells expressing either inactive ADAM8 with a Glu330 to Gln exchange (EQ-A8), or active ADAM10 and ADAM17. Consequently, processing of CHL1 was hardly detectable in brain extracts of ADAM8-deficient mice but enhanced in a neurodegenerative mouse mutant. CHL1 processed by ADAM8 in supernatants of COS-7 cells and in co-culture with cerebellar granule neurons was very potent in stimulating neurite outgrowth and suppressing neuronal cell death, not observed in cells co-transfected with CHL1/EQ-A8, CHL1/ADAM10, or CHL1/ADAM17. Taken together, we propose that ADAM8 plays an important role in physiological and pathological cell interactions by a specific release of functional CHL1 from the cell surface. The neural cell adhesion molecule "close homologue of L1," termed CHL1, has functional importance in the nervous system. CHL1 is expressed as a transmembrane protein of 185 kDa, and ectodomain shedding releases soluble fragments relevant for its physiological function. Here we describe that ADAM8, a member of the family of metalloprotease disintegrins cleaves a CHL1-Fc fusion protein in vitro at two sites corresponding to release of the extracellular domain of CHL1 in fibronectin (FN) domains II (125 kDa) and V (165 kDa), inhibited by batimastat (BB-94). Cleavage of CHL1-Fc in the 125-kDa fragment was not detectable under non-reducing conditions arguing that cleavage resulting in the 165-kDa fragment is more relevant in releasing soluble CHL1 in vivo. In cells transfected with full-length ADAM8, membrane proximal cleavage of CHL1 was similar and not stimulated by phorbol ester 12-O-tetradecanoylphorbol-13-acetate and pervanadate. No cleavage of CHL1 was observed in cells expressing either inactive ADAM8 with a Glu330 to Gln exchange (EQ-A8), or active ADAM10 and ADAM17. Consequently, processing of CHL1 was hardly detectable in brain extracts of ADAM8-deficient mice but enhanced in a neurodegenerative mouse mutant. CHL1 processed by ADAM8 in supernatants of COS-7 cells and in co-culture with cerebellar granule neurons was very potent in stimulating neurite outgrowth and suppressing neuronal cell death, not observed in cells co-transfected with CHL1/EQ-A8, CHL1/ADAM10, or CHL1/ADAM17. Taken together, we propose that ADAM8 plays an important role in physiological and pathological cell interactions by a specific release of functional CHL1 from the cell surface. Many membrane-anchored proteins are subjected to proteolytic processing thereby releasing their extracellular domains, 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: 1281-1284Crossref Scopus (1355) Google Scholar). This modification causes qualitative and irreversible changes in the function of these molecules. As demonstrated with genetic and biochemical means, enzymes capable of these functions are ADAMs 1The abbreviations used are: ADAM, a disintegrin and metalloprotease domain; CHL1, close homologue of L1; L1, neural recognition molecule L1; NCAM, neural cell adhesion molecule; OPT, 1,10-ortho-phenanthroline; MBP, myelin basic protein; TNF, tumor necrosis factor; BB-94, batimastat; TIMP, tissue inhibitor of metalloproteases; FN, fibronectin; PLL, poly-l-lysine; MeSH, mercaptoethanol. TPA, 12-O-tetradecanoylphorbol-13-acetate; TACE, TNF-α convertase; EGF, epidermal growth factor; HB-EGF, heparin-binding EGF; Ig, immunoglobulin; ConA, concanavalin A. 1The abbreviations used are: ADAM, a disintegrin and metalloprotease domain; CHL1, close homologue of L1; L1, neural recognition molecule L1; NCAM, neural cell adhesion molecule; OPT, 1,10-ortho-phenanthroline; MBP, myelin basic protein; TNF, tumor necrosis factor; BB-94, batimastat; TIMP, tissue inhibitor of metalloproteases; FN, fibronectin; PLL, poly-l-lysine; MeSH, mercaptoethanol. TPA, 12-O-tetradecanoylphorbol-13-acetate; TACE, TNF-α convertase; EGF, epidermal growth factor; HB-EGF, heparin-binding EGF; Ig, immunoglobulin; ConA, concanavalin A. (for "adisintegrin and metalloprotease domain"), proteins that constitute a family of transmembrane glycoproteins with essential physiological roles in fertilization, myogenesis, and neurogenesis (2Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (880) Google Scholar). These functions are due to distinct protein domains involved in either cell-cell fusion or cell-cell interaction, or to their zinc-coordinating metalloprotease domain. To date, the family of ADAM proteinases comprises more than 30 members in different species (3Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (224) Google Scholar, 4Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Fourteen of the murine ADAMs contain the catalytic consensus sequence HEXXHXXGXXHD in their metalloprotease domains and are therefore predictably proteolytically active (3Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (224) Google Scholar, 4Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). The cleavage of myelin basic protein (MBP) by ADAM10/MADM (mammalian disintegrin-metalloprotease) was the first demonstration of proteolysis by ADAMs (5Chantry A. Gregson N.A. Glynn P. J. Biol. Chem. 1989; 264: 21603-21607Abstract Full Text PDF PubMed Google Scholar). The TNF-α convertase (TACE/ADAM17) was purified on the basis of its ability to cleave TNF-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: 733-736Crossref PubMed Scopus (1466) Google 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: 729-733Crossref PubMed Scopus (2674) Google 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: 1281-1284Crossref Scopus (1355) Google 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: 207-216Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). TACE is also implicated in the shedding of factors in the EGF ligand family, such as heparin-binding epidermal growth factor (HB-EGF) and amphiregulin (reviewed in Ref. 2Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (880) Google Scholar), and the physiological importance of this cleavage was demonstrated by similar defects in TACE and HB-EGF-deficient mice (9Jackson L.F. Qiu T.H. Sunnarborg S.W. Chang A. Zhang C. Patterson C. Lee D.C. EMBO J. 2003; 22: 2704-2716Crossref PubMed Scopus (338) Google Scholar, 10Iwamoto R. Yamazaki S. Asakura M. Takashima S. Hasuwa H. Miyado K. Adachi S. Kitakaze M. Hashimoto K. Raab G. Nanba D. Higashiyama S. Hori M. Klagsbrun M. Mekada E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3221-3226Crossref PubMed Scopus (293) Google Scholar).In the CNS, proteolytic processing of the amyloid precursor protein (APP (11Buxbaum 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: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar)) is an essential event in the pathology of Alzheimer's disease. As α-secretases, a number of ADAMs have been described to cleave APP at the respective α-cleavage site, ADAM9, ADAM10, and ADAM17, suggesting that ADAM family members serve essential roles in the CNS.Recently, ADAM8, an ADAM originally cloned from monocytic cells (12Yoshida S. Setoguchi M. Higuchi Y. Akizuki S. Yamamoto S. Int. Immunol. 1990; 2: 585-591Crossref PubMed Scopus (106) Google Scholar), has been shown to be expressed in the CNS in neurons, reactive glia cells (astrocytes and microglia), and oligodendrocytes (13Schlomann U. Rathke-Hartlieb S. Yamamoto S. Jockusch H. Bartsch J.W. J. Neurosci. 2000; 20: 7964-7971Crossref PubMed Google Scholar) and was described as a sheddase of the low affinity IgE receptor CD23 (14Fourie A.M. Coles F. Moreno V. Karlsson L. J. Biol. Chem. 2003; 278: 30469-30477Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In addition to substrates of ADAM8 in the immune response, a specific substrate of ADAM8 in the CNS has not yet been described. From these examples it is clear, that for each substrate and tissue, the physiological relevance of processing has to be proven by a series of biochemical methods.In the CNS, neural recognition molecules play important roles in specifying cell interactions during development, regeneration, and modification of synaptic activity. One subfamily of recognition molecules is the immunoglobulin (Ig) superfamily. In their extracellular parts these molecules contain IgG-like domains with essential cysteine residues and four to five fibronectin repeats (FN). Within these FN domains, cell adhesion molecules can be proteolytically processed and released from the cell membrane, thereby exerting important biological functions. The first identified member of the Ig family was L1 (15Rathjen F.G. Schachner M. EMBO J. 1984; 3: 1-10Crossref PubMed Scopus (568) Google Scholar). L1 is a 200- to 220-kDa type I transmembrane protein. In the mouse brain, L1 protein is detectable in four distinct molecular forms with sizes of 200, 180, 140, and 80 kDa (16Sadoul K. Sadoul R. Faissner A. Schachner M. J. Neurochem. 1988; 50: 510-521Crossref PubMed Scopus (76) Google Scholar). It was recently shown that L1 is processed by ADAM10 into 85- and 32-kDa fragments (17Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Postina R. Fahrenholz F. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Crossref PubMed Scopus (27) Google Scholar), but also the proprotein convertase PC5A plays an essential role in L1 processing leading to processed fragments of 180- and 140-kDa size (18Kalus I. Schnegelsberg B. Seidah N.G. Kleene R. Schachner M. J. Biol. Chem. 2003; 278: 10381-10388Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar).Based on cross-reactivity of L1 antibodies, the close homologue of L1, termed CHL1, was cloned (19Holm J. Hillenbrand R. Steuber V. Bartsch U. Moos M. Lubbert H. Montag D. Schachner M. Eur. J. Neurosci. 1996; 8: 1613-1629Crossref PubMed Scopus (92) Google Scholar). In brain extracts, CHL1 is detectable in three distinct fragments of 185, 165, and 125 kDa. The 185-kDa fragment was only weakly detectable in non-detergent soluble brain extracts indicating that this might be the membrane-spanning form of CHL1, whereas the 165- and 125-kDa fragments are considered as proteolytically released fragments (20Hillenbrand R. Molthagen M. Montag D. Schachner M. Eur. J. Neurosci. 1999; 11: 813-826Crossref PubMed Scopus (102) Google Scholar). The soluble forms of L1 and CHL1, in which the extracellular portion was fused to an Fc tail, promote neuronal survival and control neurite outgrowth (21Chen S. Mantei N. Dong L. Schachner M. J. Neurobiol. 1999; 38: 428-439Crossref PubMed Scopus (146) Google Scholar). As demonstrated for processed L1, soluble forms can interact effectively with the extracellular matrix to regulate migration by interactions with the integrin αvβ5 (17Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Postina R. Fahrenholz F. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Crossref PubMed Scopus (27) Google Scholar, 22Thelen K. Kedar V. Panicker A.K. Schmid R.S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Crossref PubMed Google Scholar) or by unknown ligands. Similar observations have been made for CHL1 (23Buhusi M. Midkiff B.R. Gates A.M. Richter M. Schachner M. Maness P.F. J. Biol. Chem. 2003; 278: 25024-25031Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Expression of CHL1 in injured and regenerating central and peripheral neurons is strongly up-regulated (24Chaisuksunt V. Zhang Y. Anderson P.N. Campbell G. Vaudano E. Schachner M. Lieberman A.R. Neuroscience. 2000; 100: 87-108Crossref PubMed Scopus (88) Google Scholar, 25Rolf B. Lang D. Hillenbrand R. Richter M. Schachner M. Bartsch U. J. Neurosci. Res. 2003; 71: 835-843Crossref PubMed Scopus (25) Google Scholar), but its functional role in regeneration remains to be defined. Because specific proteolytic processing of CHL1 may be of functional importance for neuronal survival, neurite outgrowth, and cell migration, we screened for the ability of several members of the family of ADAM metalloproteases to process CHL1 into its naturally occurring fragments.MATERIALS AND METHODSAnimals—Breeding and experimental use of mice were performed in agreement with the German law on the protection of animals, with a permit by the local authorities. The wr mutation was maintained on a C57BL/6 background. 30-day-old mutant mice with a manifest WR phenotype were used for biochemical analysis. Mice deficient in ADAM8 were received from Dr. A. J. P. Docherty (Celltech, Slough, UK) and were maintained on a C57BL/6 background. Genotype analysis was done by PCR diagnosis on tail tip DNA. A more detailed description of these mice will be given elsewhere.Reagents—Taq DNA polymerase (Master-Mix) was obtained from Qiagen (Hilden, Germany), and Pfu polymerase was from Stratagene (Heidelberg, Germany). The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was obtained from Sigma (Deisenhofen, Germany). Pervanadate was prepared by mixing 100 μl of 50 mm Na3VO4 with 30 μl of 30% H2O2 for 5 min at room temperature, followed by adding 520 μl of water. Recombinant tissue inhibitors of metalloproteases (TIMPs) 1 and 2, the inhibitory domain of TIMP4, and the broad range matrix metalloprotease inhibitor batimastat (BB-94) were kindly provided by Dr. Harald Tschesche (Bielefeld University). TIMP3 was obtained from Chemicon (Heidelberg, Germany). As antibodies, polyclonal CHL1 antibody (25Rolf B. Lang D. Hillenbrand R. Richter M. Schachner M. Bartsch U. J. Neurosci. Res. 2003; 71: 835-843Crossref PubMed Scopus (25) Google Scholar), monoclonal antibody 4A6 directed against a birch pollen profilin tag, kindly provided by Dr. B. M. Jockusch (Braunschweig University) (26Rüdiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Crossref PubMed Scopus (30) Google Scholar, 27Schlomann U. Wildeboer D. Webster A. Antropova O. Zeuschner D. Docherty A.J. McLoughlin S. Skelton L. Jockusch H. Bartsch J.W. J. Biol. Chem. 2002; 277: 48210-48219Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and affinity-purified polyclonal antibodies against the cytoplasmic domain of ADAM8 (13Schlomann U. Rathke-Hartlieb S. Yamamoto S. Jockusch H. Bartsch J.W. J. Neurosci. 2000; 20: 7964-7971Crossref PubMed Google Scholar) and ADAM10 (epitope: amino acids 727–741 of mouse ADAM10) were used. For sample normalization, we used mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (Chemicon).Cloning of Expression Constructs—The prodomain and the catalytic domain of mouse ADAM8 were amplified by PCR with the primers mXhoFLA8MET (5′-CTC GAG ATC AGA TCT CCT GAG GCT TAA ACT GAG GGA AGG ACA CGA ACC GGT TGA CAT-3′) and mEcoREnA8Sta (5′-GAA TTC GAC GAC GAC GAC AAG ATG CTT GGC CTC TGG CTG CTC-3′). The resulting PCR product was ligated into the respective cloning sites (XhoI at the 5′ end; EcoRI at the 3′ end) of pSecTag2B vector (Invitrogen) to generate pA8MPSecTag. Full-length mouse ADAMs10 and 17 cDNAs were generated by reverse transcription-PCR with mRNA from mouse brain and spleen, respectively. Amplification of cDNAs by PCR was performed with Pfu polymerase with proofreading activity and the following primers: ADAM10s, 5′-ATG GTG TTG CCG ACA GTG TTA ATT CT-3′; ADAM10as, 5′-TTA GCG TCG CAT GTG TCC CAT TTG A-3′. For ADAM17 containing a Bi-Pro tag (26Rüdiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Crossref PubMed Scopus (30) Google Scholar), we used the following primers: ADAM17s, 5′-ATG AGG CAG TCT CTC CTA TTC CTG-3′; ADAM17as (with Bi-Pro tag sequence), 5′-TCA GAT CTC CTG AGG CTT AAA CTG AGG GAA GGA GCA CTC TGT TTC TTT GCT GTC A-3′. The resulting PCR products were cloned in the mammalian expression vector pTARGET (Promega, Heidelberg) containing a cytomegalovirus promoter. Insertion of correct sequences was verified by sequencing.Cell Culture—COS-7 cells were grown in Dulbecco's modified Eagle's medium in the presence of 10% fetal calf serum and 1% glutamine. Transient transfections were performed with LipofectAMINE (Invitrogen, Groningen, Netherlands). Stable transfected CHOK1 cells for the production of Fc fusion proteins were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplied with 5% ultra low IgG fetal calf serum, 1% glutamine, 1% non-essential amino acids, and HT-Supplement. From these cells, Fc fusion proteins were prepared as described previously (21Chen S. Mantei N. Dong L. Schachner M. J. Neurobiol. 1999; 38: 428-439Crossref PubMed Scopus (146) Google Scholar).Western Blot Analysis—The supernatants of transiently transfected cells were collected after addition of serum-free Dulbecco's modified Eagle's medium 36–48 h before harvest and were concentrated ∼10-fold by using Amicon Ultra Centrifugal Filter Devices 10K (Millipore, Eschborn, Germany). To obtain cell lysates, cells were lysed in radioimmune precipitation assay buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing Complete™ EDTA-free inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany) and 10 mm OPT. After incubation on ice for 1 h, samples were sonicated. Freshly prepared tissues were homogenized in radioimmune precipitation assay buffer containing the inhibitor mixture. After incubation on ice for 1 h, samples were centrifuged for 5 min at 13,000 × g at 4 °C. Supernatants were used for analysis. All samples were concentrated by affinity chromatography with ConA-Sepharose (Amersham Biosciences, Braunschweig, Germany). Proteins bound to ConA-Sepharose were washed twice with 20 mm Tris-HCl (pH 7.4), 0.5 m NaCl, 1 mm MnCl2, 1 mm CaCl2 and were eluted with 1× SDS-PAGE loading buffer (for reducing conditions: 60 mm Tris-HCl, pH 6.8, 2% SDS, 9% glycerol, 0.0025% Bromphenol Blue, and 0.36 m mercaptoethanol; for non-reducing running conditions, mercaptoethanol was omitted). Where indicated, cell supernatants were subjected to a high speed spin in an ultracentrifuge for 30 min at 100,000 × g to remove released membrane vesicles. For detection of CHL1 and ADAM8 proteins, samples were subjected to 5% and 7.5% SDS-PAGE, respectively. Samples were blotted onto nitrocellulose transfer membranes (Protran®, Schleicher & Schüll, Dassel, Germany) by semi-dry electroblotting. After staining with 0.1% Ponceau S solution, proteins were analyzed by immunoblotting after blocking with 5% milk powder for 2 h. Antibodies were used in a 1:10,000 dilution (crude serum of a polyclonal anti-CHL1 antibody), 1:25 (monoclonal anti-Bi-Pro antibody), 1:1,000 (polyclonal anti-ADAM8-CD antibody), and 1:1,000 (polyclonal anti-ADAM10 antibody). Detection of the proteins was performed with anti-rabbit-IgG-horseradish peroxidase (1:5000; Sigma) or anti-mouse-IgG-horseradish peroxidase (1:5,000; Jackson ImmunoResearch, West Grove, PA) using LumiLight Plus (Roche Molecular Biochemicals) as chemiluminescent substrate. For quantification of band intensities, silver-stained gels were scanned, and intensities were determined using Quantiscan software (Bio-Rad, Göttingen, Germany).Purification of Soluble ADAM8 Catalytic Domain—COS-7 cells were transiently transfected with the construct pA8MPSecTag (Invitrogen, Heidelberg, Germany) containing the pro- and the catalytic domain with a C-terminal His tag and an N-terminal secretion signal (Sec tag). For purification of ADAM8 protease, COS-7 supernatants were collected 36–48 h after addition of serum-free Dulbecco's modified Eagle's medium. Samples were concentrated with Amicon Ultra Centrifugal Filter Devices 10K (Millipore) and further purified by affinity purification with nickel-nitrilotriacetic acid chromatography (ProBond™ Purification System, Invitrogen). After elution, the supernatants were concentrated a second time by ultrafiltration ∼100-fold compared with harvested supernatants. The purity of soluble protease was confirmed by SDS-PAGE, concentrations were determined using the BCA reagent (Perbio Science, Bonn, Germany), and proteolytic activity was monitored by a fluorescent cleavage assay in which ADAM8 cleaves the quenched fluorescent peptide Suc-H-(Mcp)-GSLPQKSH-K(Dpa)-R-amide (27Schlomann U. Wildeboer D. Webster A. Antropova O. Zeuschner D. Docherty A.J. McLoughlin S. Skelton L. Jockusch H. Bartsch J.W. J. Biol. Chem. 2002; 277: 48210-48219Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar).Protease Assays—Purified ADAM8 was kept in 20 mm Tris-Cl (pH 7.4) in the presence of 5 mm CaCl2, 100 μm ZnCl2, 100 mm NaCl, Complete™ EDTA-free inhibitor mixture (Roche Molecular Biochemicals). For inhibition experiments, ADAM8 was preincubated with 10 mm 1,10-ortho-phenantroline (OPT) or 200 nm batimastat (BB-94) or 500 nm of each TIMP for 1 h before substrates were added. Cleavage assays were usually performed with 1 μg of recombinant Fc-tagged proteins L1 (L1-Fc), CHL1 (CHL1-Fc), or NCAM (NCAM-Fc), described in Ref. 21Chen S. Mantei N. Dong L. Schachner M. J. Neurobiol. 1999; 38: 428-439Crossref PubMed Scopus (146) Google Scholar, and were mixed with 1 μg of purified ADAM8 protease. Samples were incubated 3 h to overnight at 37 °C. Cleavage products were analyzed by 5% SDS-PAGE and either silver stained (28Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3729) Google Scholar) or blotted for immunostaining with anti-Bi-Pro (for ADAM8) and anti-CHL1.Analysis of Protease Activities in Cell Lysates—24 h after transfection of Chinese hamster ovary or COS-7 cells with the constructs encoding full-length ADAM proteases, the cells were washed 1× with phosphate-buffered saline and lysed with a buffer containing 20 mm HEPES, pH 7.4, 1% Nonidet P-40, and 0.5% deoxycholate. After centrifugation at 15,000 × g for 2 min, the resulting cell pellet containing cell-bound proteases was directly assayed for protease activity by adding 150 μl of mixture consisting of 50 μm TNF-α substrate (Dnp-SPLAQAVRSSSRNH2), 1× protease inhibitor mixture in fluorescamine assay buffer (0.05 m HEPES, pH 7.4, 0.2 m NaCl, 0.01 m CaCl2, 0.01% Brij 35). After 1–2 h at 37 °C, 25 μl of the sample was removed and mixed with 5 μl of 1% fluorescamine in Me2SO and 70 μl of fluorescamine assay buffer. About 50 μl of this mixture was diluted with 150 μl of assay buffer, and fluorescence (excitation: 386 nm; emission: 477 nm) was measured in a PerkinElmer Life Sciences LS50B Luminescence spectrometer in black-coated 96-well plates (Nunc, Wiesbaden, Germany). For activity measurements with the MBP substrate, the cell pellets were mixed with 50 μl of 100 μm MBP substrate (Suc-H-(Mcp)-GSLPQKSH-K(Dpa)-R-amide (26Rüdiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Crossref PubMed Scopus (30) Google Scholar)) and 50 μl of assay buffer (20 mm HEPES, pH 7.4, and 0.0015% Brij 35). Activity was measured at 340-nm excitation and 405-nm emission.N-terminal Sequence Analysis—For preparation of samples for sequencing, ∼10 μg of CHL1-Fc protein was incubated overnight with 10 μg of soluble, recombinant ADAM8. The samples were run on an SDS-PAGE gel and washed in borate buffer (50 mm H3BO3, pH 9.0, 20% ethanol, 1 mm dithiothreitol) after electrophoresis. Samples were electroblotted onto polyvinylidene difluoride membranes (Porablot, Macherey and Nagel, Düren, Germany) and stained with 0.2% Coomassie Brilliant Blue in 45.5% EtOH, 9% acetic acid, and 1 mm dithiothreitol. For N-terminal sequencing, the respective bands were excised and applied to a protein sequencer (Knauer, Germany). Five cycles of automated Edman degradation were run to obtain sufficient sequence information.Neurite Outgrowth Assays—Cerebellar granule neurons were prepared from 6- to 8-day-old CD1 mice as described previously (29Keilhauer G. Faissner A. Schachner M. Nature. 1985; 316: 728-730Crossref PubMed Scopus (411) Google Scholar). Cells were seeded onto poly-l-lysine (PLL)-coated coverslips and were maintained in serum-free medium (30Fischer G. Kunemund V. Schachner M. J. Neurosci. 1986; 6: 605-612Crossref PubMed Google Scholar). 1–2 h after seeding the neurons, CHL1-Fc protein was added to the medium as a positive control. To determine neurite outgrowth in the presence of supernatants from transfected COS-7 cells, serum-free medium was applied 12 h after co-transfection of COS-7 cells with CHL1/ADAM constructs and was left on the cells for 24 h. The COS-7 supernatants were applied to cerebellar neurons, and 18 h after addition of either CHL1-Fc protein or supernatants of transfected COS-7 cells, neurite outgrowth was determined. To visualize neurite outgrowth, cells were fixed with glutaraldehyde (4%) for 5 min and stained with either Richardson's Blue or with a polyclonal anti-NF200 antibody (Sigma, Germany). For each experimental setup, neurites of at least 50 cells with processes longer than the cell body diameter were measured using a Zeiss Axiophot microscope with digital equipment.Neurite outgrowth assays in co-cultures of cerebellar neurons with transfected COS-7 cells were performed by plating 5 × 104 COS-7 cells in 30-mm plates on PLL-coated coverslips. One day after transfection of COS-7 cells, freshly prepared cerebellar neurons were added at densities of 105 cells/plate, and the co-culture was maintained in serum-free medium. After 24 h, the number of attached neurons per visual field and neurite lengths were determined as described above.Each neurite outgrowth experiment was performed with cerebella from 3 mice, and each condition was determined in triplicates. The experiments were repeated at least 3 (n = 9) or 4 (n = 12) times as indicated. Data are given as means ± S.E.Cell Survival Assay—Cerebellar granule neurons were seeded in 96-well plates coated with PLL at densities of 105/well. After cell attachment, either serum-free medium or supernatants from transfected COS-7 cells were applied. After days 1, 3, and 5, the numbers of surviving cells were counted by Trypan blue exclusion staining. Data are from three independent experiments in triplicates and as means ± S.E. A Student t test was used to determine the significance, and a p value of <0.01 was considered as highly significant.RESULTSCleavage of Recombinant CHL1 by ADAM8 —Recombinant ADAM8 was expressed as a secreted soluble form from COS-7 cells containing the pro- and metalloprotease domain (A8-MP). To monitor for catalytic activity, a parallel assay with a fluorogenic substrate was performed (27Schlomann U. Wildeboer D. Webster A. Antropova O. Zeuschner D. Docherty A.J. McLoughlin S. Skelton L. Jockusch H. Bartsch J.W. J. Biol. Chem. 2002; 277: 48210-48219Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Extracellular domains of NCAM, L1, and CHL1 (Fig. 1) fused to an Fc tag (21Chen S. Mantei N. Dong L. Schachner M. J. Neurobiol. 1999; 38: 428-439Crossref PubMed Scopus (146) Google Scholar) were incubated with purified ADAM8 (Fig. 2A). Whereas NCAM and L1 were not cleaved by ADAM8, CHL1-Fc was cleaved into two products: a prominent 165-kDa fragment corresponding to the entire extracellular portion of CHL1 and a less prominent 125-kDa fragment. Whereas the 165-kDa fragment was detectable under reducing and non-reducing conditions, the 125-kDa fragment disappeared under non-reducing conditions (Fig. 2A, right panel). Specificity of the cleavage was confirmed by immunostaining using an antibody against the extracellular portion of CHL1 (Fig. 2B), and equal loading as well as processing of pro-ADAM8 into active protease by autocatalytic prodomain removal was confirmed by Western blot analysis (Fig. 2C). Usually, about 50% of he ADAM8 protein is processed into the active form. In Western blot experiments with anti-CHL1, the specific cleavage of CHL1 was confirmed by

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