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• W2140797812 abstract "Carnosine (β-alanyl-l-histidine) and homocarnosine (γ-aminobutyric acid-l-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = βAla (carnosine, K m 1.2 mm),N-methyl βAla, Ala, Gly, and γ-aminobutyric acid (homocarnosine, K m 200 μm), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn2+ for full activity, and is sensitive to inhibition by bestatin (IC50 7 nm). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC 3.4.13.20), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC 3.4.13.18). Carnosine (β-alanyl-l-histidine) and homocarnosine (γ-aminobutyric acid-l-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = βAla (carnosine, K m 1.2 mm),N-methyl βAla, Ala, Gly, and γ-aminobutyric acid (homocarnosine, K m 200 μm), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn2+ for full activity, and is sensitive to inhibition by bestatin (IC50 7 nm). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC 3.4.13.20), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC 3.4.13.18). The dipeptide carnosine (β-alanyl-l-histidine) was first isolated in 1900 from meat extracts (1Gulewitsch W. Amiradgibi S. Ber. Dtsch. Chem. Ges. 1900; 33: 1902-1903Google Scholar) and subsequently found to be widely distributed in excitable central and peripheral vertebrate tissues (for review see Ref. 2Quinn P. Boldyrev A. Formazuyk V. Mol. Aspects Med. 1992; 13: 379-444Google Scholar). This compound is the archetype of a variety of aminoacyl histidine dipeptides such as homocarnosine (γ-amino-butyryl-histidine, GABA 1The abbreviations used are: GABA, γ-aminobutyric acid; AGE, advanced glycation end products; CSF, cerebrospinal fluid; EST, expressed sequence tag; MDA, malondialdehyde; OPA, o-pthaldialdehyde; CHO, Chinese hamster ovary; HGS, Human Genome Sciences; HMM, hidden Markov model; ORF, open reading frame; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight 1The abbreviations used are: GABA, γ-aminobutyric acid; AGE, advanced glycation end products; CSF, cerebrospinal fluid; EST, expressed sequence tag; MDA, malondialdehyde; OPA, o-pthaldialdehyde; CHO, Chinese hamster ovary; HGS, Human Genome Sciences; HMM, hidden Markov model; ORF, open reading frame; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight-His) or anserine (β-alanyl-l-1-methylhistidine). Whereas the role of many of the carnosine-related dipeptides is poorly understood, the function of carnosine has been studied intensively. In peripheral tissues this compound can be found at high levels (e.g. muscle tissues, 1–20 mm) (3Mannion A.F. Jakeman P.M. Dunnett M. Harris R.C. Willan P.L. Eur. J. Appl. Physiol. 1992; 64: 47-50Google Scholar) suggesting a crucial role as cytosolic buffer to neutralize lactic acid (4Bale-Smith E.C. J. Phys. 1938; 92: 336-343Google Scholar) (for review see Ref. 5Abe H. Biochemistry (Mosc.). 2000; 65: 757-765Google Scholar). In the central nervous system carnosine meets many criteria for a neurotransmitter modulating synaptic processes but also appears to be involved in neuroprotection (6Tabakman R. Lazarovici P. Kohen R. J. Neurosci. Res. 2002; 68: 463-469Google Scholar). The dipeptide has antioxidant and free radical scavenger properties, through complexation of transition metals such as zinc or copper (7Baran E.J. Biochemistry (Mosc.). 2000; 65: 789-797Google Scholar, 8Tromblay P.Q. Horning M.S. Blakemore L.J. Biochemistry (Mosc.). 2000; 65: 807-816Google Scholar); it retards senescence of cultured fibroblasts (9McFarland G. Holliday R. Exp. Cell Res. 1994; 167: 175-184Google Scholar) and is an anti-glycation agent (for review see Ref. 10Hipkiss A.R. Int. J. Biochem. Cell Biol. 1998; 30: 863-868Google Scholar). Homocarnosine was suggested to be a precursor for the neurotransmitter GABA. Being controlled by one or several carnosinases, it acts as a GABA reservoir and may mediate the antiseizure effects of GABAergic therapies (11Petroff O.A.C. Hyder F. Rothman D.L. Mattson R.H. Neurology. 2001; 56: 709-715Google Scholar, 12Petroff O.A.C. Hyder F. Rothman D.L. Mattson R.H. Neurology. 1999; 52: 473-478Google Scholar). Carnosine is synthesized by carnosine synthase (EC 6.3.2.11; see Ref.13Horinishi H. Grillo M. Margolis F.L. J. Neurochem. 1978; 31: 909-919Google Scholar) from β-alanine and histidine in many tissues and degraded by intra- or extracellular dipeptidases, also named carnosinases, all belonging to the large family of metalloproteases. A cytosolic form previously named tissue carnosinase (EC 3.4.13.18) was first isolated from porcine kidney by Hanson and Smith (14Hanson H.T. Smith E.L. J. Biol. Chem. 1949; 179: 789-801Google Scholar) in 1949 and subsequently found widely distributed in tissues of rodents and higher mammals (15Wood T. Nature. 1957; 180: 39-40Google Scholar, 16Margolis F.L. Grillo M. Grannot-Reisfeld N. Farbman A.I. Biochim. Biophys. Acta. 1983; 744: 237-248Google Scholar, 17Wolos A. Piekarska K. Glogowski J. Koieczka I. Int. J. Biochem. 1978; 9: 57-62Google Scholar, 18Kunze N. Kleinkauf H. Bauer K. Eur. J. Biochem. 1986; 160: 605-613Google Scholar). The human isoform purified from kidney was described by Lenney et al. (19Lenney J.F. Peppers S.C. Kucero-Ozallo C.M. George R.P. Biochem. J. 1985; 228: 653-660Google Scholar). The authors suggested, however, that “human tissue carnosinase” acts as a cytosolic nonspecific dipeptidase rather than a selective carnosinase based on its broad substrate specificity and the strong inhibition by bestatin (20Lenney J.F. Biol. Chem. Hoppe-Seyler. 1990; 371: 167-171Google Scholar). A secreted form of human carnosinase was first described by Perryet al. (21Perry T.L. Hansen S. Love D.L. Lancet. 1968; 8: 1229-1230Google Scholar) in patients with carnosinemia and was first purified from human placenta (22Zoch E. Muller H. Enzymologica. 1971; 40: 199-208Google Scholar). The enzyme was also isolated from human plasma and originally named human serum carnosinase (EC3.4.13.20; see Ref. 23Lenney J.F. George R.P. Weiss A.M. Kucera C.M. Chan P.W.H. Rinzler G.S. Clin. Chim. Acta. 1982; 123: 221-231Google Scholar). It was distinguished from its cytosolic counterpart because of its particular distribution in human plasma and brain, its unique capability to degrade homocarnosine, and absence in non-primate mammals except for the Syrian golden hamster (24Jackson M.C. Kucera C.M. Lenney J.F. Clin. Chim. Acta. 1991; 196: 193-206Google Scholar). In this study the discovery of two novel genes CN1 and CN2 coding for metallopeptidases of the M20 family is described. We demonstrate that CN1 corresponds to the secreted human carnosinase and CN2 to the cytosolic nonspecific dipeptidase previously named tissue carnosinase. The pcDNA3 vector was purchased from Clontech. pCRII was from New England Biolabs. Oligonucleotides were synthesized on an Applied Biosystems DNA/RNA synthesizer model 392. DNA sequence analysis was performed using an Applied Biosystems model 373A DNA sequencer with the Big Dye dyedeoxy terminator cycle reagents (Applied Biosystems, Foster City, CA). Amplification steps using PCR were carried out with a Thermal Cycler 480 (PerkinElmer Life Sciences) using the GeneAmp kit reagents (Applied Biosystems). Heparanized human plasma was obtained from the Centre Regional de Transfusion Sanguine de Strasbourg. LLC-PK1 and CHO-K1 were from the American Type Culture Collection (Manassas, VA). Human serum was obtained from the Centre de Transfusion Strasbourg, France. Protein concentrations were determined using a protein assay kit (Bio-Rad). SDS-PAGE was carried out using 8% Tris/glycine polyacrylamide gels (Invitrogen). Proteins were visualized by silver staining of the gels using a commercial kit (Silver Express; Invitrogen). Carnosine and homocarnosine were from Sigma, Ala-Ala, βAla-Ala, Ala-His, Gly-His, Ser-His, Tyr-His, His-His, Glu-His, Ile-His, Met-His, Val-His, pGlu-His, βAsp-His, Leu-His, GABA-His, Gly-His-Gly, Gly-Gly-His, Ser-Pro, Ala-Pro, Gly-Gly, and Gly-Leu were from Bachem. N-Methylcarnosine was synthesized by Neosystem (Strasbourg, France). Pepstatin, Bestatin, AEBSF, E64, Phosphoramidone, and leupeptin were from Roche Diagnostics.o-Pthaldialdehyde 1,10-o-phenantrolin,p-hydromercurybenzoate, and other chemicals were from Sigma. A particularly abundant set of ESTs with overlapping sequences was identified in the Human Genome Sciences (HGS) human brain cDNA libraries. The 2.0-kb cDNA insert of the EST clone 999021 from this set (shown below to bear the full-length sequence of CN1) was sequenced, and the amino acid structure was deduced using standard algorithms. The insert was excised from pBluescript byEcoRI/XhoI digestion and subcloned into the cytomegalovirus promoter-based plasmid pcDNA3.1(+) to obtain pCN1. The insert sequence was compared with public and HGS data bases. Another set of homologous ESTs was identified in the HGS data base, and the EST clone 2831791 (bearing CN2) from HGS was sequenced, and the 2.6-kb cDNA insert was subcloned into pcDNA3.1(−) after excision by EcoRI/BamHI digestion and subsequently named pCN2. The 2-kb insert of mouse EST clone AI 746479 carrying the mCN1 gene was identified in the NCBI data base and sequenced, and the amino acid sequence was deduced using standard algorithms. A rat homologue of CN1 (EST clone AA925553) was identified in the GenBankTM data base and sequenced. Sequence analysis was performed using the Genetic Computer Group Sequence (www.gcg.com). For HMM profiling the CN1, CN2, and orthologues derived from the Drosophila melanogaster and Caenorhabditis elegans, together with the sequence of a yeast protein with unknown function (Swiss-Prot entry YFL4_YEAST), were used as a seed to build a hidden Markov model profile with HMMer (hmmer.wustl.edu), which was progressively refined during an iterative search of Swiss-Prot and TrEMBL. Fold recognition was carried out with the ProCeryon (www.proceryon.com/) software package. Three point mutations, M1, M2, M3, and a double mutant, M3M4, corresponding, respectively, to amino acid changes H133A, D166A, E201A, and E201A+D229A of CN1 ORF, were generated. Mutants were generated by PCR using full-length CN1 cDNA as template and the following oligonucleotides: for M1, 5′-AAA GGC ACC GTG TGC TTC TAC GGC GCT TTG GAC-3′ and 5′-CTC GAG GCG GCC GCT CAT TAG TGA TGG TGA TGG TGA TGG AGC TGG GCC AT-3′; for M2, 5′-ACG GAG GTA GAC GGG AAA CTT TAT GGA CGA GGA GCG ACC GCC AAC AAA-3′ and 5′-CTC GAG GCG GCC GCT CAT TAG TGA TGG TGA TGG TGA TGG AGC TGG GCC AT-3′; for M3, 5′-CTG GAG CAA GAT CTT CCT GTG AAT ATC AAA TTC ATC ATT GAG GGG ATG GAA GCT GCT GGC-3′ and 5′-CTC GAG GCG GCC GCT CAT TAG TGA TGG TGA TGG TGA TGG AGC TGG GCC AT-3′; and for M4, 5′-GCT GTT CCC CCG GGT TCC ATA AGT GAT TGC TGG CTT CCT TTG GCT GAT CCA CAG GTT GGC TGA AAT-3′ and 5′-GAA TTC GTC GAC ATG GAT CCC AAA-3′. In addition, a His6 domain was incorporated during amplification to follow expression of mutant proteins. Amplified fragments were inserted into pCRII and fully sequenced. Mutated fragments were reintroduced into full-length carnosinase by fragment reassembly. Briefly, M1 mutated fragment was digested byDraIII and XhoI to obtain 3′ end harboring the mutation (1149 bp) and ligated with theEcoRI-DraIII CN1 fragment (378 bp) corresponding to the 5′ end. For M2, M2 mutated fragment was digested withAccI to obtain a central fragment containing the mutation (391 bp) that was ligated, together with theEcoRI-AccI 5′ end CN1 fragment (463 bp) and with the AccI-XhoI 3′ end CN1 fragment (673 bp). For M3, M3 mutated fragment was digested with BglII andXhoI to obtain the 3′ end mutated fragment (969 bp) and ligated to the CN1 EcoRI-BglII fragment. Finally the double mutant M3M4 was obtained after restriction of M3M4 mutated fragment by EcoRI and SmaI to obtain 5′ end mutated region (731 bp), which was ligated to theSmaI-XhoI CN1 3′ end. All mutants were inserted into pcDNA3 for expression, and full-length reconstructed mutants were verified by DNA sequencing. Multiple tissue expression array and Northern blots were from a commercial source (Clontech). Hybridization and washing were performed according to the manufacturer's specifications using 32P-labeled cDNA probes. The probe CN1 was synthesized by PCR with the primers 5′-TTTCAGATAACCTGTGGATCA and 5′-GAACATTTT GGCAATTGGAATG covering nucleotides 680 to 1374 of the coding sequence. The CN2 probe was synthesized by PCR with the primers 5′-TTCCCTCTTTCCTTTCCCTC and 5′-GCATACACCACCATGTCTG, located in the 3′-untranslated region. A commercial multiple mouse tissue Northern blot (Clontech) was hybridized to a 1022-bp32P-labeled probe obtained from amplification of the mouse CN1 EST clone (accession number AI746749) and the primers 5′-GTGGTGGAGAAACAGGTAAC and 5′-CCAAAGGTTCCTGAGTGGAA. A commercial multiple rat tissue Northern blot (Clontech) has been hybridized to a 276-bp 32P-labeled probe obtained from amplification of rat cDNA and the primers 5′-TTACACCACCAAGCCCAATC and 5′-CCTCCCACTCCG TCAGTAAA, designed from the rat EST (accession number AA 925553) containing predicted rat CN1. Hybridization signals were detected with a PhosphorImager (Molecular Dynamics). Two synthetic peptides containing an N-terminal cystein and CN1 residues 256–272 (peptide 256C17E272) and 312–329 (peptide 312Y18K329) were used to raise polyclonal rabbit antibodies (C17E and Y18K) at Eurogentech followed by affinity purification with immobilized antigenic peptides. Purified antibodies were stored at −20 °C in 50% glycerol at a final concentration of 0.5–1 mg/ml. The same protocol was applied to produce antibodies against a synthetic peptide223S16E238 (S16E) of human CN2. Immunochemical detection of CN1 and CN2 was carried out by Western blotting of commercial multiple tissue blot (Oncogene, Boston, MA) or of 20 μg of human brain extracts (Clontech), CSF, and plasma using an immunopurified anti-CN1 polyclonal antibody (Y18K 1:1000) and a standard protocol. Tissue distribution of CN2 was studied using a multiple tissue blot (Oncogene) with purified anti-S16E antibody (1:1000). Specific immunocomplexes were visualized using a commercial kit (ECL; AmershamBiosciences). Human brain sections of 7 μm were deparaffinized and processed using the microwave antigen retrieval approach in target unmasking fluid buffer (Pharmingen) according to the manufacturer's protocol. The sections were pre-treated with 10% of normal goat serum in phosphate-buffered saline and then incubated with primary antibodies diluted in phosphate-buffered saline/2% normal goat serum for 24–48h at 4 °C. Antibody dilutions were 1:1000 for anti-C17E and 1:500 for anti-Y18K. Specific staining was detected with a commercial kit (ABC; Vector Laboratories) according to the manufacturer's instructions. Slides were examined with a Nikon Diaphot 300 microscope, and images were captured with an Eastman Kodak Co. digital camera. In control experiments primary antibodies were omitted and replaced by phosphate-buffered saline/2% normal goat serum. The specificity of the anti-C17E antibody was evaluated by pre-incubation with the corresponding antigenic peptide before proceeding to immunostaining of tissue sections. Immunostaining with each antibody was repeated at least twice. CHO cell line K1 (ATCC) was cultured according to ATCC instructions. The cells were plated a day before transfection into a 10-cm culture plate (5 × 106 cells per plate). Cells were transfected with 15 μg of pcDNA expression vectors carrying wild-type CN1, M1, M2, M3, or M3M4 or CN2 genes using the LipofectAMINE2000 protocol according to the manufacturer's instructions (Invitrogen). Cells and culture supernatants were harvested after 48 h of incubation in Dulbecco's modified Eagle's medium/H12 medium supplemented with 10% fetal calf serum and analyzed for enzyme activity as described below. The expression of recombinant proteins was verified by Western blot analysis. A stable CHO-K1 cell line expressing CN1 was constructed using the LipofectAmine 2000 protocol (Invitrogen) according to the manufacturer's instructions. Subclones were obtained by the limiting dilution technique (25Clarke R. Davis J.M. Basic Cell Culture, A Practical Approach. IRL Press, Cambridge, MA1994: 223-242Google Scholar). Positive selection was based on the presence of secreted active enzyme and confirmed by Western blot using Y18K antibodies. Large scale production using the stably expressing CHO cell line was performed with Cell Factory plates (Nunc). 9.5 × 107cells were plated per unit, amplified for 5 days in Dulbecco's modified Eagle's medium/H12 medium supplemented with 10% fetal calf serum, and incubated for 3 days in serum-free MSSL medium (500 ml per plate), minimum Eagle's medium/HF12 (1:1), 2 mmGlutamaxI, 1% nonessential amino acids, 0.5 mm sodium pyruvate, d-glucose (0.5 g/liter) concentrated lipids (0.1%), ITS supplement (1%), and 0.83 μg/ml FeSO4 (Invitrogen). To the medium, 0.5 mmsodium butyrate was added to further trigger the overexpression of the enzyme by 30% (26Gorman C.M. Howard B.H. Reeves R. Nucleic Acids Res. 1983; 11: 7631-7648Google Scholar). The cell culture medium was concentrated 10-fold by ultrafiltration prior to protein purification. Carnosinase activity was assayed according to a method described by Bando et al. (27Bando K. Shimotsuji T. Toyoshima H. Hayashi C. Miyai K. Ann. Clin. Biochem. 1984; 21: 510-514Google Scholar) modified and adapted to 96-well plates. Briefly, substrate hydrolysis was carried out in 50 mmTris-HCl buffer, pH 7.5, 1 mm carnosine in a 100-μl final volume using 0.25–0.5 μg of cell extract or 10 ng of purified enzyme. To assay for carnosinase activity in mammalian cells, LLC-PK1 and CHO-K1 cells were cultivated as recommended by ATCC. A soluble cell extract was obtained by centrifugation (15 min at 50000 ×g at 4 °C) of cell lysates from 10 × 108 sonified cells. Carnosinase activity in cell culture supernatants or heparanized human plasma was determined with a 5-μl sample. The reaction was initiated by addition of substrate and stopped after 60 min of incubation at 30 °C by adding 50 μl of 1% trichloroacetic acid. Liberated histidine was derivatized by adding 50 μl of 5 mg/ml o-pthaldialdehyde (OPA) dissolved in 2m NaOH and 30 min of incubation at 30 °C. Fluorescence was read using a MicroTek plate reader (λExc: 360 nm and λEm: 460 nm). Reaction blank values were obtained by adding the trichloroacetic acid stop solution 1 min prior to substrate addition. Reactions were carried out in triplicate. The activity of CN2 was assayed as described above except that 50 mmTris-sarcosine, pH 9.5, 0.1 mm MnCl2 was used as buffer. For the determination of V max andK m an HPLC-based method, which detects liberated amino acids by precolumn derivatization with OPA, was used (28Lindroth P. Mopper L. Anal. Chem. 1979; 51: 1667-1674Google Scholar); 10 μl of enzyme solution (diluted to 1 ng/μl) was added to 50 μl of 100 mm Tris-HCl, pH 7.5, and the volume was adjusted to 90 μl by adding H2O. The reaction was started by addition of 10 μl of carnosine (0.1–50 mm), homocarnosine, βAla-Ala, Ala-Ala, Pro-Ala, and Ser-Gln, as well as the tripeptides Gly-Gly-His or Gly-His-Gly, and stopped after 60 min of incubation at 30 °C by adding 400 μl of 96% ethanol. After 15 min of centrifugation at 15000 rpm at room temperature, supernatants were transferred to a fresh tube and dried under vacuum using a SpeedVac. The pellet was resuspended in 1 ml of 100 mm sodium borate buffer, pH 9.6. Prior to HPLC separation the samples were derivatized with OPA using an automated method programmed on a HP1100 HPLC system (Agilent, Waldbronn, Germany). Freshly prepared OPA reagent was added 1 min prior to injection, and the derivatized amino acids were separated at 0.3 ml/min on a Kromasil C18 column (2 × 250 mm; Merck) equilibrated in Eluent A (99.5:0.5 of 0.012 m sodium acetate:tetrahydrofuran, pH 6.5) thermostated at 25 °C. A linear gradient was applied from 50–100% amino acid Eluent B (50:35:15 of 0.012 m sodium acetate:methanol:acetonitrile, pH 6.5) for 16 min, and a step elution of 100% amino acid Eluent B was applied for 7 min. Derivatized amino acids were detected by their fluorescence (λExc: 340 nm, λEm: 450 nm). Calculation of kinetic constants was done with MICROCAL ORIGIN™ (Microcal Software Inc., Northampton, MA) using a direct fit of the Michaelis equation to the experimental data. Five liters of filtered cell culture supernatant were bound to 500 ml of DEAE-Sephacel (Amersham Biosciences) equilibrated with Buffer A (50 mmTris-HCl, pH 7.5, containing 0.1 mm MnCl2) and packed into a chromatography column (XK 50/50; Amersham Biosciences). The column was washed at 10 ml/min with 4 column volumes of Buffer A. A linear NaCl gradient from 0 to 500 mm in Buffer A over 3.5 column volumes followed by a step to 1 m NaCl was applied to elute the bound material, and 10-ml fractions were collected. Fractions containing carnosinase activity were pooled and concentrated by ultrafiltration on an AMICON YM100 membrane (Millipore, Waltham, MA). The concentrated sample was injected on a Hi-Load Superdex 200 prep grade HR26/60 (Amersham Biosciences) size exclusion column. Proteins were eluted in Buffer A at 3 ml/min, and 3-ml fractions were collected. Pooled fractions were concentrated by ultrafiltration as described above. The pooled fractions from the size exclusion step were loaded after 2-fold dilution with Buffer A onto a MonoQ HR5/5 column (Amersham Biosciences) equilibrated with the same buffer. Non-specifically bound proteins were eluted applying a step of Buffer A containing 100 mm NaCl followed by a linear gradient from 100 to 200 mm NaCl over 5 column volumes. Then a linear NaCl gradient between 200 and 400 mm in Buffer A over 13 column volumes was applied, followed by a second gradient of 5 column volumes to obtain 500 mm NaCl. Finally a 1 m NaCl step was applied for 5 column volumes. 5 × 108 CHO-K1 cells transfected with pCN2 were resuspended in 15 ml of ice-cold resuspension buffer (10 mm Tris-HCl buffer, pH 7.0, containing 100 μmMnCl2), sonicated 5 times for 10 s, and the lysate was cleared by centrifugation at 20000 × g for 15 min at 4 °C (S20 fraction). The S20 supernatant was loaded on a POROS50 DEAETM column equilibrated with resuspension buffer (10 × 100 mm; Applied Biosystems) connected to a BioCAD® work station (Applied Biosystems). Unbound protein was washed with 5 column volumes of resuspension buffer, and the CN2 protein was eluted with a linear gradient from 0 to 500 mm NaCl over 20 column volumes. Fractions of 3 ml were collected, and enzyme activity was assayed with 10 μl of each fraction. Fractions containing carnosinase activity were pooled and concentrated by ultrafiltration on an AMICON YM10 membrane (Millipore). The concentrated sample was injected on a Hi-Load Superdex™ 200 prep grade HR26/60 (AmershamBiosciences) size exclusion column equilibrated with Buffer A containing 150 mm NaCl. Proteins were eluted in that buffer at 3 ml/min, and 3-ml fractions were collected. Pooled fractions were concentrated by ultrafiltration as described above. The pooled fractions from the size exclusion step were loaded after 2-fold dilution with Buffer C (20 mm Tris-HCl, pH 7.5, 0.1 mm MnCl2,10% (v/v) glycerol) onto a MonoQ column (HR5/5; Amersham Biosciences) equilibrated with the same buffer. Unspecific bound proteins were eluted with Buffer C containing 100 mm NaCl. Then a linear NaCl gradient between 100 and 400 mm NaCl in Buffer C over 20 column volumes was applied, followed by a step of 0.5 mNaCl in Buffer C for 5 column volumes. The pooled fraction (100 μl) of CN1 or CN2 obtained from the MonoQ purification step were injected on a C4 column (2.1 × 100 mm, Aquabore; Applied Biosystems) equilibrated with 5% acetonitrile containing 0.1% trifluoroacetic acid using the INTEGRAL™ microanalytical work station (Applied Biosystems). The column was washed at 250 μl/min with 20 column volumes of this solvent and then a gradient 5–80% acetonitrile in 0.1% trifluoroacetic acid over 20 column volumes was applied. UV absorption was monitored at 280 nm. MALDI-TOF mass spectrometry was performed on a VOYAGER DEPRO instrument (Applied Biosystems). Matrix (α-cyano-hydroxycinnamic acid or sinapinic acid; Fluka) was freshly prepared in 30% acetonitrile, 0.3% trifluoroacetic acid at a concentration of 25 mg/ml. Crystals containing the protein sample were obtained by the drying droplet method. 1 μl of HPLC fraction was applied to the MALDI sample plate, and 0.5 μl of matrix was added. Spectra were recorded by accumulating 100 shots using the linear mode at 20000-V acceleration voltage, with 1 μs of delayed extraction. Purified CN1 (∼300 pmol) were subjected to automated Edman degradation using a 492HT protein sequencer (Applied Biosystems) and standard protocols. The sequence of CN2 was verified using internal peptides obtained from a trypsin digestion of purified enzyme. Peptides were separated by reversed phase HPLC. 100 pmol of purified peptides were subjected to automated Edman degradation as described above. 100 ng of purified CN1 were treated with PNGase F (Oxford Glycosciences) using the following procedure: 200-μl protein samples after MonoQ were dried under vacuum using a SpeedVac. The dried pellet was resuspended in 100 μl of deglycosylation buffer (20 mm NaPO4, 50 mm EDTA, 0.5% SDS, 1 mm β− mercaptoethanol) and then heated to 95 °C for 5 min before 10 μl of Nonidet P-40 were added. The experiment was started by the addition of 30 μl of PNGAseF solution and followed at 37 °C for 24 h. The sample was subsequently analyzed by SDS-PAGE using an 8% polyacrylamide gel (Invitrogen), and proteins were visualized after silver staining of the gel using a commercial kit (Invitrogen). SH-SY5Y neuroblastoma cells (ATCC) were cultured according to ATCC instructions. The cells were plated a day before transfection into a 10-cm culture plate (5 × 106 cells per plate). Cells were transfected with 15 μg of pCN1 using a Lipofectamine2000 (Invitrogen) protocol according to the manufacturer's instructions. 5 to 6 h after transfection, cells were transferred into 96-well culture plates at a density of 2 × 104 cells/well and cultured for 48 h before treatment with malondialdehyde (MDA). MDA (3 mm) and carnosine treatment were performed as described previously (29Hipkiss A.R. Preston J.E. Himswoth D.T.M. Worthington V.C. Abbot N.J. Neurosci. Lett. 1997; 238: 135-138Google Scholar). Cell viability was determined 16 h after treatment by assaying mitochondrial dehydrogenases as measured by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg/ml, 4 h). In control experiments cells were transfected with empty vector pcDNA3.1. Experiments were done in triplicate and repeated five times. An assembly of overlapping ESTs (contig) derived from human brain cDNA libraries were identified in the Human Genome Sciences data base, and an open reading frame of 1524 nucleotides could be defined (Fig. 1 A). One of the clones (EST 999021), containing the full-length sequence coding for an unknown polypeptide of 508 amino acids, was identified and named CN1. The putative ATG translation start codon is in a favorable context for translation initiation (30Kozak M. J. Cell Biol. 1989; 108: 229-241Google Scholar), and no ATG codons were detected further upstream. Sequence analysis predicted a protein of a molecular mass of 56803 Da with an isoelectric point of pH 4.4, carrying a typical signal peptide sequence and three N-glycosylation sites. Further analysis of the HGS data base revealed EST 2831719 from human kidney cont" @default.
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