Purification and sequence identification of anserinase Shoji Yamada, Yoshito Tanaka and Seiichi Ando Faculty of Fisheries, Kagoshima University, Japan Na-Acetylhistidine is found in high concentration exclusively in the brain, retina, lens, and occasionally the heart of poikilothermic vertebrates (bony fishes, amphibians and reptiles) excluding jawless and cartila- ginous fishes, but is absent from these tissues in homothermic vertebrates (birds and mammals) [1–3]. However, little is known about its biological roles in poikilothermic vertebrates. It is synthesized from l-His and acetyl-CoA by histidine acetyltransferase (EC 2.3.1.33) in the brain and lens [4], and hydrolyzed to histidine by anserinase (Xaa-methyl-His dipeptidase, EC 3.4.13.5) in the brain and eye [5,6]. Baslow & Lenney [5] isolated the enzyme that deacetylates Na-acetylhistidine from the brain of skipjack tuna Katsuwonus pelamis, and thus this enzyme was tempor- arily classified as ‘Na-acetylhistidine deacetylase’ (EC Keywords acetylhistidine; anserinase; carnosinase; cytosolic nonspecific dipeptidase; MEROPS M20A metallopeptidase Correspondence S. Yamada, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan Fax: +81 99 2864015 Tel: +81 99 2864172 E-mail: yamada@fish.kagoshima-u.ac.jp Enzyme EC 3.4.13.5; recommended name: Xaa- methyl-His dipeptidase; other names: anserinase, aminoacyl-methylhistidine dipeptidase, acetylhistidine deacetylase, N-acetylhistidine deacetylase, a-N-acetyl- L-histidine aminohydrolase, X-methyl-His dipeptidase Note The nucleotide sequences reported in this paper have been submitted to DDBJ ⁄ EMBL ⁄ GenBank databank with accession numbers AB179777 for anserinase and AB219566 for CNDP-like protein. (Received 7 August 2005, revised 2 September 2005, accepted 23 September 2005) doi:10.1111/j.1742-4658.2005.04991.x Anserinase (Xaa-methyl-His dipeptidase, EC 3.4.13.5) is a dipeptidase that mainly catalyzes the hydrolysis of Na-acetylhistidine in the brain, retina and vitreous body of all poikilothermic vertebrates. The gene encoding anserinase has not been previously identified. We report the molecular identification of anserinase, purified from brain of Nile tilapia Oreochromis niloticus. The determination of the N-terminal sequence of the purified anserinase allowed the design of primers permitting the corresponding cDNA to be cloned by PCR. The anserinase cDNA has an ORF of 1485 nucleotides and encodes a signal peptide of 18 amino acids and a mature protein of 476 amino acids with a predicted molecular mass of 53.3 kDa. Sequence analysis showed that anserinase is a member of the M20A metal- lopeptidase subfamily in MEROPS peptidase database, to which ‘serum’ carnosinase (EC 3.4.13.20) and cytosolic nonspecific dipeptidase (EC 3.4.13.18, CNDP) belong. A cDNA encoding CNDP-like protein was also isolated from tilapia brain. Whereas anserinase mRNA was detected only in brain, retina, kidney and skeletal muscle, CNDP-like protein mRNA was detected in all tissues examined. Abbreviations CNDP, cytosolic nonspecific dipeptidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6001 3.5.1.34). Subsequently, Lenney et al. [6] reported that the substrate specificity of Na-acetylhistidine deacety- lase purified from brain and eye of skipjack tuna resembled that of anserinase purified from skeletal muscle of codling Gadus callarias. In 1981, therefore, Na-acetylhistidine deacetylase was judged to be identi- cal with anserinase by NC-IUB, and its EC number was deleted. Anserinase was discovered by Jones [7,8], who found that anserine (b-alanyl-1-methylhistidine) was hydrolyzed by this enzyme in skeletal muscle of G. callarias. Anserinase is activated by bivalent metal ions and has broad specificity, with ability to hydrolyze many kinds of substrates such as Na-ace- tylhistidine, N-acetylmethionine, anserine, carnosine, homocarnosine (c-aminobutyrylhistidine), alanylhisti- dine, glycyl-leucine and leucylglycine [6,8,9]. Previ- ously, we reported that this enzyme, purified from the brain of rainbow trout Oncorhynchus mykiss to appar- ent homogeneity, is a homodimeric protein with a subunit of 55 kDa [9]. It is commonly believed that anserinase is universally distributed in poikilothermic animals containing Na-acetylhistidine in their tissues [5,6,9–11]. Mammalian tissues contain another peptidase called carnosinase. Carnosinase resembles anserinase in hydrolytic ability against carnosine, anserine and homo- carnosine, which are unusual dipeptides containing non-a-amino acids (i.e. b-alanine and c-aminobutyric acid). No other enzymes except anserinase and carno- sinase can hydrolyze these three dipeptides. Human tis- sues were aggressively investigated for carnosinase because its deficiency has been associated with neuro- logical deficits including intermittent seizures and men- tal retardation [12,13]. Carnosinase exists as two types: ‘tissue’ carnosinase (Xaa-His dipeptidase, EC 3.4.13.3) and ‘serum’ carnosinase (b-Ala-His dipeptidase, EC 3.4.13.20) [14–16]. As ‘tissue’ carnosinase with broad specificity is present in every human tissue, it has been suggested that this enzyme is identical with cytosolic nonspecific dipeptidase (CNDP, EC 3.4.13.18) [16]. On the other hand, ‘serum’ carnosinase with a narrow specificity is a glycoprotein and found in human serum, as well as in the brain and spinal fluid [17]. Recently, Teufel et al. [18] reported the molecular iden- tification of the two types of carnosinase in human, and the sequence analyses revealed that both CNDP and ‘serum’ carnosinase belong to the M20A metallo- peptidase subfamily in MEROPS database [19]. Unlike carnosinase, the gene encoding anserinase has not been previously identified. We report here for the first time the molecular identification of anserinase. Results and Discussion Purification and characterization of anserinase from Nile tilapia brain The procedure for the purification of the enzyme from Nile tilapia is summarized in Table 1. The brains were collected from  1000 specimens of Nile tilapia (com- mercial size). Crude extracts from the fish brains were first subjected to ammonium sulfate fractionation. Anserinase was recovered from the 50–60% saturated ammonium sulfate fraction. The active fraction was then subjected to hydrophobic interaction chromato- graphy on octyl-Sepharose CL-4B. When the fractions were screened for hydrolysis against Na-acetylhistidine, the activity was recovered as an unbound fraction (data not shown). The unbound fraction containing anserinase was then subjected to gel filtration using Superdex 200 HR (Fig. 1A). The molecular mass of anserinase as determined by gel filtration was 120 kDa. The active fractions were pooled, and subjec- ted to anion-exchange chromatography using Resource Q. The bound enzyme was eluted from the column as a single peak of enzyme activity when the salt concen- tration was  0.30 m (Fig. 1B). The active fractions were concentrated, and applied to a preparative native PAGE (Fig. 2). As shown in Fig. 2A, several protein bands were detected on the native PAGE. When the samples extracted from the gel slices were assayed for hydrolysis against Na-acetylhistidine, the activity was Table 1. Purification of anserinase from brain of Nile tilapia. One enzyme unit is defined as that activity of enzyme that catalyzes the hydro- lysis of 1 lmol Na-acetylhistidine in 1 h under the standard conditions. Step Fraction Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Purification (fold) Yield (%) 1 Crude extract 10479 6949 0.7 1 100 2 Ammonium sulfate precipitation 992 2811 2.8 4 40 3 Octyl-Sepharose CL-4B 125 1936 15 21 28 4 Superdex 200 HR 32 1494 47 67 21 5 Resource Q 7.5 651 87 124 9 6 Preparative native PAGE 0.52 289 556 794 4 7 Preparative SDS ⁄ PAGE 0.11 – – – – Purification and sequence identification of anserinase S. Yamada et al. 6002 FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing mainly recovered from gel slice numbers 13 and 14 (Fig. 2B). Gel slices 11–15 were subjected to SDS ⁄ PAGE under reducing conditions. Protein bands were visualized with silver staining (Fig. 2C). The visualized intensity of the 55-kDa protein band, indicated by an arrow in Fig. 2C, correlated with enzyme activity levels shown in Fig. 2B. Moreover, we previously reported that anserinase purified from brain of rainbow trout consists of a subunit of 55 kDa [9]. Therefore, the 55-kDa protein was assumed to be a single subunit of Nile tilapia anserinase. As the molecular mass of anserinase determined by gel filtration is 120 kDa in the present study, Nile tilapia anserinase, like the trout enzyme [9], is apparently a homodimeric protein. The sample solutions from gel slices 13 and 14 containing high enzyme activity were pooled and concentrated. The separation procedures by preparative native PAGE (step 6) resulted in 794-fold purification and 4% of the enzyme activity. However, the enzyme was not homogeneous, as there were several protein bands besides anserinase visualized from gel slices 13 and 14, as shown in Fig. 2A. Therefore, the final purification was conducted using preparative SDS ⁄ PAGE (step 7). The purified enzyme obtained from step 7 showed a single protein band (55 kDa) on SDS ⁄ PAGE (Fig. 3). The final recovery of the anserinase protein was 110 lg from 345 g fish brain. The sequence of the N-terminal 20 amino acids for the purified Nile tilapia anserinase, determined by automated Edman degrada- tion, was FXYMDLAQYVDSXQDEYVEM. In the N-terminal sequence, two amino acids expressed as ‘X’ at the 2nd and 13th residue from the N-terminus failed to be identified for unknown reasons. In Table 2 the substrate specificities of the Nile til- apia anserinase obtained by step 6 were compared with the previous data obtained from trout anserinase [9], which was purified from the trout brain to apparent homogeneity. The Nile tilapia and rainbow trout enzymes had similar broad specificities. Both enzymes showed strong hydrolytic activity against Na-chloro- acetyl-l-Leu and Gly-Gly. Anserine, carnosine and homocarnosine were also hydrolyzed. The Nile tilapia enzyme, however, hydrolyzed l-Ala-l-His, l-Leu-Gly and l-Pro-Gly at a much higher rate than the trout enzyme. Moreover, the Nile tilapia enzyme hydrolyzed both l-His-Gly and l-Ala-l-Pro, which were hardly cleaved at all by the trout enzyme. From these results, it is likely that the specificity of Nile tilapia anserinase is broader than that of rainbow trout anserinase. Molecular cloning of anserinase and CNDP-like protein Database search Initially we searched for a candidate for anserinase in the GenBank database by blastp using the N-terminal sequence of Nile tilapia anserinase. An ‘unnamed pro- tein’ product (DDBJ ⁄ EMBL ⁄ GenBank accession num- ber CAF95589) of the spotted river puffer Tetraodon nigroviridis was extracted from the database (Fig. 4). The N-terminal amino-acid sequence of Nile tilapia anserinase displayed 13 of 18 residues (excluding two residues of X) identical with the deduced amino-acid sequence 19–38 from the N-terminus of the Tetraodon Fig. 1. Gel filtration and anion-exchange chromatography of Nile til- apia anserinase. (A) Gel filtration on a Superdex 200 HR column (step 4). The concentrated sample from octyl-Sepharose CL-4B chromatography was applied to a Superdex 200 HR 10 ⁄ 30 column equilibrated with 50 m M sodium phosphate buffer, pH 7.0, contain- ing 150 m M NaCl. Fractions of 200 lL each were collected at a flow rate of 0.4 mLÆmin )1 . Standard proteins (molecular mass in parentheses) were aldolase (158 kDa), BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa). (B) Chromatography on a Resource Q column (step 5). The pooled active fraction from Superdex 200 HR chromatography was applied to a Resource Q column (1 mL size) equilibrated with 20 m M Tris ⁄ HCl buffer, pH 7.8. The column was washed with 10 mL of the equilibration buffer. A linear NaCl gradient (0–0.5 M; 20 mL of 20 m M Tris ⁄ HCl buffer, pH 7.8) was then applied. Frac- tions of 1 mL each were collected at a flow rate of 0.4 mLÆmin )1 . S. Yamada et al. Purification and sequence identification of anserinase FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6003 ‘unnamed protein’. The organization principle of the MEROPS peptidase database is a hierarchical classifi- cation in which homologous sets of the proteins of interest are grouped into families, and the homologous families are grouped into clans [19]. Therefore, the blastp program of the MEROPS database was used to search homologous peptidase genes to the Tetrao- don ‘unnamed protein’. Members of CNDP (MEROPS ID M20.005) and ‘serum’ carnosinase (MEROPS ID M20.006) in the M20A metallopeptidase family ⁄ sub- family of the MH clan were extracted from the data- base. Multiple sequence alignments of the extracted genes of vertebrates were performed using the clustal w program to reveal highly conserved amino-acid sequences for designing degenerate primers for PCR amplification (data not shown). Cloning of anserinase cDNA PCR was performed using a set of primers (A and B) and the first-strand cDNA for 3¢ rapid amplification of cDNA ends (RACE), prepared from total RNA of Nile tilapia brain, as the template. The first-round PCR product was then used as a template for nested PCR amplification using a set of nested primers (A and C). As a result, the 281-bp PCR product was spe- cifically amplified. A full-length cDNA sequence of Nile tilapia anserinase was finally obtained by 3¢ and 5¢ RACE PCR (Fig. 5). The cDNA contained 1840-bp nucleotides including 56 bp of 5¢ UTR, 1482 bp of ORF, and 299 bp of 3¢ UTR. The coding region of the sequence was translated into 494 amino acids, which included a typical signal peptide of 18 amino acids and two potential N-glycosylation sites at the 104th and 134th residue (Asn) from the N-terminus. The predic- ted N-terminal amino-acid sequence of the ORF exclu- ding signal peptide completely matched the sequence determined by automated Edman degradation. cDNA sequence analysis predicted that two unknown amino acids at the 2nd and 13th residue from the N-terminus of the purified protein were both histidines. The cal- culated molecular mass and isoelectric point of the mature protein, excluding the signal peptide, were 53 311 Da and pH 5.3, respectively. A B C Fig. 2. Preparative native PAGE (step 6) of Nile tilapia anserinase. (A) Preparative native PAGE (7.5% running gel and 4.5% stacking gel) was performed as described in Experi- mental procedures. Proteins were stained with Coomassie Brilliant Blue R-250. (B) Un- stained gels were cut into 30 gel slices (numbered 1–30), and each fraction was assayed for hydrolytic activity against Na- acetylhistidine. (C) The samples of gel slices 11–15 were subjected to SDS ⁄ PAGE (7.5% running gel and 4.5% stacking gel) under reducing conditions. Protein bands were visualized with silver staining. Standard pro- teins (STD) were phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), and car- bonic anhydrase (30 kDa). The arrow indi- cates the position of the 55-kDa protein that proved to be anserinase. Purification and sequence identification of anserinase S. Yamada et al. 6004 FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing Cloning of the CNDP-like protein cDNA We also cloned a full-length cDNA encoding Nile til- apia CNDP-like protein using a partial nucleotide sequence of Mozambique tilapia (Oreochromis mossam- bicus) CNDP-like protein for primer design. PCR was performed using a set of primers (D and E) and the first-strand cDNA for 3¢ RACE as the template. As a result, the 527-bp PCR product was specifically ampli- fied. To obtain the 3¢ and 5¢ terminal segments of the cDNA, 3¢ and 5¢ RACE were then performed. The ORF of CNDP-like protein coded for a cytoplasmic protein (no signal peptide) of 474 amino acids with a calculated molecular mass of 52 807 Da and isoelectric point of 5.6 (data not shown). The deduced amino-acid sequence of CNDP-like protein showed 52% identity with and 66% similarity to that of anserinase from Nile tilapia (Fig. 6). Tissue distribution of the mRNAs for anserinase and CNDP-like protein in Nile tilapia The possibility of genomic contamination was elimin- ated by the observation of amplifications spanning the exon–intron boundaries, which were based on the information of the two scaffold files (M001527 for Fig. 3. SDS ⁄ PAGE of purified anserinase (step 7). The purified enzyme obtained from preparative SDS ⁄ PAGE was subjected to SDS ⁄ PAGE (12.5% running gel and 4.5% stacking gel) under redu- cing conditions. Protein bands were visualized with Coomassie Bril- liant Blue R-250. Standard proteins (STD) were phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa). Table 2. Substrate specificity of Nile tilapia anserinase. Substrate Relative rates of hydrolysis Nile tilapia a Rainbow trout b Na-Acetyl-L-His 100 100 Na-Acetyl- L-Asp 0 3 Na-Acetyl- L-Glu 0 23 Na-Acetyl- L-Met 116 254 Na-Acetyl- L-Cys 0 0 Na-Acetyl- L-Trp 3 30 Na-Acetyl- L-Leu 58 110 Na-Chloroacetyl- L-Leu 755 1235 L-Leu-b-naphthylamide 0 0 Anserine 39 16 Carnosine 74 83 Homocarnosine 155 67 L-Ala-L-His 447 105 Gly- L-His 327 166 L-His-Gly 58 6 Gly- L-Leu 443 823 Gly- D-Leu 0 0 L-Leu-Gly 360 101 L-His-L-Leu 26 0 Gly-Gly 543 385 L-Cys-Gly 0 0 L-Pro-Gly 301 76 L-Ala-L-Pro 46 0 Gly-Gly- L-Leu 0 0 Gly- L-Leu-L-Tyr 0 0 L-Leu-Gly-Gly 0 0 a The data from this study. The semipurified Nile tilapia enzyme obtained from step 6 (Table 1) was incubated at 30 °C for 1 h with 1m M substrate in 150 mM N-ethylmorpholine ⁄ HCl buffer, pH 6.5, containing 1 m M CoSO 4 . b The data from the previous study [9]. The anserinase of rainbow trout was purified to apparent homogeneity, and incubated under the same conditions as in the present study. Fig. 4. Alignment of amino-acid sequences of Nile tilapia anserinase N-terminus and ‘unnamed protein’ product (DDBJ ⁄ EMBL ⁄ GenBank accession number CAF95589) of spotted river puffer Tetraodon nigroviridis. Vertical lines indicate amino-acid identities, whereas colons indicate conservative substitutions. In the N-terminal sequence of Nile tilapia anserinase, two amino acids expressed as ‘X’ at the 2nd and 13th residue from the N-terminus were not able to be analyzed for unknown reasons. S. Yamada et al. Purification and sequence identification of anserinase FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6005 anserinase-like protein and M001163 for CNDP-like protein) extracted from the Fugu genome database. The RT-PCR products of the expected size for CNDP- like protein were observed in all tissues examined (Fig. 7). These distributions of CNDP-like protein in the fish are exactly consistent with those of human CNDP. In mouse, Otani et al. [20] recently reported that Western blotting analysis using the antibody against the recombinant carnosine-hydrolyzing protein, which is identical with CNDP-like protein, revealed the presence of the protein in kidney, liver, brain and spleen, and weakly in heart muscle and skeletal muscle. Although no enzymological information for CNDP in fish is at present available, it is likely that fish CNDP- like protein plays the same role as mammalian CNDP. On the other hand, anserinase mRNA was expressed strongly in brain, retina, skeletal muscle and kidney, and slightly in spleen (Fig. 7). We could not detect the RT-PCR products of the expected size for anserinase mRNA in any other tissues. According to our previous works [11,21], the enzymatic activity of anserinase was detected strongly in kidney, brain, liver and ocular fluid, and weakly in skeletal muscle and spleen of Nile tilapia. The mRNA expression in the brain and kidney obtained in this study are therefore consistent with the distribution of the enzyme activity. It seems likely that the enzyme activity in the ocular fluid originates from anserinase secretion from the retina, in which anseri- nase mRNA was strongly expressed. Interestingly, anserinase mRNA was not expressed in the liver, which contained the enzyme activity. Therefore, the question arises which tissue is the origin of liver anseri- nase. Whereas human ‘serum’ carnosinase is expressed only in central nervous system, rat and mouse ortho- logues are found exclusively in the kidney and are not expressed in the central nervous system [18]. Margolis et al. [22,23] revealed that tissues with carnosinase activity can be divided into two groups: kidney, uterus and olfactory mucosa represent one group, and central nervous system, muscle, spleen, etc. represent the sec- ond. Therefore, it can be suggested that mouse kidney carnosinase is translated from a gene orthologous to Fig. 5. Nucleotide sequence and predicted amino-acid sequence of cDNA encoding Nile tilapia anserinase. The putative signal sequence is shown in italics, and the N-terminal amino acids as determined by protein sequencing are underlined. Potential N-glycosylation sites are surrounded by rectangles. The active site residues are surrounded by ovals, and the metal binding sites are also highlighted in black. The terminal stop codon is marked with an asterisk. Purification and sequence identification of anserinase S. Yamada et al. 6006 FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing human ‘serum’ carnosinase. Interestingly, the mouse protein predicted from ‘serum’ carnosinase cDNA, unlike the human ‘serum’ carnosinase, does not have a typical N-terminal signal peptide. The gene expression and protein distribution of either anserinase or ‘serum’ carnosinase are therefore very complicated in verteb- rate animals. Molecular phylogenetic analysis We aligned the deduced amino-acid sequences of ver- tebrate M20A genes including the gene of Nile tilapia anserinase and constructed the unrooted phylogenetic tree shown in Fig. 8. Gene sequences from ascidian and vertebrate animals cluster within three distinct groups, CNDP-like, ‘serum’ carnosinase-like, and anse- rinase-like types. Only one gene was extracted from the databases for the ascidian Ciona intestinalis.It is likely that the ascidian gene is grouped as a CNDP-like type. The primary structures of all Fig. 6. Amino-acid alignment of Nile tilapia anserinase and CNDP-like genes. The putative signal sequence is underlined. Identical amino acids are indicated by an asterisk, and chemically similar amino acids are indicated by dots. Gaps inserted into the sequences are indicated by dashed lines. The active-site and metal-binding-site residues are highlighted in gray and black, respectively. The deduced amino-acid sequence of the ORF-encoded anserinase was aligned with the encoded CNDP-like protein using CLUSTAL W, showing 52% sequence identity and 66% similarity. Fig. 7. Tissue distribution of the mRNAs for anserinase and CNDP- like protein. Total RNA was prepared from Nile tilapia tissues, and RT-PCR was performed using specific primers. The expected sizes of the amplified bands of anserinase, CNDP-like protein and GAPDH were 530, 395 and 517 bp, respectively. S. Yamada et al. Purification and sequence identification of anserinase FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6007 CNDP-like proteins are relatively conserved; however, the function of these proteins is unknown except in humans [18]. In both African clawed frog Xenopus laevis and Atlantic salmon Salmo salar, three genes, which are separately grouped into CNDP-like, ‘serum’ carnosinase-like, and anserinase-like types, were extrac- ted from the databases. Darmin, the function of which is unknown, is a secreted protein expressed during endoderm development in African clawed frog [24]. From our phylogenetic analysis, Darmin protein is grouped as an anserinase-like type. However, as the phylogenetic divergence of Xenopus Darmin protein is a relatively long way from fish anserinase, as shown in Fig. 8, the enzymatic properties of Darmin protein need to be investigated and compared with those of fish anserinase. Another hypothetical MGC68563 protein of African clawed frog is closely related to a human ‘serum’ carnosinase. A homologous gene to ‘serum’ carnosinase in Atlantic salmon was also obtained by assembling three EST clones (TC33189, CX353277 and TC29012) extracted from the databases. We therefore suggest that before the vertebrate–ascidian divergence, an ancestral CNDP gene was first duplica- ted to form the original CNDP and the copied CNDP genes. The copied CNDP gene was further dupli- cated to form ‘serum’ carnosinase and anserinase genes. This second gene duplication event occurred before the divergence of ray-finned fish and tetrapod lineages. In conclusion, we report the molecular identification of anserinase, and demonstrate that the enzyme is a member of the M20A metallopeptidase subfamily, as well as ‘serum’ carnosinase and CNDP. The anserinase Fig. 8. Phylogenetic tree of anserinase and M20A genes of vertebrate animals. The tree was constructed by neighbor-joining distance analy- sis. Bootstrap values of 1000 resampling are indicated for all nodes on the tree. The scale bar corresponds to the estimated evolutionary dis- tance units. In Fugu Takifugu rubripes, the homologous genes to anserinase-like and CNDP-like proteins were located on scaffolds M001527 and M001163, respectively. The accession numbers of the homologues extracted from the DDBJ ⁄ EMBL ⁄ GenBank or the TIGR (http:// www.tigr.org/tdb/tgi/) databases are as follows: human Homo sapiens, ‘serum’ carnosinase (NM_032649) and CNDP (BC003176); mouse Mus musculus, ‘serum’ carnosinase-like (NM_177450) and CNDP-like (NM_023149); chicken Gallus gallus, ‘serum’ carnosinase-like (BX931960) and CNDP-like (TC188297); African clawed frog Xenopus laevis, MGC68563 protein (BC060450), Darmin protein (AY166869) and CNDP-like (BC056069); zebrafish Danio rerio, CNDP-like (AY391414); medaka Oryzias latipes, CNDP-like (TC30957); salmon Salmo salar, ‘serum’ carnosinase-like (assembled using TC33189, CX353277 and TC29012), anserinase-like (assembled using CK873786 and TC31285) and CNDP-like (assembled using CK884742, CX352802 and TC22931); ascidian Ciona intestinalis, CNDP-like (TC64855). Purification and sequence identification of anserinase S. Yamada et al. 6008 FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing mRNA was expressed strongly in brain, retina, skeletal muscle and kidney of Nile tilapia, whereas the CNDP mRNA was expressed in all tissues. It is also expected that a set of three genes, CNDP-like, anserinase-like and ‘serum’ carnosinase-like genes, exists in tetrapods (African clawed frog) and fish (Atlantic salmon). Fur- ther studies are therefore required to extensively investigate the existence of anserinase-like and ‘serum’ carnosinase-like genes in vertebrates. Experimental procedures Enzyme assay As Na-acetylhistidine is a major physiological substrate for anserinase in brain and eye of fish, we used it instead of anserine as a substrate for the anserinase assay throughout this study. Enzyme activity was assayed as follows: sample containing enzyme was incubated at 30 °C for 1 h with 1mm Na-acetylhistidine in 150 mm N-ethylmorpholine ⁄ HCl buffer, pH 6.5, containing 1 mm CoSO 4 [9]. The reaction was terminated by the addition of HClO 4 at a final concen- tration of 5% (w ⁄ v). The sample was then centrifuged for 15 min at 8000 g to precipitate the protein. Released histi- dine in the supernatant was quantified by HPLC using the o-phthalaldehyde post-column labeling method [21]. Protein determination Protein concentration was calculated as the sum of amino- acid contents after acid hydrolysis (6 m HCl, 24 h). Amino acid content was determined by HPLC as above. Analytical SDS/PAGE Electrophoresis in the presence of SDS and 2-mercaptoeth- anol was performed by the method of Laemmli [25], with a 7.5% or a 12.5% polyacrylamide running gel and a 4.5% polyacrylamide stacking gel. Proteins were stained with 0.25% Coomassie Brilliant Blue R-250 in 50% methanol containing 10% acetic acid or Silver Stain II Kit (Wako Pure Chemical Industries, Osaka, Japan). Purification of brain anserinase All operations were conducted at 0–4 °C unless otherwise mentioned. Fresh brains (345 g) of Nile tilapia O. niloticus were stored at )20 ° C. Step 1: extraction The frozen brains were homogenized with a Polytron homo- genizer in 10 vol. 10 mm sodium phosphate buffer, pH 7.8. The crude homogenate was centrifuged at 20 000 g for 1 h. Step 2: ammonium sulfate precipitation The supernatant was brought to 50% saturation with solid ammonium sulfate and left overnight. The precipi- tate was removed by centrifugation (20 000 g, 1 h) and discarded. The supernatant was precipitated by increasing ammonium sulfate to 60% saturation and left for 1 h. The precipitate was collected by centrifugation (20 000 g, 1 h), dissolved in 67 mL 10 mm N-ethylmorpholine ⁄ HCl buffer, pH 7.2, containing 0.1 mm CoSO 4 and brought to 30% saturation with solid ammonium sulfate. Insoluble material was removed by centrifugation (20 000 g,1h) and discarded. Step 3: octyl-Sepharose CL-4B chromatography The supernatant was applied to a column (2.6 · 40 cm) of octyl-Sepharose CL-4B (Amersham Pharmacia Biotech) previously equilibrated with 10 mm N-ethylmorpholine ⁄ HCl buffer, pH 7.2, containing 30% saturated ammonium sul- fate and 0.1 mm CoSO 4 at 7 °C. The column was washed with 500 mL of the equilibration buffer at a flow rate of 1.5 mLÆmin )1 , and a linear ammonium sulfate gradient (30–0% saturation; 2 L) was applied. The effluent was fractionated into 15-mL portions. The active fractions were pooled and concentrated to 1.2 mL by ultrafiltration through a PM-10 membrane (Amicon, Inc.). Step 4: Superdex 200 HR gel filtration The sample (200 lL) was injected at room temperature into a Superdex 200 HR 10 ⁄ 30 column (10 · 300 mm; Amersham Pharmacia Biotech) equilibrated with 50 mm sodium phosphate buffer, pH 7.0, containing 150 mm NaCl at a flow rate of 0.4 mLÆmin )1 . Fractions of 200 lL each were collected. The active fractions were combined and concentrated to 800 lL using a Centricon 10 (Amicon, Inc.). This separation step was separately performed six times (200 lL · 6). Step 5: Resource Q chromatography The sample was applied at room temperature to a Resource Q column (1 mL; Amersham Pharmacia Biotech) equilibrated with 20 mm Tris ⁄ HCl buffer, pH 7.8, at a flow rate of 1.0 mLÆmin )1 . The column was washed with 10 mL of the equilibration buffer for 10 min. A linear NaCl gradient (0–0.5 m; 20mL 20mm Tris ⁄ HCl buffer, pH 7.8) was applied, and 1-mL fractions were collected. The active fractions containing the enzyme were concentrated, and the buffer was replaced with 10 mm N-ethylmorpholine ⁄ HCl buffer, pH 7.2, containing 30% glycerol, using a centrifugal concentrator (Centricon- 10). S. Yamada et al. Purification and sequence identification of anserinase FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6009 Step 6: preparative native PAGE Preparative native PAGE (7.5% running gel and 4.5% stacking gel) was conducted according to a modification of the method of Lenney et al. [26]. The sample was applied to a 11 · 14 · 0.2-cm gel (five slabs). After electrophoresis for 2.5 h at 30 mA, the gel was cut into 3-mm slices. Each slice was crushed in 5 mL 10 mm N-ethylmorpholine ⁄ HCl buffer, pH 7.2, containing 0.1 mm CoSO 4 using a Potter- Elvehjem homogenizer. After centrifugation at 20 000 g for 30 min, the supernatant was concentrated, and the buffer was completely replaced with 10 mm N-ethylmorpho- line ⁄ HCl buffer, pH 7.2, using a Centricon-10. An aliquot of the sample obtained from each gel slice was assayed for enzyme activity. The active fractions of gel slices were con- centrated to 400 lL using a Centricon-10. Step 7: preparative SDS ⁄ PAGE The concentrate was applied over the width of a gel slab (11 · 14 · 0.1 cm, three slabs) and subjected to SDS ⁄ PAGE (7.5% running gel and 4.5% stacking gel) as described by Laemmli [25]. After electrophoresis for 2.5 h at 30 mA, 1-cm vertical strips were cut from the right and left sides of the slab using a cheese knife with a zig- zag shaped blade; these were immediately stained with Quick-CBB (Wako Pure Chemical Industries). The stained gel strips were replaced to each original position on the slab joining along the zigzag edge. The horizontal strip containing the anserinase band was excised from the unstained gel slab. Elution of the protein from the gel strips was performed electrophoretically using Electro- Eluter model 422 (Bio-Rad Laboratories), according to the manufacturer’s instructions. The sample solution was concentrated, and the buffer was completely replaced with distilled water, using a Centricon-10 for N-terminal sequence analysis. N-Terminal sequence analysis and BLAST search Edman degradation was performed on an automated pro- tein sequencer (model 491; Applied Biosystems). Protein Sequence Databases were searched for homologies with N-terminal sequence of anserinase using the world wide web-based blastp search engine of GenBank (http:// www.ncbi.nlm.nih.gov/BLAST/). A further blastp search was conducted by an engine of the MEROPS database (http://merops.sanger.ac.uk) [19] using ‘unnamed protein’ sequence (DDBJ ⁄ EMBL ⁄ GenBank accession number CAF95589) of spotted river puffer Tetraodon nigroviridis, which was extracted from blastp for N-terminal sequence of anserinase. Multiple sequence alignments were performed using the clustal w program (http://align.genome.jp/) to find highly conserved amino-acid sequences. Molecular cloning of Nile tilapia anserinase and CNDP-like protein cDNAs Isolation of a partial cDNA encoding anserinase Brain (193 mg) was dissected from Nile tilapia weighing  100 g and immediately stored in liquid nitrogen. Total RNA was isolated from the frozen brain using 2 mL TRI- zol Reagent (Invitrogen Corp., Carlsbad, CA, USA) follow- ing the manufacturer’s instructions. The first-strand cDNA for 3¢ RACE was synthesized from 1 lg total RNA using 200 U SuperScript II reverse transcriptase, the 35-mer oligo (dT)-adaptor (10 pmol) 5¢-GGCCACGCGTCGACTAG TACTTTTTTTTTTTTTTT-3¢, and reverse transcriptase buffer (20 lL) of the first-strand cDNA synthesis kit (Invi- trogen Corp.). The synthesis reaction was performed at 42 °C for 50 min. The forward primer (A) was designed for PCR; 21-mer degenerate oligonucleotide 5¢-CAR GAYGARTAYGTNGARATG-3¢ corresponding to N-ter- minal amino-acid sequences 14–20 (QDEYVEM) of Nile tilapia anserinase. Multiple sequence alignment of the genes belonging to CNDP (MEROPS ID M20.005) and ‘serum’ carnosinase (MEROPS ID M20.006), which were extracted from the MEROPS blastp search using the sequence of Tetraodon ‘unnamed protein’, revealed two highly con- served regions suitable for designing degenerate oligonucleo- tides for amplification of anserinase gene fragments (data not shown). Therefore, two reverse primers were designed for PCR; the 23-mer oligonucleotide 5¢-GAG CCNGWYTCYTCCATBCCYTC-3¢ corresponding to one consensus sequence (EGMEES ⁄ TGS) as the outer pri- mer (B), and the 23-mer oligonucleotide 5¢-TCCAG GYTDGCNGGCTGVACRTC-3¢ corresponding to another consensus sequence (DVQPAN ⁄ SLD ⁄ E) as the inner pri- mer (C). The first-round PCR was performed using a set of the primers (A and B), and the second-round nested PCR was primed with first-round PCR product and as a tem- plate a set of the primers (A and C). PCR amplification was carried out in a total volume of 50 lL containing 0.75 lL of a template, 150 pmol of a forward primer (A), 150 pmol of a reverse primer (B or C), 1 · G-Taq buffer, 10 nmol each of dATP, dGTP, dCTP and dTTP, and 0.5 U G-Taq DNA Polymerase (Cosmo Genetech Co., Seoul, Korea). For PCR the following conditions were used: initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 50 °C for 30 s, and extension at 72 °C for 1 min, final extension step at 72 ° C for 7 min. Isolation of a partial cDNA encoding CNDP-like protein A partial sequence of CNDP-like protein (DDBJ ⁄ EMBL ⁄ GenBank accession number AY260749) of Mozambique Purification and sequence identification of anserinase S. Yamada et al. 6010 FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing [...]... FEBS ⁄ Blackwell publishing 6011 Purification and sequence identification of anserinase protein, and GAPDH, respectively PCR amplification was performed in a total volume of 50 lL containing 0.5 lL of a template, 15 pmol of a forward primer, 15 pmol of a reverse primer, 1· PCR Gold buffer containing 2.5 mm MgCl2, 10 nmol each of dATP, dGTP, dCTP and dTTP, and 1 U AmpliTaq Gold (Applied Biosystems Japan... (http://genes.mit.edu/GENSCAN.html) was then used to Purification and sequence identification of anserinase predict Fugu anserinase- like protein and CNDP-like protein genes in scaffold files extracted from the genome database The homologous genes to anserinase- like and CNDP-like proteins were located on scaffolds M001527 and M001163, respectively The accession numbers of the homologues extracted from the DDBJ ⁄ EMBL... The free amino acids of fish; 1-methylhistidine and b-alanine liberation by skeletal muscle anserinase of codling (Gadus callarias) Biochem J 60, 81–87 8 Jones NR (1956) Anserinase and other dipeptidase activity in skeletal muscle of codling (Gadus callarias) Biochem J 64, 20 9 Yamada S, Tanaka Y, Sameshima M & Furuichi M (1993) Properties of Na-acetylhistidine deacetylase in brain of rainbow trout Oncorhynchus... Cleavage of structural proteins during assembly of the head of bacteriophage T4 Nature 227, 680–685 26 Lenney JF, Kan SC, Siu K & Sugiyama GH (1977) Homocarnosinase: a hog kidney dipeptidase with a Purification and sequence identification of anserinase broader specificity than carnosinase Arch Biochem Biophys 184, 257–266 27 Nielsen H, Engelbrecht J, Brunak S & Heijne G (1997) Identification of prokaryotic and. .. twice a day for 1 month at a water temperature of 25 °C Fish was killed by decapitation under anesthesia (50 mgÆL)1 of benzocaine) and tissues were then collected Total RNA was prepared from tissues of the fish using the TRIzol Reagent, and the first-strand cDNA was synthesized using the 35-mer oligo(dT)-adaptor as described above Anserinase, CNDP-like protein and glyceraldehyde-3-phosphate dehydrogenase... amplification of a partial cDNA for CNDP-like protein of Nile tilapia, the following primers were synthesized; the 22-mer forward oligonucleotide (D) 5¢-CTGTAAAGATGGTGGAGTTGGC-3¢ and the 22-mer reverse oligonucleotide (E) 5¢-GCAGCTTGACTCCCTGA ATGTA-3¢ corresponding to cDNA sequences 2–23 and 507–528, respectively, of Mozambique tilapia CNDP-like protein 3¢ and 5¢ RACE To obtain the 3¢ end, the first-strand cDNA... services/NetNGlyc/) A phylogenetic tree based on the amino-acid sequences was constructed by the neighbor-joining method of the clustal w program as described above The robustness of internal branches was estimated by 1000 bootstrap resamplings Tissue distribution of the mRNA for anserinase and CNDP-like protein The ethical guidelines from the Animal Ethics Committee of Kagoshima University on animal care were followed... N-acetyl-l-histidine and distribution in aquatic vertebrates Zoologica 50, 63–66 2 Erspamer V, Roseghini M & Anastasi A (1965) Occurrence and distribution of N-acetylhistidine in brain and extracerebral tissues of poikilothermal vertebrates J Neurochem 12, 123–130 3 Yamada S & Furuichi M (1990) Na-Acetylhistidine metabolism in fish 1 Identification of Na-acetylhistidine in the heart of rainbow trout Salmo... purification and characterization of histidine acetyltransferase in brain of Nile tilapia (Oreochromis niloticus) Biochim Biophys Acta 1245, 239–247 5 Baslow MH & Lenney JF (1967) Na-Acetyl-L-histidine amidohydrolase activity from the brain of the skipjack tuna Katsuwonus pelamis Can J Biochem 45, 337–340 6 Lenney JF, Baslow MH & Sugiyama GH (1978) Similarity of tuna N-acetylhistidine deacetylase and cod fish anserinase. .. volume of 10 lL containing 0.68 lg total RNA, 200 U SuperScript II reverse transcriptase, 5¢ RACE cDNA synthesis primer (10 pmol), and SMART II oligonucleotide (10 pmol) The synthesis reaction was performed at 42 °C for 90 min, and the 30-mer oligonucleotide 5¢-CCCGCGT ACTCTGCGTTGTTACCACTGCTT-3¢ was finally ligated to the 3¢ end of the first-strand cDNA Aliquots (0.25 lL) of the reaction mixture (first-strand . Purification and sequence identification of anserinase Shoji Yamada, Yoshito Tanaka and Seiichi Ando Faculty of Fisheries, Kagoshima University,. that of rainbow trout anserinase. Molecular cloning of anserinase and CNDP-like protein Database search Initially we searched for a candidate for anserinase