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Characterization of CA XIII, a Novel Member of the Carbonic Anhydrase Isozyme Family

2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês

10.1074/jbc.m308984200

1083-351X

Jonna Lehtonen, Bairong Shen, Mauno Vihinen, Angela Casini, Andrea Scozzafava, Claudiu T. Supuran, Anna‐Kaisa Parkkila, Juha Saarnio, Antti Kivelä, Abdül Waheed, William S. Sly, Seppo Parkkila,

Chemical synthesis and alkaloids

The carbonic anhydrase (CA) gene family has been reported to consist of at least 11 enzymatically active members and a few inactive homologous proteins. Recent analyses of human and mouse databases provided evidence that human and mouse genomes contain genes for still another novel CA isozyme hereby named CA XIII. In the present study, we modeled the structure of human CA XIII. This model revealed a globular molecule with high structural similarity to cytosolic isozymes, CA I, II, and III. Recombinant mouse CA XIII showed catalytic activity similar to those of mitochondrial CA V and cytosolic CA I, with kcat/Km of 4.3 × 107m–1 s–1, and kcat of 8.3 × 104 s–1. It is very susceptible to inhibition by sulfonamide and anionic inhibitors, with inhibition constants of 17 nm for acetazolamide, a clinically used sulfonamide, and of 0.25 μm, for cyanate, respectively. Using panels of cDNAs we evaluated human and mouse CA13 gene expression in a number of different tissues. In human tissues, positive signals were identified in the thymus, small intestine, spleen, prostate, ovary, colon, and testis. In mouse, positive tissues included the spleen, lung, kidney, heart, brain, skeletal muscle, and testis. We also investigated the cellular and subcellular localization of CA XIII in human and mouse tissues using an antibody raised against a polypeptide of 14 amino acids common for both human and mouse orthologues. Immunohistochemical staining showed a unique and widespread distribution pattern for CA XIII compared with the other cytosolic CA isozymes. In conclusion, the predicted amino acid sequence, structural model, distribution, and activity data suggest that CA XIII represents a novel enzyme, which may play important physiological roles in several organs. The carbonic anhydrase (CA) gene family has been reported to consist of at least 11 enzymatically active members and a few inactive homologous proteins. Recent analyses of human and mouse databases provided evidence that human and mouse genomes contain genes for still another novel CA isozyme hereby named CA XIII. In the present study, we modeled the structure of human CA XIII. This model revealed a globular molecule with high structural similarity to cytosolic isozymes, CA I, II, and III. Recombinant mouse CA XIII showed catalytic activity similar to those of mitochondrial CA V and cytosolic CA I, with kcat/Km of 4.3 × 107m–1 s–1, and kcat of 8.3 × 104 s–1. It is very susceptible to inhibition by sulfonamide and anionic inhibitors, with inhibition constants of 17 nm for acetazolamide, a clinically used sulfonamide, and of 0.25 μm, for cyanate, respectively. Using panels of cDNAs we evaluated human and mouse CA13 gene expression in a number of different tissues. In human tissues, positive signals were identified in the thymus, small intestine, spleen, prostate, ovary, colon, and testis. In mouse, positive tissues included the spleen, lung, kidney, heart, brain, skeletal muscle, and testis. We also investigated the cellular and subcellular localization of CA XIII in human and mouse tissues using an antibody raised against a polypeptide of 14 amino acids common for both human and mouse orthologues. Immunohistochemical staining showed a unique and widespread distribution pattern for CA XIII compared with the other cytosolic CA isozymes. In conclusion, the predicted amino acid sequence, structural model, distribution, and activity data suggest that CA XIII represents a novel enzyme, which may play important physiological roles in several organs. Carbonic anhydrases (CAs) 1The abbreviations used are: CAs, carbonic anhydrases; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DAB, 3,3′-diaminobenzidine tetrahydrochloride. are zinc-containing metalloenzymes that catalyze reversible hydration of carbon dioxide in a reaction CO2+H2O⇆H++HCO3-. CAs are produced in a variety of tissues where they participate in several important biological processes such as acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, body fluid generation, and lipogenesis (1.Sly W.S. Hu P.Y. Annu. Rev. Biochem. 1995; 64: 375-401Crossref PubMed Scopus (737) Google Scholar, 2.Parkkila S. Parkkila A.-K. Scand. J. Gastroenterol. 1996; 31: 305-319Crossref PubMed Scopus (87) Google Scholar). The CA isozymes differ in their kinetic properties, their tissue distribution, and subcellular localization. The mammalian α-CA gene family has been reported to include at least eleven enzymatically active isoforms with different structural and catalytic properties (1.Sly W.S. Hu P.Y. Annu. Rev. Biochem. 1995; 64: 375-401Crossref PubMed Scopus (737) Google Scholar, 3.Hewett-Emmett D. Tashian R.E. Mol. Phylogenet. Evol. 1996; 5: 50-77Crossref PubMed Scopus (520) Google Scholar). Four of the active CA isozymes are cytosolic (CA I, II, III, and VII), four are membrane-associated (CA IV, IX, XII, and XIV), two are mitochondrial (CA VA and -VB), and one is a secretory form (CA VI). In addition to these isozymes with measurable CA activity, there are several proteins with homologous "CA-like" domains. These include e.g. carbonic anhydrase-related proteins (CA-RP)-VIII, CA-RP-X, CA-RP-XI, and receptor-type protein-tyrosine phosphatases (RPTP) β and γ (3.Hewett-Emmett D. Tashian R.E. Mol. Phylogenet. Evol. 1996; 5: 50-77Crossref PubMed Scopus (520) Google Scholar, 4.Krueger N.X. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7417-7421Crossref PubMed Scopus (201) Google Scholar, 5.Skaggs L.A. Bergenhem N.C.H. Venta P.J. Tashian R.E. Gene (Amst.). 1993; 126: 291-292Crossref PubMed Scopus (36) Google Scholar, 6.Levy J.B. Canoll P.D. Silvennoinen O. Barnea G. Morse B. Honegger A.M. Huang J.-T. Cannizzaro L.A. Park S.-H. Druck T. Huebner K. Sap J. Ehrlich M. Musacchio J.M. Schlessinger J. J. Biol. Chem. 1993; 268: 10573-10581Abstract Full Text PDF PubMed Google Scholar, 7.Barnea G. Silvennoinen O. Shaanan B. Honegger A.M. Canoll P.D. D'Eustachio P. Morse B. Levy J.B. Laforgia S. Huebner K. Musacchio J.M. Sap J. Schlessinger J. Mol. Cell. Biol. 1993; 13: 1497-1506Crossref PubMed Google Scholar, 8.Tashian R.E. Hewett-Emmett D. Carter N.D. Bergenhem N.C.H. Chegwidden W.R. Carter N.D. Edwards Y.H. The Carbonic Anhydrases New Horizons. Birkhäuser Verlag Basel, Switzerland2000: 105-120Google Scholar). A common feature for these CA-like proteins is that some of the critical histidine residues in the active site are missing, and the zinc ion, which is necessary for the enzymatic activity, is not bound to the molecule. Recent exploitation of mammalian genomic data has allowed researchers to search for novel proteins including new isoforms of the existing protein families. The mouse CA13 cDNA sequence had been submitted to the National Center for Biotechnology Information (NCBI) data base by the Dr. Hewett-Emmett group in 2000. By comparing sequences, we also identified a human cDNA sequence, which appeared to encode human CA XIII protein. Based on the predicted amino acid sequence of human CA XIII and the known tertiary structure of CA I (9.Chakravarty S. Kannan K.K. J. Mol. Biol. 1994; 243: 298-309Crossref PubMed Scopus (105) Google Scholar), we designed a computer-based model for the structure of CA XIII. We also studied the distribution of CA XIII gene and protein expression in a number of human and mouse tissues. Mouse CA XIII was produced in Escherichia coli, and the kinetic and inhibition data for the recombinant enzyme was measured using a colorimetric method. Phylogenetic Analysis—The sequence alignment of CAs was performed with ClustalW (10.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55989) Google Scholar). The phylogenetic analysis was done with PAUP* 4.0 (11.Swofford, D. (2001) PAUP* 4.0 Sinauor AssociatesGoogle Scholar). The majority rule consensus tree was obtained by three bootstrap runs with different random seeds. The dataset was bootstrapped 1000 times for each run. Structure Modeling—The model of human CA XIII was based on the structure of the human CA I at 2.0 Å resolution (9.Chakravarty S. Kannan K.K. J. Mol. Biol. 1994; 243: 298-309Crossref PubMed Scopus (105) Google Scholar) (PDB entry 1CZM). The model was built with the program InsightII (Accelrys Inc., San Diego, CA). Amino acid substitutions were built using a side chain rotamer library. The initial model was refined with Discover™ in a stepwise manner by energy minimization using the Amber™ force field. First, the newly built loops were refined with 500 steps of minimization with a fixed and a free backbone, respectively. Then, all side chains with a constrained backbone were minimized for 500 steps, followed by another 1000 steps of minimization for the whole model. Kinetic and Inhibition Measurements—Initial rates of CO2 hydration were measured with an SX.18MV-R Applied Photophysics stopped-flow instrument (Oxford, UK) by the changing pH indicator method (12.Khalifah R.G. J. Biol. Chem. 1971; 246: 2561-2573Abstract Full Text PDF PubMed Google Scholar). Phenol red (at a concentration of 0.2 mm) was used as an indicator, working at the absorbance maximum of 557 nm, with 10 mm Hepes (pH 7.5), 10 mm Tris, and 0.1 m Na2SO4, following the CA-catalyzed CO2 hydration reaction for a period of 10–100 s. Saturated CO2 solutions in water at 25 °C were used as substrate (12.Khalifah R.G. J. Biol. Chem. 1971; 246: 2561-2573Abstract Full Text PDF PubMed Google Scholar). Stock solutions of inhibitors (1 mm) were prepared in distilled-deionized water with 10–20% (v/v) Me2SO (which is not inhibitory at these concentrations), and dilutions up to 0.1 nm were made thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the enzyme-inhibitor complex. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the study are the mean of such results. Inhibition constants were calculated as described earlier (13.Scozzafava A. Menabuoni L. Mincione F. Briganti F. Mincione G. Supuran C.T. J. Med. Chem. 2000; 43: 4542-4551Crossref PubMed Scopus (149) Google Scholar). Antibodies—The anti-mouse CA XIII serum was raised by Innovagen AB (Lund, Sweden) in a rabbit against a synthetic peptide (AC-) DG-DQQSPIEIKTKEC (-COOH), which was designed based on the predicted amino acid sequence of mouse CA XIII. This peptide was found to be identical in the predicted human CA XIII sequence (Fig. 1), and thus, the same antibody was also used to detect the human orthologue. Both mouse and human CA13 cDNA sequences are available in GenBank™ (accession numbers AF231123 and AK095314). Rabbit antisera against human CA I and II have been produced and characterized earlier (12.Khalifah R.G. J. Biol. Chem. 1971; 246: 2561-2573Abstract Full Text PDF PubMed Google Scholar). Rabbit anti-mouse CA II antibody was raised against mouse CA II produced in E. coli. The specificity of the antibody was confirmed using blood lysates from both normal and CA II-deficient (Car-2–/–) mice (data not shown). Western Blotting—30 μg of protein from human colon, human liver, and mouse colon total homogenates or 20 μl of purified fraction from ProBond™ or inhibitor affinity chromatography were analyzed by SDS-PAGE under reducing conditions according to Laemmli (13.Scozzafava A. Menabuoni L. Mincione F. Briganti F. Mincione G. Supuran C.T. J. Med. Chem. 2000; 43: 4542-4551Crossref PubMed Scopus (149) Google Scholar). The separated proteins were stained by Colloidal Coomassie Blue (Invitrogen, Carlsbad, CA) or transferred electrophoretically from the gel to a polyvinylidene fluoride membrane (Invitrogen) in a Novex Xcell II blot module (Invitrogen). After the transblotting, the sample lines were detected by WesternBreeze chromogenic immunodetection system (Invitrogen) or ECL method (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. The first antibodies were usually diluted 1:1000. His-tagged CA XIII was detected using anti-CA XIII serum or anti-HisG antibody (1:5000). In the control experiments, preimmune serum (1:1000) or a mixture of anti-human CA I, anti-human CA II, and anti-mouse CA II sera were used instead of the anti-CA XIII serum. Additional control experiments were also performed in the presence of the blocking peptide (1:1000 diluted antibody plus CA XIII peptide, 100 μg in 10 ml). Immunohistochemistry—Immunoperoxidase staining for human CA XIII was performed using the biotin-streptavidin complex method. In the mouse tissues, the localization of CA XIII was examined by both immunoperoxidase and immunofluorescence methods. Human and mouse tissue samples fixed in Carnoy's fluid and embedded in paraffin were cut at 5 μm sections and placed on microscope slides. The biotin-streptavidin complex method included the following steps: (a) pretreatment of the sections with undiluted cow colostral whey (Biotop, Oulu, Finland) for 30 min and rinsing in phosphate-buffered saline (PBS); (b) incubation for 1 h with the primary antiserum or preimmune serum diluted 1:100 in 1% bovine serum albumin-PBS solution (BSA-PBS); (c) incubation for 10 min with biotinylated second antibody (Histostain-plus, Zymed Laboratories Inc., South San Francisco, CA); (d) incubation for 10 min with peroxidase-conjugated streptavidin (Histostain-plus, Zymed Laboratories Inc.); (e) incubation for 3 min with 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution (Liquid DAB substrate kit, Zymed Laboratories Inc.). The sections were washed three times for 5 min in PBS after incubation steps b and c and four times for 5 min after incubation step d. All of the incubations and washings were carried out at room temperature. Anti-human CA II serum was used as a positive control. Additional control staining was performed in the presence of the corresponding peptide (1:100 diluted anti-CA XIII serum plus 100 μg of peptide/100 μl of BSA-PBS). The sections were finally mounted in Neo-Mount (Merck, Darmstadt, Germany). The stained sections were examined and photographed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany). Mouse CA XIII was immunostained by the biotin-streptavidin complex method by employing the following steps: (a) pretreatment of the sections with undiluted cow colostral whey (Biotop) for 40 min and rinsing in PBS; (b) incubation for 1 h with the primary antiserum or preimmune serum diluted 1:100 in 1% BSA-PBS; (c) incubation for 1 h with biotinylated second antibody diluted 1:300 in 1% BSA-PBS (ZyMax goat anti-rabbit IgG, Zymed Laboratories Inc.); (d) incubation for 30 min with peroxidase-conjugated streptavidin diluted 1:750 in PBS (ZyMax, Zymed Laboratories Inc.); (e) incubation for 1 min with DAB solution (9 mg of DAB in 15 ml of PBS plus 5 μl of H2O2). The sections were washed three times for 10 min in PBS after incubation steps b and c and four times for 5 min after incubation step d. All of the incubations and washings were carried out at room temperature. Preimmune serum and anti-mouse CA II serum were used for control purposes. Mouse tissue sections were also stained by the immunofluorescence method and analyzed by confocal laser scanning microscopy. The steps in the immunofluorescence staining were as follows: (a) pretreatment of the sections with undiluted cow colostral whey (Biotop) for 40 min and rinsing in PBS; (b) incubation for 1 h with the primary antiserum or preimmune serum diluted 1:100 in 1% BSA-PBS; (c) incubation for 1 h with fluorescein-conjugated second antibody diluted 1:200 in 1% BSA-PBS (Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG (H+L), Molecular Probes, Eugene, OR). The sections were washed three times for 10 min in PBS after incubation step b and four times for 5 min after incubation step c. The sections were finally mounted in glycerol. The immunofluorescent sections were examined with a confocal laser scanning microscopy (PerkinElmer Life Sciences, Boston, MA). PCR Methods—The expression of human and mouse CA XIII mRNAs were examined using cDNA kits purchased from BD Biosciences (Palo Alto, CA). The cDNAs included in MTC™ panels were used as templates for polymerase chain reaction (PCR) using CA XIII gene-specific primers. The human MTC™ panel II and mouse MTC™ panel I (BD Biosciences) contained first strand cDNA preparations produced from total poly(A) RNAs isolated from a number of different tissues. Two primers for amplifying human CA13 cDNA were chosen based on the human cDNA sequence (accession number AK095314), which appeared to encode human CA XIII; forward 5′-ACAACGGTCCTATTCACT-3′ (nucleotides 83–100) and reverse 5′-CAGGATATGTCCAGTAGT-3′ (nucleotides 623–640), which generated a 557-bp product. The primers were produced by Sigma Genosys. To amplify mouse CA13 cDNA, three primers (Sigma Genosys) were chosen based on the mouse CA13 sequence published in GenBank™ (AF231123); forward (F1) 5′-GTCCCTGCCACAGGCTCT-3′ (nucleotides 3–20) and reverse (R1) 5′-TGCACAAGAGGCTTCGGA-3′ (nucleotides 712–729), which generated a 730-bp product. Forward primer F1 together with reverse primer (R2) 5′-ATACATTGGGGCAAATCT-3′ (nucleotides 993–1010) generated a full-length 1008-bp amplification product. Primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH, BD Biosciences) were used to monitor the quality of the cDNA samples. 5 ng of total cDNA was used as a template for PCR. All the reagents for PCR were from BD Biosciences. The PCR reactions were amplified on the thermal cycler (Gene Amp PCR system 9700, Applied Biosystems, Foster City, CA). The PCR amplification for human CA13 was performed using a three-step method. The PCR cycling parameters consisted of denaturation at 94 °C for 2 min, followed by 33 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 1 min 30 s, followed by final extension at 72 °C for 3 min. The PCR amplification for mouse CA13 with primer F1 and R1 was performed using a two-step method recommended by the manufacturer. The PCR cycling parameters consisted of denaturation at 94 °C for 2 min, followed by 33 cycles of denaturation at 94 °C for 30 s and extension and annealing at 68 °C for 2 min, followed by final extension at 68 °C for 3 min. The PCR amplifications for mouse spleen and 17-day-old embryo with primers F1 and R2 were also performed using a three-step method consisting cycling parameters of denaturation at 94 °C for 2 min, followed by 33 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min 30 s, followed by final extension at 72 °C for 10 min. The PCR products were analyzed by electrophoresis on 1.2% agarose gel containing 0.1 μg/ml ethidium bromide with DNA standard (100 bp DNA Ladder, New England Biolabs, Beverly, MA). Sequencing the PCR Products—The PCR products from the human thymus, mouse spleen, and mouse 17-day-embryo were purified from agarose gel using GFX™ PCR DNA and Gel Band Purification kit (Amersham Biosciences). The sequencing was performed using ABI PRISM™ Big Dye Terminator Cycle Sequencing Ready Reactions Kit, version 2.0 (Applied Biosystems) following the protocol of the manufacturer. First, 5 μl of DNA template was mixed with 4 μl of Terminator Ready Reaction Mix (Applied Biosystems) and 1.6 pmol of the primer was added to the reaction mixture. Second, the reactions were amplified by cycle sequencing on the Gene Amp PCR system 9700 thermal cycler (Applied Biosystems). The cycling parameters consisted of 25 cycles of denaturation at 94 °C for 10 s, annealing at 50 °C for 5 s and extension at 60 °C for 4 min. Third, after cycle sequencing, the extension products were purified by ethanol precipitation method recommended by the manufacturer. After purification the samples were resuspended to Template Suppression Reagent (Applied Biosystems) and denatured according to the manufacturer's protocol. The sequencing was finally performed on the ABI PRISM™ 3100 Genetic Analyser instrument (Applied Biosystems). Expression and Purification of the His-tagged CA XIII—The full-length PCR product from mouse 17-day-embryo was cloned into the E. coli TOP 10 bacterium using pTrcHis TOPO TA expression kit (Invitrogen) following the manufacturer's instructions. First, the PCR product was cloned into the TOPO vector and then the recombinant vector was transformed into E. coli. The transformed bacteria were incubated overnight at 37 °C on LB (Luria Bertani) plates containing 50 μg/ml ampicillin and 0.5% glucose. Several colonies were picked for analysis of positive clones. First, the colonies were cultured overnight in LB medium containing 50 μg/ml ampicillin and 0.5% glucose at 37 °C. Then the plasmids were isolated using QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The isolated plasmids were analyzed by restriction analysis and by sequencing. The restriction analysis was performed using two restriction enzymes, BamHI and HindIII (New England Biolabs), according to the manufacturer's protocol. The sequencing was performed using ABI PRISM™ Big Dye Terminator Cycle Sequencing Ready Reactions Kit, version 2.0 (Applied Biosystems) following the protocol of the manufacturer. The sequence primers, Xpress Forward primer and pTrcHis Reverse primer were from Invitrogen. E. coli TOP 10 strain transformed with pTrcHis plasmid containing mouse CA XIII cDNA was grown at 37 °C in 50 ml of LB medium containing 50 μg/ml ampicillin and 0.5% glucose to an O.D of 0.6 at 600 nm. The inducer, isopropyl-1-thio-β-d-galactopyranoside, was then added to a final concentration of 1 mm and then the culture was incubated at 37 °C. The cells were harvested after 5 h by centrifugation, and the pellet was frozen at –80 °C for later use. For large scale expression the bacteria were grown in 1 liter of LB medium. The bacterial cell pellet was resuspended in 160 ml of lysing buffer (50 mm NaHPO4, 0.5 M NaCl, pH 8.0) and 160 mg lysozyme (Sigma) was added. The resuspended cells were incubated on ice 30 min and lysed by pipetting. Bacterial cell components were removed by centrifugation (13,000 rpm, 15 min, +4 °C). The supernatant was directly applied onto either Invitrogen′s ProBond™ -purification system or conventional CA-inhibitor affinity chromatography purification. The ProBond™ is a nickel-charged agarose resin that has been designed to purify recombinant proteins containing a polyhistidine sequence. The ProBond™ purification was performed under native conditions according to Invitrogen's protocol. The bound enzyme was eluted using 250 mm imidazole, pH 8.0. The inhibitor affinity chromatography was performed using the carboxymethyl (CM) Bio-Gel A (BioRad Laboratories, Richmond, CA) coupled to p-aminomethylbenzene-sulfonamide. The gel was washed using 10 mm HEPES buffer (pH 7.5) containing 150 mm NaCl and the bound enzyme was eluted using 0.1 m sodium acetate, pH 5.6, containing 0.5 m sodium perchlorate. The purified His-tagged CA XIII was visualized by two methods: Colloidal Coomassie Blue protein stain (Invitrogen) and Western blotting. Phylogenesis and Structural Model of CA XIII—Fig. 1 shows the amino acid sequence alignment of human CAs including the novel isozyme, CA XIII. All three histidine residues (His-94, -96, and -119) known to be critical for CA enzymatic activity are conserved in the predicted human and mouse (data not shown) CA XIII sequences. The phylogenetic analysis (Fig. 2) showed that CAs form three distinct clusters. CA I, II, III, V, and VII are clustered together as cytosolic/intracellular CAs. The second cluster includes extracellular and membrane-bound isozymes CA IV, VI, IX, XII, and XIV. The CA-RP VIII, X, and XI belong to the third cluster. The novel CA XIII is most closely related to CA I, II, and III with about 60% sequence identity, and thus, is clustered as cytosolic/intracellular isoform. In Fig. 3, the surfaces of three CAs (human CA I (1CZM), human CA II (1CNX), and human CA XIII) are colored according to the hydrophobic/hydrophilic properties of amino acids. The superimposition of these structures indicates that the location of the inhibitor binding pocket is conserved. The sulfonamide-type inhibitors are located inside the cavity and are bound to the zinc and the hydrophobic patch of the active site. The sequence similarity between the template (human CA I) and the target (human CA XIII) was 77%. A sequence identity above 50% generally guarantees that the modeled protein structure is correct at a level comparable to that of an x-ray crystal structure (16.Sanchez R. Sali A. Curr. Opin. Struct. Biol. 1997; 7: 206-214Crossref PubMed Scopus (267) Google Scholar). The sequence alignment showed that the three Zn2+ coordinating histidine residues are conserved in CA XIII molecule. Therefore, the Zn2+ ion was directly merged into the structural model. The quality of the final model was checked using the three-dimensional profiles verification (17.Luthy R. Bowie J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2610) Google Scholar) and PROCHECK (18.Rodriguez R. Chinea G. Lopez N. Pons T. Vriend G. Bioinformatics. 1998; 14: 523-528Crossref PubMed Scopus (312) Google Scholar). The three-dimensional profiles verification method showed that the three-dimensional/one-dimensional scores of our model were always positive, which are within the range of scores for x-ray structure determinations. Additionally, PROCHECK confirmed that the model of human CA XIII had good geometric structural parameters. Catalytic Activity and Inhibition by Sulfonamides and Anions—Table I shows the kinetic parameters for the CO2 hydration reaction catalyzed by several α-CAs including recombinant mouse CA XIII. Inhibition data with acetazolamide, a clinically used CA inhibitor of the sulfonamide type, and cyanate, an anion inhibitor, are also presented.Table IKinetic parameters for different α-CA isozymesIsozymeActivity levelaAt 25 °C, pH 7.5 in the following buffer system: Hepes 0.01 M, Tris 0.01 M, Na2SO4 0.1 M.kcatkcat/KmK1AcetazolamideCyanates-1M-1 × s-1nMμMhCA Ibh, human.Moderate2 × 1055 × 1072000.7hCA IIHigh1.4 × 1061.5 × 1081230hCA IIILow1 × 1043 × 105300,000ntcnt, not tested.hCA IVHigh1.1 × 1065 × 1074030mCA VModerate7 × 1043 × 1076031hCA IXHigh3.8 × 1055.5 × 1072543mCA XIIIdm, murine isozyme.Moderate8.3 × 1044.3 × 107170.25a At 25 °C, pH 7.5 in the following buffer system: Hepes 0.01 M, Tris 0.01 M, Na2SO4 0.1 M.b h, human.c nt, not tested.d m, murine isozyme. Open table in a new tab CA13 Gene Expression—CA13 gene expression was investigated by PCR amplification of commercially available sets of cDNAs produced for selected human and mouse tissues. Human CA XIII mRNA was expressed in the thymus and small intestine, testis, spleen, prostate, ovary, and colon (Fig. 4). No CA XIII transcript was detected in the human leukocytes. In the mouse tissues, the positive signals were detected in the spleen, lung, kidney, heart, brain, skeletal muscle, testis, 7-day-old embryo, 11-day-old embryo, 15-day-old embryo, and 17-day-old embryo (Fig. 5). No CA XIII transcript was detected in the mouse liver.Fig. 5Mouse CA XIII mRNA expression analyzed by PCR amplification. The strongest signals were detected in the spleen, lung, kidney, 7-day-old embryo, and 17-day-old embryo, followed by the heart, brain, skeletal muscle, testis, 11-day-old embryo, and 15-day-old embryo. No signal was detected in the liver. All cDNAs were amplified using intrinsic primers (A) and spleen and 17-day-old embryo were selected to amplify a 1008-bp fragment representing full-length CA13 cDNA (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Sequencing the PCR Amplification Products—Automated sequencing of the PCR products confirmed that correct CA13 sequences were amplified from the cDNAs of human thymus, mouse spleen, and 17-day-old mouse embryo. The primers for mouse CA13 were designed to obtain a full-length cDNA, while the corresponding human PCR product represented only a partial one. For sequencing the PCR product of human CA13 cDNA was amplified forward from the 5′-end, while mouse CA13 was amplified from both the 5′- and 3′-ends covering the whole coding sequence. The results clearly indicated that the PCR fragments represented CA13 cDNAs in both species. Localization of CA XIII in Human and Mouse Tissues— Immunoreactivity of anti-CA XIII serum was confirmed by Western blotting. Single, 30-kDa polypeptides were detected in the human and mouse colon, while the signal in the human liver was barely apparent (Fig. 6). The control experiments using preimmune serum or peptide blocking were negative. The immunolocalization studies for CA XIII revealed three major features on this novel enzyme: First, CA XIII showed an intracellular staining pattern, typical for cytosolic proteins. Second, there were distinct differences in the distribution of CA XIII between human and mouse tissues. Third, CA XIII was found in a number of different tissues in both species. Although the distribution of CA XIII resembles that of CA II in several tissues, we found some differences that make CA XIII unique with respect to its localization. In the human alimentary tract, CA XIII was found in a number of tissues from the salivary glands to the large intestine. In the submandibular gland, CA XIII was expressed in serous acinar cells and duct epithelial cells (Fig. 7). Compared with CA II, CA XIII showed stronger signal in the duct epithelium. In the gastric mucosa, CA XIII showed only very weak staining in the surface epithelial cells of the body and