Molecular Cloning and Characterization of Carbonic Anhydrase XII ...

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MolecularCloning Cloningand andCharacterization Characterizationof ofCarbonic Carbonic Molecular AnhydraseXII XIIfrom fromPufferfish Pufferfish(Takifugu (Takifugurubripes) rubripes) Anhydrase KanijRukshana RukshanaSumi Sumi11,,Soo SooCheol CheolKim Kim22,, Jewel Jewel Howlader Howlader 33,, Won Won Kyo Kyo Lee Lee 11,, Kap Kap Seong Seong Choi Choi 44,, Kanij 3 3 3 1, Hoy-TaekKim Kim3 , ,Jong-In Jong-InPark Park3 , ,Ill-Sup Ill-SupNou Nou3 and and Kang Kang Hee Hee Kho Kho 1,** Hoy-Taek Department Department of of Fisheries Fisheries Science, Science, College College of of Fisheries Fisheries and and Ocean Ocean Sciences, Sciences, Chonnam Chonnam National National University, University, 50, Daehak-ro, Yeosu, Jeonnam 59626, Korea; [email protected] (K.R.S.); [email protected] 50, Daehak-ro, Yeosu, Jeonnam 59626, Korea; [email protected] (K.R.S.); [email protected] (W.K.L.) (W.K.L.) 2 National 2 Department Department of of Biomedical Biomedical and and Electronic Electronic Engineering, Engineering, College College of of Engineering, Engineering, Chonnam Chonnam National University, Jeonnam 59626, 59626, Korea; Korea; [email protected] [email protected] University, Yeosu, Yeosu, Jeonnam 3 of Horticulture, College of Life Sunchon National National University, University, 3 Department Department of Horticulture, College of Life Science Science and and Natural Natural Resources, Resources, Sunchon 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Korea; [email protected] (J.H.); 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Korea; [email protected] (J.H.); [email protected] [email protected] (I.-S.N.) [email protected] (H.-T.K.); (H.-T.K.); [email protected] [email protected] (J.-I.P.); (J.-I.P.); [email protected] (I.-S.N.) of Food Science, College of Life Science and Natural University, 4 4 Department Department of Food Science, College of Life Science and Natural Resources, Resources, Sunchon Sunchon National National University, 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Korea; [email protected] 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Korea; [email protected] * * Correspondence: Correspondence: [email protected]; [email protected]; Tel: Tel.:+82-61-659-7168; +82-61-659-7168;Fax: Fax:+82-61-659-7169 +82-61-659-7169 1

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Received:22February February2018; 2018;Accepted: Accepted:12 12March March2018; 2018;Published: Published:13 13March March2018 2018 Received:

Abstract: In In this this study, sequence was cloned from the study, an an 1888-bp 1888-bp carbonic carbonicanhydrase anhydraseXII XII(CA (CAXII) XII) sequence was cloned from Abstract: brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of 1470-bp, the brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of which was predicted to translate into a protein of 490 amino residues. The predicted protein 1470-bp, which was predicted to translate into a protein of 490 acid amino acid residues. The predicted showed between 68–56% identity with the large yellow croaker (Larimichthys crocea), tilapia protein showed between 68–56% identity with the large yellow croaker (Larimichthys crocea), tilapia (Oreochromis niloticus), and Asian arowana (Scleropages formosus) CA XII proteins. It also exhibited (Oreochromis niloticus), and Asian arowana (Scleropages formosus) CA XII proteins. It also exhibited 36%and and53% 53%identity identitywith withhuman humanCA CAIIIIand andCA CAXII, XII,respectively. respectively.The Thecloned clonedsequence sequencecontained containedaa 36% 22 amino acid NH 2 -terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which 22 amino acid NH2 -terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, Cys-355, andtwo Cys-358), two(Cys-21 of which and Cys-201) couldform potentially formbond. a disulfide bond. and Cys-358), of which and(Cys-21 Cys-201) could potentially a disulfide A 22-amino A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase protein kinase A was also sequence found. The cloned might beprotein, a transmembrane protein, as Abywas also found. The cloned might be sequence a transmembrane as predicted from in predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its showed its active sitehighly residues were highly tilapia CA XII protein. Homology active sitethat residues were conserved with conserved tilapia CAwith XII protein. Homology modeling of modeling of the CA XII wasthe done usingstructure the crystal of the extracellular domain the pufferfish CApufferfish XII was done using crystal of structure the extracellular domain of human of human carbonicXII anhydrase at 1.55 as Å aresolution a template. Semi-quantitative reverse carbonic anhydrase at 1.55 ÅXII resolution template.as Semi-quantitative reverse transcription transcription (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed that pufferfish CA XIIthat is pufferfish CA XIIinisthe highly expressed in the brain. highly expressed brain. Keywords:pufferfish; pufferfish;Takifugu Takifugurubripes; rubripes;brain braintissue; tissue;CA CAXII XII Keywords:

Introduction 1.1.Introduction Carbonicanhydrases anhydrases(CAs) (CAs)catalyze catalyzethe thereversible reversibleconversion conversionof ofcarbon carbondioxide dioxideand andwater watertoto Carbonic − + − + bicarbonateand andproton protonininthe thefollowing followingreaction: reaction:CO CO22 ++ H H22O ⇄ HCO33 ++HH [1]. are bicarbonate [1].CAs CAs areinvolved involvedin ion transport, transport, inseveral severalphysiological physiologicalprocesses processessuch suchas asacid-base acid-base homeostasis, homeostasis, electrolyte secretion, ion biosynthetic reactions, bone resorption, and tumorigenicity [2 – 4]. Moreover, many CA isoforms biosynthetic reactions, bone resorption, and tumorigenicity [2–4]. Moreover, many CA isoforms havebeen beenused usedas aspotential potentialtargets targetsfor forthe thedesign designofofthe thefunctional functionalpharmacological pharmacologicaldrugs, drugs,such suchas as have antiglaucoma,anticonvulsant, anticonvulsant, antiurolithic, andthefor the treatment Thirteen antiglaucoma, antiurolithic, and for treatment of obesityof [5].obesity Thirteen[5]. catalytically catalytically active CAs (cytosolic: CAsand I, II,XIII; III, VII, and XIII; membrane-associated: CAs IV,and IX,XV; XII, active CAs (cytosolic: CAs I, II, III, VII, membrane-associated: CAs IV, IX, XII, XIV, XIV, and XV; mitochondrial: VA and VB; VI);inactive and three inactive CAs X, (CA VIII, X, mitochondrial: CAs VA and CAs VB; secreted CA secreted VI); andCA three CAs (CA VIII, and XI), Int. J. Mol. Sci.2018, 19, x; doi:FOR PEER REVIEW Int. J. Mol. Sci. 2018, 19, 842; doi:10.3390/ijms19030842

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all belonging to the α-CA gene family, are found in the animal kingdom [4,6]. This study focuses on CA XII found in pufferfish (Takifugu rubripes). CA XII is a membrane-bound anhydrase that was independently isolated and characterized from human cells by Ivanov et al. [7] and Türeci et al. [8]. CA XII is a type I transmembrane protein and has three zinc-binding histidine residues in its active extracellular domain. Thus, it resembles other active CAs. It also has two potential sites for asparagine glycosylation. CA XII shows 30–42% sequence identity with other CAs [8]. The presence of an NH2 -terminal signal peptide and a short COOH-terminal cytoplasmic tail is a characteristic feature of human CA XII [8]. The crystal structure of human CA XII has been described at a 1.55 Å resolution [9]. In humans, CA XII is expressed in many tissues, including the aorta, bladder, brain, colon, esophagus, kidneys, liver, lungs, lymph nodes, mammary glands, ovaries, prostate, pancreas, peripheral blood lymphocytes, rectum, stomach, skeletal muscles, skin, spleen, testis, trachea, and uterus [7,8,10]. Halmi [11] reported that the expression of mammalian CA XII is upregulated in some cancers and that its expression might be induced by hypoxia. Although the GenBank database of the National Center for Biotechnology Information (NCBI) documents the sequences of CA XII in the genomes of many fish species, such predictions are only based on gene homology and no published literature describes the isolation and characterization of CA XII in fish. We used the information available on the NCBI platform to isolate and molecularly characterize the gene for the CA XII from pufferfish (T. rubripes). 2. Results 2.1. Molecular Cloning and Characterization of CA XII from Pufferfish The NCBI CA XII sequence of T. rubripes was used for molecular cloning of CA from pufferfish. The conformance of the cloned product to the 1888-bp nucleotide sequence (GenBank accession no. MF134897) was checked by 50 and 30 -rapid amplification of cDNA ends (RACE)-PCR. The 1888-bp transcript obtained upon RACE PCR contained an open reading frame (ORF) of 1470-bp, including a 25-bp 50 -untranslated region (UTR) and a 390-bp 30 -UTR (Figure 1). Although little difference was observed between the NCBI CA XII and the cloned CA sequences, two regions (46-bp 50 -UTR and 342-bp 30 -UTR) were found to be unique to the NCBI CA XII sequence. Moreover, in the cloned sequence, an adenine/guanine substitution was observed at the 1649 position in the 30 -UTR. The deduced amino acid sequence of 490 residues from ORF showed that the cloned CA protein from pufferfish exhibited 36% and 53% identity with human CA II and CA XII proteins, respectively. It also exhibited 68% identity with large yellow croaker (Larimichthys crocea), 65% identity with tilapia (Oreochromis niloticus), and 56% identity with Asian arowana (Scleropages formosus) CA XII proteins. A 22-amino acid NH2 -terminal signal sequence was predicted to be present in the cloned CA sequence (Figure 1). Three potential Asn-Xaa-Ser/Thr sequons were also observed in the sequence. One located at position 149 potentially served as an N-linked glycosylation site, with a probability of over 0.5. Moreover, four cysteine residues (Cys-21, Cys-201, Cys-355, and Cys-358) were also identified in the cloned CA sequence (Figure 1). Two of these cysteine residues (Cys-21 and Cys-201) likely formed a disulfide bond based on folding (Figure S1). A 22 amino acid COOH-terminal cytoplasmic tail that contained a potential site for phosphorylation by protein kinase A was also found. The predicted molecular mass of the cloned CA sequence was 55.64 kDa and it was predicted to be inserted into the plasma membrane.

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Figure 1. Cont.

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1. The nucleotide and deduced amino acid sequences of pufferfish CA XII. The deduced FigureFigure 1. The nucleotide and deduced amino acid sequences of pufferfish CA XII. The deduced amino acid sequence is reported in one-letter code. The start, stop codon (asterisks), and the potential amino acid sequence is reported in one-letter code. The start, stop codon (asterisks), and the potential polyadenylation signal are in bold font. The regions used for designing primers for RT-PCR and polyadenylation signal are in bold font. The regions used for designing primers for RT-PCR and q-PCR q-PCR are underlined in the sequence. The N-terminal signal peptide is double underlined. The are underlined in the sequence. The N-terminal signal peptide is double underlined. The potential potential glycosylation site is underlined with a dotted line. The four cysteine residues (Cys-21, glycosylation is underlined withare a dotted line. The fourtwo cysteine residues (Cys-21, Cys-355, Cys-201,site Cys-355, and Cys-358) highlighted in grey; of them (Cys-21, Cys-201)Cys-201, likely form a and Cys-358) highlighted in grey; two oftail them (Cys-21, Cys-201) likely form a disulfide bond. disulfideare bond. The C-terminal cytoplasmic is highlighted in light grey. The C-terminal cytoplasmic tail is highlighted in light grey. The amino acid residues in the active sites of CA proteins in human and fish are highly conserved (Figure 2). The cloned CA protein had 28 and 30 amino acid residues identical with the The amino acid residues in the active sites of CA proteins in human and fish are highly conserved putative active site pockets of human CA II and human CA XII proteins, respectively. In pufferfish (Figure 2). The cloned CA protein had 28 and 30 amino acid residues identical with the putative CA XII, substitutions were observed at positions 65, 66, 67, 69, 91, 204, and 206 when compared with active human site pockets of human100% CA II and human CAsite XIIresidues proteins, In pufferfish CA II. Moreover, similarity of active wasrespectively. observed between pufferfish CA and XII, substitutions were observed at positions 65, 66, 67, 69, 91, 204, and 206 when compared with human tilapia CA XII proteins, while only two and four amino acid residues differed between the pufferfish CA II. CA Moreover, 100% similarity of active residues observed pufferfish and tilapia XII and the large yellow croaker CA site XII and Asian was arowana CA XII,between respectively.

CA XII proteins, while only two and four amino acid residues differed between the pufferfish CA XII and the large yellow croaker CA XII and Asian arowana CA XII, respectively.

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Figure 2. Comparison of putative active sites in CA II,active CA IX, andinin CA XII sequences derived from Figure 2.2.Comparison ofofputative sites CA II,II,CA CA sequences Figure Comparison putative active sites CA CAIX, IX,and and CAXII XII sequencesderived derivedfrom from human and several species of fish. The CA IX and XII sequences were aligned against human CA II human and several species of fish. The CA IX and XII sequences were aligned against human CA human and several species of fish. The CA IX and XII sequences were aligned against human CAIIII (GenBank accession(GenBank no. NP_000058.1) by Clustal Omega by to by identify putative active sites. The active coloractive accession no.no. NP_000058.1) Clustal Omega to identify putative sites. sites. The color (GenBank accession NP_000058.1) Clustal Omega to identify putative The was color was given according to default color scheme used for alignments in Clustal X, where blue: given to default scheme for alignments in Clustal X,in where blue:X,hydrophobic was according given according to color default colorused scheme used for alignments Clustal where blue: hydrophobic residues; red: positive charge residues; magenta: negative charge residues; green: polar residues; red: residues; positive charge residues; magenta: negative charge residues; green: polar residues; hydrophobic red: positive charge residues; magenta: negative charge residues; green: polar residues; pink: cysteines; orange: glycines; yellow: prolines; cyan: aromatic residues; white: pink: cysteines; orange: glycines; yellow: prolines; cyan: aromatic residues; white: unconserved residues; pink: cysteines; orange: glycines; yellow: prolines; cyan: aromatic residues; white: unconserved residues. Numbers below the Numbers alignment represent the in the active active siterelative residues. Numbers below the alignment represent thepositions positions in the site to the human unconserved residues. below the alignment represent the positions in the active site relative to the human CA II reference sequence [12]. Notations full-forms are as : putative CA II reference sequence [12]. Notations full-forms are as follows: putative active site pockets; relative to the human CA II reference sequence [12]. Notations full-forms are as follows: : putative active site pockets; z: z:active zincbinding binding site; +: proton shuttling ~: substrate associated *: associated zinc site; +: z: proton shuttling site;site; ~: proton substrate associated pocket; *: Thr-199 loop site.pocket; The red*: site pockets; zinc binding site; +: shuttling site; ~: pocket; substrate Thr-199 loop site. The red colored box represents the potential carbonic anhydrase domain of colored box represents the potential carbonic anhydrase domain of pufferfish CA XII. The pufferfish Thr-199 loop site. The red colored box represents the potential carbonic anhydrase domain of pufferfish CA XII. The pufferfish XII cloned in theis present studyinin isbold highlighted in bold font. CA XII cloned inXII. the present study highlighted font. GenBank accession numbers for font. the pufferfish CACA The pufferfish CA XII cloned the present study is highlighted in bold GenBank accession numbers for the sequences are as follows: CA IX (human, NP_001207.2); CA XII sequences are as follows: CA IX (human, NP_001207.2); CA XII (human, NP_996808.1; large yellow GenBank accession numbers for the sequences are as follows: CA IX (human, NP_001207.2); CA XII (human, NP_996808.1; largeKKF18125.1; yellow croaker, KKF18125.1; tilapia, XP_005470754.1); CAXP_005470754.1); XII-like croaker, tilapia, XP_005470754.1); CAKKF18125.1; XII-like sequence (Asian arowana, KPP78156.1). (human, NP_996808.1; large yellow croaker, tilapia, CA XII-like sequence (Asian arowana, KPP78156.1). sequence (Asian arowana, KPP78156.1).

Several clades were formed during phylogenetic analysis of the broad class of transmembrane Several cladesCAs. wereMammalian formedclades during phylogenetic analysis of the broad class transmembrane Several formed during phylogenetic analysis of the broad class transmembrane CAwere IX and XII formed one monophyletic clade,of whereas all CA XIIof from fish formed CAs. Mammalian aCA IX and XII formed one monophyletic clade, whereas all CA XIIwhereas from fish CAs. Mammalian IX and one all 3). CAThe XIIfish from fish separate clade. TheCA human CAXII IV formed was used as amonophyletic monophyleticclade, outgroup (Figure clade formed a separateshowed clade. The human CA IV was used as a monophyletic outgroup (Figure 3). The formeddivergence a separatewith clade. The human was as ashowed monophyletic outgroup (Figure 3). the The evolution over CA time.IVThe garused CA XII the earliest divergence, while fish clade showed divergence with evolution over time. The the garover CAtime. XII fish cladeCA showed divergence with evolution The garthe CAearliest XII showed theCA earliest pufferfish XII grouped more closely with tilapia CAshowed XII and large yellow croaker XII divergence, while(Figure the pufferfish CA XII closely with more the tilapia CAwith XII the andtilapia large CA XII and large divergence, while the grouped pufferfishmore CA XII grouped closely 3). yellow croaker CA yellow XII (Figure 3). CA XII (Figure 3). croaker

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74

puffer XII

87

Mexican tetra XII

99

Asian arowana XII-like

96

gar XII rat XII 100

human XII elephant shark IX

Transmembrane clade

97 99

rat IX

96 100

human IX human IV

0.1

Figure analysis of carbonic anhydrase (CA) proteins present present in mammalian (human, Figure 3.3.Phylogenetic Phylogenetic analysis of carbonic anhydrase (CA) proteins in mammalian Homo sapiens; rat, Rattus norvegicus) and several fish species (pufferfish, Takifugu rubripes; (human, Homo sapiens; rat, Rattus norvegicus) and several fish species (pufferfish, Takifugu rubripes;tilapia, tilapia, Oreochromis niloticus;large large yellow croaker, Larimichthys gar, Lepisosteus Asian Oreochromis niloticus; yellow croaker, Larimichthys crocea;crocea; gar, Lepisosteus oculatus; oculatus; Asian arowana, arowana, Mexican Astyanax mexicanus; elephant shark,Callorhinchus Callorhinchus milii). ScleropagesScleropages formosus;formosus; Mexican tetra, tetra, Astyanax mexicanus; elephant shark, milii). The is is highlighted in bold font. The The phylogenetic tree The pufferfish pufferfish CA CAXII XIIcloned clonedininthe thepresent presentstudy study highlighted in bold font. phylogenetic was constructed using the neighbor-joining method. The results were confirmed by bootstrapping. tree was constructed using the neighbor-joining method. The results were confirmed by bootstrapping. Values given at the nodes indicate bootstrap percentages percentages for for the the given given split split among among 1000 1000 repetitions. repetitions. GenBank accession accessionnumbers numbersforfor sequences used to generate theare tree are as follows: CA IV thethe sequences used to generate the tree as follows: CA IV (human, (human, NP_000708.1); CA IX (human, NP_001207.2; rat, XP_008761898.1; elephant shark, NP_000708.1); CA IX (human, NP_001207.2; rat, XP_008761898.1; elephant shark, XP_007895370.1); XP_007895370.1); CA XII (human, NP_996808.1; rat,large XP_006243372.1; large yellow croaker, CA XII (human, NP_996808.1; rat, XP_006243372.1; yellow croaker, KKF18125.1; tilapia, KKF18125.1; tilapia, XP_005470754.1; Mexican tetra, XP_007228316.1; gar, XP_015199103.1); CA XP_005470754.1; Mexican tetra, XP_007228316.1; gar, XP_015199103.1); CA XII-like sequence XII-like sequenceKPP78156.1). (Asian arowana, KPP78156.1). (Asian arowana,

The structureofofthe the amino sequence the cloned from pufferfish was The structure 490490 amino acidacid sequence of theofcloned CA XII CA fromXII pufferfish was predicted predicted by homology modeling. The template for modeling was selected by BLAST searches of by homology modeling. The template for modeling was selected by BLAST searches of NCBI (available NCBI online: https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the protein structure databases online:(available https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the protein structure databases (available online: (available online: http://www.rcsb.org). Crystal structure of the extracellular domain of the human http://www.rcsb.org). Crystal structure of the extracellular domain of the human carbonic anhydrase carbonic ID: as 1JCZ) was chosen as the of template because(53%) of high (53%) XII (PDBanhydrase ID: 1JCZ) XII was(PDB chosen the template because high identity andidentity low E-value −103 and E-value (9 × 10Chimera ). TheModeller UCSF Chimera was used for modeling. (9 × low 10−103 ). The UCSF interfaceModeller was usedinterface for modeling. The model was builtThe by 2+) ligand. The modeling program model was built by assuming co-crystallization of the zinc ion (Zn assuming co-crystallization of the zinc ion (Zn2+ ) ligand. The modeling program conforms the cloned conforms the cloned CA template XII sequence ontoBased the template sequence. Based on(GA341 different CA XII sequence onto the sequence. on different statistical scores andstatistical zDOPE), scores (GA341 and zDOPE), the best model of CA XII was selected. To further optimize the model, the best model of CA XII was selected. To further optimize the model, stereoscopic properties were stereoscopic properties were validated with the ProQ, Verify3D, and ERRAT tools. The evaluation validated with the ProQ, Verify3D, and ERRAT tools. The evaluation results for the CA XII model results for the CA XII model were as follows. ProQ, LGscore: 5.873 (value >4 implies an extremely were as follows. ProQ, LGscore: 5.873 (value >4 implies an extremely good model) and MaxSub: good model) and MaxSub: 0.585 (values >0.5–0.5–