Abstract
In this study, an 1888-bp carbonic anhydrase XII (CA XII) sequence was cloned from the brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted 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 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH2-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358), two of which (Cys-21 and Cys-201) could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed that pufferfish CA XII is highly expressed in the brain.
Highlights
The 1888-bp transcript obtained upon rapid amplification of complementary DNA (cDNA) ends (RACE) PCR contained an open reading frame (ORF) of 1470-bp, including a 25-bp 5 -untranslated region (UTR) and a 390-bp 3 -UTR (Figure 1)
A BLAST search indicated that the 490 amino acid translated protein shows 56–68% identity with large yellow croaker, tilapia, and Asian arowana CA XII proteins
Similar conclusions can be made based on the high similarity of pufferfish CA XII active site residues with tilapia CA XII active site residues
Summary
AAbbsstrtaracct:t:IInn tthhiiss ssttuuddyy,, aann 11888888--bbppccaarrbboonnicicaannhhyyddraraseseXXIIII(C(CAAXIXI)IIs)esqeuqeunecnecwe aws acslocnleodnefdrofmrotmhe thberabinraoifnthoef pthueffperufifsfehr,fiTsahk,ifuTagkuifruugburipruesb.rTiphees.clTohneedclsoenqeudensecequcoenntcaeinceodntaacinoeddinag creogdiionngorfe1g4io70n-bopf , 1w47h0i-cbhpw, washipcrhedwiactsepdretoditcrtaendsltaotetrianntsolaatepirnottoeian porfo4t9e0inaomf i4n9o0 aacmidinroesaicdiuderse.sTidhueepsr.eTdhicetpedrepdricotteedin psrhootewinedshboewtwedeebnetw68e–e5n66%8–i5d6e%ntiidtyenwtitiythwtihthe thlaerlgaergyeeylleolwlowcrcoroakakerer((LLaarriimmiicchhtthhyyss ccrroocceeaa)),, titlialappiaia (O(Oreroeocchhrorommisisnniliolotitcicuuss)),,aannddAAssiaiannaarorowwaannaa(S(Scclelreoroppaaggeessfoforrmmoossuuss))CCAAXXIIIIpproroteteininss. .ItItaalslsooeexxhhibibitietedd 3366%%aanndd5533%%idideenntittiytywwitihthhhuummaannCCAAIIIIaannddCCAAXXIIII,,rreessppeecctitviveelyly. .TThheecclolonneeddsseeqquueenncceeccoonntatainineeddaa 2222amamininooacaidcidNHN2H-t2e-rtemrminianlasligsniganl saelqsueeqnuceenacnedanthdretehAresen-AXsana--XSaera/-STehrr/Tsheqrusoenqsu,oanms,onagmwonhgichwohniceh woansepwotaesntpiaoltleyngtilaylclyosygllaytceods.yFlaotuedr .cyFsotueirnecyrsetseiidnueesrewsiedrueeaslswo eidreenatlisfioedid(eCnytsif-i2e1d, C(Cysy-s2-0211,, CCyyss--325051,, aCndysC-3y5s5-3, 5a8n)d, tCwyoso-3f5w8)h,itcwh o(Coyfsw-2h1iacnhd(CCyyss--22101a)ncdouClydsp-2o0t1e)nctioaulllydfopromtenatdiaislluylfifodremboanddi.suAlf2i2d-eamboinnod. aAcid22C-OamOiHn-otearcmidinCalOcOytHop-tlearsmmiinc atalicl ycotonptalainsmingicatapioltceonntitaalisniitnegfoar pphootesnpthiaolrysliatetiofonrbpyhporsoptehionrkyilnaatisoen Abywpasroatlesion fkoiunnadse. 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. 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)
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