Báo cáo Y học: RING finger, B-box, and coiled-coil (RBCC) protein expression in branchial epithelial cells of Japanese eel, Anguilla japonica pot

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RING finger, B-box, and coiled-coil (RBCC) protein expressionin branchial epithelial cells of Japanese eel,Anguilla japonicaKentaro Miyamoto1, Nobuhiro Nakamura1, Masahide Kashiwagi1, Shinji Honda1, Akira Kato1,Sanae Hasegawa2, Yoshio Takei2and Shigehisa Hirose11Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan;2Ocean Research Institute, The Universityof Tokyo, Tokyo, JapanAn RBCC (RING finger, B-box, and coiled-coil) proteinwas identified that belongs to the superfamily of zinc-bindingproteins and is specifically expressed in the gill of eel,Anguilla japonica. Euryhaline fishes such as eels can migratebetween freshwater and seawater, which is considered to beaccomplished by efficient remodeling of the architecture andfunction of the gill, a major osmoregulatory organ. Toidentify molecules involved in such adaptive changes, weperformed differential display using mRNA preparationsfrom freshwater and seawater eel gills and obtained anRBCC clone among several differentially expressed clones.The clone encoded a protein of 514 amino acid residues withstructural features characteristic of the RBCC protein; wetherefore named it eRBCC (e for eel). eRBCC mRNA wasspecifically expressed in the gills with a greater extent in thegills of freshwater eels. Immunohistochemistry revealed thatthe expression of eRBCC is confined to particular epithelialcells of the gills including freshwater-specific lamellarchloride cells. The RING finger of eRBCC was found tohave a ubiquitin ligase activity, suggesting an importantregulatory role of eRBCC in the remodeling of branchialcells.Keywords: freshwater adaptation; gill; RBCC protein;RING finger; ubiquitin ligase.RING finger, B-box, and coiled-coil (RBCC) proteins are agroup of zinc-binding proteins that belong to the RINGfinger family. They are so called because they have anN-terminal RING finger motif defined by one histidine andseven cysteine residues (C3HC4) followed by one or twoadditional zinc-binding domains (B-box), and a putativeleucine coiled-coil region. The RING finger coordinates twozinc atoms and is found almost exclusively in the N-terminalposition in RBCC proteins. The second motif or the B-boxis defined by the consensus sequence CHC3H2and bindsone zinc atom. Members of the RBCC protein familyinclude PML [1], TIF1 [2], KAP-1 [3], the MID1 geneproduct [4], XNF7 [5], RFP [6], SS-A/Ro [7], Rpt-1 [8],Staf50 [9], and HT2A [10] which are known to playimportant roles in regulating gene expression and cellproliferation [11–14]. Consistent with these functions, manyof RBCC proteins have been defined as potential proto-oncogenes. We were interested in the RBCC protein familywhen we found a member of the family among cDNAclones that are differentially expressed between freshwaterand seawater eels while attempting clarification of themechanism of adaptation of euryhaline fishes. Euryhalinefishes can survive in both freshwater and seawater. Movingfrom freshwater to seawater or vice versa is expected to beaccompanied by massive reorganization of the moleculararchitecture of gill cells or changes of their types. Tounderstand the molecular basis for such extraordinaryability of adaptation, identification and characterization ofregulatory proteins, such as RBCC family members, areessential.The RBCC protein identified here is unique not only in itsC-terminal sequence but also in its restricted expression: It ishighly expressed in the gill but not in detectable amounts inother tissues and furthermore it is expressed much morehighly in freshwater than in seawater eels, suggesting thatthe eRBCC may play an important role in the differenti-ation and maintenance of freshwater gill cells. In support ofthis potential regulatory role, we show here that the eel gillRBCC protein has an E3 ubiquitin ligase activity. Theubiquitin system targets a wide array of short-lived regu-latory proteins and incorporates into them a ubiquitin tagfor degradation through a three-step mechanism involvingubiquitin activating (E1), conjugating (E2), and ligating(E3) enzymes [15].EXPERIMENTAL PROCEDURESAnimalJapanese eels (Anguilla japonica) weighing approximately200 g were purchased from a local dealer. They were rearedunfed in a freshwater tank for 2 weeks (freshwater-adaptedeels). Some eels were transferred to a seawater tank andCorrespondence to S. Hirose, Department of Biological Sciences,Tokyo Institute of Technology, 4259 Nagatsuta-cho,Midoriku, Yokohama, Japan 226–8501.Fax: + 81 45 9245824, Tel.: + 81 45 9245726,E-mail: shirose@bio.titech.ac.jpAbbreviations: GSt, glutathione S-transferase; RBCC, RING finger,B-box, and coiled-coil; TPEN, tetrakis-(2-pyridylmethyl)ethylene-diamine; Ub, ubiquitin.Note: The novel nucleotide sequence data published here have beendeposited with the DDBJ/GenBank/EMBL data bank and areavailable under accession number AB086259.(Received 15 August 2002, revised 18 October 2002,accepted 23 October 2002)Eur. J. Biochem. 269, 6152–6161 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03332.xacclimated there for 2 weeks before use (seawater-adaptedeels). The water temperature was maintained at 18–22 °C.All eels were anaesthetized by immersion in 0.1% ethylm-aminobenzoate methanesulfonate (MS222) before beingkilled by decapitation. The various tissues for RNAextraction were dissected out, snap-frozen in liquid nitrogenandstoredat)80 °C until use.Differential displayDifferential display was performed following the protocol ofLiang and Pardee [16,17]. Total RNA was isolated by theguanidinium thiocyanate/cesium chloride method [18] froma pool of gill tissues from five freshwater- and five seawater-adapted eels, and then mRNA was prepared using anoligo(dT)-cellulose column (Amersham Pharmacia Bio-tech). One microgram of mRNA was used for cDNAsynthesis with a Superscript kit (Life Technologies, Inc.)together with a single arbitrary primer. Differential displayPCR was performed using 5 ng of cDNA, 1 lMsamearbitrary primer, 0.5 mMdNTPs, 0.7 MBq of [a-32P]dCTP(Amersham Pharmacia Biotech), and 2.5 units of Taqpolymerase (Takara). The mixture was cycled first at 94 °Cfor 1 min, 36 °Cfor5 min,and72 °C for 5 min followed by40 cycles at 94 °Cfor1min,60°Cfor2min,and72°Cfor2 min. An aliquot of each amplification mixture wassubjected to electrophoresis in a 7.5% polyacrylamide gel,exposed to an imaging plate for 8 h and the result wasanalyzed with a BAS-2000 image analyzer (Fuji Film).Differentially expressed bands of interest were extractedfromthegelandreamplifiedandthenclonedintopBlue-script II vector (Stratagene). DNA sequence analysis fromboth strands was performed using a SequiThermTMcyclesequencing kit (Epicentre Technologies). The DNAsequence was compared with the GenBankTM/EMBL/DDBJ databases using the BLAST network service at theNational Center for Biotechnological Information.Northern blot analysisPoly(A)-rich RNA (3 lg) from a pool of gill tissues from fivefreshwater- and five seawater-adapted eels was denatured ina2.2-Mformaldehyde, 50% (v/v) formamide buffer and thenseparated on 1% (w/v) agarose gel containing 2.2Mformal-dehyde. Size-fractionated RNAs were then transferred to anylon membrane (MagnaGraph, Micron Separations Inc.).The eRBCC cDNA was32P-labeled by random priming andhybridized to the RNA filters in 50% formamide, 5 · SSPE(SSPE ¼ 0.15 mMNaCl, 1 mMEDTA, and 10 mMNaH2PO4,pH7.4),2· Denhardt’s solution, and 0.5%SDS for 16 h at 42 °C. After hybridization, the membranewas rinsed twice in 2 · NaCl/Cit (1 · NaCl/Cit contains0.15 mMNaCl and 0.015Msodium citrate) containing 0.1%SDS for 30 min at 50 °C, washed with 0.5 · NaCl/Citcontaining 0.1% SDS for 1 h at 55 °C.Themembranewasexposed to an imaging plate for 8 h and the result wasanalyzed with a BAS-2000 image analyzer (Fuji Film).Screening and sequencingThe freshwater-adapted eel gill cDNA library in kZAP II(Stratagene) was prepared as described [19]. The library wasplated out at a density of 3 · 104plaque-forming units/150-mm plate. Phage plaques were lifted onto nitrocellulosefilters (Schleicher & Schuell), and the filters were prehy-bridized for 2 h at 42 °C in a solution containing 50% (v/v)formamide, 5 · SSPE, 0.1% SDS, and 5 · Denhardt’ssolution. The probe was labeled with [a-32P]dCTP usingrandom primers. Hybridization was performed for 16 h at42 °C. To identify positive clones, filters were washed andthen exposed to Kodak X-Omat film at )80 °Covernightwith intensifying screens. Positive plaques were isolated andrescreened after dilution. Conditions for secondary andtertiary screening were identical to primary screening. Theobtained positive clones were excised with R408 helperphage (Stratagene) and sequenced using a SequiThermTMcycle sequencing kit (Epicentre Technologies).Rapid amplification of cDNA ends (RACE) PCRTo obtain the 5¢ end of the eRBCC cDNA, 5¢-RACE PCRwas conducted using the 5¢/3¢-RACE kit (Roche MolecularBiochemicals). One microgram of poly(A)-rich RNA fromfreshwater-adapted eel gill was reverse-transcribed using thegene-specific antisense primer, 5¢-CTTGAAGTGCTCGGT-3¢, complementary to nucleotides 450–464 of theeRBCC cDNA sequence by AMV reverse transcriptase.First strand cDNA was purified and oligo(dA)-tailedaccording to the manufacturer’s protocol. The resultingcDNA was then PCR-amplified using a second gene-specificantisense primer, 5¢-ATCTCCTTCAGGGTGCGGTT-3¢,complementary to a eRBCC cDNA nucleotides 429–448 ofthe eRBCC cDNA and an oligo(dT) anchor primersupplied by the manufacturer. Second PCR was performedusing a third gene-specific antisense primer, 5¢-ATGTGCAGGCAGGGCCTCTT-3¢, complementary to nucleo-tides 408–427 of the eRBCC cDNA and a PCR anchorprimer supplied by the manufacturer. The PCR productswere cloned into pBluescript II vector (Stratagene). DNAsequence analysis was performed using a SequiThermTMcycle sequencing kit (Epicentre Technologies).RNase protection analysisRNase protection assays were performed using an RPA IIkit (Ambion) according to the manufacturer’s protocol. A540-bp PCR fragment of eRBCC cDNA (1233–1772) and a138-bp PCR fragment of eel b-actin cDNA were subclonedinto the pBluescript II vector and used to generate cRNAprobes. The probes were synthesized with T7 RNA poly-merase and an RNA transcription kit (Stratagene) in thepresence of [32P]UTP (Amersham Pharmacia Biotech). TheRNA probe was treated with DNase I, purified by SephadexG-50 chromatography and ethanol precipitation, and1.7 · 102kBq of the probe was hybridized to 10 lgoftotalRNA from pools of various tissues from five freshwater- orfive seawater-adapted eels for 16 h at 42 °C. After digestionwith RNase A/T1, protected fragments were electrophore-sed on 5% polyacrylamide, 8Murea denaturing gels andexposed to an imaging plate for 16 h and the result wasanalyzed with a BAS-2000 image analyzer (Fuji Film).Transfer experimentToexaminethetime-coursechangesinthelevelsofeRBCCmRNA following freshwater entry, seawater-adapted eelsÓ FEBS 2002 RBCC protein expression in Anguilla japonica (Eur. J. Biochem. 269) 6153(n ¼ 36) were transferred directly to freshwater and the gillswere sampled from six eels on days 0, 1/8 (3 h), 1/2 (12 h), 1,3 and 7 for RNase protection assay. Six seawater eels thatwere kept in seawater for 7 days served as time controls. Thechanges in the levels of Na+,K+-ATPasemRNAwerealsoexamined in parallel with those of RBCC. The data served asreference controls because its expression may be down-regulated in contrast to the expected up-regulation of RBCC.The changes in plasma Na+concentration were monitoredduring the course of freshwater adaptation. The collected gilltissues were immediately frozen in liquid nitrogen, and totalRNA was isolated as mentioned above. RNase protectionassay was performed with 10 lg of each RNA as describedabove. Optical densities of the protected fragments for eachgill were measured and normalized to the b-actin bands. Themean normalized values were plotted ± SE. Student’s t-testwas used to determine the significance of any differencesbetween two groups, P < 0.05 was considered significant.Antibody productionA PCR fragment of the eRBCC cDNA (corresponding toaminoacidresidues1–514)wassubclonedintothebacterial expression vector pRSET-A (Invitrogen). Afterinduction with 1 mMisopropyl-1-thio-b-D-galactopyrano-side, the fusion protein was expressed in Escherichia colistrain BL21 and purified in a denaturing buffer (8Murea,50 mMNa2HPO4and 300 mMNaCl, pH 7.6) by affinitycolumn chromatography using Ni-NTA agarose (Qiagen)and dialyzed against phosphate-buffered saline (NaCl/Pi¼ 100 mMNaCl, 10 mMNaH2PO4,pH7.4)at4°C.About 100 lg of the fusion protein emulsified in completeFreund’s adjuvant (1 : 1) was injected into rats to raisepolyclonal antibodies. The rats were injected three timesat 2-week intervals and bled 7 days after the thirdimmunization.Affinity purification of anti-eRBCC IgThe polyclonal rat serum was purified on an affinitycolumn. The affinity column was prepared by coupling1mgofHis6-eRBCC fusion protein to an Affi-Gel 10 solidsupport, according to the manufacturer’s instruction (Bio-Rad)andthen10mLofanti-eRBCCserum(diluted1:10in NaCl/Pi) was applied to the column and incubate at 4 °Cfor 24 h. The bound antibody was eluted with 10 mL of100 mMglycine (pH 2.5) and dialyzed against NaCl/Pi.Cell culture and plasmid transfectionCOS-7 cells were cultured in Dulbecco’s modified Eagle’smedium (Sigma) containing 10% (v/v) fetal bovine serumand 100 unitsÆmL)1penicillin. The cells were maintained inhumidified atmosphere with 5% (v/v) CO2at 37 °C. TheeRBCC cDNA was introduced into the pcDNA3 vector.Cells were transfected with the plasmid using LipofectAMINE (Life Technologies, Inc.) according to the manu-facturer’s instruction.Western blottingThe COS-7 cells expressing eRBCC or mock transfectedcells were washed three times with NaCl/Piand solubilizedwith Laemmli buffer. The cell lysate was separated by SDS/PAGE and transferred onto polyvinylidene difluoridemembrane. Nonspecific binding was blocked with 10%(v/v) fetal bovine serum in TBS-T (TBS-T ¼ 100 mMTris/HCl, pH 7.5, 150 mMNaCl, and 0.1% Tween 20).The membrane was then incubated with the affinity purifiedanti-eRBCC Ig at 1 : 200 dilution overnight at 4 °C. Afterwashing the membranes in a TBS-T, blots were incubatedwith horseradish peroxidase-linked secondary antibodyfollowed by enhanced chemiluminescence detection usingthe ECL-Plus reagent according to the manufacturer’sinstruction (Amersham Pharmacia Biotech).ImmunohistochemistryTen eels were first acclimated in seawater for 2 weeks andfive of them were then transferred to freshwater. On day 7after transfer, gills were removed from freshwater andseawater eels and fixed for 2 h in NaCl/Picontaining 4%(w/v) paraformaldehyde at 4 °C. After incubation in NaCl/Picontaining 20% (w/v) sucrose for 1 h at 4 °C, thespecimen was frozen in Tissue Tek OCT Compound on acryostat holder. Sections (5 lm) were prepared at )20 °Cina cryostat and mounted on Vectabond-treated glass slidesand dried in air for 1 h. After washing with NaCl/Pi,sections were permeabilized by incubating in NaCl/Picontaining 0.1% (v/v) Triton X-100 at room temperaturefor 5 min and then incubation with NaCl/Picontaining0.3% (v/v) H2O2for 30 min at room temperature. Forstaining, sections were incubated with affinity-purified anti-eRBCC Ig (1 : 200), anti-eRBCC serum (1 : 2000), preim-mune serum (1 : 2000) or anti-eRBCC Ig preabsorbed withthe corresponding antigen (1 : 2000) or anti-(Na+,K+-ATPase a-subunit) serum (1 : 10 000) [20] at 4 °Cover-night. Bound antibodies were detected by incubation withbiotinylated second antibody (diluted 1 : 200) and avidin–peroxidase conjugate using the Vectastain ABC kit (VectorLaboratories) following the manual supplied.ImmunofluorescenceGills form freshwater-adapted eels (n ¼ 5) were fixed for4hinNaCl/Picontaining 4% (w/v) paraformaldehyde at4 °C, immersed in NaCl/Picontaining 20% (w/v) sucrosefor 1 h at 4 °C, and frozen in Tissue Tek OCT Compound.Sections (7 lm) were cut and permeabilized as describedabove. After incubation for 1 h in NaCl/Picontaining 2%(w/v) fetal bovine serum, sections were incubated withaffinity-purified anti-eRBCC Ig (1 : 200) and anti-(Na+,K+-ATPase a-subunit) serum (1 : 10 000) [20] at4 °C overnight. Bound antibodies were detected by incuba-tion with anti-rat IgG Cy3-conjugated (Jackson Immuno-Research Laboratories; 1 : 400) and anti-rabbit IgGAlexa488-conjugated (Molecular Probes; 1 : 1000) secon-dary antibodies together with Hoechst 33342 (MolecularProbes; 100 ngÆmL)1). Immunofluorescence microscopywas carried out using an Olympus IX70 microscope(Olympus).In vitroubiquitination assayA glutathione S-transferase (GSt) fusion of eRBCC wasexpressed in E. coli and assessed for its ubiquitination6154 K. Miyamoto et al. (Eur. J. Biochem. 269) Ó FEBS 2002activity in vitro as described [21,22] with some modifications.Reaction mixtures were assembled in 20 lLofabuffercontaining 0.1 lgofrabbitE1,1lgofE2,1lg of GSt-Ub,25 mMTris/HCl (pH 7.5), 120 mMNaCl, 2 mMATP,1mMMgCl2,0.3mMdithiothreitol, 1 mMcreatine phos-phokinase, 100 lMMG-132, and 100 ng of GSt-eRBCC.E2s (UbcH2, UbcH5C, UbcH7, UbcH8, and UbcH9) usedin ubiquitination assay were expressed as recombinantproteins in E. coli. After incubation at 30 °C for 4 h, thesamples were processed for SDS/PAGE on 10% gelsand Western blot with mouse monoclonal antibody toubiquitin. As a negative control, ubiquitination assay with2mMN,N,N¢,N¢-tetrakis(2-pyridylmethyl)-ethylenediamine(TPEN) was performed.RESULTSIdentification of a novel RBCC protein by differentialdisplayIn a differential display using mRNA preparations fromfreshwater and seawater eel gills, we identified an RBCCprotein as a potential regulator of differentiation of gill cells.A strong differentially displayed band of 1600 bp (data notshown) was subcloned into pBluescript II, amplified inE. coli, and sequenced. Computer-assisted analysis of thesequence confirmed that the clone encodes a member of thefamily of RBCC proteins. The RBCC protein was namedeRBCC (e for eel).Cloning of full-length cDNA and its sequence analysisAfter confirming its differential expression by Northern blotanalysis (Fig. 1), a full-length eRBCC cDNA was isolatedfrom an eel gill cDNA library that was constructed usingmRNA from freshwater eel gills. Figure 2 shows thenucleotide sequence of the longest clone and the deducedamino acid sequence. eRBCC consists of 514 amino acidresidues and has motifs characteristic of the RBCC proteinat the N terminus: a RING finger of the C3HC4type; aB-box, another form of zinc finger; and a coiled-coil domain(Figs 3 and 4). Although the third Cys of the consensussequence of the B-box (CHC3H2) is not conserved ineRBCC (CHC2H2, Fig. 4), the zinc-coordinating Cys andHis residues are conserved. The C-terminal domain exhi-bited significant similarity (62–63%) to the B30.2-likedomains of other known members including newt PwA33[23], frog Xnf7 [5], and mammalian RFP [6] (Fig. 3). TheB30.2-like domain is a conserved region of 170 amino acidresidues usually found in the C-terminal position [24]. Thesestructural features and the unique tissue distributionindicate that eRBCC is a novel member of the C-terminal-domain-containing subgroup of the RBCC group of RINGfinger proteins.Although the first Met codon is in a perfect Kozakconsensus environment (GGCATGG) [25], no stop codoncould be found in frame upstream of the start codon.Therefore we performed 5¢-RACE to confirm the positionof the initiator Met codon. Most of the RACE productsterminated at the position almost identical to that of thelongest cDNA clone, rendering the possibility of theexistence of another ATG codon upstream of position + 1unlikely.Confirmation of freshwater- and gill-specific expressionby RNase protection analysisUsing total RNA preparations from various tissues offreshwater and seawater eels, we performed RNase protec-tion analysis, a method capable of detecting specific RNAspecies with high sensitivity and accuracy [26,27], todetermine the tissue distribution of eRBCC mRNA.Expression of the eRBCC message was highly restricted tothe gill (Fig. 5). Compared to the levels in seawater eel gills,its levels in freshwater eel gills were much higher.Time course of induction during freshwater adaptationAfter transfer of seawater eels to freshwater, the expressionof RBCC mRNA in the gill was induced and maximalinduction occurred after 12 h to approximately fivefoldcompared with the seawater level (Fig. 6A). Significantincreases in RBCC mRNA continued thereafter for 7 days.The levels of RBCC mRNA did not change in eels kept inseawater for 7 days. In contrast to the up-regulation ofRBCC mRNA, the levels of Na+,K+-ATPase mRNAdecreased gradually to a level that was about half theoriginal seawater level (Fig. 6B). The high levels ofNa+,K+-ATPase mRNA in seawater persisted for 7 daysin time controls. Plasma Na+concentration decreasedgradually and reached equilibrium within 7 days aftertransfer to freshwater, thereby confirming successful adap-tation to freshwater environments (Fig. 6C).Fig. 1. Differential expression of eRBCC mRNA in gills of freshwater-and seawater-adapted eels. Northern blot analysis was performed usingmRNA preparations from eels adapted to freshwater or seawater.Poly(A)-rich RNA (3 lg) from seawater and freshwater was electro-phoresed on a 1% agarose-formaldehyde gel, transferred to a nylonmembrane, and hybridized with eRBCC32P-labeled cDNA probe.Position of 2.6 kb and 1.8 kb are as noted in figure. Hybridization toan eel b-actin probe demonstrated equal loading of the lanes. Datarepresent two separate experiments that yielded similar results.Ó FEBS 2002 RBCC protein expression in Anguilla japonica (Eur. J. Biochem. 269) 6155Immunohistochemical localization of eRBCCTo perform immunohistochemistry, we raised antiserumagainst recombinant eRBCC, purified it by affinity chro-matography, and confirmed its specificity by Western blotanalysis using extracts of COS-7 cells expressing exogenouseRBCC (Fig. 7). Affinity purification of the antiserum waseffective to eliminate nonspecific staining of the cartilagin-ous support of the primary lamella, which was seen togetherwith specific staining in the secondary lamella when thecrude antiserum was applied to gill sections (Fig. 8A, panelsa and b). The secondary lamella staining was absent whenpreimmune serum (Fig. 8A, panel c) or preabsorbedantiserum (Fig. 8A, panel d) was used. Using the purifiedantibody, we next performed immunohistochemistry onsections of freshwater and seawater eel gills to determine thetype of cells expressing eRBCC. Serial sections were stainedwith anti-eRBCC and anti-(Na+,K+-ATPase). In fresh-water specimens, anti-eRBCC immunostaining wasobserved mainly in epithelial cells of the secondary lamella(Fig. 8, panels a and e). The staining pattern was reminis-centofthatoffreshwater-typechloridecellsthathaverecently been shown to migrate from the basal area to theouter surface of the secondary lamella in salmon [28] and eel[29]. We therefore stained consecutive sections with anantiserum against Na+,K+-ATPase, a marker enzyme ofchloride cells [30,31]. Significant overlapping was observedbetween the eRBCC-positive cells (Fig. 8B, panel e) and thechloride cells decorated with anti-(Na+,K+-ATPase)(Fig. 8B, panel g; arrowheads). In seawater eel gill sections,eRBCC signals were weak and less abundant (Fig. 8B,panel f).Fig. 2. Nucleotide and deduced amino acid sequences of eRBCC cDNA.The nucleotide sequence was derived from the longest clone. The first98-bp nucleotides were isolated by 5¢-RACE. The deduced amino acidsare shown below their respective codons. Numbers to the right refer tothe last amino acids on the lines, and the numbers to the left refer to thefirst nucleotides on the lines. The putative initiation codon (ATG) andan upstream stop codon (TGA) are underlined. Conserved cysteine/histidine residues in the RING finger domain and B box domain arecircled. The potential coiled-coil and B30.2 domain are underlined.Potential polyadenylation site in the 3¢-untranslated region is boxed.Asterisks indicate stop codons.Fig. 3. Schematic representation of the relationship between eRBCCand several other RBCC proteins. The RING finger, B-box, coiled-coil,and B30.2 domains are shown as distinctive boxes. The overall identity(Ident.) and similarity [Sim.] of amino acids for each protein relative toeRBCC are shown under the name of the protein. The identity andsimilarity of the B30.2 domains are also shown. Proteins comparedwith eRBCC are PwA33 [23], Xnf7 [5], and mouse RFP (mRFP) [6].NLS, nuclear localization signal (open box).Fig. 4. Alignment of amino acid sequences of eRBCC, mRFP, PwA33,and Xnf7 proteins. The alignment of the amino acid sequence of theeRBCC RING finger domain and B-box domain with several mem-bers of the RBCC family is shown. The conserved Cys and His residuesare shown with asterisks. The zinc-coordinating Cys and His residuesof the B-box that binds one Zn atom are indicated by arrowheads.6156 K. Miyamoto et al. (Eur. J. Biochem. 269) Ó FEBS 2002Figure 9 shows simultaneous immunofluorescence stain-ing of freshwater eel gill sections with anti-eRBCC(Fig. 9B), anti-(Na+,K+-ATPase) (Fig. 9C), and theDNA-selective dye Hoechst 33342 (Fig. 9D). As seen fromthe merged image (Fig. 9A), the majority of eRBCCappears to be present in the nucleus of the epithelial cellsof the secondary lamella including the chloride cells andpavement cells whose nuclei are labeled by arrows anddouble arrowheads, respectively, in Fig. 9D. The nuclei ofthe pillar cells were not stained with anti-eRBCC (arrow-heads). The mechanism of nuclear localization of eRBCCremains to be clarified as it has no apparent nuclearlocalization signal.Ubiquitin ligase activity of eRBCCAs it has recently been realized that the RING finger motifhas a general role in ubiquitination, we determined whethereRBCC has a ubiquitin ligase activity using recombinantproteins generated in E. coli that do not express compo-nents of the ubiquitin-conjugating system. When GSt-eRBCCwasmixedwithUbcH5C,anE2enzyme,andGSt-Ub in the presence of rabbit E1, ubiquitinated productsof higher molecular weights were detected (Fig. 10A, lane2). The bands were not observed in control experiments withTPEN, a zinc-cheleting agent, suggesting that the ubiqui-tination reaction was mediated by the E3 action of eRBCC(Fig. 10A, lane 3). To determine the specificity of eRBCC,we next prepared a number of recombinant E2 enzymes andexamined their interaction with eRBCC. The ubiquitinationreaction was observed only in the case of UbcH5C,demonstrating that eRBCC is relatively specific to UbcH5C(Fig. 10B).DISCUSSIONIn the present study, we identified an eel mRNA speciesthat encodes an RBCC protein (eRBCC), is specificallyexpressed in the gill, and is therefore considered to beinvolved in the differentiation and maintenance of gillcells. The gill cell-restricted and fresh water-enhancedexpression of eRBCC, first suggested by differentialdisplay, was confirmed by Northern blot analysis(Fig. 1) and RNase protection analysis (Fig. 5). Immu-nohistochemistry suggested that the eRBCC-expressingcells are mainly located in the outer surface of thesecondary lamella (Fig. 8). Colocalization studies with anantiserum against Na+,K+-ATPase, a marker protein forthe chloride cells, further revealed a significant overlapbetween eRBCC-positive cells and Na+,K+-ATPase-posit-ive cells. This is interesting in relation to the recent findingFig. 6. Changes in the levels of eRBCC (A) and Na+,K+-ATPase (B)mRNA following transfer from seawater to freshwater. Seawater-adapted eels were transferred to freshwater and their RNA wasisolated from gills of each eel separately (n ¼ 4–6). RNase protectionassay was performed as described under ÔExperimental proceduresÕ.Optical densities of the protected fragments were measured and nor-malized to the b-actin bands. In C, plasma Na+concentrations areshown. The mean normalized values were plotted ± SE. Asterisksindicate significant differences from the initial values (SW, day 0):*P <0.05.SW,seawater;FW,freshwater.Fig. 5. eRBCC mRNA levels in various eel tissues in freshwater andseawater condition. Eels were adapted to freshwater or seawater for2 weeks, and total RNA was isolated from the indicated tissues. Anautoradiogram of an RNase protection assay (10 lgÆlane)1)wasper-formed with the indicated32P-labeled cRNA probe as described underÔExperimental proceduresÕ. In addition to the indicated tissues, we alsoanalyzed total RNA preparations from the atrium, ventricle, stomach,and bladder, but they gave no signals (data not shown). Probe, labeledriboprobe alone; F, RNA preparation from freshwater-adapted eels; S,RNA preparation from seawater-adapted eels. A representative dataset is shown from three separate experiments.Ó FEBS 2002 RBCC protein expression in Anguilla japonica (Eur. J. Biochem. 269) 6157of Uchida et al.[28]andSasaiet al. [29]. They demon-strated that the chloride cells can be classified into twotypes based on the locations in the gill: filament chloridecells and lamellar chloride cells. The lamellar chloride cellsare considered to play a pivotal role in freshwateradaptation as they appear in freshwater and disappearin seawater [28,29]. The chloride cells are mainly located inthe gill and involved in osmoregulation of teleost fish.Reflecting their extraordinary power of ion transport,chloride cells are rich in mitochondria and Na+,K+-ATPase and their surface areas are tremendouslyincreased by extensive invaginations of the basolateralmembrane [30,31]. Although circumstantial, our resultssuggest that eRBCC plays a key role in the differentiationand maintenance of certain epithelial cells, at least somepopulations of the lamellar chloride cells, of the freshwatereel gills. Identification, by future studies, of the moleculeswith which eRBCC interacts is essential for understandingthe function of eRBCC.eRBCC belongs to a newly emerging family of modularproteins consisting of a C3HC4-type RING finger motif,one or two B-box(es), and one or two coiled-coil region(s).Members of the RBCC family [14,32,33] of proteins canbe classified into several groups based on the numbers andlocations of the B-box and coiled-coil regions and also bythe presence or absence of a C-terminal domain. Theknown members of the C-terminal domain-containinggroup to which eRBCC belongs include newt A33 [23],frog Xnf7 [5], and mammalian RFP [6] (Fig. 3). The factthat (a) all these proteins have been implicated in theregulation of cell differentiation and (b) among themembers, the C-terminal regions are relatively highlyconserved suggests that eRBCC also has a similarfunctional role.The RING finger motif has recently been shown in manycases to function as an E3 ubiquitin ligase [34–37]. However,the RING finger of this subfamily of the RBCC family hasnot been characterized except a recent report on Efp, atarget gene product of estrogen receptor a essential forestrogen-dependent cell proliferation and organ develop-ment [38]. In the present study, we demonstrated thateRBCC has an E3 activity, which is dependent on, amongthe E2s examined, UbcH5C, an E2 enzyme that isconsidered to be involved in the stress response and play acentral role in the targeting of short-lived regulatoryproteins for degradation [39]. The finding may open a newavenue leading to better understanding of the mode ofaction of not only eRBCC but also other members of theRBCC family through identification of their cellularsubstrates.Fig. 8. Immunohistochemistry of eRBCC in freshwater and seawater eelgills. (A) Serial sections of freshwater eel gill were stained with affinity-purified anti-eRBCC Ig (a), antiserum against eRBCC (b), preimmuneserum (c) and antiserum against eRBCC preabsorbed with the cor-responding antigen (d). (B) Serial sections of freshwater (e, g) andseawater (f, h) eel gills were stained with affinity-purified anti-eRBCCantibody (e, f) and antiserum against Na+,K+-ATPase a-subunit (g,h). The arrowheads indicate the eRBCC positive chloride cells. PL,primary lamella; SL, secondary lamella. Scale bar represents 20 lm.Staining was repeated 10 times, with similar results, on gill sectionsfrom five different sets of freshwater and seawater eels.Fig. 7. Western blot analysis of eRBCC protein expressed in COS-7cells. COS-7 cells expressing eRBCC or mock transfected cells weresolubilized with the Laemmli buffer and analyzed by Western blottingas described under ÔExperimental proceduresÕ.6158 K. Miyamoto et al. (Eur. J. Biochem. 269) Ó FEBS 2002Concerning physiological roles of RING finger proteinsin fishes facing osmotic stress, a paper has recently beenappeared reporting identification of Shop21, a salmonhomolog of the E3 ubiquitin ligase Rbx1, whose expressionis highly induced in branchial lamella when salmon isexposed to seawater [40]. Shop21 identified by Pan et al. [40]and eRBCC identified here may be one of the essentialregulators for seawater and freshwater adaptation ofeuryhaline fishes. The proteins may contribute to remode-ling of the gill architecture and its maintenance by targeting,for degradation via the proteasomal pathway, a group ofregulatory and structural proteins that are not necessary foradaptation to new osmotic environments.ACKNOWLEDGMENTSWe thank Setsuko Sato for secretarial assistance. This work wassupported by Grants-in-Aid for Scientific Research (09102008 and14104002) from the Ministry of Education, Science, Sport and Cultureof Japan.Fig. 9. Immunofluorescence localization ofeRBCC and Na+,K+-ATPase in freshwatereel gill. Freshwater eel gill sections werestained with Cy3–conjugated antibody toeRBCC (B), Alexa488–conjugated antibodyto Na+,K+-ATPase (C), and Hoechst 33342(D). A merge of B, C and D is shown in A.Arrows point to chloride cells; doublearrowheads, pavement cells; and arrowheads,pillar cells. Scale bar represents 50 lm. Datarepresent three separate experiments. Similarresults were obtained in two others.Fig. 10. E3 activity of eRBCC. (A) Demon-stration of ubiquitin ligase (E3) activity ofeRBCC. GSt-eRBCC fusion protein wasevaluated for its E3 activity in the presence ofrecombinant E2, UbcH5C, and GSt-Ub withor without TPEN, a Zn2+-chelating agent(lanes 1–3). (B) E2 preference of eRBCCproteins. Ubiquitination assay was performedwith GSt-eRBCC protein in the presence ofthe indicated E2 proteins (lanes 4–9). Bargraphs in A and B represent the results ofquantitative analysis. 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RING finger, B-box, and coiled-coil (RBCC) protein expression in branchial epithelial cells of Japanese eel, Anguilla japonica Kentaro Miyamoto1,. branchial cells. Keywords: freshwater adaptation; gill; RBCC protein; RING finger; ubiquitin ligase. RING finger, B-box, and coiled-coil (RBCC) proteins are agroup of zinc-binding
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