红细胞生成素受体6

The zebrafish erythropoietin: and biochemical c -De ha , Nati nica, 1 iology 20 Jul ne 10 asayu the treatment of peptide-N-glycosidase F (PNGase F). Using the morpholino approach, we showed that zepo morphants displayed severe anemia leading to high mortality during development. Such an effect can be significantly rescued by zepo RNA. Furthermore, topoietic progenitor cells [2]. Upon binding of Epo to its recep- N-glycosylation sites by replacing the asparagines (Asn) with glutamines (Gln) was secreted poorly from COS-1 and CHO FEBS Letters 581 (2007) 4265–4271 E-mail address: cjibc@gate.sinica.edu.tw (C.-J. Huang). tor, dimerization of Epor leads to the activation of the JAK/ STAT signaling pathway [3]. The main function of Epo is to inhibit the apoptosis of erythroid precursor cells and to increase their survival. Currently, recombinant human Epo (rhEpo) has been used to treat anemia caused by chronic kidney disease [4]. Moreover, rhEpo is also used to treat cancer patients with ane- mia induced by chemotherapy or radiotherapy [5]. The first erythropoietin gene from non-mammalian verte- brate has been cloned from fugu and the encoded protein shows only 32% identity to human Epo [6]. Human Epo is a glycoprotein and contains three N-linked sugar chains on 2.2. Total RNA isolation and RT-PCR analysis of zebrafish erythropoietin mRNA Total RNA was isolated from the fertilized eggs at different stages and various tissues of zebrafish (Danio rerio), using the RNAzol reagent (Tel-Test, Friendswood, TX, USA) according to the instruc- tions of the manufacturer. After treatment with RQ1 RNase-Free DNaseI (Promega), 50–100 lg of total RNA was subjected to the first strand cDNA synthesis. PCR amplifications were performed with zepo primers (zEPO-RT-F, 5 0-GTG CCT CTC ACT GAG TTC TTG GAA G-3 0 and zEPO-RT-R, 5 0-CTC GTT CAG CAT GTG TAA GCC TGA C-3 0) or b-actin primers (zAct-F, 5 0-CCT CCG GTC GTA CCA CTG GTA T-3 0 and zAct-R, 5 0-CAA CGG AAG GTC TCA TTG CCG ATC GTG-3 0) and the cDNA as a template. 2.3. Cloning of the full-length cDNAs encoding zEpo and hEpo The full-length cDNA encoding zEpo was isolated by PCR amplifi- cation using gene-specific primers (zEPO-F, 5 0-ATG TTT CAC GGT TCA GGA CTC-3 0; zEPO-R, 5 0-GCT GAC ACC CTG TCG ACA *Corresponding author. Address: Institute of Biological Chemistry, Academia Sinica, 128, Sec 2, Academia Road, Taipei 115, Taiwan. Fax: +886 2 2788 9759. in the absence of functional zEpo, the expression of specific mark- ers for adult globin genes, such as aA1- and bA1-globin, but not the embryonic be1-globin, was affected. � 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Erythropoietin; Zebrafish; Morpholino oligonucleotide; Glycoprotein; Secretion; Globin staining 1. Introduction Erythropoietin (Epo) is a glycoprotein hormone with the function as a principal regulator of erythropoiesis. In mam- mals, the initial production organ is liver and is shifted to the kidney after birth [1]. Effects of Epo are mediated by binding to Epo receptor (Epor), which is primarily expressed in hema- Cheng-Ying Chua,b, Chia-Hsiung Chengb, Gen Kai-Yun Huangb, C a Graduate Institute of Biochemical Sciences b Institute of Biological Chemistry, Academia Si c Institute of Cellular and Organismic B Received 9 May 2007; revised Available onli Edited by M Abstract In the present study, the zebrafish epo cDNA was cloned. The encoded protein displays 90%, 55% and 32% identity to the Epo from carp, fugu and human, respectively. Through RT- PCR, the expression of zepo mRNA was mainly in the heart and liver. In the COS-1 cell transfection experiments, the recombinant zEpo-HA protein was efficiently secreted into the culture medium as a glycoprotein and the carbohydrate moiety can be cleaved by 0014-5793/$32.00 � 2007 Federation of European Biochemical Societies. Pu doi:10.1016/j.febslet.2007.07.073 cells [8]. The secretion ability of the fugu Epo in cultured cells has not been determined yet. In this study, we isolated a gene encoding zebrafish Epo (zEpo) that shows 55% and 32% identity to the Epo from fugu and human, respectively. Unlike the fugu Epo, zEpo contains two N-glycosylation sites and it was efficiently secreted into the culture medium as a glycoprotein. Through RT-PCR, the expression of zepo mRNA was mainly in the heart and liver. Using morpholino approach, we showed that zEpo morphants displayed severe anemia leading to high mortality during development. Furthermore, in the absence of functional zEpo, the expression of erythroid specific markers, such as adult glo- bin genes and the embryonic be1-globin gene were examined. 2. Materials and methods 2.1. Fish Zebrafish were raised and maintained under standard conditions. Embryos were incubated at 28 �C and the different developmental stages were determined according to the descriptions in the Zebrafish Book [9]. Functional identification haracterization r Chenb, Yi-Chung Chenb, Chin-Chun Hungb, ng-Jen Huanga,b,c,* onal Taiwan University, Taipei 106, Taiwan 28, Sec 2, Academia Road, Taipei 115, Taiwan , Academia Sinica, Taipei 115, Taiwan y 2007; accepted 30 July 2007 August 2007 ki Miyasaka Asn24, Asn38 and Asn83 as well as one O-linked sugar on Ser126 [7]. The fugu Epo has no N-linked glycosylation site, but it is postulated to have an O-linked glycosylation site on Ser117 [6]. Site-directed mutagenesis analysis has indicated that the rhEpo variant with either a double mutation (Gln38,83) or a triple mutation (Gln24,38,83) on the three blished by Elsevier B.V. All rights reserved. pattern is consistent with that of fugu epo mRNA, which is predominantly expressed in heart and moderate in liver and 4266 C.-Y. Chu et al. / FEBS Letters 581 (2007) 4265–4271 GAC-3 0) according to the sequence with GenBank accession number EF426727. The full-length cDNA encoding hEpo was isolated by PCR amplification using gene-specific primers (hEPO-F, 5 0-ATG GGG GTG CAC GAA TGT CCT GCC-3 0; hEPO-R, 5 0-TCT GTC CCC TGT CCT GCA GGC CTC-3 0) according to the sequence with GenBank accession number NM000799 from human kidney tissue cDNA. The cDNA encoding zEpo or hEpo was subcloned into the BamHI and EcoRI site of pHA-YUN vector [10] to generate pCMV-zEpo-HA or pCMV-hEpo-HA, respectively. The plasmid pCMV-GFP-HA [10] was constructed by inserting the green fluores- cence protein (GFP) coding region into pHA-YUN. 2.4. Cell cultures Monkey kidney fibroblast COS-1 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum (HyClone, UT, USA) in a humidified atmosphere of 5% CO2 at 37 �C. Cell transfection was carried out using Lipofect- amine Plus reagent (Life Technologies) according to the manufac- turer’s protocol. After transfection for 12 h, cells were incubated in serum-free medium for an additional 36 h. Conditioned media were then collected and concentrated with YM-10 MW Centricon (Milli- pore, Bedford, MA). The concentrated supernatant was digested with 1 U of peptide N-glycosidase F (PNGase F) (Roche) at 37 �C for 18 h. Untreated and N-glycosidase-treated culture supernatants were ana- lyzed by SDS–PAGE followed by detection with Western blotting. 2.5. Western blot and immunocytochemistry assay Cells were harvested at 48 h post-transfection for Western blot and at 24 h post-transfection for immunocytochemistry analysis. Western blot analyses were performed with anti-HA monoclonal antibody (1:3000; Santa Cruz, CA) at 4 �C overnight. The signals were detected using enhanced chemiluminescence (ECL) (NEN Life Science Prod- ucts, MA). For immunocytochemistry analysis, cells were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. Immu- nostaining was performed using anti-HA antibody (1:1000) at 4 �C overnight, followed by Cy3-conjugated goat anti-mouse antibody and co-stained with DAPI for 30 min at room temperature. Photo- images were prepared by using a laser scanning confocal microscope (LSM 510 Meta, Carl Zeiss Inc., Germany). 2.6. Morpholino injection Antisense morpholinos (MOs) were synthesized by Gene Tools (Philomath, OR, USA). The sequence of zepo-MO was as follows: 5 0-TGA AAC ATT CGC AAA ACA ACT TGG C-3 0. The MO was dissolved in 1 · Danieau solution containing 0.5% phenol red to 0.3 mM and 3.2 ng per embryo was injected into embryos at the 1–2 cell stage. The embryos were processed for whole-mount in situ hybrid- ization at 24 hpf or o-dianisidine staining at 48 hpf. 2.7. Morpholino rescue experiment by mRNA injection Capped zepo sense RNA was synthesized with the mMESSAGE mMACHINE T7 Ultra kit (Ambion, Austin, USA) according to the manufacturer’s protocol from linearized pT7TS plasmids containing the entire coding region of zepo cDNA. Ten picograms to 30 pg of sense zepo RNA was co-injected with 3.2 ng zEpo-MO per embryo at 1–2 cell stage. 2.8. o-Dianisidine staining MO-injected or uninjected embryos at 48 hpf were dechorionated and fixed with 4% paraformaldehyde overnight. Fixed embryos were washed in PBS for three times and then incubated in the staining buffer (0.6 mg/mL o-dianisidine, 10 mM sodium acetate (pH 5.2), 0.65% hydrogen peroxide, and 40% ethanol) for 15 min in the dark. 2.9. Whole-mount in situ hybridization Digoxigenin-labeled antisense RNA probes were generated by in vitro transcription using linearized plasmids as template. Whole- mount in situ hybridization were performed as previously described [10]. Specific primers for epo (5 0-GTG CCT CTC ACT GAG TTC TTG GAA G-3 0 and 5 0-CTC GTT CAG CAT GTG TAA GCC TGA C-3 0), gata1 (5 0-ACC TGA GGC TCG TGA ATG TG-3 0 and 5 0-GCT CAT CTG GAG GTG CCA TGT-3 0), scl (5 0-TCC TAG CAA TCG AGT CAA GCG-30 and 5 0-TGG ACT CCA CTG ATG brain [6]. During development, the zepo transcript was detected in all stages (Fig. 2B). We also examined the temporal and spatial patterns of zepo mRNA expression using whole-mount in situ hybridization. In 24, 48, 72, 96 and 120 hpf embryos (Fig. 2C), zepo mRNA was expressed in forebrain, midbrain, hindbrain and brachial arch. ited in GenBank with the accession number of EF426727. To determine the genomic structure of zebrafish erythropoietin (zepo) gene, we used this cDNA as a bait to perform an online BLAST search of the GenBank database and matched five non- contiguous regions in the 200556 bp zebrafish BAC clone DKEY-46E6. Subsequently, we compared the sequences be- tween this BAC clone and the zepo cDNA. The result indicates that the epo cDNA is contained within five putative exons and four introns spanning approximately 13.4 kb. The deduced protein sequences were aligned with other fish Erythropoietins and mammalian Erythropoietins. The zEpo shows an overall identity of 90%, 55%, 32% and 35% to Epo from carp, Fugu, human and mouse, respectively (Fig. 1B). In a previous report, Fugu Epo also shows 32% identity to human Epo [6]. The signal peptide of human Epo has been assigned [7] and the corresponding regions of zEpo were also predicted (Fig. 1B). There are four cysteine residues in mature human Epo that form two disulphide bridges important for the stability and function of the mature protein [11]. These cys- teine residues are conserved in all fish Erythropoietins (Fig. 1B). Moreover, human Epo is a glycoprotein and con- tains three N-linked sugar chains on Asn24, Asn38 and Asn83 [7]. Similarly, the zEpo contains two N-linked glycosyl- ation sites, 38NVT and 81NQT. 3.2. Expression profiles of zepo transcript in different developmental stages and adult tissues in zebrafish To investigate the expression pattern of zepo transcript, zeb- rafish embryos at different developmental stages and various tissues from adult zebrafish were collected for cDNA prepara- tion and subjected to RT-PCR. In adult zebrafish, the epo mRNA is predominantly expressed in the heart and liver, while low expression is observed in the brain, gill, eye, intestine and kidney (Fig. 2A). On the contrary, the kidney is the primary organ to synthesize Epo in adult mammals [1]. This expression AGT CCT G-3 0), be1-globin (5 0-ACA TGG TTG TGT GGA CAG ACT TCG AG-30 and 5 0-TTA GTG GTA CTG TCT TCC CAG AGC GG-3 0), aA1-globin (5 0-ACG CAG CGA TGA GTC TCT CTG ATA C-3 0 and 5 0-GCA CAG TGT TGT TGT CAG TGA ATA T-3 0) and bA1-globin (5 0-ACA TGG TTG AGT GGA CAG ATG CCG-3 0 and 5 0-CAT TGG CGA TGA GAC TCT AGT GGT- 3 0) were used to amplify the DNA as template. 3. Results 3.1. Cloning of the erythropoietin gene from zebrafish To identify zebrafish cDNA related to Fugu epo, we used the coding region of the Fugu epo (GenBank accession number AY303753) to search the GenBank for related expression sequence tag (EST) sequences by using the program tBLAST. Two zebrafish EST clones (GenBank accession numbers DN903417 and EB886593) were found and assembled to obtain the full-length cDNA. Their sequences have been depos- C.-Y. Chu et al. / FEBS Letters 581 (2007) 4265–4271 4267 zEpo 5896 bp I zEpo CcEpo TrEpo hEpo mEpo A B In addition, zepo transcript was detected in early embryos at 12 hpf. 3.3. Secretion of zebrafish Epo in COS-1 cells Epo is a secreted glycoprotein and the signal peptide of human Epo has been assigned [7]. The putative signal peptide of zEpo contains 23 amino acid residues and those of human and mouse Epo have 27 amino acid residues. Interestingly, eight amino acids of the N-terminal 12 amino acids, AP- PRLICDSRVL, of mature human Epo are highly conserved between fish and mammalian Erythropoietins. In order to confirm the secretion ability of zEpo, a HA-tag was added to the C-terminal end of zEpo. At 48 h post-trans- fection of pCMV-zEpo-HA into COS-1 cells, the fusion pro- tein in cell lysate or conditioned medium was detected by SDS–PAGE and Western blot analysis using a monoclonal antibody against HA-tag. As shown in Fig. 3A, the zEpo- zEpo CcEpo TrEpo hEpo mEpo zEpo CcEpo TrEpo hEpo mEpo zEpo CcEpo TrEpo hEpo mEpo Fig. 1. Genomic organization of the zebrafish erythropoietin gene and alignm Genomic structure of the zepo gene. Exons are indicated by the boxes num untranslated regions (open boxes). Introns and the 5 0-flanking regions are ind mammals using CLUSTAL X: The amino acid sequence of the zebrafish DQ278877), Fugu (trEpo; AY303753), human (hEpo; NM_000799) and m highlighted. Signal peptide and putative N-linked glycosylation sites are unde by arrows. 2052 bp 84 bp 4735 bp II III IV V HA was present in both conditioned medium and cell lysate. The molecular mass of secreted zEpo-HA was reduced when it was treated with PNGase F, suggesting that this fusion pro- tein undergoes post-translational modification of glycosyla- tion. As a positive control, the HA-tagged human Epo was also expressed in both conditioned medium and cell lysate and was glycosylated in COS-1 cells. Due to the properties of secretion and glycosylation, zEpo- HA and hEpo-HA are expected to be localized in the ER/Gol- gi. Immunofluorescence studies of COS-1 cells transfected with pCMV-zEpo-HA using the anti-HA monoclonal antibody re- vealed that zEpo-HA was localized in the perinuclear region and displayed a punctate, non-uniform expression pattern, suggesting that it is localized to ER/Golgi (Fig. 3B). Similarly, hEpo-HA was localized to ER/Golgi of COS-1 cells as well. As a control, GFP-HA was localized to the cytosol with a uni- form distribution. ent of amino acid sequences of zEpo with Epos from other species. (A) bered from 1 to 5, including the coding regions (solid boxes) and the icated by the solid lines. (B) Multiple alignment of Epos from fish and Epo (zEpo; EF426727) was compared with those from carp (ccEpo; ouse (mEpo; NM_007942). Identical residues in three proteins are rlined or overlined. The conserved four cysteine residues are indicated ount e use n situ hybridization with antisense zepo at different developmental stages was to the left and dorsal to the top. fb, forebrain; mb, midbrain; hb, hindbrain; 4268 C.-Y. Chu et al. / FEBS Letters 581 (2007) 4265–4271 3.4. Knockdown of zepo resulted in strong suppression of hemoglobin production in zebrafish embryos and reduction Fig. 2. Expression profiles of zepo mRNA by RT-PCR and by whole-m pair of primers producing a DNA fragment of 438 bp. b-actin bands wer and from embryos at different developmental stages (B). Whole-mount i performed (C). The images were taken from a lateral view, with anterior ba, brachial arch. of erythroid-specific gene expression Morpholino (MO)-mediated knockdown of genes in zebra- fish embryos has become a routine and efficient method to pro- vide information about gene function in vivo [12]. To examine the function of zEpo in the regulation of hemoglobin produc- tion, we injected zepo-MO into zebrafish embryos at 1–2 cells stage. In Fig. 4A, the presence of hemoglobin in zebrafish em- bryos was detected by o-dianisidine staining [13]. Embryos in- jected with zepo-MO at 48 hpf displayed significant loss of hemoglobin (panels b and b 0), as compared to that of wild-type embryos (panels a and a 0). Moreover, the mortality rate of zepo-MO-injected embryos was very high (80% mortality, n = 250), possibly due to the significant loss of hemoglobin and other Epo-responsive effect during development [14]. In addition, obvious pericardial edema was observed in the EPO-MO-injected embryos. To test whether zepo RNA can rescue this defect, we co-injected zepo-MO and zepo RNA into zebrafish embryos at the one-cell stage. Our results showed that the mortality rate was reduced to approximately 30% (n = 150) and zepo RNA-injected embryos displayed obvious hemoglobins stained by o-dianisidine (panels c and c 0). To further examine the role of Epo during primitive erythro- genesis, we analyzed the effect of zepo-MO injection on the expression of erythroid-specific genes, such as aA1-globin, bA1-globin, be1-globin, scl and gata1 by whole-mount in situ hybridization. There are five adult globin genes in zebrafish, three a-globin, and two b-globin genes [15], while only one embryonic globin gene has been identified, a b-like globin gene (be1) [16]. In addition, the stem cell marker scl [17] and the immature erythroblast marker gata1 [13] were also examined. As shown in Fig. 4B, there was no difference in the expression of scl, gata1 and be1-globin in both zepo-MO-injected and in situ hybridization. RT-PCR of zepo transcript was performed with a d to normalize the amount of cDNA prepared from different tissues (A) wild-type embryos at 24 hpf. However, the expression of aA1-globin and bA1-globin was reduced significantly in zepo- MO-injected embryos at 24 hpf. 4. Discussion In this study, the zepo gene and its cDNA were cloned and characterized. The cDNA encodes a protein of 183 amino acids, which displays 55% and 32% identity to the fugu Epo and human Epo. RT-PCR analyses showed that zepo mRNA was primarily expressed in the heart and liver. Unlike the fugu Epo, zEpo contains two glycosylation sites and the recombi- nant zEpo was efficiently secreted into the culture medium as a glycoprotein. Knockdown of zEpo caused severe anemia leading to high mortality in zebrafish embryos and the expres- sions of specific markers for adult globin genes, such as aA1- globin and bA1-globin, were affected. The genomic structure of epo genes from human and fugu have been determined [6]. In this study, the zebrafish epo gene consists of five exons and four introns spanning approximately 13.4 kb (Fig. 1). For comparison, the human epo gene spans only about 2.9 kb, while the fugu epo gene spans about 5.9 kb. The first intron of fugu and zebrafish epo gene is 3,105 bp and 5896 bp in length respectively, which is larger than that of human epo gene for only 584 bp in length. The second and the last introns of zEPO gene are also large, which are 2052 bp and 4735 bp in length, compared to those of human EPO gene with the length of 258 bp