Pesq. Vet. Bras. 32(10):1050-1054, outubro 2012 1050 RESUMO.- [Sequenciamento e expressão do RNA men- sageiro da hepcidina no �ígado de jumentos (Equus asi- nus).] A hipoferremia observada durante os processos in�la- matórios sistêmicos é mediada pela hepcidina, um peptídeo que é sintetizado predominantemente no �ígado de mamí- feros. A hepcidina desempenha um papel chave no metabo- lismo do ferro e no sistema imune. O aumento da expressão da hepcidina é particularmente útil durante a in�lamação Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus) liver1 José P. Oliveira-Filho2, Jessica A. Marques2, Paulo Henrique J. Cunha3, Gildenor X. Medeiros4, Franklin Riet-Correa4, Vânia Maria V. Machado2 and Alexandre S. Borges2* ABSTRACT.- Oliveira-Filho J.P., Marques J.A., Cunha P.H.J., Medeiros G.X., Riet-Correa F., Machado V.M.V. & Borges A.S. 2012. Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus) liver. Pesquisa Veterinária Brasileira 32(10):1050-1054. Departamento de Clínica Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Uni- versidade Estadual Paulista, Campus de Botucatu, Distrito de Rubião Júnior s/n, Botucatu, SP 18618-000, Brazil. E-mail: asborges@fmvz.unesp.br The hypoferremia that is observed during systemic in�lammatory processes is mediated by hepcidin, which is a peptide that is mainly synthesized in the livers of several mamma- lian species. Hepcidin plays a key role in iron metabolism and in the innate immune system. It’s up-regulation is particularly useful during acute in�lammation, and it restricts the iron availability that is necessary for the growth of pathogenic microorganisms. In this study, the hepcidin mRNA of Equus asinus has been characterized, and the expression of don- key hepcidin in the liver has been determined. The donkey hepcidin sequence has an open reading frame (ORF) of 261 nucleotides, and the deduced corresponding protein sequence has 86 amino acids. The amino acid sequence of donkey hepcidin was most homologous to Equus caballus (98%). The mature donkey hepcidin sequence (25 amino acids) was 100% homologous to the equine mature hepcidin and has eight conserved cysteine residues that are found in all of the investigated hepcidin sequences. The expression pro�ile of donkey hepcidin in the liver was high and was similar to the reference gene expression. The donkey hepcidin sequence was deposited in GenBankTM (HQ902884) and may be useful for addi- tional studies on iron metabolism and the in�lammatory process in this species. INDEXS TERMS: Hepcidin, mRNA, sequencing, expression, Equus asinus. 1 Received on August 10, 2011. Accepted for publication on April 15, 2012. 2 Departamento de Clínica Veterinária, Faculdade de Medicina Vete- rinária e Zootecnia, Universidade Estadual Paulista (Unesp), Campus de Botucatu, Distrito de Rubião Júnior s/n, Botucatu, SP 18618-000, Brazil. *Corresponding author: asborges@fmvz.unesp.br 3 Departamento de Medicina Veterinária, Escola de Veterinária, Univer- sidade Federal de Goiás (UFG), Campus Samambaia (Campus II), Cx. Postal 131, Goiânia, GO 74001-970, Brazil. 4 Hospital Veterinário, Campus de Patos da Universidade Federal de Campina Grande (UFCG), Patos, PB 58700-000, Brazil. aguda, pois restringe a disponibilidade de ferro, necessária para o crescimento de microorganismos patogênicos. Neste estudo, o RNA mensageiro da hepcidina asinina foi caracteri- zado e sua expressão foi determinada em �ígado de jumentos (Equus asinus). A sequência da hepcidina asinina tem uma janela de leitura de 261 nucleotídeos e a proteína correspon- dente é formada por 86 aminoácidos. A sequência de amino- ácidos da hepcidina asinina foi mais homóloga à sequência da hepcidina equina (98%). A hepcidina madura (25 amino- ácidos) foi 100% idêntica à hepcidina madura equina e pos- suía as oito cisteínas conservadas nas demais sequências de hepcidinas analisadas. O per�il de expressão da hepcidina no �ígado de jumentos saudáveis foi alto e similar ao per�il de expressão do gene de referência. A sequência da hepcidina asinina foi depositada no GenBankTM (HQ902884) e será útil para o desenvolvimento de estudos adicionais sobre o meta- bolismo de ferro e in�lamação nesta espécie. TERMOS DE INDEXAÇÃO: Hepcidina, mRNA, sequenciamento e expressão gênica, Equus asinus. Pesq. Vet. Bras. 32(10):1050-1054, outubro 2012 1051Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus) liver INTRODUCTION Hepcidin is a peptide of 25 amino acids (aa) in length that is primarily expressed in the liver. Hepcidin has activity against some Gram-positive and Gram-negative bacteria and yeasts (Krause et al. 2000, Park et al. 2001) and plays an important role in the metabolism of iron in the mamma- lian species (Ganz & Nemeth 2006). Hepcidin binds to the iron exporter ferroportin (Fpn) and promotes its interna- lization and degradation (De Domenico et al. 2009). Thus, there is an inhibition of the iron in�lux into plasma from fer- roportin-rich tissues and a decrease in dietary iron absorp- tion (Ganz & Nemeth 2006). The resulting hypoferremia is a common response to systemic infections or generalized in�lammatory disorders (Nemeth et al. 2004), and it pri- marily serves as a host defense mechanism that limits iron availability to invading microorganisms (Andrews 2004). The hepcidin gene has been previously characterized in several mammalian species, e.g., humans (Krause et al. 2000), dog (Fry et al. 2004), swine (Sang et al. 2006), cattle (Hilton & Lambert 2008), sheep (Badial et al. 2011), water buffalo (Khangembam & Kumar 2011), and horse (Oliveira Filho et al. 2010); however, there are no reports concerning the donkey hepcidin sequence. Therefore, we describe the complete nucleotide sequence of the open reading frame (ORF) of donkey hepcidin and show differences between the donkey and horse nucleotide and protein sequences. A hepcidin expression analysis of the liver of a clinically heal- thy donkey was also performed. MATERIALS AND METHODS Liver tissue samples. Six adult donkeys (Equus asinus) were obtained from three farms and were used in this study. The donkeys were all sedated by an intravenous injection of xylazine hydrochloride (1mg/kg of body weight) and were administered local anesthesia (2ml of 2% lidocaine). Ultrasound-guided liver biopsies were obtained from the right 11th or 12th intercostal spa- ce in a line that had been drawn from the point of the shoulder to the tuber coxae. Liver biopsies were taken using a 14-G Tru-cut® biopsy needle (Biomedical, Italy). Immediately after harvesting, all of the biopsy samples were frozen in liquid nitrogen and sto- red at -80°C until RNA puri�ication. All experiments were carried out according to the Univ Estadual Paulista (Unesp) Institutional Animal Care and Use Committee (03/2010 CEUA). RNA isolation, reverse transcription-polymerase chain reaction (RT-PCR) and sequence analysis. Total RNA was iso- lated from a donkey liver sample using the RNeasy® Mini Kit (Qiagen, USA), following the manufacturer’s instructions. The relative purity and quality of the isolated RNA was determined by a Nanodrop® 2000 Spectrophotometer (Thermo Scienti�icTM, USA). Brie�ly, the total RNA, which had been treated with RQ1 RNase-Free DNase (Promega, USA) in order to eliminate genomic DNA from the samples, was used for �irst-strand cDNA synthesis. The cDNA was prepared using 500 ng of total RNA per 60 μl of the reaction solution using random hexamers and the ImProm- -IITM Reverse Transcription System (Promega, USA), following the manufacturer’s instructions. The RT-PCR was performed using primer sets that had previously been designed for the sequence of equine hepcidin (Oliveira Filho et al. 2010). The RT-PCR was per- formed in duplicate using 20.0-μl reaction solution that contained 0.4μM of each primer, 2.0μl of template cDNA, 10.0μl of GoTaq® Green Master Mix (Promega, USA) and 6.4μl of nuclease-free wa- ter. In addition, a ‘‘ no template’’ control was performed in order to demonstrate the absence of contamination. The PCR conditions were established as follows: initial denaturation at 94°C for 5 min and 35 cycles at 94°C for 15 s (denaturation), 60°C for 30 s (an- nealing) and 72°C for 60 s (extension) and then a �inal extension at 72°C for 5 min. The PCR products were analyzed via 1.5% agarose gel elec- trophoresis (Invitrogen®, USA), stained by GelRedTM (BiotiumTM, USA) and visualized under an ultraviolet light. The molecular wei- ghts of the PCR products were estimated by comparing them to a known molecular weight marker of 100 bp in length (Norgen®, Canada). The PCR products with predicted sizes of ~450 bp were puri�ied using the QIAquick® PCR Puri�ication Kit (Qiagen®, USA). An automated direct sequence analysis was performed in quadru- plicate via 3500 Genetic Analyzers (Applied BiosystemsTM, USA) and the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied BiosystemsTM, USA) using 5.0μl of each primer and 10.0μl of the PCR product. The sequences and electropherograms obtained were analyzed using Sequencing Analysis 5.3.1 software (Applied BiosystemsTM, USA). The obtained sequence was blasted (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) in order to verify sequence ho- mology against equine hepcidin and six other mammalian hepci- din sequences that have previously been deposited in GenBankTM (http://www.ncbi.nlm.nih.gov/). Expression analysis of hepatic donkey hepcidin by real- -time RT-PCR (qRT-PCR). The primer sets that were used in the qRT-PCR (amplifying an ~81 bp (base pair) fragment of the hep- cidin mRNA and an ~86 bp fragment of the β-actin mRNA as a reference gene) have previously been described (Oliveira Filho et al. 2010). The relative quanti�ication of the donkey hepatic hep- cidin mRNA was performed using the comparative Ct method (2-ΔΔCt method) for relative real-time PCR data processing (Livak & Schmittgen 2001) via a 7500 Real-Time PCR System (Applied BiosystemsTM, USA) and the Power SYBR® Green PCR Master Mix (Applied BiosystemsTM, USA). Each qRT-PCR reaction was perfor- med in triplicate using 20.0-μl reaction solutions, which contai- ned 0.3mM of each forward and reverse primer, 2.0μl of template cDNA, 10.0μl of the qRT-PCR master mix and 6.8μl of nuclease- -free water. In addition, two ‘‘no template’’ controls were included on each plate in order to demonstrate the absence of amplicon contamination. The PCR conditions were established as follows: initial denaturation at 95°C for 10 min and 40 cycles at 95°C for 15 s and 60°C for 60 s, followed by a melting curve. The expression of basal donkey hepcidin mRNA was compared to the expression of β-actin mRNA. RESULTS The ultrasound-guided liver biopsies were safe and w ere successfully used to obtain puri�ied RNA. No adverse effects were observed in the donkeys after the procedu- re. The A260/A280 of the isolated RNA exceeded 2.0 for all of the preparations, which indicates the purity of the RNA. The ampli�ication of the cDNA using equine hepcidin primers worked well and facilitated the sequencing of the hepcidin that was obtained from the donkeys. The cDNA of the donkey hepcidin that is presented here is 354 bp in length, which includes 19 bp in the 5’-untranslated region (UTR) and 74 bp in the 3’-UTR. The ORF of the obtained preprohepcidin sequence has 261 bp (Fig.1) and was sub- mitted to the NCBI (accession number HQ902884). The base composition of the ORF L-strand consists of 20.7% adenine, 34.1% cytosine, 26.8% guanine, and 18.4% thy- mine. The nucleotide sequences of the preprohepcidin Pesq. Vet. Bras. 32(10):1050-1054, outubro 2012 1052 José P. Oliveira-Filho et al. cDNA of donkey and equine (GQ_253624) exhibited a to- tal difference of three nucleotides at positions 15, 121 and 143, wherein the bases guanine, guanine and cytosine in the equine were replaced by adenine, adenine and thymine, respectively, in the donkey. The donkey preprohepcidin protein sequence is 86 aa long and contains a putative 23-aa-long signal peptide, a 38-aa-long pro-peptide region and a 25-aa-long mature hepcidin peptide (Fig.2). The donkey and equine hepcidin sequences have similar putative signal peptide cleavage sites between a glycine (G23) and serine (S24) and similar putative propeptide cleavage recognition sites that consist of the motif HQLRRR56–61. Upon alignment, the donkey pre- prohepcidin amino acid sequence was observed to share a 98% identity with the equine hepcidin (ACZ04924.1) and show two different amino acids at positions 41 (the threo- nine in the donkey sequence was replaced by alanine in the equine sequence) and 48 (the valine in the donkey sequen- ce was replaced by alanine in the equine sequence). No in- sertions or deletions were observed between the donkey and the equine preprohepcidin sequences. The donkey, equine and six other preprohepcidin amino acid sequences were aligned using the CLUSTAL X program (Larkin et al. 2007), and the phylogenetic analyses were conducted using MEGA4 software (Tamura et al. 2007). The evolutionary history was inferred using the neighbor-joining method (Saitou and Nei 1987), a complete deletion of gaps, and 1,000 bootstrap interactions (Felsenstein 1985) (Fig.3). The experimentally determined donkey hepcidin sequence had a 75% identity to Bos taurus (AAI11659.1) and Bubalus bubalis (ABY81280.2), 74% to Ovis aries (NP001182241.1), 73% to Sus scrofa (AAM77745.1), 69% to Canis familiaris (NP001007141.1) and 68% to Homo sapiens (NP066998.1) (Fig.4). The putative 25-aa-long mature peptide sequence of donkey hepcidin is 100% identical to the equine mature hep- cidin; 76% identical to the canine, porcine and human matu- re hepcidin sequences and 72% identical to the cattle and bu- ffalo mature hepcidin sequences. Although the �irst six amino acids62-67 that were observed in the donkey mature hepcidin sequence was highly conserved among the compared mam- malian species, eight cysteine (C) residues in the mature hep- cidin and an additional cysteine residue at position 11 of the preprohepcidin were also conserved in donkey hepcidin. The equine hepcidin and β-actin mRNA primers (Oli- veira Filho et al. 2010) that were used in the expression analysis of hepcidin mRNA donkey liver ampli�ied speci�ic transcripts, which were con�irmed by melting curve pro�i- les that were generated at the end of each run. Quantita- tive PCR analysis demonstrated that expression pro�ile of donkey hepcidin in liver was high and was similar to the cycle threshold (CT) values that have been obtained for the β-actin housekeeping gene. Fig.1. Open reading frame (top) and deduced amino acid (bottom) sequences of donkey hepcidin. Start and stop codons are in italics. Bold nucleotides and underlined amino acids indicate differences with equine sequence. Fig.4. Alignment of amino acid sequence of donkey hepcidin (HQ902884) with hepcidin of different species. The se- quence alignment was constructed using the Clustal X. NCBI accession number: Equus caballus, ACZ04924.1; Homo sa- piens, NP066998.1; Bos taurus, AAI11659.1; Bubalus bu- balis, ABY81280.2; Ovis aries, NP001182241.1; Sus scrofa, AAM77745.1 and Canis familiaris, NP001007141.1. Fig.2. Amino acid sequence of the donkey and equine signal pep- tide, pro-region and mature hepcidin. Conserved cysteine (C) residues in the signal peptide and mature hepcidin are indi- cated in gray. Residues in parenthesis indicate amino acids different between both sequences. Bold sequence indicates the predicted furin cleavage site. Underlined residues indicate essential amino acid sequence for bidding to ferroportin. The sequence alignment was constructed using the Clustal X. Fig.3. Phylogenic tree showing the relationship of the donkey hep- cidin (HAMP) amino acid sequence with previously identi�ied hepcidin sequences. The phylogeny was constructed using Clustal X program, and the evolutionary history was infer- red using the Neighbor-Joining method. The numbers next to the branches show bootstrap majority consensus values on 1000 replicates in percent. All positions containing gaps and missing data were eliminated from the dataset. Phylogenetic analyses were conducted in MEGA4. Pesq. Vet. Bras. 32(10):1050-1054, outubro 2012 1053Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus) liver DISCUSSION Donkeys and horses belong to the order Perissodactyla, fa- mily Equidae and genus Equus (Grubb 1993), and although there are similar morphologic aspects between these two species, the mitochondrial DNA differences suggest that the evolutionary separation of these two species occurred around nine million years ago (Xu et al. 1996). In our study, the amino acid sequence of the hepcidin ORF of the donkey showed a greater identity (98%) with the horse (Oliveira Filho et al. 2010) in comparison to other mammalian hepci- din sequences (Krause et al. 2000, Park et al. 2001, Fry et al. 2004, Sang et al. 2006, Hilton & Lambert 2008, Badial et al. 2011, Khangembam & Kumar 2011). Xu et al. (1996) also demonstrated an average identity of 98% (91.2-99.8%) be- tween the 13 mitochondrial DNA and amino acid sequen- ces of the donkey and horse. Human hepcidin is initially synthesized as a larger pre- cursor protein (preprohepcidin) that undergoes two clea- vages (the signal peptide and then the propeptide) befo- re being secreted from the cell (Valore & Ganz 2008). The horse hepcidin precursor protein also has three segments (signal peptide, pro-region and mature hepcidin) (Oliveira Filho et al. 2010). Similar to the horse, the donkey hepci- din precursor protein is divided into three segments. The number of amino acids in each of the segments and cleava- ge points of the donkey hepcidin protein are similar to the equine hepcidin. Furin is the proprotein convertase for the human hepcidin (Valore & Ganz 2008), which is most likely also the case for horse hepcidin (Oliveira Filho et al. 2010). In addition, donkey and equine have the same putative pro- peptide cleavage motif, which is different from the cleavage motifs that are found in other hepcidin sequences, sugges- ting that furin may also be the proprotein convertase for donkey hepcidin. As is the case for other species, the mature donkey hep- cidin sequence is 25-aa-long and is 100% homologous to the mature equine hepcidin. The �irst six amino acids62-67 that were observed in the mature donkey hepcidin and are conserved in other hepcidins (Krause et al. 2000, Park et al. 2001, Fry et al. 2004, Sang et al. 2006, Hilton & Lam- bert 2008, Badial et al. 2011, Khangembam & Kumar 2011) appear to be essential to the binding of ferroportin (Hilton & Lambert 2008). The mature donkey hepcidin sequence was observed to contain eight cysteines at amino acid po- sitions that are absolutely conserved across mammalian species (Krause et al. 2000, Park et al. 2001, Fry et al. 2004, Sang et al. 2006, Hilton & Lambert 2008, Badial et al. 2011, Khangembam & Kumar 2011). These eight conserved resi- dues are important for disul�ide bonding and the mainte- nance of the structural shape of the hepcidin protein (Hun- ter et al. 2002, Fry et al. 2004). This result con�irms that mature hepcidin sequences are highly conserved among mammalian species (Hilton & Lambert 2008, Oliveira Filho et al. 2010). The expression analysis of hepcidin mRNA from donkey liver by real-time PCR was performed with primers that had been previously designed for the equine hepcidin sequen- ce (Oliveira Filho et al. 2010), and the speci�icity of these primers was tested via Blast analysis, resulting in a 100% identity and complete annealing to the sequenced donkey hepcidin gene. In addition, the ampli�ication of speci�ic transcripts was con�irmed by melting curve pro�iles. Hep- cidin was �irst identi�ied as an antimicrobial peptide from human plasma ultra�iltrate (Krause et al. 2000) and urine (Park et al. 2001). Its expression levels in human (Krause et al. 2000) and equine (Oliveira Filho et al. 2010) livers are comparable to those of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin housekeeping genes, respectively. This result is consistent with the �indings of other authors that have shown that hepcidin is predomi- nantly synthesized in the livers of dogs (Fry et al. 2004), pigs (Sang et al. 2006) and sheep (Badial et al. 2011). In summary, to the best of our knowledge, this report is the �irst to describe the nucleotide and amino acid sequen- ce (GenBankTM accession number HQ902884) of hepcidin and its expression in the livers of healthy donkeys. This sequence will be helpful for additional studies on iron me- tabolism and in�lammatory processes in donkeys. Further research is needed to increase our knowledge concerning the role of hepcidin in the regulation of iron metabolism in donkeys. Acknowledgements.- This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Proc.07/07344-6 e 10/00058-0). 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