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K⁺-ATPase isoform α4
Biochimie 158 (2019) 149e155
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Research paper
Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoform a4 Knud Larsen*, Carina Henriksen, Kaja Kjær Kristensen, Jamal Momeni, Leila Farajzadeh Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers All e 3, DK-8000, Aarhus C, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 August 2018 Accepted 5 January 2019 Available online 8 January 2019
Naþ/Kþ-ATPase is responsible for maintaining electrochemical gradients of Naþ and Kþ, which is essential for a variety of cellular functions including neuronal activity. The a-subunit of the Naþ/KþATPase is composed of four different polypeptides (a1ea4) encoded by different genes. Na,K-ATPase a4, encoded by the ATP1A4 gene, is expressed in testis and in male germ cells of humans, rats and mice. The a4 polypeptide has an important role in sperm motility, and is essential for male fertility. Here we present the RT-PCR cloning and characterization of the porcine ATP1A4 cDNA coding for Na⁺/ K⁺-ATPase polypeptide a4. The Na⁺/K⁺-ATPase polypeptide a4, consisting of 1030 amino acids, displays a high homology with its human counterpart (86%). Phylogenetic analysis demonstrated that porcine Na⁺/ K⁺-ATPase polypeptide a4 is closely related to other mammalian counterparts. In addition, the genomic structure of the porcine ATP1A4 gene was determined, and the intron-exon organization was found to be similar to that of the human ATP1A4 gene. The promoter sequence for the porcine ATP1A4 gene was also identified. Investigation of the genetic variation in the porcine ATP1A4 gene revealed a missense A/G SNP in exon 18. This A/G polymorphism results in a substitution of a methionine to a glycine residue (M888G). A very high overall DNA methylation rate of the ATP1A4 gene, 70e80%, was observed in both brain and liver. Expression analysis demonstrated that the porcine ATP1A4 gene is predominantly expressed in testis. The sequence of the porcine ATP1A4 cDNA encoding the Na⁺/K⁺-ATPase a4 protein has been submitted to GenBank under the accession number GenBank Accession No. MG587082. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: ATP1A4 Methylation Na⁺/K⁺-ATPase Pig SNP Testis
1. Introduction Naþ,Kþ-ATPase, initially described by Skou in 1957 [1], is an integral membrane protein which is responsible for establishing and maintaining electrochemical gradients of Naþ and Kþ across the plasma membrane [2,3]. This enzyme catalyzes the hydrolysis of ATP coupled with the exchange of sodium and potassium ions across the plasma membrane. The gradients created hereby are essential for electrical excitability of nerves and muscles, for sodium-coupled transport of various organics and inorganics molecules and for osmoregulation. The Naþ,Kþ-ATPase is composed of two subunits, a which is the catalytic subunit, a glycosylated b
* Corresponding author. E-mail addresses: [email protected] (K. Larsen), Carina.Henriksen@mbg. au.dk (C. Henriksen), [email protected] (K.K. Kristensen), Jamal.Momeni@mbg. au.dk (J. Momeni), [email protected] (L. Farajzadeh).
subunit and in many tissues a regulatory subunit belonging to the FXYD protein family. The a subunit of Naþ,Kþ-ATPase is encoded by four different genes, ATP1A1, ATP1A2, ATP1A3 and ATP1A4 [4,5]. The ATP1A4 gene encodes the Naþ,Kþ-ATPase a4 subunit. The human ATP1A4 gene, composed of 22 exons and spanning approx. 35 kb, is located on chromosome 1q23.2. The ATP1A4 gene is specifically expressed in testis and sperm cells [6,7]. However, low levels of ATP1A4 mRNA can be detected in human and mouse skeletal muscle [8]. Naþ,Kþ-ATPase a4 protein is expressed in mature sperm. Three fourth of total Na,K-ATPase activity in sperm cells is attributed to the a4 polypeptide and the remaining 25% is attributed to the a1 polypeptide [9]. The Na,KATPase a4 polypeptide is essential for sperm fertility and loss of a4 leads to complete male sterility in mice [10]. Sperm cells from a4 knockout mice have unaffected viability but are unable to fertilize eggs in vitro [10]. The loss of a4 results in reduced sperm motility, depolarization of the membrane potential and increased intracellular sodium [10]. Overexpression of the a4 polypeptide enhances
https://doi.org/10.1016/j.biochi.2019.01.003 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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sperm motility in transgenic mice [11,12]. Recently, is was shown in cattle that Naþ/KþATPase regulates sperm capacitation through a mechanism involving kinases, protein kinases A and C, and redistribution of ATP1A4 [13]. Inhibition of Naþ/KþATPase induces tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. In Holstein bulls ATP1A4 content and activity, ROS, F-actin and calcium are parameters significantly correlated with fertility [14]. The a4 polypeptide is responsible for sperm motility. Inhibition of a4 with ouabain inhibits rat sperm cell characteristics including total and progressive motility and other kinetic parameters [15,16]. Similar inhibitory effects of ouabain was seen with human sperm cells [17]. Decrease in Na,K-ATPase activity and sperm motility was observed in sperm cells from men with a normal sperm profile treated with ouabain [17]. In oligoasthenospermic males the sperm motility was almost completely lost by addition of ouabain [17]. The role of ATP1A4 in regulation of sperm motility and capacitation in cattle has also been investigated [14,18]. Capacitation is a prerequisite for fertilization in mammals. Mammalian sperm cells undergo a series of structural and functional modifications to render them competent to fertilize an oocyte. Disease causing mutations have been identified in Na,KATPase alpha polypeptides a1, a2 and a3, resulting in diseases like hypertension, familial hemiplegic migraine, rapid-onset dystonia Parkinsonism and alternating hemiplegia of childhood [19]. The Na,K-ATPase a4 polypeptide is essential for sperm function, and therefore mutations in the ATP1A4 gene are very likely to affect the male bearers' fertility. Animal models for male infertility including genetically modified and chemical-induced point mutant mice have aided the identification of infertility causing mutations. Studies with mouse models have identified more than 400 genes essential for male fertility [20]. However, thousands of genes are estimated to be involved in the regulation of the complex process of male fertility, so many genes remain to be characterized. With a view to future use of pig as an animal model in studies of human diseases caused by Naþ/Kþ-ATPase mutations, we have cloned and characterized the ATP1A4 gene encoding the Naþ/Kþ-ATPase a4 polypeptide. This includes the determination of its genomic organization and chromosomal localization, and expression pattern. 2. Materials and methods 2.1. Ethics statement The pigs used in the present study were housed and used in compliance with European Community animal care guidelines. The experimental procedures were approved by the National Ethical Committee in Denmark (Approval No. 2010/561e1891). 2.2. Animals One year old Danish Landrace pigs were used in this study. Pigs were sacrificed by an injection with 30 mg/kg Pentobarbital (Vipidan, Denmark). 2.3. Cloning of the porcine ATP1A4 cDNA The cDNA fragment encoding ATP1A4 was amplified by PCR using a testis cDNA and ATP1A4 specific primers derived from genomic sequences. The various pig tissues used for cloning and expression analysis were dissected and pulverized in liquid nitrogen. Total RNA was isolated by the RNeasy method (Qiagen) and RNA integrity was verified by ethidium bromide staining of 1% agarose gels. Synthesis of cDNA was carried out with 5 mg of total RNA isolated from pig testis and other tissues and organs using
SuperScript II RNase H reverse transcriptase (Invitrogen) and oligo(dT)12e18 primers according to the manufacturer's recommendations. For the molecular cloning of the ATP1A4 cDNA, RNA was isolated from testis. The PCR reaction mix contained: 2.5 ml cDNA, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 mM of each primer ATP1A4F and ATP1A4-R (Table S1) and 1 U Phusion DNA polymerase (Finnzymes), in a total volume of 25 ml. The PCR conditions was as follows: 95 C for 2 min, 10 touchdown cycles of 95 C for 20 s, 60 C for 30 s, 72 C for 45 s, followed by 25 cycles of 95 C for 20 s, 55 C for 30 s, 72 C for 45 s and finally an elongation at 72 C for 5 min. A PCR product of approx. 3000 bp was visualized and isolated from an ethidium bromide stained 1% agarose gel. The recovered cDNA amplicon was cloned directly into the pCR TOPO 2.1 vector (Invitrogen) and sequenced in both directions. DNA sequencing was performed, as previously described [21]. 2.4. Genomic organization and promoter of ATP1A4 To identify the porcine ATP1A4 gene, the cloned ATP1A4 cDNA sequence was used in a blast search in Ensembl pig genome data (Sus scrofa 11.1; https://www.ensembl.org/Sus_scrofa/Info/Index). The prediction software Genscan (http://genes.mit.edu/GENSCAN. html) and the Expasy translate tool (https://web.expasy.org/ translate/) was used to identify the exon-intron organization. 2.5. SNP discovery Fourteen boars were examined for SNPs in exon 18 of the ATP1A4 gene. An oligonucleotide primer set, ATP1A4-EX18F and ATP1A4-EX18R, was designed to cover the individual exon 18 (Table S1). A 269 bp fragment was amplified using 0.5 mM primer, 0.2 mM dNTP, 1 Phusion buffer, 2 U Phusion DNA Polymerase (Finnzymes), and 2 ml genomic cDNA in a total volume of 10 ml. The cycling conditions employed were: denaturation at 98 C for 2 min denaturation, 30 cycles of 98 C for 10 s, 60 C for 30 s and 72 C for 30 s, with a final extension of 72 C for 10 min. The AcycloPrime-FP SNP detection kit (Perkin Elmer) was used to remove residual primers and dNTP from the PCR fragments. Six microliters PCR product, 1 PCR Clean-Up dilution buffer (Perkin Elmer), and 0.05 ml PCR Clean-up reagent (Perkin Elmer) were mixed. The reaction was incubated at 37 C for 90 min and 80 C for 15 min. The products were then sequenced using the BigDye terminator v3.1 Cycle Sequencing Kit and the SNP primer set for the detection of possible SNPs. The obtained sequences were analyzed in Consed. 2.6. Methylation status of ATP1A4 The methylation status of ATP1A4 was determined by library preparation, sequencing, mapping and analysis as described [22]. For determination of the methylation percentage of specific genes or sequences from our methylome data file, we used Tabix [23]. 2.7. Expression analysis Real time RT-PCR analysis of porcine ATP1A4 mRNA expression was performed on cDNA synthesized from total RNA isolated from the following porcine organs: testis, lung, spleen, kidney, cerebellum, parietal cortex, bladder, brain stem, frontal cortex, heart, spinal cord, occipital cortex, temporal lobe and hypothalamus. Three separate tissues were applied for each type of brain tissue. Tissue samples were collected from three Danish Landrace pigs, aged 1e2 years and weighing 125e200 kg ATP1A4-specific primers and 18S-specific primers were designed to amplify a DNA sequence of exon 18 of the porcine ATP1A4 gene and the 18S ribosomal gene using the EXIQON Human ProbeLibrary. The ATP1A4 primers
K. Larsen et al. / Biochimie 158 (2019) 149e155
produce an amplicon of 60 bp by PCR amplification. Probes for ATP1A4: 50 -CCCAGCAG-30 (# 3 in the Roche Human probe library) and 18S ribosomal gene were designed using either the ProbeFinder web tool (www.roche-applied-science.com) or the Primer Express software program (Applied Biosystems). Primers and probe sequences are listed in TableS1. Each reaction was performed in technical and biological triplicates. Real-time quantitative RT-PCR was performed as previously described [24]. 3. Results and discussion 3.1. Characterization of the porcine ATP1A4 cDNA Porcine testis RNA and ATP1A4 specific oligonucleotide primers was used to perform RT-PCR cloning of the cDNA representing the entire ATP1A4 open reading frame. The amplicon was sequenced and homology search confirmed the identity of the porcine ATP1A4 cDNA. The RT-PCR cloning identified two porcine ATP1A4 cDNAs.
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Both were 3140 bp in length with the translation start at nucleotide 45 and the TGA stop codon at nucleotide 3135. The two identified ATP1A4 cDNAs, with a calculated G þ C content of 54%, encode proteins with an estimated molecular mass of 114 kDa. The porcine ATP1A4 cDNAs display highly homology (86%) with the human counterpart throughout the coding sequence. The ATP1A4/Na,KATPase a4 proteins, deduced from the isolated cDNAs, differ in only one amino acid. One quantitatively dominating isoform is found with a methionine residue at amino acid position 888 and another rare isoform with a glycine residue at the same position (Fig. 1). Amino acid sequence similarity between porcine Na,K-ATPase a4 protein and its human, bovine, mouse and rat counterparts was examined by the Clustal method (Fig. 1). The encoded porcine Na,K-ATPase a4 protein and other Na,K-ATPase a4 proteins exhibit significant sequence identity (82e90%) with highest similarity with the bovine sequence. The alignment showed that the human ATP1A4/Na,K-ATPase a4 protein is one amino acid shorter than the
Fig. 1. Amino acid alignment of Na⁺/K⁺-ATPase a4 sequences from pig (MG587082), cow, (NM_001144103) human (NM_144699), rat (NM_022848) and mouse (NM_013734). Sequence alignment was performed with the Clustal W program. The numbers in the right margin represent the positions of the amino acids in the aligned protein sequences. Complete identity at amino acid positions among all sequences are indicated by asterisks. The E1-E2 ATPase P site is shown as a boxed sequence. Overlined blue bars indicate the deduced transmembrane domains TM1-TM10. The amino acid at position 888 affected by a SNP identified in the porcine ATP1A4 sequence is indicated by a bold and underlined letter. A synonymous SNP is indicated by underling. The abbreviations for species names used are: Bt, Bos taurus; Ss, Sus scrofa; Hs, Homo sapiens; Rn, Rattus norvegicus; Mm, Mus musculus.
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porcine a4 protein. As shown in Fig. 1, the porcine Na,K-ATPase a4 protein shares common structures and functional elements with the human ATP1A4/Na,K-a4 protein molecule. Very high amino acid homology is observed in the in the carboxy-terminal end of the Na,K-ATPase a4 protein. The sequence in the very amino terminal is much more variable. The sequence of the E1-E2 ATPase P site, shown as a boxed sequence in Fig. 1, is completely conserved between the five species analyzed. In addition, there is a high sequence similarity within the ten transmembrane sequences (TM1 - TM10) as shown in the alignment. Especially transmembrane domains 6 and 7 show complete homology by comparison of the a4 sequences form the five species. The a4 polypeptide is the most divergent of the Na,K-ATPase a subunits in comparison with a1, a2 and a3 [19,21]. In addition, when comparing the individual a polypeptides between species the highest divergence is found for the a4 subunit. A phylogenetic analysis with ten different species demonstrated that the porcine Na⁺/K⁺-ATPase polypeptide a4 is closely related to other mammalian counterparts (Fig.S1). As expected, the closest relatives are the cow and camel, both members of the mammalian order Artiodactyla, and the two whale species. 3.2. Genomic organization of the porcine ATP1A4 gene The genomic organization, with the introneexon structure, of the porcine ATP1A4 gene was established by a blast search in the Pig Genome v.10.2 sequence database using the porcine ATP1A4 cDNA sequence (GenBank ID: MG587082). The blast search identified a 46 kb sequence covering the entire ATP1A4 gene. The porcine ATP1A4 gene is localized on chromosome 4. The human counterpart was mapped to chromosome 1q23.2. This localization of the ATP1A4 gene in the two species genomes is in agreement with the synteny described earlier [25]. The porcine ATP1A2 gene is localized on the same chromosome 4: 90013253e90649588. The mouse ATP1A4 gene is located proximal to ATP1A2 on the mouse chromosome 1 region, which corresponds to human 1q23 [26]. The intron-exon structure of ATP1A4 is illustrated in Table 1. Exonintron boundaries were determined by alignment of the ATP1A4 cDNA with ATP1A4 sequences identified in the published pig genome Sscrofa10.2. The porcine ATP1A4 gene is organized in 22 exons with sizes from 60 to 269 nucleotides (Table 1). The identified splice acceptor and donor sites are in accordance with the consensus GT-AG rule (Table 1). All exons, except for exon 1, of the porcine and human ATP1A4 genes have identical length of coding sequence. The coding sequence of porcine exon 1 is three nucleotides longer than that of its human counterpart. The lengths of most introns of the porcine ATP1A4 gene were of similar size as in the human counterpart. However, introns 12, 14 and 15 were significantly longer in the porcine gene, all being approx. 3 times longer. 3.3. Examination of the 50 flanking region of the porcine ATP1A4 gene In order to investigate the putative promoter of the porcine ATP1A4 gene we cloned and sequenced a 1800 bp amplicon containing sequence upstream of the transcription start site (TSS). The putative ATP1A4 promoter sequence was analyzed for the presence of transcription factor binding sites using the computer-based MatInspector and TFSEARCH program (http://molsun1.cbrc.aist.go. jp/htbin/nph-tfsearch). Within the region - 490 to - 1800, recognition sites for several different transcription factors were found: SRY, GATA-1, HFH-2, GATA-2, AML-1a, MZF1 and, interestingly a CRE recognition sequence (GTCA) for the cyclic AMP response element modulator CREM (Fig.S2). The CRE recognition site is also reported for the mouse ATP1A4 promoter [26]. Rodova et al. [27], who examined a region of 1 kb upstream of the translation start
Table 1 Exon/intron structure of the porcine ATP1A4 gene. Human
Porcine Size (bp)
50 -sequence
30 -sequence
Size (bp)
Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Intron 3 Exon 4 Intron 4 Exon 5 Intron 5 Exon 6 Intron 6 Exon 7 Intron 7 Exon 8 Intron 8 Exon 9 Intron 9 Exon 10 Intron 10 Exon 11 Intron 11 Exon 12 Intron 12 Exon 13 Intron 13 Exon 14 Intron 14 Exon 15 Intron 15 Exon 16 Intron 16 Exon 17 Intron 17 Exon 18 Intron 18 Exon 19 Intron 19 Exon 20 Intron 20 Exon 21 Intron 21 Exon 22
150a (486b) 183 60 2346 204 1061 114 2623 135 480 118 5121 269 2292 199 236 110 174 135 4251 190 140 173 5175 137 392 151 926 169 3530 155 127 124 1523 146 3879 131 105 102 2939 92 224 29a
CCACCTGCC GTGAGAACG GATGATCAC GTGAGTTAA GGCCATAGC GTGAGTCCG CTGTACCTG GTAAGACCG CACGCTCTG GTGAGGGGA GTGGACAAC GTAAATATC GAACCGCCC GTGAGTAGC GTGTGTCTG GTGATGAGT GGAATTCGT GTGTCAGGG TGGGCGACG GTACAGAAC ATGTCCATC GTGAGGGGG GGTTCTGCT GTAAACATC GTGATTATG GTGAGACCC GGACGTCAA GTAAGGGAG GGAGCCATT GTGAGGAAG GCCGCCTGA GTGAGGCCC GTCCCTGCT GTGCGGCCA GCATGATCC GTGAGTGAA ACCTATGAG GTGAGCGCC AAATAAGAT GTGAGTAAG GATAACCTG GTATGCTCC GCTGGCTGG
TGGTCCTG TCCCCTCAG CTGAACAAG TCCACACAG AAAGATAAT TCCCCCCAG GTCCCTCAG TTTCCACAG GGATGTAAG CCATCCCAG GTGTGGAAG CACCCACAG ACCGTCACG ATGCCTTAG AACAGACTG TCTCCTTAG ATCTCTAAG TCCCTGCAG AAGTACCAG TCTCCCTAG GCGTGCTAG CCTCTCCAG GGGATTAAG CCAACCTAG CAATGCCAG TTCCTTCAG CAGAGGCTG TTCACCCAG TGGAGGAGG CCTCCCCAG ACCGACATG CTTCTCCAG GACAGATTG CACTCACAG CAGCAGTGG CTCCCCCAG GGGCATGAA TCTTCTCAG CCCACTCAA TCTCTCCAG ATCCTGGCG CTTGCACAG TGTGACGCT
147a (618b) 977 60 1820 204 796 114 2843 135 272 118 4629 269 2103 199 241 110 200 135 3838 190 144 173 1823 137 393 151 317 169 1.344 155 232 124 916 146 4019 131 115 102 4244 92 303 29 (307b)
a b
Coding sequence. Untranslated þ coding sequence.
codon, studied the transcriptional control of the human ATP1A4 gene. They revealed that the transcription factor CREMtau binds to the CRE sequences and acts together with cAMP as a regulator of ATP1A4 expression [27]. Potential binding sites for the transcription factors AP-1, AP-4, GATA-3, NF-Y, MYOD, NF1 and NFAT are identified in the human and mouse ATP1A4 promoters [8]. Hence, some of these factors seem to be conserved between the three compared species. Among the binding sites identified in the porcine promoter (Fig.S2), GATA and NF binding sequences are also found in the human promoter. The computer analysis also revealed a two potential TATA boxes in the 50 -flanking sequence of porcine ATP1A4 (pos. - 490 to - 498 and - 1161 to - 1167), and two CAAT boxes at positions - 91 and - 714 upstream of the TSS (Fig.S2). Neither the human nor the mouse ATP1A4 promoters contain a TATA box sequence [28]. However, a CAAT sequence has been identified in these promoters [8]. A testis-specific exon was observed in the pituitary adenylate cyclase-activating polypeptide, PACAP, a pleiotropic neuropeptide expressed in brain and testis [29]. The potential promoter driving expression of the testis-specific exon contains a TATA box and recognition sequences for the transcription factors GATA-1, GATA-2, GATA-3, SRY and AML-1a [29]. These sites, except for GATA3, are also found in the ATP1A4 promoter
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indicating that they are contributing to the testis-specific expression. Two tandemly arranged AML-1a binding sequence element in the PACAP promoter is suggested to be part of a minimal key region for the testis-specific promoter activity. One of the AML-1a sequences, TGGGT, found in the human PACAP promoter has 100% sequence identity with that found in the porcine ATP1A4 promoter. 3.4. SNP detection By the RT-PCR cloning we identified two different ATP1A4 cDNA clones differing in only nucleotide in exon 18. To perform a more detailed investigation of this genetic variation in the porcine ATP1A4 gene we PCR cloned and sequenced exon 18 of ATP1A4. A panel consisting of 14 unrelated boars was included in the SNP analysis of exon 18. The PCR amplicons had the expected size of 269 bp, and were subjected to sequence analysis. Sequencing results revealed two single-nucleotide polymorphisms (SNPs) within exon 18. One was a missense G/A SNP, shown in Fig. 2, (nucleotide position 2706 in GenBank ID: MG587082). This non-synonymous A/G SNP results in a substitution of a methionine to a glycine residue (M888G) in the porcine Na,K-ATPase a4 protein sequence. As the genotype G/G was only identified in one boar, boar C, among 14 different boars examined, the allele A/A genotype is predominantly more frequent than the G/G genotype. The amino acid at position 888 in the a4 protein is localized between two transmembrane domains TM7 and TM8 as shown in Fig. 1. The alignment also shows that this particular position is less conserved/more variable than the surrounding amino acid positions. Judged from a predicted three-dimensional structure of the a4 protein it is unlikely that the methionine to glycine substitution would have any deleterious consequences (pers. comm. Hanne Poulsen). A second, silent, C/T SNP was found in exon 18 at amino acid position Ala 881 (C2687T in GenBank ID: MG587082). No mutations in the human ATP1A4 have been reported.
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resulted in a dataset of 1926 and 1302 million reads for liver and occipital cortex, respectively. Reads were mapped and methylation levels for individual genes were determined as described [22]. The methylation status for the coding sequence of the ATP1A4 gene (3679 bp) was determined and the analysis detected 13,555 methylated CpG reads in the occipital cortex out of 16,811 reads, resulting in a methylation degree of 80% (Table 2). In the liver tissue, 17,705 methylated reads in a total of 25,453 reads, results in a methylation degree of 70%. In conclusion, the methylation degree is very high in both brain tissue and liver. Comparison of the very high methylation values for both liver and occipital cortex with ATP1A4 mRNA expression (Fig.S3) reveals no inverse correlation between methylation and expression. The tissue with the highest expression, occipital cortex, also has the highest methylation degree. This suggests that methylation of the coding region is doubtful as a determinant for regulation of ATP1A4 expression. Unfortunately, we did not include the testis in this methylation analysis. Recent studies by Kumar et al. [30] revealed that methylation of both the ATP1A4 promoter and an intragenic region of the ATP1A4 gene very likely is a determinant of its testis-specific expression. Both methylation-dependent and independent regulatory regions in the ATP1A4 were identified which may influence the testis-specific expression of the gene. Kumar et al. [30] have examined the methylation status of the mouse ATP1A4 gene in sperm cells and kidney. The methylation pattern of the promoter (900 nucleotides) and an intragenic CpG island was determined. A very high methylation level, 88%, was found in ATP1A4 promoter in both sperm cells and kidney. In contrast, a low level of methylation, around 17%, was detected in the intragenic region in sperm cell, whereas a high level was found in kidney. It was concluded from the study that both the promoter and the intragenic CpG island have methylation-dependent and methylation-independent effects on ATP1A4 expression [31]. 3.6. Spatial expression of the ATP1A4 transcript
3.5. Methylation status of the ATP1A4 gene As a part of the characterization of the porcine ATP1A4 gene, we determined the methylation status of the ATP1A4 coding sequence in two porcine tissues: occipital cortex and liver. The data was retrieved from a global study resulting from bisulfite sequencing on an Illumina HiSeq platform [22]. The global bisulfite sequencing
The expression level of the ATP1A4 transcript was determined in multiple pig organs and tissues using quantitative real-time RTPCR. Among the fourteen tissues analyzed, expression of ATP1A4 mRNA was only detected in testis (Fig. 3). The spatial expression of ATP1A4 mRNA was also determined by sequencing of RNA from ten different organs and tissues (Fig.S3). A high level of ATP1A4 mRNA
Fig. 2. SNP analysis of ATP1A4 exon 18. The presented electropherogram of a selected nucleotide sequence identifies a G > A transition within exon 18 in boar C. The partial nucleotide sequence of exon 18 and the amino acid of the affected codon (Met > Gly) and are shown. In addition, a synonymous C/T SNP is shown in boar C. Top: section of sequence representing genotype G/G. Bottom: section of sequence with a A/A genotype.
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Table 2 Methylation status of the coding sequence of the porcine ATP1A4 gene in liver and brain (Sus scrofa 10.2). Gene
Length (bp)
Chr
Start
End
Tissue
Methylated reads
Total reads
Methylation percentage (%)
ATP1A4
3679
4
98232544
98273950
Brain Liver
13555 17705
16811 25453
80.6 69.6
expression was detected in cerebellum, occipital cortex, hypothalamus and skeletal muscle (longissiumus dorsii), whereas a lower level of transcript was found in and spleen. No ATP1A4 transcript was expressed in frontal cortex, heart, kidney, liver and lung (Fig.S3). Unfortunately, testis was not included in this experiment. The ATP1A4 gene is specifically expressed in human and mouse testis and sperm cells [5e7]. Thus, the observed differential pig ATP1A4 mRNA expression in various organs and tissues is consistent with the results for human and mouse counterparts. More than 50 years ago Uesugi and Yamazue [31] demonstrated presence of Na,K-ATPase activity in boar epididymal spermatozoa. Several reports showing Na,K-ATPase a4 expression in testis and sperm cells [5,32]. Blanco [33] measured the functional expression of the Na⁺/ K⁺-ATPase isoform a4 in rat testis and found expression as early as seven days after birth. Na⁺/K⁺-ATPase isoform a4 activity was detected in spermatogonia, spermatocytes, spermatids and spermatozoa [33]. The spatial and temporal expression of the a4 subunit has also been investigated by expression of the GFP structural gene fused to an ATP1A4 promoter in transgenic mice [7]. This study confirmed the testis-specific expression of a4. The regulation of ATP1A4 expression seem to be rather complex. Recently, Zhou et al. [34] demonstrated down-regulation of ATP1A4 a4 protein in EMC10 knockout mice. EMC10 encodes a subunit of endoplasmic reticulum (ER) membrane complex, and elimination of its function leads to ion imbalance in spermatozoa. Interestingly, the EMC deletion leads to loss of ATP1A4 expression in sperm cells. In an earlier study Jimenez et al. [10] reported that ATP1A4 knockout mice are completely sterile. In addition, the sperm cells of EMC10 knockout mice and ATP1A4 knockout mice share similar phenotype. 4. Conclusion Our study provides valuable molecular information about the porcine ATP1A4 gene and the encoded Na⁺/K⁺-ATPase a4 subunit. The ATP1A4 cDNA was cloned from pig testis, sequenced and characterized. The genomic organization of the porcine ATP1A4 was
very similar to that of the human homolog. In addition, some sequences in the ATP1A4 promoter, including CRE recognition sites, are conserved between these two species. Two SNPs were identified in the pig ATP1A4 sequence of which one is non-synonymous and result in an amino acid substitution of a methionine to a glycine at amino acid position 888. It is not known whether this change affects the fertility of the boar. The spatial expression profile, with a testis and sperm-specific expression for the ATP1A4 transcript, was similar to that described for human, mice and rat. As demonstrated in our study, there are very similar molecular properties between human and pig ATP1A4 and the encoded a4 isoform. This supports the idea that the pig could serve as a potential model to study infertility associated with dysregulation of this gene. Infertility is a growing problem and involves approx. one in seven couples wishing to establish a family. Often, the diagnosis is simply unexplained, while in other cases a variety of reasons have been identified including lack of ovulation, mechanical stoppage, sperm deficiencies and dysfunctions and parental age. Male infertility in humans is a growing problem and around 7% of men suffer from male factor infertility. In a fraction of 25% of infertile men, the etiology remains unknown. In some cases gene-gene and geneenvironment, interactions may be causative of male infertility. A genetic approach that involves single candidate genes and incorporates biological information from an animal model e.g. the pig, is likely to be valuable in explaining the genetic basis of male fertility. Genes playing essential roles in sperm function, such as ATP1A4, can be studied in the model organism. CRISPR/Cas9 technology could be utilized to produce inactivating mutations in the ATP1A4 gene of domestic pigs to study. Elimination of ATP1A4 expression in a pig model might contribute to increased understanding of sperm function. In future prospect, this could contribute to a better diagnosis and development of treatments of male infertility. Conflicts of interest The authors declare no conflict of interest. Acknowledgements The authors wish to thank Bente Flügel, Hanne Jørgensen and Mahesha Perera for excellent technical assistance. The authors also acknowledge associate professor Hanne Poulsen for the valuable discussions on a4 protein structure and conformation. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2019.01.003. References
Fig. 3. Spatial expression of porcine ATP1A4 mRNA. Relative expression of ATP1A4 in fourteen different porcine organs and tissues. TES, testis; LUN, lung; HEA, heart; BLA, bladder; KID, kidney; HYP, hypothalamus, BST, brain stem; FCO, frontal cortex; CBE, cerebellum; PCO, parietal cortex; OCC, occipital cortex; TEMPL, temporal lobe; SPC, spinal cord; SPL, spleen. The 18S ribosomal subunit was used as a reference gene.
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Research paper
Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoform a4 Knud Larsen*, Carina Henriksen, Kaja Kjær Kristensen, Jamal Momeni, Leila Farajzadeh Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers All e 3, DK-8000, Aarhus C, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 August 2018 Accepted 5 January 2019 Available online 8 January 2019
Naþ/Kþ-ATPase is responsible for maintaining electrochemical gradients of Naþ and Kþ, which is essential for a variety of cellular functions including neuronal activity. The a-subunit of the Naþ/KþATPase is composed of four different polypeptides (a1ea4) encoded by different genes. Na,K-ATPase a4, encoded by the ATP1A4 gene, is expressed in testis and in male germ cells of humans, rats and mice. The a4 polypeptide has an important role in sperm motility, and is essential for male fertility. Here we present the RT-PCR cloning and characterization of the porcine ATP1A4 cDNA coding for Na⁺/ K⁺-ATPase polypeptide a4. The Na⁺/K⁺-ATPase polypeptide a4, consisting of 1030 amino acids, displays a high homology with its human counterpart (86%). Phylogenetic analysis demonstrated that porcine Na⁺/ K⁺-ATPase polypeptide a4 is closely related to other mammalian counterparts. In addition, the genomic structure of the porcine ATP1A4 gene was determined, and the intron-exon organization was found to be similar to that of the human ATP1A4 gene. The promoter sequence for the porcine ATP1A4 gene was also identified. Investigation of the genetic variation in the porcine ATP1A4 gene revealed a missense A/G SNP in exon 18. This A/G polymorphism results in a substitution of a methionine to a glycine residue (M888G). A very high overall DNA methylation rate of the ATP1A4 gene, 70e80%, was observed in both brain and liver. Expression analysis demonstrated that the porcine ATP1A4 gene is predominantly expressed in testis. The sequence of the porcine ATP1A4 cDNA encoding the Na⁺/K⁺-ATPase a4 protein has been submitted to GenBank under the accession number GenBank Accession No. MG587082. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: ATP1A4 Methylation Na⁺/K⁺-ATPase Pig SNP Testis
1. Introduction Naþ,Kþ-ATPase, initially described by Skou in 1957 [1], is an integral membrane protein which is responsible for establishing and maintaining electrochemical gradients of Naþ and Kþ across the plasma membrane [2,3]. This enzyme catalyzes the hydrolysis of ATP coupled with the exchange of sodium and potassium ions across the plasma membrane. The gradients created hereby are essential for electrical excitability of nerves and muscles, for sodium-coupled transport of various organics and inorganics molecules and for osmoregulation. The Naþ,Kþ-ATPase is composed of two subunits, a which is the catalytic subunit, a glycosylated b
* Corresponding author. E-mail addresses: [email protected] (K. Larsen), Carina.Henriksen@mbg. au.dk (C. Henriksen), [email protected] (K.K. Kristensen), Jamal.Momeni@mbg. au.dk (J. Momeni), [email protected] (L. Farajzadeh).
subunit and in many tissues a regulatory subunit belonging to the FXYD protein family. The a subunit of Naþ,Kþ-ATPase is encoded by four different genes, ATP1A1, ATP1A2, ATP1A3 and ATP1A4 [4,5]. The ATP1A4 gene encodes the Naþ,Kþ-ATPase a4 subunit. The human ATP1A4 gene, composed of 22 exons and spanning approx. 35 kb, is located on chromosome 1q23.2. The ATP1A4 gene is specifically expressed in testis and sperm cells [6,7]. However, low levels of ATP1A4 mRNA can be detected in human and mouse skeletal muscle [8]. Naþ,Kþ-ATPase a4 protein is expressed in mature sperm. Three fourth of total Na,K-ATPase activity in sperm cells is attributed to the a4 polypeptide and the remaining 25% is attributed to the a1 polypeptide [9]. The Na,KATPase a4 polypeptide is essential for sperm fertility and loss of a4 leads to complete male sterility in mice [10]. Sperm cells from a4 knockout mice have unaffected viability but are unable to fertilize eggs in vitro [10]. The loss of a4 results in reduced sperm motility, depolarization of the membrane potential and increased intracellular sodium [10]. Overexpression of the a4 polypeptide enhances
https://doi.org/10.1016/j.biochi.2019.01.003 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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sperm motility in transgenic mice [11,12]. Recently, is was shown in cattle that Naþ/KþATPase regulates sperm capacitation through a mechanism involving kinases, protein kinases A and C, and redistribution of ATP1A4 [13]. Inhibition of Naþ/KþATPase induces tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. In Holstein bulls ATP1A4 content and activity, ROS, F-actin and calcium are parameters significantly correlated with fertility [14]. The a4 polypeptide is responsible for sperm motility. Inhibition of a4 with ouabain inhibits rat sperm cell characteristics including total and progressive motility and other kinetic parameters [15,16]. Similar inhibitory effects of ouabain was seen with human sperm cells [17]. Decrease in Na,K-ATPase activity and sperm motility was observed in sperm cells from men with a normal sperm profile treated with ouabain [17]. In oligoasthenospermic males the sperm motility was almost completely lost by addition of ouabain [17]. The role of ATP1A4 in regulation of sperm motility and capacitation in cattle has also been investigated [14,18]. Capacitation is a prerequisite for fertilization in mammals. Mammalian sperm cells undergo a series of structural and functional modifications to render them competent to fertilize an oocyte. Disease causing mutations have been identified in Na,KATPase alpha polypeptides a1, a2 and a3, resulting in diseases like hypertension, familial hemiplegic migraine, rapid-onset dystonia Parkinsonism and alternating hemiplegia of childhood [19]. The Na,K-ATPase a4 polypeptide is essential for sperm function, and therefore mutations in the ATP1A4 gene are very likely to affect the male bearers' fertility. Animal models for male infertility including genetically modified and chemical-induced point mutant mice have aided the identification of infertility causing mutations. Studies with mouse models have identified more than 400 genes essential for male fertility [20]. However, thousands of genes are estimated to be involved in the regulation of the complex process of male fertility, so many genes remain to be characterized. With a view to future use of pig as an animal model in studies of human diseases caused by Naþ/Kþ-ATPase mutations, we have cloned and characterized the ATP1A4 gene encoding the Naþ/Kþ-ATPase a4 polypeptide. This includes the determination of its genomic organization and chromosomal localization, and expression pattern. 2. Materials and methods 2.1. Ethics statement The pigs used in the present study were housed and used in compliance with European Community animal care guidelines. The experimental procedures were approved by the National Ethical Committee in Denmark (Approval No. 2010/561e1891). 2.2. Animals One year old Danish Landrace pigs were used in this study. Pigs were sacrificed by an injection with 30 mg/kg Pentobarbital (Vipidan, Denmark). 2.3. Cloning of the porcine ATP1A4 cDNA The cDNA fragment encoding ATP1A4 was amplified by PCR using a testis cDNA and ATP1A4 specific primers derived from genomic sequences. The various pig tissues used for cloning and expression analysis were dissected and pulverized in liquid nitrogen. Total RNA was isolated by the RNeasy method (Qiagen) and RNA integrity was verified by ethidium bromide staining of 1% agarose gels. Synthesis of cDNA was carried out with 5 mg of total RNA isolated from pig testis and other tissues and organs using
SuperScript II RNase H reverse transcriptase (Invitrogen) and oligo(dT)12e18 primers according to the manufacturer's recommendations. For the molecular cloning of the ATP1A4 cDNA, RNA was isolated from testis. The PCR reaction mix contained: 2.5 ml cDNA, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 mM of each primer ATP1A4F and ATP1A4-R (Table S1) and 1 U Phusion DNA polymerase (Finnzymes), in a total volume of 25 ml. The PCR conditions was as follows: 95 C for 2 min, 10 touchdown cycles of 95 C for 20 s, 60 C for 30 s, 72 C for 45 s, followed by 25 cycles of 95 C for 20 s, 55 C for 30 s, 72 C for 45 s and finally an elongation at 72 C for 5 min. A PCR product of approx. 3000 bp was visualized and isolated from an ethidium bromide stained 1% agarose gel. The recovered cDNA amplicon was cloned directly into the pCR TOPO 2.1 vector (Invitrogen) and sequenced in both directions. DNA sequencing was performed, as previously described [21]. 2.4. Genomic organization and promoter of ATP1A4 To identify the porcine ATP1A4 gene, the cloned ATP1A4 cDNA sequence was used in a blast search in Ensembl pig genome data (Sus scrofa 11.1; https://www.ensembl.org/Sus_scrofa/Info/Index). The prediction software Genscan (http://genes.mit.edu/GENSCAN. html) and the Expasy translate tool (https://web.expasy.org/ translate/) was used to identify the exon-intron organization. 2.5. SNP discovery Fourteen boars were examined for SNPs in exon 18 of the ATP1A4 gene. An oligonucleotide primer set, ATP1A4-EX18F and ATP1A4-EX18R, was designed to cover the individual exon 18 (Table S1). A 269 bp fragment was amplified using 0.5 mM primer, 0.2 mM dNTP, 1 Phusion buffer, 2 U Phusion DNA Polymerase (Finnzymes), and 2 ml genomic cDNA in a total volume of 10 ml. The cycling conditions employed were: denaturation at 98 C for 2 min denaturation, 30 cycles of 98 C for 10 s, 60 C for 30 s and 72 C for 30 s, with a final extension of 72 C for 10 min. The AcycloPrime-FP SNP detection kit (Perkin Elmer) was used to remove residual primers and dNTP from the PCR fragments. Six microliters PCR product, 1 PCR Clean-Up dilution buffer (Perkin Elmer), and 0.05 ml PCR Clean-up reagent (Perkin Elmer) were mixed. The reaction was incubated at 37 C for 90 min and 80 C for 15 min. The products were then sequenced using the BigDye terminator v3.1 Cycle Sequencing Kit and the SNP primer set for the detection of possible SNPs. The obtained sequences were analyzed in Consed. 2.6. Methylation status of ATP1A4 The methylation status of ATP1A4 was determined by library preparation, sequencing, mapping and analysis as described [22]. For determination of the methylation percentage of specific genes or sequences from our methylome data file, we used Tabix [23]. 2.7. Expression analysis Real time RT-PCR analysis of porcine ATP1A4 mRNA expression was performed on cDNA synthesized from total RNA isolated from the following porcine organs: testis, lung, spleen, kidney, cerebellum, parietal cortex, bladder, brain stem, frontal cortex, heart, spinal cord, occipital cortex, temporal lobe and hypothalamus. Three separate tissues were applied for each type of brain tissue. Tissue samples were collected from three Danish Landrace pigs, aged 1e2 years and weighing 125e200 kg ATP1A4-specific primers and 18S-specific primers were designed to amplify a DNA sequence of exon 18 of the porcine ATP1A4 gene and the 18S ribosomal gene using the EXIQON Human ProbeLibrary. The ATP1A4 primers
K. Larsen et al. / Biochimie 158 (2019) 149e155
produce an amplicon of 60 bp by PCR amplification. Probes for ATP1A4: 50 -CCCAGCAG-30 (# 3 in the Roche Human probe library) and 18S ribosomal gene were designed using either the ProbeFinder web tool (www.roche-applied-science.com) or the Primer Express software program (Applied Biosystems). Primers and probe sequences are listed in TableS1. Each reaction was performed in technical and biological triplicates. Real-time quantitative RT-PCR was performed as previously described [24]. 3. Results and discussion 3.1. Characterization of the porcine ATP1A4 cDNA Porcine testis RNA and ATP1A4 specific oligonucleotide primers was used to perform RT-PCR cloning of the cDNA representing the entire ATP1A4 open reading frame. The amplicon was sequenced and homology search confirmed the identity of the porcine ATP1A4 cDNA. The RT-PCR cloning identified two porcine ATP1A4 cDNAs.
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Both were 3140 bp in length with the translation start at nucleotide 45 and the TGA stop codon at nucleotide 3135. The two identified ATP1A4 cDNAs, with a calculated G þ C content of 54%, encode proteins with an estimated molecular mass of 114 kDa. The porcine ATP1A4 cDNAs display highly homology (86%) with the human counterpart throughout the coding sequence. The ATP1A4/Na,KATPase a4 proteins, deduced from the isolated cDNAs, differ in only one amino acid. One quantitatively dominating isoform is found with a methionine residue at amino acid position 888 and another rare isoform with a glycine residue at the same position (Fig. 1). Amino acid sequence similarity between porcine Na,K-ATPase a4 protein and its human, bovine, mouse and rat counterparts was examined by the Clustal method (Fig. 1). The encoded porcine Na,K-ATPase a4 protein and other Na,K-ATPase a4 proteins exhibit significant sequence identity (82e90%) with highest similarity with the bovine sequence. The alignment showed that the human ATP1A4/Na,K-ATPase a4 protein is one amino acid shorter than the
Fig. 1. Amino acid alignment of Na⁺/K⁺-ATPase a4 sequences from pig (MG587082), cow, (NM_001144103) human (NM_144699), rat (NM_022848) and mouse (NM_013734). Sequence alignment was performed with the Clustal W program. The numbers in the right margin represent the positions of the amino acids in the aligned protein sequences. Complete identity at amino acid positions among all sequences are indicated by asterisks. The E1-E2 ATPase P site is shown as a boxed sequence. Overlined blue bars indicate the deduced transmembrane domains TM1-TM10. The amino acid at position 888 affected by a SNP identified in the porcine ATP1A4 sequence is indicated by a bold and underlined letter. A synonymous SNP is indicated by underling. The abbreviations for species names used are: Bt, Bos taurus; Ss, Sus scrofa; Hs, Homo sapiens; Rn, Rattus norvegicus; Mm, Mus musculus.
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porcine a4 protein. As shown in Fig. 1, the porcine Na,K-ATPase a4 protein shares common structures and functional elements with the human ATP1A4/Na,K-a4 protein molecule. Very high amino acid homology is observed in the in the carboxy-terminal end of the Na,K-ATPase a4 protein. The sequence in the very amino terminal is much more variable. The sequence of the E1-E2 ATPase P site, shown as a boxed sequence in Fig. 1, is completely conserved between the five species analyzed. In addition, there is a high sequence similarity within the ten transmembrane sequences (TM1 - TM10) as shown in the alignment. Especially transmembrane domains 6 and 7 show complete homology by comparison of the a4 sequences form the five species. The a4 polypeptide is the most divergent of the Na,K-ATPase a subunits in comparison with a1, a2 and a3 [19,21]. In addition, when comparing the individual a polypeptides between species the highest divergence is found for the a4 subunit. A phylogenetic analysis with ten different species demonstrated that the porcine Na⁺/K⁺-ATPase polypeptide a4 is closely related to other mammalian counterparts (Fig.S1). As expected, the closest relatives are the cow and camel, both members of the mammalian order Artiodactyla, and the two whale species. 3.2. Genomic organization of the porcine ATP1A4 gene The genomic organization, with the introneexon structure, of the porcine ATP1A4 gene was established by a blast search in the Pig Genome v.10.2 sequence database using the porcine ATP1A4 cDNA sequence (GenBank ID: MG587082). The blast search identified a 46 kb sequence covering the entire ATP1A4 gene. The porcine ATP1A4 gene is localized on chromosome 4. The human counterpart was mapped to chromosome 1q23.2. This localization of the ATP1A4 gene in the two species genomes is in agreement with the synteny described earlier [25]. The porcine ATP1A2 gene is localized on the same chromosome 4: 90013253e90649588. The mouse ATP1A4 gene is located proximal to ATP1A2 on the mouse chromosome 1 region, which corresponds to human 1q23 [26]. The intron-exon structure of ATP1A4 is illustrated in Table 1. Exonintron boundaries were determined by alignment of the ATP1A4 cDNA with ATP1A4 sequences identified in the published pig genome Sscrofa10.2. The porcine ATP1A4 gene is organized in 22 exons with sizes from 60 to 269 nucleotides (Table 1). The identified splice acceptor and donor sites are in accordance with the consensus GT-AG rule (Table 1). All exons, except for exon 1, of the porcine and human ATP1A4 genes have identical length of coding sequence. The coding sequence of porcine exon 1 is three nucleotides longer than that of its human counterpart. The lengths of most introns of the porcine ATP1A4 gene were of similar size as in the human counterpart. However, introns 12, 14 and 15 were significantly longer in the porcine gene, all being approx. 3 times longer. 3.3. Examination of the 50 flanking region of the porcine ATP1A4 gene In order to investigate the putative promoter of the porcine ATP1A4 gene we cloned and sequenced a 1800 bp amplicon containing sequence upstream of the transcription start site (TSS). The putative ATP1A4 promoter sequence was analyzed for the presence of transcription factor binding sites using the computer-based MatInspector and TFSEARCH program (http://molsun1.cbrc.aist.go. jp/htbin/nph-tfsearch). Within the region - 490 to - 1800, recognition sites for several different transcription factors were found: SRY, GATA-1, HFH-2, GATA-2, AML-1a, MZF1 and, interestingly a CRE recognition sequence (GTCA) for the cyclic AMP response element modulator CREM (Fig.S2). The CRE recognition site is also reported for the mouse ATP1A4 promoter [26]. Rodova et al. [27], who examined a region of 1 kb upstream of the translation start
Table 1 Exon/intron structure of the porcine ATP1A4 gene. Human
Porcine Size (bp)
50 -sequence
30 -sequence
Size (bp)
Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Intron 3 Exon 4 Intron 4 Exon 5 Intron 5 Exon 6 Intron 6 Exon 7 Intron 7 Exon 8 Intron 8 Exon 9 Intron 9 Exon 10 Intron 10 Exon 11 Intron 11 Exon 12 Intron 12 Exon 13 Intron 13 Exon 14 Intron 14 Exon 15 Intron 15 Exon 16 Intron 16 Exon 17 Intron 17 Exon 18 Intron 18 Exon 19 Intron 19 Exon 20 Intron 20 Exon 21 Intron 21 Exon 22
150a (486b) 183 60 2346 204 1061 114 2623 135 480 118 5121 269 2292 199 236 110 174 135 4251 190 140 173 5175 137 392 151 926 169 3530 155 127 124 1523 146 3879 131 105 102 2939 92 224 29a
CCACCTGCC GTGAGAACG GATGATCAC GTGAGTTAA GGCCATAGC GTGAGTCCG CTGTACCTG GTAAGACCG CACGCTCTG GTGAGGGGA GTGGACAAC GTAAATATC GAACCGCCC GTGAGTAGC GTGTGTCTG GTGATGAGT GGAATTCGT GTGTCAGGG TGGGCGACG GTACAGAAC ATGTCCATC GTGAGGGGG GGTTCTGCT GTAAACATC GTGATTATG GTGAGACCC GGACGTCAA GTAAGGGAG GGAGCCATT GTGAGGAAG GCCGCCTGA GTGAGGCCC GTCCCTGCT GTGCGGCCA GCATGATCC GTGAGTGAA ACCTATGAG GTGAGCGCC AAATAAGAT GTGAGTAAG GATAACCTG GTATGCTCC GCTGGCTGG
TGGTCCTG TCCCCTCAG CTGAACAAG TCCACACAG AAAGATAAT TCCCCCCAG GTCCCTCAG TTTCCACAG GGATGTAAG CCATCCCAG GTGTGGAAG CACCCACAG ACCGTCACG ATGCCTTAG AACAGACTG TCTCCTTAG ATCTCTAAG TCCCTGCAG AAGTACCAG TCTCCCTAG GCGTGCTAG CCTCTCCAG GGGATTAAG CCAACCTAG CAATGCCAG TTCCTTCAG CAGAGGCTG TTCACCCAG TGGAGGAGG CCTCCCCAG ACCGACATG CTTCTCCAG GACAGATTG CACTCACAG CAGCAGTGG CTCCCCCAG GGGCATGAA TCTTCTCAG CCCACTCAA TCTCTCCAG ATCCTGGCG CTTGCACAG TGTGACGCT
147a (618b) 977 60 1820 204 796 114 2843 135 272 118 4629 269 2103 199 241 110 200 135 3838 190 144 173 1823 137 393 151 317 169 1.344 155 232 124 916 146 4019 131 115 102 4244 92 303 29 (307b)
a b
Coding sequence. Untranslated þ coding sequence.
codon, studied the transcriptional control of the human ATP1A4 gene. They revealed that the transcription factor CREMtau binds to the CRE sequences and acts together with cAMP as a regulator of ATP1A4 expression [27]. Potential binding sites for the transcription factors AP-1, AP-4, GATA-3, NF-Y, MYOD, NF1 and NFAT are identified in the human and mouse ATP1A4 promoters [8]. Hence, some of these factors seem to be conserved between the three compared species. Among the binding sites identified in the porcine promoter (Fig.S2), GATA and NF binding sequences are also found in the human promoter. The computer analysis also revealed a two potential TATA boxes in the 50 -flanking sequence of porcine ATP1A4 (pos. - 490 to - 498 and - 1161 to - 1167), and two CAAT boxes at positions - 91 and - 714 upstream of the TSS (Fig.S2). Neither the human nor the mouse ATP1A4 promoters contain a TATA box sequence [28]. However, a CAAT sequence has been identified in these promoters [8]. A testis-specific exon was observed in the pituitary adenylate cyclase-activating polypeptide, PACAP, a pleiotropic neuropeptide expressed in brain and testis [29]. The potential promoter driving expression of the testis-specific exon contains a TATA box and recognition sequences for the transcription factors GATA-1, GATA-2, GATA-3, SRY and AML-1a [29]. These sites, except for GATA3, are also found in the ATP1A4 promoter
K. Larsen et al. / Biochimie 158 (2019) 149e155
indicating that they are contributing to the testis-specific expression. Two tandemly arranged AML-1a binding sequence element in the PACAP promoter is suggested to be part of a minimal key region for the testis-specific promoter activity. One of the AML-1a sequences, TGGGT, found in the human PACAP promoter has 100% sequence identity with that found in the porcine ATP1A4 promoter. 3.4. SNP detection By the RT-PCR cloning we identified two different ATP1A4 cDNA clones differing in only nucleotide in exon 18. To perform a more detailed investigation of this genetic variation in the porcine ATP1A4 gene we PCR cloned and sequenced exon 18 of ATP1A4. A panel consisting of 14 unrelated boars was included in the SNP analysis of exon 18. The PCR amplicons had the expected size of 269 bp, and were subjected to sequence analysis. Sequencing results revealed two single-nucleotide polymorphisms (SNPs) within exon 18. One was a missense G/A SNP, shown in Fig. 2, (nucleotide position 2706 in GenBank ID: MG587082). This non-synonymous A/G SNP results in a substitution of a methionine to a glycine residue (M888G) in the porcine Na,K-ATPase a4 protein sequence. As the genotype G/G was only identified in one boar, boar C, among 14 different boars examined, the allele A/A genotype is predominantly more frequent than the G/G genotype. The amino acid at position 888 in the a4 protein is localized between two transmembrane domains TM7 and TM8 as shown in Fig. 1. The alignment also shows that this particular position is less conserved/more variable than the surrounding amino acid positions. Judged from a predicted three-dimensional structure of the a4 protein it is unlikely that the methionine to glycine substitution would have any deleterious consequences (pers. comm. Hanne Poulsen). A second, silent, C/T SNP was found in exon 18 at amino acid position Ala 881 (C2687T in GenBank ID: MG587082). No mutations in the human ATP1A4 have been reported.
153
resulted in a dataset of 1926 and 1302 million reads for liver and occipital cortex, respectively. Reads were mapped and methylation levels for individual genes were determined as described [22]. The methylation status for the coding sequence of the ATP1A4 gene (3679 bp) was determined and the analysis detected 13,555 methylated CpG reads in the occipital cortex out of 16,811 reads, resulting in a methylation degree of 80% (Table 2). In the liver tissue, 17,705 methylated reads in a total of 25,453 reads, results in a methylation degree of 70%. In conclusion, the methylation degree is very high in both brain tissue and liver. Comparison of the very high methylation values for both liver and occipital cortex with ATP1A4 mRNA expression (Fig.S3) reveals no inverse correlation between methylation and expression. The tissue with the highest expression, occipital cortex, also has the highest methylation degree. This suggests that methylation of the coding region is doubtful as a determinant for regulation of ATP1A4 expression. Unfortunately, we did not include the testis in this methylation analysis. Recent studies by Kumar et al. [30] revealed that methylation of both the ATP1A4 promoter and an intragenic region of the ATP1A4 gene very likely is a determinant of its testis-specific expression. Both methylation-dependent and independent regulatory regions in the ATP1A4 were identified which may influence the testis-specific expression of the gene. Kumar et al. [30] have examined the methylation status of the mouse ATP1A4 gene in sperm cells and kidney. The methylation pattern of the promoter (900 nucleotides) and an intragenic CpG island was determined. A very high methylation level, 88%, was found in ATP1A4 promoter in both sperm cells and kidney. In contrast, a low level of methylation, around 17%, was detected in the intragenic region in sperm cell, whereas a high level was found in kidney. It was concluded from the study that both the promoter and the intragenic CpG island have methylation-dependent and methylation-independent effects on ATP1A4 expression [31]. 3.6. Spatial expression of the ATP1A4 transcript
3.5. Methylation status of the ATP1A4 gene As a part of the characterization of the porcine ATP1A4 gene, we determined the methylation status of the ATP1A4 coding sequence in two porcine tissues: occipital cortex and liver. The data was retrieved from a global study resulting from bisulfite sequencing on an Illumina HiSeq platform [22]. The global bisulfite sequencing
The expression level of the ATP1A4 transcript was determined in multiple pig organs and tissues using quantitative real-time RTPCR. Among the fourteen tissues analyzed, expression of ATP1A4 mRNA was only detected in testis (Fig. 3). The spatial expression of ATP1A4 mRNA was also determined by sequencing of RNA from ten different organs and tissues (Fig.S3). A high level of ATP1A4 mRNA
Fig. 2. SNP analysis of ATP1A4 exon 18. The presented electropherogram of a selected nucleotide sequence identifies a G > A transition within exon 18 in boar C. The partial nucleotide sequence of exon 18 and the amino acid of the affected codon (Met > Gly) and are shown. In addition, a synonymous C/T SNP is shown in boar C. Top: section of sequence representing genotype G/G. Bottom: section of sequence with a A/A genotype.
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K. Larsen et al. / Biochimie 158 (2019) 149e155
Table 2 Methylation status of the coding sequence of the porcine ATP1A4 gene in liver and brain (Sus scrofa 10.2). Gene
Length (bp)
Chr
Start
End
Tissue
Methylated reads
Total reads
Methylation percentage (%)
ATP1A4
3679
4
98232544
98273950
Brain Liver
13555 17705
16811 25453
80.6 69.6
expression was detected in cerebellum, occipital cortex, hypothalamus and skeletal muscle (longissiumus dorsii), whereas a lower level of transcript was found in and spleen. No ATP1A4 transcript was expressed in frontal cortex, heart, kidney, liver and lung (Fig.S3). Unfortunately, testis was not included in this experiment. The ATP1A4 gene is specifically expressed in human and mouse testis and sperm cells [5e7]. Thus, the observed differential pig ATP1A4 mRNA expression in various organs and tissues is consistent with the results for human and mouse counterparts. More than 50 years ago Uesugi and Yamazue [31] demonstrated presence of Na,K-ATPase activity in boar epididymal spermatozoa. Several reports showing Na,K-ATPase a4 expression in testis and sperm cells [5,32]. Blanco [33] measured the functional expression of the Na⁺/ K⁺-ATPase isoform a4 in rat testis and found expression as early as seven days after birth. Na⁺/K⁺-ATPase isoform a4 activity was detected in spermatogonia, spermatocytes, spermatids and spermatozoa [33]. The spatial and temporal expression of the a4 subunit has also been investigated by expression of the GFP structural gene fused to an ATP1A4 promoter in transgenic mice [7]. This study confirmed the testis-specific expression of a4. The regulation of ATP1A4 expression seem to be rather complex. Recently, Zhou et al. [34] demonstrated down-regulation of ATP1A4 a4 protein in EMC10 knockout mice. EMC10 encodes a subunit of endoplasmic reticulum (ER) membrane complex, and elimination of its function leads to ion imbalance in spermatozoa. Interestingly, the EMC deletion leads to loss of ATP1A4 expression in sperm cells. In an earlier study Jimenez et al. [10] reported that ATP1A4 knockout mice are completely sterile. In addition, the sperm cells of EMC10 knockout mice and ATP1A4 knockout mice share similar phenotype. 4. Conclusion Our study provides valuable molecular information about the porcine ATP1A4 gene and the encoded Na⁺/K⁺-ATPase a4 subunit. The ATP1A4 cDNA was cloned from pig testis, sequenced and characterized. The genomic organization of the porcine ATP1A4 was
very similar to that of the human homolog. In addition, some sequences in the ATP1A4 promoter, including CRE recognition sites, are conserved between these two species. Two SNPs were identified in the pig ATP1A4 sequence of which one is non-synonymous and result in an amino acid substitution of a methionine to a glycine at amino acid position 888. It is not known whether this change affects the fertility of the boar. The spatial expression profile, with a testis and sperm-specific expression for the ATP1A4 transcript, was similar to that described for human, mice and rat. As demonstrated in our study, there are very similar molecular properties between human and pig ATP1A4 and the encoded a4 isoform. This supports the idea that the pig could serve as a potential model to study infertility associated with dysregulation of this gene. Infertility is a growing problem and involves approx. one in seven couples wishing to establish a family. Often, the diagnosis is simply unexplained, while in other cases a variety of reasons have been identified including lack of ovulation, mechanical stoppage, sperm deficiencies and dysfunctions and parental age. Male infertility in humans is a growing problem and around 7% of men suffer from male factor infertility. In a fraction of 25% of infertile men, the etiology remains unknown. In some cases gene-gene and geneenvironment, interactions may be causative of male infertility. A genetic approach that involves single candidate genes and incorporates biological information from an animal model e.g. the pig, is likely to be valuable in explaining the genetic basis of male fertility. Genes playing essential roles in sperm function, such as ATP1A4, can be studied in the model organism. CRISPR/Cas9 technology could be utilized to produce inactivating mutations in the ATP1A4 gene of domestic pigs to study. Elimination of ATP1A4 expression in a pig model might contribute to increased understanding of sperm function. In future prospect, this could contribute to a better diagnosis and development of treatments of male infertility. Conflicts of interest The authors declare no conflict of interest. Acknowledgements The authors wish to thank Bente Flügel, Hanne Jørgensen and Mahesha Perera for excellent technical assistance. The authors also acknowledge associate professor Hanne Poulsen for the valuable discussions on a4 protein structure and conformation. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2019.01.003. References
Fig. 3. Spatial expression of porcine ATP1A4 mRNA. Relative expression of ATP1A4 in fourteen different porcine organs and tissues. TES, testis; LUN, lung; HEA, heart; BLA, bladder; KID, kidney; HYP, hypothalamus, BST, brain stem; FCO, frontal cortex; CBE, cerebellum; PCO, parietal cortex; OCC, occipital cortex; TEMPL, temporal lobe; SPC, spinal cord; SPL, spleen. The 18S ribosomal subunit was used as a reference gene.
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