key: cord-1022996-wuicmcwy
authors: Bora, Roop Singh; Kanamori, Akiko; Hirabayashi, Yoshio
title: Cloning and characterization of a putative mouse acetyl-CoA transporter cDNA
date: 1999-10-01
journal: Gene
DOI: 10.1016/s0378-1119(99)00361-3
sha: 0b159ec402f822d502c0a2a478f5e08c1212acb5
doc_id: 1022996
cord_uid: wuicmcwy

A mouse acetyl-CoA transporter (Acatn) cDNA was isolated by PCR cloning. Mouse Acatn exhibited 92% homology with human sequence on the basis of amino-acid sequence. The predicted gene product of Acatn is a 61 kDa hydrophobic protein with six to 10 transmembrane domains. Transfection of mouse Acatn cDNA into HeLa/GT3(+) cells resulted in significant increase in the amount of 9-O-acetylated gangliosides, suggesting that Acatn does play an important role in the acetylation of gangliosides. Northern blot analysis of Acatn mRNA suggested that transcript of Acatn is widely distributed in various adult tissues. Expression of Acatn was found to be developmentally regulated, with high expression levels during early embryonic stages, and then there was a subsequent decrease in expression levels in the later embryonic stages.

significant effects on the action of bacterial sialidases (Corfield et al., 1986; Varki and Diaz, 1983) . It also Gangliosides are a diverse series of sialic acid-containaffects virus binding, cell adhesion and the immunogeing glycosphingolipids present on the plasma membrane nicity of sialic acid residues of gangliosides ( Varki, of most vertebrate cells. They are particularly abundant 1992). Expression of O-acetylated gangliosides on the in the central nervous system. Sialic acid residues on cell surface can alter the binding of pathogenic viruses. gangliosides are sometimes modified by O-acetylation In most cases, it confers protection to the host from the at the 9-position. Some biological properties are found corresponding pathogen. Interestingly, Influenza C and to be associated with the modification of sialic acids by Corona viruses bind specifically to 9-O-acetylated sialic O-acetylation. The expression of 9-O-acetylated ganglioacids; however, these are relatively benign pathogens sides is apparently associated with neural cell differenticompared to the Influenza A and B viruses, whose ation and migration (Mendez-Otero and Cavalcante, binding to sialic acids is abrogated by O-acetylation 1996; Constantine Paton et al., 1986) . Expression of O- ( Higa et al., 1985; Rogers et al., 1986) . O-Acetylation acetylated sialic acids on cell surfaces can also cause of sialic acids on murine erythrocytes appears to inhibit binding of the malarial parasites (Reuter et al., 1991) . These data are supportive that O-acetylation of sialic ( Varki, 1992; Schlosshauer et al., 1988; Zhang et al., fax: +81-48-467-9614. 1997 acids might be involved in segmentation of the embryo, and the late expression in specific organs caused develop-2.2. DNA transfection and immunocytochemical analysis mental abnormalities ( Varki et al., 1991) .

A BamHI-XbaI fragment of mouse Acatn cDNA In spite of its importance, the O-acetylation mechacontaining the entire coding sequence was subcloned nism is poorly understood at the molecular and genetic into BamHI and XbaI sites of mammalian expression levels. The molecular cloning of genes encoding for vector pcDNA3.1 (Invitrogen, CA, USA) and the resulvarious factors that take part in the biosynthesis of tant plasmid was designated as pcDNA3.1-Acatn. Stable acetylated gangliosides is necessary to understand the transfectant of HeLa cells expressing gangliosides GD3 biological functions of O-acetylated gangliosides during and GT3, named HeLa/GT3+ was isolated as reported development. Previously, we have reported the isolation ( Kanamori et al., 1997) and used as recipient cells for of a novel cDNA encoding for a putative acetyl-CoA transient expression of Acatn cDNA. Cells were cultured transporter that is required for the formation of Oon coverslips in Dulbecco's modified Eagle medium acetylated gangliosides ( Kanamori et al., 1997) . Since (DMEM ) containing 10% fetal-bovine serum (FBS ) expression of O-acetylated gangliosides is developmenand grown in a humidified 5% CO 2 incubator at 37°C. tally regulated, it is necessary to analyze the expression HeLa/GT3+ cells were transfected with 10 mg of plasmid of acetyl-CoA transporter protein during embryonic DNA using Super Fect Transfection reagent (QIAGEN, development. As mouse is a model experimental system, Germany) following the manufacturer's instructions. the study of mouse genes is becoming increasingly

Immunocytochemical analysis was carried out as important with the advent of gene-targeting technology.

described previously ( Kanamori et al., 1997) . Briefly, However, mouse acetyl-CoA transporter cDNA has not 40 h after transfection, the cells were fixed with 2% been isolated so far. In this paper we report, for the paraformaldehyde for 20 min at room temperature and first time, cDNA cloning of putative mouse acetyl-CoA incubated with mAb493D4 (obtained from S. Fujita, transporter, and study its expression during embryonic Mitsubishi Kasei Institute of Life Sciences) for 2 h at development. room temperature, followed by incubation with Alexaconjugated anti mouse IgG (Molecular probes, Eugene, USA) for 1 h at room temperature. Fluorescence labeling was detected by using a Zeiss Axioplan Fluorescence microscope.

Normal adult tissues were obtained from 8-10 week old male Balb/c mice. mRNA was isolated from the PCR cloning was performed to isolate mouse acetyladult tissues using Poly A Tract mRNA isolation kit CoA transporter cDNA. A mouse melanoma cDNA (Promega, WI, USA) following the manufacturer's library in Uni-ZAP@XR vector (Stratagene, La Jolla, instructions. Approximately 2 mg of mRNA was electro-CA, USA) was used directly as a template. Forward phoresed through 1% agarose-formaldehyde gel and primer, 5∞-ATGTCACCCACCATCTCCCACAAG-3∞ transferred to nylon membrane according to Sambrook corresponding to nucleotide position 388-412 and et al. (1989) . Mouse Embryo Multiple Tissue blot reverse primer, 5∞-TTAATTGTTCCTTTTGCATTT-(Clontech, CA, USA) was used for analysis of Acatn CCAC-3∞, corresponding to nucleotide position 2012expression during developmental stages. Digoxigenin-2037 of human Acatn cDNA sequence, were used for labeled Acatn antisense RNA probe corresponding to DNA amplification. Expand High Fidelity PCR system nucleotide position between 1 and 688 was generated by (Boehringer Mannheim, Germany) was used according SP6 RNA polymerase, using DIG-RNA labeling kit to the manufacturer's instructions. Thirty PCR cycles (Boehringer Mannheim, Germany). Hybridization was were carried out on 1 ml of mouse melanoma cDNA carried out at 68°C for 14 h. The membrane was washed library (1.5×1010 pfu/ml ). Each PCR cycle consisted of with 2×SSC/0.5% SDS and 0.1×SSC/0.5% SDS each denaturation at 94°C for 1 min, annealing at 55°C for for 30 min at 68°C. Bound RNA probes were revealed 1 min and extension at 72°C for 2 min. The amplified by incubation with anti-digoxigenin-alkaline phosphafragment of 1.65 kb was subcloned into pZErO-1 vector tase conjugate, followed by chemiluminescence detec-(Invitrogen CA, USA) after digestion with EcoRV.

tion according to the manufacturer's instructions Nucleotide sequence was determined in both directions (Boehringer Mannheim, Germany). using cycle sequencing kit (Amersham Life Technologies, USA) based on the dideoxy chain termination 2.4. In situ hybridization method (Sanger et al., 1977) . A LI-COR 4000L Sequencer (LI-COR, NE, USA) was used for the Rat Hybrid-Ready tissues (Novagen, WI, USA) were used to detect the Acatn mRNA expression during analysis. developmental stages, since expression of O-acetylated gangliosides is well characterized in rat system ( Varki, 1992) . Rat embryos, cut into sagittal sections, were from strain Sprague-Dawley. Digoxigenin-labeled Acatn antisense RNA probe corresponding to nucleotide position between 1 and 688 was generated by SP6 RNA polymerase, using DIG-RNA labeling kit. In situ hybridization was carried out using modified protocol of Mutter and Wolgemuth (1987) . Hybridization was carried out at 42°C in the presence of 50% formamide for 14 h, followed by washing in 2×SSC at 42°C. Sections were then incubated with anti-DIG-alkaline phosphatase conjugate for 2 h at room temperature, and the bound antibody was detected by a standard immuno-alkaline phosphatase reaction using nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate as substrate.

Mouse acetyl-CoA transporter (Acatn) cDNA was isolated by PCR cloning using mouse melanoma cDNA library in Uni-ZAP@XR vector as a template. A fulllength coding region (nt 1-1653) was amplified using forward and reverse primers specific to human Acatn cDNA. The amplified fragment was cloned into pZErO-1 vector and the nucleotide sequence was determined in both directions. The nucleotide sequence and deduced amino-acid sequence of mouse Acatn cDNA are shown in Fig. 1 . The nucleotide sequence of mouse Acatn cDNA was further confirmed by cloning and sequencing the exon sequences of mouse Acatn gene (unpublished data). Mouse Acatn cDNA encodes for a protein of 550 amino acids, with a predicted molecular mass of 61 kDa. The mouse Acatn cDNA sequence was highly homologous with the human cDNA, and it exhibited 87% homology at nucleotide sequence level and 92% homology on the basis of amino acid sequence. Hydropathy analysis revealed a highly hydrophobic, transmembrane protein with approximately 6 to 10 transmembrane domains, similar to human Acatn protein (data not shown). Mouse Acatn protein was also found to contain a leucine zipper motif in the transmem- available nucleotide and protein databases revealed two hypothetical proteins with high degree of homology: a putative transmembrane protein of Saccharomyces cere-Caenorhabditis elegans T26C5.3 ( EMBL, accession No. Z50859) that has 632 amino acids with 49% homology visiae ( EMBL, accession No. Z36088) that has 560 amino acids with 34% homology, and a protein from on the basis of amino-acid sequence. As shown in Fig. 2,  Fig. 2 . Amino-acid sequence alignment of mouse, human Acatn and the homologs. Gaps have been introduced to maximize alignments. The identical amino-acid residues among all the species are enclosed in boxes, and the asterisks indicate conserved residues in three species.

there are highly conserved segments among acetyl-CoA pcDNA3.1-Acatn, was transfected into HeLa/GT3+ cells. Expression of 9-O-acetylated gangliosides in transporter and its homologs from different organisms. In particular, maximum homology is seen in the regions HeLa/GT3+ cells was detected by immunostaining with mAb 493D4, specific for 9-O-acetylated GT3. As corresponding to amino acid residues 72-246 and 431-482 of mouse Acatn, suggesting that these might be shown in Fig. 3 , expression of 9-O-acetylated gangliosides was strongly detected with mAb 493D4 on Acatn functionally more significant domains of Acatn protein.

The function of these homologous proteins in C. elegans transfected cells as compared with the cells transfected with vector pcDNA3.1 alone, indicating that Acatn and S. cerevisiae is not yet known. Since these organisms lack both sialic acids and gangliosides, it is unlikely that protein does play an important role in acetylation of gangliosides. Our previous studies on the expression Acatn is involved solely in acetylation of sialic acids. Thus Acatn (and its homologs) might be involved in of human Acatn cDNA in COS-1/GD3+ and HeLa/GT3+ cells also showed a high level expression other acetylation processes as well. In addition to these two proteins, mouse Acatn protein also exhibited low of 9-O-acetylated GT3 and GD3 in the transfected cells ( Kanamori et al., 1997) . Based on these results and also similarity to Amp G protein of Escherichia coli ( EMBL, accession No. X82158) with 22% identity on the basis earlier studies on in vitro transport activity for acetyl-CoA ( Kanamori et al., 1997) , the protein encoded by of amino acid sequence (data not shown), indicating an evolutionary relationship between these two proteins.

Acatn is suggested to be an acetyl-CoA transporter that is involved in the process of O-acetylation. Interestingly, like other homologs, there are several conserved amino-acid residues in the region corresponding to amino-acid position between 66 and 237 of mouse 3.3. Tissue distribution of Acatn mRNA Acatn. The gene product of ampG is a 53 kDa hydrophobic protein with 7 to10 transmembrane domains,

The expression of Acatn mRNA in various mouse tissues was examined by Northern blot analysis, using and is found to be involved in the transport of muropeptides of bacterial cell wall inside the cell and also in the mRNA purified from adult mouse tissues. A major transcript of 3.0 kb was detected in all the tissues regulation of beta-lactamase induction (Lindquist et al., 1993; Jacobs et al., 1994) . At present, the role of AmpG examined, including brain, heart, kidney, liver and spleen, as shown in Fig. 4A . The maximum expression protein in acetylation process is not known.

of Acatn was observed in kidney and liver. In contrast to human, which expressed two species of the mRNA 3.2. Expression of 9-O-acetylated gangliosides after introduction of mouse Acatn cDNA into HeLa/ GT3+ corresponding to 3.3 and 4.3 kb respectively ( Kanamori et al., 1997) , mouse expressed only a single transcript cells of 3.0 kb. Mouse Acatn cDNA was introduced into HeLa/GT3+ recipient cells, that express precursor gan-3.4. Tissue-specific and developmentally regulated expression of mouse Acatn gene gliosides GT3 but lack 9-O-acetylated GT3. A BamHI-Xba I fragment of mouse Acatn cDNA (nt 1-1653) containing the entire coding sequence was subcloned Expression of Acatn mRNA was examined during embryonic development by Northern blot analysis. High into BamHI and XbaI sites of mammalian expression vector pcDNA3.1. The resultant plasmid, designated as level of expression was observed in early embryonic stage ED7, and then there was a subsequent decrease in 4. Conclusion the expression level up to embryonic day 17, as shown

We have isolated a mouse cDNA encoding for an in Fig. 4C , suggesting a highly specific role for Acatn acetyl-CoA transporter which exhibited 87% homology protein during early embryonic development. A major with the human cDNA at nucleotide sequence level. transcript of 3.0 kb was detected in all the embryonic Acatn is found to be evolutionarily conserved as its stages. Expression of Acatn mRNA during developmenhomologs are detected in various organisms including tal stages was also examined by in situ hybridization C. elegans, S. cerevisiae and E. coli, exhibiting 49%, using rat embryos, because expression of acetylated 34% and 22% homology, respectively, with mouse Acatn gangliosides is very well characterized in rat system protein.

In particular, maximum homology is seen in ( Varki, 1992; Schlosshauer et al., 1988) . In the 10 day the regions corresponding to amino acid residues 72old embryo, high level of mRNA expression was 246 and 431-482 of mouse Acatn protein. Transfection detected in the neural tube and neural crest cells using of Acatn cDNA into HeLa/GT3+ cells resulted in high Acatn antisense RNA probe (data not shown). In the expression of 9-O-acetylated gangliosides, indicating 13 day old embryo, maximum expression was detected that it plays an important role in the acetylation of in the dorsal root ganglia (derived from neural crest) gangliosides. During embryonic development, Acatn with Acatn antisense RNA probe ( Fig. 5A ). In the expression levels were high during early embryonic 16 day old embryo, expression of Acatn mRNA was stages such as ED7 and there was a subsequent decrease also detected in dorsal root ganglia, but the expression in expression levels in later stages, suggesting that Acatn level was low as compared with that in the 13 day old expression is developmentally regulated. Expression of embryo ( Fig. 5C ). With the control sense probe, no Acatn was also found to be tissue-specific, as it was signal was detected in the rat embryos (Fig. 5B, D) . In detected in neural tube and neural crest cells in early the 18 day old embryo, Acatn expression was barely embryonic stages of development and, in later stages, detected in dorsal root ganglia (data not shown), sugthe expression was detected in dorsal root ganglia. In gesting that there is a decrease in Acatn expression levels adult stages, transcript of Acatn was detected in all the at later stages of embryonic development, as also tissues, with higher expression levels in kidney and liver. observed by Northern blot analysis. Earlier studies have Although transcript of Acatn is more widely distributed indicated the developmentally regulated expression of in adult stages, until now, acetylated gangliosides have 9-O-acetylated gangliosides in rat. In early embryonic been reported only in adult kidney, brain and adrenal stages, ED8-11, expression of 9-O-acetylated gangliomedulla (Reivinen et al., 1992; Leclerc et al., 1992 ; sides was observed in neural tube and neural crest cells. Schlosshauer et al., 1988) , suggesting that some other In embryonic stages from ED13-18, expression of 9-Ofactors also might be involved in regulation of the acetylated gangliosides was detected in dorsal root ganglia and germinal cells in the ventricular zones ( Varki, expression of acetylated gangliosides. In fact, another cDNA clone that is involved in O-acetylation of ganglio-1992). Hence, the expression of Acatn mRNA is in accordance with the expression of O-acetylated ganglio-sides had been isolated from rat brain (Ogura et al., 1996) . The predicted sequence of the protein does not sides during embryonic development. share any similarity with Acatn protein. The function required to further elucidate the biological functions of Acatn during embryonic development. of this protein as an O-acetyltransferase is not yet clear. Homologs of Acatn protein have been identified in C. elegans and S. cerevisiae. Since these organisms have neither gangliosides nor sialic acids, it is unlikely that

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Acatn is involved solely in acetylation of sialic acids. Thus Acatn protein might be involved in other acetyla-We thank Dr Yoshitaka Nagai for generous help and encouragement. This work was supported by a Frontier tion processes in addition to the acetylation of sialic acids in gangliosides. Our earlier studies have suggested Research Program Grant of Science and Technology Agency and a Grant-in-Aid for Scientific Research of that Acatn protein functions as an acetyl-CoA transporter ( Kanamori et al., 1997) ; development of transgenic Priority Area from the Ministry of Education, Science and culture (No. 05274106) to Y.H. mice with both the alleles of Acatn disrupted will be