Molecular Cloning, Bioinformatics Analysis and Expression Profiling of aGene Encoding Vacuolar-type H+-ATP Synthetase (V-ATPase) c Subunitfrom Bombyx mori

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Molecular Cloning, Bioinformatics Analysis and Expression Profiling of a Gene Encoding Vacuolar-type H

⁺

-ATP Synthetase (V-ATPase) c Subunit from Bombyx mori

Peng Lü, Keping Chen¹*, Qin Yao and Huajun Yang

Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, P. R. China (Received 7 November 2007; Accepted 3 December 2007)

As the genome of B.mori is available in GenBank and the EST database of B.mori is expanding, identifica- tion of novel genes of B.mori is conceivable by data- mining techniques. We used the in silico cloning method to get the vacuolar-type H⁺-ATP synthetase (V-ATPase) c subunit (16 kDa proteolipid subunit) gene of B.mori and analysed with bioinformatics tools.

The result was confirmed by RT-PCR and sequencing.

The V-ATPase c subunit cDNA contains a 468 bp ORF.

The ORF encoded a 155-residue protein that showed extensive homology with V-ATPase c subunits from other 15 species and contained four membrane-span- ning helices. Tissue expression pattern analysis revealed that V-ATPase c expressed strongly in Mal- pighian tubules, not in fat body. This gene has been registered in GenBank under the accession number EU082222.

Key words: 16 kDa proteolipid, RT-PCR, sequencing, tissue expression

Introduction

The vacuolar-type H⁺-ATP synthetase (V-ATPase) was discovered in various endomembranes system in the early 1980(Harvey and Nelson, 1992). V-ATPases played an important role in acidification of diverse intracellular compartments in eukaryotic cells, extensively existing in endosomes, lysosomes, calthrin-coated vesicles, secretary vesicles, chromaffin granules of eukaryotic cells (Nelson,

1989) and were also found in the central vacuoles of plants and fungi(Takase et al., 1994). The V-ATPase is a multi-subunit enzyme that consists of a peripheral V1

complex and an integral V0 complex. V1 complex is responsible for hydrolysis of ATP, and V0 complex is responsible for the translocation of protons across endomembrane or plasma membranes(Forgac, 1999;

Nishi and Forgac, 2002; Stevens and Forgac, 1997).

The 16-17 kDa subunit c of the V-ATPase belongs to the integral V0 complex, forming a hexameric complex with subunits c’ and c’’ as a core of the V0 complex(Wilkens et al., 2004). It is known that the 16 kDa proteolipid encoded by this gene is one of the most conserved membrane proteins, with greater than 65% identity between all species(Finbow and Harrison, 1997). The genes from diverse species have been cloned and studied(Finbow and Har- rison, 1997), they are vital for the assembly of the V- ATPase(Nelson and Klionsky, 1996) and forms the prin- cipal pathway for proton translocation(Sun et al., 1987;

Zhang et al., 1994; Zhang et al., 1992). In addition, they have been concerned with cell-cell communication as a connexins (or ductin) of gap junctions(Finbow and Meagher, 1992) and with neurotransmitter release as a component of the mediatophore(Birman et al., 1990).

Some studies also demonstrated that it is a target for some unusual viral oncoproteins(Finbow et al., 1995; Galli et al., 1996).

In this study, we employed repeated EST searching, multiple sequence comparisons and other data-mining techniques to obtain the B.mori V-ATPase c subunit gene for the first time, then it was confirmed by RT-PCR and sequencing. The expression profiles of V-ATPase c subunit gene in various tissues were also investigated, facil- itating future work to realize the function of V-ATPase c subunit.

Industrial Entomology

*To whom the correspondence addressed

Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, P. R. China. Tel: 86-511-88791923;

Fax: 86-511-88791923; E-mail: kpchen@ujs.edu.cn

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Materials and methods

Animal materials and RNA isolation

The silkworm (Bombyx mori) was bred in our laboratory.

Silkworm strain 306 was used in this study. The whole B.mori midguts were dissected from feeding fifth instar larva for total RNA isolation. Testis, ovary, blood-lymph, fat body, midgut, silk gland and Malpighian tubules were also obtained from feeding fifth instar larvae and imme- diately used for RNA extraction. Total RNA was isolated according to the protocol of the RNeasy Mini Kit (QIAGEN).

Data extraction of cDNA sequence of B.mori V-ATPase c subunit

The NCBI’s EST database is a popular starting point for identifying expressed sequence tags (ESTs) of different species, and more than 110,000 B.mori EST sequences are currently available in GenBank. Data-mining techniques and bioinformatics tools were applied to search for cDNA sequence of the B.mori V-ATPase c subunit gene by repeated cycles of assembling and extending EST sequences, and the computationally predicted sequence has been deposited at GenBank under number EU082222.

Comparative and bioinformatic analysis

Analysis of the nucleotide sequcence, deduced amino acid sequence and open reading frame (ORF) were carried out online at the websites (http://www.ncbi.nlm.nih.gov and http://cn.expasy.org). In order to realize the genomic orga- nization, the cDNA sequence was blasted to the contigs of B.mori genome in GenBank, and SIM4 (http://pbil.univ- lyon1.fr/sim4.php) (Florea et al., 1998) was used to align the cDNA sequence with the genomic sequences to search potential introns. Splice Site Prediction by Neural Net- work (NNSSP) (http://www.fruitfly.org/seqtools/splice.

html) was used to predict the potential splice sites. The sequence comparison was conducted through database search using BLAST program (National Center for Bio- technology Services, http://www.ncbi.nlm.nih.gov). The phylogenetic analysis of B.mori V-ATPase c subunit (the 16 kDa subunit) and V-ATPase 16 kDa subunits from other species were aligned with CLUSTAL W using default parameters. A phylogenetic tree was constructed using MEGA version 3.1(Kumar et al., 2001) form CLUSTAL W alignments. The neighbor-joining method(Saitou and Nei, 1987) was used to construct the tree. The three-dimensional (3D) structural modeling of B.mori V-ATPase c subunit was accomplished by Swiss-Modeling and WebLab ViewerLite(Guex and Peitsch, 1997; Schwede et al., 2003) was used for 3Dstructure displaying.

Cloning and sequencing of B.mori V-ATPase c subunit geneSingle-strand cDNAs were synthesized from midgut total RNA with M-MLV reverse transcriptase (Promega) and an oligo(dT)17 primer (Songon). The single-strand cDNA mixtures were used as templates for PCR amplification of V-ATPase c subunit gene from B.mori. Two V-ATPase c subunit gene specific primers, Vc-F (forward, 5’-GGG- GAATTCATGGCTGAAAATAATCCAATCT-3’) contained a EcoR restriction site (underlined), and Vc-R (reverse, 5’-GGGAAGCTTTTATTTTGTGTACAGGTA- GATGG-3’) contained a Hind restriction site (underlined) were designed according to the predicted sequence (submitted under number EU082222). The PCR reaction was carried out for 40 amplification cycles (94^oC/60 s, 58^oC/

45 s, and 72^oC/45 s) in a Gene Amp 2400 System ther- mocycler.

The PCR product was ligated into pMD18-T vector (Takara) using T4 DNA ligase (Takara) and then trans- formed into Escherichia coli strain TG1 followed by sequencing. The sequencing was performed using ABI PRISM 3730 with BigDye terminator v3.1.

Expression profile analysis

The expression profiles of B.mori V-ATPase c subunit in different tissues including testis, ovary, blood-lymph, fat body, midgut, silk gland and Malpighian tubules were measured by semi-quantitative RT-PCR. The methods of RNA isolation, single-strand cDNA synthesization and amplification with two V-ATPase c subunit gene specific primers were according to the “cloning and sequencing of B.mori V-ATPase c subunit full-length cDNA” above. The constitutively expressed B.mori actin gene A3 (GenBank accession number BMU49854) was used as the internal control , and two primers were AF (forward, 5’-GGAT- GTCCACGTCGCACTTCA-3’) and AR (reverse,5’GCG CGGCTACTCGTTCACTACC-3’).PCR products were separated to 1% agarose gels stained with ethidium bro- mide and intensities of the target bands measured using FMBIO^R Fluorescent Imaging System (Hitachii).

Results

Cloning of the full-length cDNA of B.mori V-ATPase c subunit using in silico clone method

Through the in silico clone method, the full-length cDNA of B.mori V-ATPase c subunit gene was obtained, which was 1923 bp. ORF Finding analysis on NCBI showed that the B.mori V-ATPase c subunit gene contained a 486 bp ORF. The full-length cDNA of B.mori V-ATPase c subunit is shown in Fig. 1.

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Fig. 1.The full-length cDNA sequence and the deduced amino acid sequence of Bombyx mori V-ATPase c subunit. The start codon (ATG) was underlined in bold and the stop codon (TAA) was underlined italically in bold. The amino acids with bold underline were the predicted transmembane regions. The bold “E” with emphasis symbol “.” was an important amino acid.

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Comparative and bioinformatics analyses of B.mori V- ATPase c subunit gene

BLASTing the cDNA to contigs of B. mori genome in GenBank revealed that only contig 001543 (GenBank accession No.AADK01001543) had high similarity.

Using SIM4, three exons and two introns were found in the relevant DNA sequence (Fig. 2). Splicing signals (exon/GU-intron-AG/e xon) were also identified by NNSSP and were shown in Fig. 2.

The deduced amino acid sequence of B. mori V-ATPase c subunit encoded a peptide of 155 amino acids, with a calculated molecular mass of 15.8 kDa, an isoelectric point of 8.96 (http://cn.expasy.org/tools/protparam.html) and four transmembrane regions (Fig. 1)(Rost, 1996). It was submitted to NCBI for PSI-BLAST (http://www.

ncbi.nlm.nih.gov/BLAST) searching and the result showed that B. mori V-ATPase c subunit gene had high identity with V-ATPase sequences from other species, with 85%

identity with V-ATPase c subunit from Homo sapiens. B.

mori V-ATPase c subunit was also highly similar to V- ATPase c subunits from Aedes aegypti (identities=91%, positives=94%), Aspergillus niger (identities=70%, positives=85%), Caenorhabditis elegans (identities=

73%, positives=81%), Candida tropicalis(identities=

70%, positives=85%), Drosophila melanogaster (identities=66%, positives=83%), Drosophila pseudoobscura (identities=92%, positives=96%), Aponica cultivar (identities=66%, positives=81%), Manduca sexta (identities=73%, positives=87%), Mus musculus (identities=82%, positives=90%), Pichia stipitis (identities=69%, positives=84%), Saccharomyces cerevisiae (identities=71%, positives=84%), Schizosaccharomyces pombe (identities=62%, positives=78%) and Xenopus laevis (identities=84%, positives=89%).

The amino acid sequences of fifteen V-ATPase c subunits were aligned according to the algorithm of the CLUSTAL W(Higgins et al., 1994). The result showed that these V-ATPase c subunits (16 kDa proteolipid subunits) had high similarity throughout the entire coding region. Two motifs were found in fifteen V-ATPase c subunits sequences. Conserved Motif-TAK ([ST].[RK]) was a protein kinase C phosphorylation site, and the second motif-SGTG ([SG.G]) was a glycosaminoglycan attachment site (Bairoch et al., 1997). A highly conserved

glutamic acid residue in C-terminal region of V-ATPase c subunits (16 kDa proteolipid subunits) was very important, it was known as the cation-binding site for translocation of protons(Fillingame, 1996). The substratebinding motif of fifteen V-ATPase c subunits (16 kDa proteolipid subunits) used in the multiple alignment analysis were picked out and the motif of B. mori V-ATPase c subunit (16 kDa proteolipid subunit) had almost the same amino acid composition with those of other V-ATPase c subunits (16 kDa proteolipid subunits) (Fig. 3). These conserved residues may function as important domains of the holo- enzyme.

To investigate B. mori V-ATPase c subunit’s evolutionary position among the phylogenetic tree of various V- ATPase c subunits, we used MEGA 3.1 from CLUSTAL W alignments, phylogenetic tree of V-ATPase c subunits was constructed from different organisms (Fig. 4).

According to the phylogenetic tree, V-ATPase c subunits’

evolution was in accordance with evolution of species. B.

mori V-ATPase c subunit had high homology with V- ATPase c subunits from other insect species (Drosophila pseudoobscura, Manduca sexta and Aedes aegypti) and mammalian (Homo sapiens and Mus musculus), and it had low homology with fungal (Saccharomyces cerevisiae) and plant (japonica), relatively.

The predicted 3D structural modeling of B. mori V- ATPase c subunit was analyzed by Swiss-Modeling on the basis of Enterococcus hirae 2b12 chain A crystal structure(Guex and Peitsch, 1997; Schwede et al., 2003) and displayed by WebLab ViewerLite (Fig. 5).The overall folding of B. mori V-ATPase c subunit, typically built from helices connected by loops, created very tight structural scaffold. The highly conserved glutamic acid residue in the fourth helix may form a cation-binding site for translocation of protons(Fillingame, 1996).

Cloning and sequencing of B.mori V-ATPase c subunit geneBased on the predicted sequence (EU082222), two specific oligonucleotide primers (Vc-R and Vc-F) were designed and used for PCR amplification of the ORF of V-ATPase c subunit cDNA from B.mori. A 486 bp product was amplified and subcloned following by sequencing, and the sequence was sequenced by ABI PRISM

Fig. 2.DNA sequence frame of Bombyx mori V-ATPase c subunit. It is based on contig 001543 (GenBank accession No.AADK01001543). The first position (14024) and the last position (17213) of the cDNA that we got in contig 001543 were indicated (the first nucleotide of contig 001543 was defined as position 1). The splicing signals (exon/GT-intron-AG/exon) were also indicated.

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Fig. 3.Multi-alignment of amino acid sequence of Bombyx mori V-ATPase c subunit (16 kDa proteolipid subunit) and other V- ATPase c subunits (16 kDa proteolipid subunits). The identical amino acids were showed in white with black background and the conserved amino acids were showed in white gray background. The conserved protein kinase C phosphorylation motif TAK ([ST].[RK]) and glycosaminoglycan attachment motif (SG.G) were underlined, and a highly conesrved amino acid Glu(E) was underlined and framed. The aligned V-ATPase c subunits (16 kDa proteolipid sununits) were from Aedes aegypti (Aa-16 kDa.p, GenBank accession no.: AAB71660), Aspergillus niger (An-16 kDa.p, GenBank accession no.:XP_001399935), Caenorhabditis elegans (Ce-16 kDa.p, GenBank accession no.: NP_500188), Candida tropicalis (Ct-16 kDa.p, GenBank accession no.:Q00607), Drosophila melanogaster (Dm-16 kDa.p, GenBank accession no.:NP_611169),Drosophila pseudoobscura (Dp-16 kDa.p, Gen- Bank accession no.:XP_001361962), Homo sapiens (Hs-16 kDa.p, GenBank accession no.: XP_001130742), japonica cultivar (Jc- 16 kDa.p, GenBank accession no.:NP_001054416), Manduca sexta (Ms-16 kDa.p, GenBank accession no.: P31403), Mus musculus (Mm-16 kDa.p, GenBank accession no.:AAH50939), Pichia stipitis (Ps–16 kDa.p, GenBank accession no.:XP_001387092), Sac- charomyces cerevisiae (Sc-16 kDa.p, GenBank accession no.:NP_010887), Schizos accharomyces pombe (Sp-16 kDa.p, GenBank accession no.:NP_593600), Xenopus laevis (Xl-16 kDa.p, GenBank accession no.:AAH43805) and Bombyx mori (Bm-16 kDa.p, GenBank accession no.:EU082222).

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3730. The result accorded with the predicted sequence.

Expression profile of B. mori V-ATPase c subunit gene in different tissues of Bombyx mori

To investigate the expression profile of B.mori, total RNA was isolated from testis, ovary, blood-lymph, fat body, midgut, silk gland and Malpighian tubules and subjected to semi-quantitative RT-PCR using primers Vc-F and Vc- R. The result showed B. mori V-ATPase c subunit gene expression could be detected in all tissues except fat body, suggesting that V-ATPase is an important expressed gene but at different expression levels, with the strong expression in Malpighian tubules(Fig. 6).

Fig. 4.Phylogenetic tree analysis of V-ATPase c subunits from other species by MEGA version 3.1 from CLUSTAL W alignments.

The neighbor-joining method was used to construct the tree. The V-ATPase c subunits used in phylogenetic tree analysis included Aedes aegypti (GenBank accession no.: AAB71660), Aspergillus niger (GenBank accession no.:XP_ 001399935), Caenorhabditis elegans (GenBank accession no.:NP_500188), Candida tropicalis (GenBank accession no.: Q00607), Drosophila melanogaster (GenBank accession no.: NP_611169), Drosophila pseudoobscura (GenBank accession no.:XP_001361962), Homo sapiens (Gen- Bank accession no.: XP_001130742), japonica cultivar-group (GenBank accession no.:NP_001054416), Manduca sexta (GenBank accession no.: P31403), Mus musculus (GenBank accession no.:AAH50939), Pichia stipitis (GenBank accession no.:XP_001387092), Saccharomyces cerevisiae (GenBank accession no.: NP_010887), Schizosaccharomyces pombe (GenBank accession no.:NP_

593600) and Xenopus laevis (GenBank accession no.: AAH43805).

Fig. 5.The 3D structure of B. mori V-ATPase c subunit estab- lished by homology-based modeling. The α-helix was indicated in red. Loops were indicated in red and green. The conserved Glu(E) residue was indicated in yellow (See www.ijie.or.kr for color context).

Fig. 6.Expression profile of B. mori V-ATPase c subunit gene in different tissues of Bombyx mori. Lanes 1-7 represented testis, ovary, blood-lymph, fat body, midgut, silk gland and Mal- pighian tubules, respectively. B.mori actin gene A3 was used as the control to show the normalization of the amount of templates in PCR reactions.

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Discussion

In this study, we identified a novel B.mori V-ATPase c subunit gene through bioinformatics approaches and was confirmed by RT-PCR and sequencing. Through nucleotide sequence analysis, we found that this gene contained a 486 bp ORF encoding a peptide of 155 amino acids, and locates within contig001543 of B.mori genome. While comparing the cDNA sequence of B.mori V-ATPase c subunit with the contigs of B.mori genome, only contig001543 had high similarity, suggested to be a single copy gene in the whole genome.

The deduced amino acid sequence of B.mori V-ATPase c subunit showed that this cDNA encoded a polypeptide of 155 amino acids with a predicted molecular mass of 15.8 kDa and an isoelectric point of 8.96. Through predicted protein sequence analysis, we found that this polypeptide is highly hydrophobic, containing four transmembrane regions interspersed by three short hydrophilic loops. The B.mori V-ATPase c subunit showed sequence identities to the other polypeptides at least 63%, it was known as one of the most conserved membrane proteins at all species(Finbow and Harrison, 1997), some studies showed that V-ATPase c subunit has two orientations in membranes apparently(Dunlop et al., 1995; Finbow et al., 1993), it may be a very important constitutive protein with multiple functions. The motif TAK at position 32 to 34 was identified as a potential protein kinase C phosphorylation site. The highly conesrved amino acid Glu(E) at position 139 in the fourth helix, was commonly thought to be a cation-binding site for translocation of protons and to be essential for proton translocation, it was also the sole site for reaction with the lipophilic inhibitor, for instance, N,N’-dicyclohexylcarbodi-imide (DCCD)(Finbow and Harrison, 1997). Recently, some people found that this subunit contained an endogenous divalent ion binding site in the cystine at position 54 from Nephrops norvegicus(Pali et al., 2006), but we did not know its function.

Some studies showed the V-ATPase c subunits of high plants and human express in different tissues with different isoforms encoded by multiple genes(Finbow and Har- rison, 1997), exsiting in the majority of tissues extensively. Our result of expression profile showed B. mori V- ATPase c subunit gene could be detected in all tissues except fat body, with the strong expression in Malpighian tubules.

The V-ATPase c subunit genes have been cloned from diverse fungal, plant, arthropod, mammalian and other vertebrate(Finbow and Harrison, 1997). However, there is no report on the cloning of V-ATPase c subunit genes from Bombyx mori. Silkworm (Bombyx mori) is an important economic insect, regarded as a model insect of Lep-

idoptera. Studies on structure and functions of certain related genes from B.mori have attracted more and more attention. As the core structure of V⁰ domain of V- ATPase, V-ATPase c subunit should play an important role, with its highly conservation and versatility. The cloning and characterization of this cDNA will help us to know how V-ATPases function, and it is helpful for our future work.

Acknowledgements

This work was supported by National Basic Research Program of China (No.2005CB121000), and Natural Science Foundation of Jiangsu Province, China (BK2006074)

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