Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes
© The Author(s). 2017
Received: 16 September 2016
Accepted: 22 December 2016
Published: 13 January 2017
Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases.
Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis.
We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.
There are more than one million species of insects in the world. Insects can adapt to any number of environmental conditions because of their small size. Studies of insects have contributed a wealth of scientific discoveries. The i5k project, which began in 2011 , aims to sequence the genomes of 5000 arthropod species. This project provides genomic information for minor insect species, such as those not used experimentally (https://www.hgsc.bcm.edu/i5k-pilot-project-summary).
Researchers can now access public databases containing the genomic information of many insects for comparative analyses . For our present study, we easily obtained gene sequences and analyses from large datasets, including RNA-seq results, from public databases. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of gene annotations in non-model organisms. Thus, we need to develop an analysis procedure for how to select functional genes from public databases.
Enolase is a key glycolytic enzyme (2-phospho-d-glycerate hydrolase; EC 220.127.116.11). Glycolysis is responsible for the majority of energy production in all organisms. In a human study, three enolase isoenzymes were identified as homodimers composed of two alpha (also known as ENO1; Online Mendelian Inheritance in Man (OMIM), 172430), two gamma (ENO2; OMIM, 131360), or two beta (ENO3; OMIM, 131370) subunits. Isoenzyme alpha is present in most tissues, whereas the beta form is localized to the muscle and the gamma form is found only in nervous tissue . A sperm-specific enolase was also identified in Mus musculus . The ENO1 and ENO 3 sequences are well conserved in vertebrates, whereas the insect Enolase 1-like sequence is well conserved across arthropods.
In recent years, many insect enolases have been discovered. Insect enolases differ from mammalian enolases in that they have relatively low conservation among insects and show species-specific functions. For example, the enolase of the parasitic wasp, Aphidius ervi is expressed on the egg surface and contributes to the digestion of host proteins by promoting plasmin generation as a plasminogen receptor . The expression of an enolase protein was up-regulated in the midgut of Aedes aegypti infected with chikungunya or dengue viruses . These reports suggest that insect enolases can have many different species-specific roles. To analyze the function of an enolase, the gene sequence of the organism of interest is required.
The silkworm Bombyx mori is a lepidopteran insect that has been used as a model insect in agricultural research for several reasons: 1) the majority of agricultural pests are lepidopterans, 2) its genome sequence is almost completely characterized, 3) various spontaneous genetic mutants are available, and 4) the silkworm is amenable to transgenic, knock-out, and microarray technologies [7–12]. However, there have been very few reports about enolase in lepidopteran insects.
In this study, we used a combination of dry and wet bench processes to identify functional enolase genes in B. mori using public databases. We found two genes and one isoform of B. mori functional enolases and characterized their functions.
Five enolase candidates were identified from B. mori datasets
All of these BmEno genes, except BmEnoC, contained both an enolase N-terminal domain (Enolase_N, pfam; PF03952) and enolase C-terminal domain (Enolase_C, pfam; PF00113) (Additional file 1).
BmEno amino acid sequence homology with enolases from other species
Therefore, we identified five putative enolase sequences from the B. mori gene datasets from Ensembl Metazoa and KAIKObase.
Analysis of B. mori enolase sequences
Alignment with enolase homologs from other species showed that the deduced BmEno amino acid sequences, except for BmEno2 and BmEnoC, contained all of the conserved Ser, Glu, and Asp residues (Fig. 2, arrows). These amino acid residues are involved in the coordination of the metal-binding domain. The BmEno2 and BmEnoC sequences contained a conserved Asn residue that replaced the Asp residue as the metal-binding amino acid residue (Fig. 2, red arrows). In Fig. 2, amino acid residues (Glu, Lys, and His) related to enolase active sites, also known as substrate binding pockets, are shown with asterisks. These amino acid residues were changed to Ser, Arg, and Asp in BmEno2 and BmEnoC (Fig. 2, asterisks).
The BmEnoX amino acid sequence corresponded well with the BmEno1 amino acid sequence except for an unknown amino acid at residue 431 (shown as “X;” Fig. 2, bottom of alignment). Furthermore, the BmEnoC amino acid sequence from position 1 to 209 corresponded well with the BmEno2 amino acid sequence at positions 225 to 433 (98.6% similarity). However, the N-terminus of BmEnoC was slightly different from that of BmEno2. The entire amino acid sequence of BmEnoY corresponded with that of H. sapiens ENO1 (NP_001419.1) (Fig. 2).
cDNA cloning of BmEnos from B. mori larvae and verification with RNA-seq analysis
Developmental stage- and tissue-specific expression patterns of BmEno mRNAs as determined by RT-PCR
Confirmation of BmEno isoforms
In this study, we obtained candidate BmEno sequences from public databases. Using a combination of dry and wet processes, we identified functional enolases in B. mori. These enolase amino acid sequences were composed of two domains. The N-terminus consisted of a shorter Enolase_N motif, and the C-terminus consisted of a longer Enolase_C motif. BmEnoC did not contain the Enolase_N motif.
The His 159, Glu 168, Glu 211, Lys 345, His 373, and Lys 396 amino acid residues are required for S. cerevisiae enolase activity. Amino acid residues Ser 39, Asp 246, Glu 295, and Asp 320 were identified as critical for metal-binding in S. cerevisiae enolase [13–16]. BmEno1 also contained these active residues (Fig. 2, asterisks) and metal-binding residues (Fig. 2, arrows). However, BmEno2 and BmEnoC had different amino residues from BmEno1 at the metal-binding and substrate-binding sites (Fig. 2, red asterisks and arrows). The Chinese oak moth (Antheraea pernyi) is a lepidopteran insect, and its enolaseI and enolaseII genes corresponded well with BmEno1 and BmEno2. A. pernyi enolaseII contains the same metal-binding and substrate-binding amino acid residues as BmEno2 and BmEnoC . Our phylogenetic analysis showed that BmEno1 was close to the cluster containing insect enolases, such as those from D. melanogaster, T. castaneum and A. mellifera. BmEno2 and BmEnoC were located in a lepidopteran-specific cluster. Sequences that belong to this cluster have residues that differ from the conserved residues necessary for enolase activity (Additional file 2). Thus, lepidopteran-specific enolases may have glycolytic enzyme activity that does not require the aforementioned conserved enolase residues.
cDNA cloning showed that the BmEno1 amino acid sequence corresponded with that of BmEnoX. We also confirmed that the chromosome positions of BmEno1 and BmEnoX were identical. As a result, only one transcript was detected as BmEno1. These results suggest that the BmEnoX sequence was misread and incorrectly registered in the public database.
The chromosome positions of BmEno2 and BmEnoC were also nearly identical. However, the BmEnoC sequence differed from that of BmEno2 by three amino acid residues. cDNA cloning and RNA-seq analysis showed BmEnoC is likely an isoform of BmEno2. RT-PCR analysis showed that the BmEno2 mRNA was mainly expressed in the testis, followed by the suboesophageal ganglion, and also a little bit in the ovary. BmEnoC was only expressed in the testis. To examine the physiological functions of BmEno2 and BmEnoC in the testis, we performed qRT-PCR on testis from day 0 fifth-instar larvae to adults. BmEno2 and BmEnoC were highly expressed from day 5 of the fifth-instar larval stage to the prepupal stage. When spermiogenesis occurs from the end of larval developmental stage to the prepupal developmental stage, the testis becomes hypertrophied in B. mori . The ecdysone titer is increased in B. mori at this time . Thus, the expression of BmEno2 and BmEnoC may correlate with spermiogenesis in B. mori.
Almost all lepidopteran insects have two kinds of sperm: apyrene and eupyrene sperm [20, 21]. Both sperm types are essential for fertilization in lepidopteran insects, but the role of this evolutionarily-conserved system in fertilization remains unclear. Furthermore, a sperm-specific enolase was reported to control sperm formation and mobility in M. musculus .
In this study, we found differences in the mRNA expression of BmEno2 and BmEnoC. The BmEno2 mRNA was expressed in the ovary and suboesophageal ganglion. Pheromone biosynthesis activating neuropeptide (PBAN) is secreted from the suboesophageal ganglion in B. mori, and affects the pheromone glands of female moths and stimulates the biosynthesis of a sex pheromone [23–25]. Diapause hormone (DH) is also secreted from the suboesophageal ganglion and promotes embryonic diapause [26–28]. DH also stimulates the prothoracic gland and promotes ecdysteroid generation, which controls molting and metamorphosis . Future studies should examine the function of BmEno2 outside of reproduction. Based on these data, BmEnoC might be an isoform of BmEno2 that has a different function.
BmEnoY mRNA expression was not detected by RT-PCR in any B. mori tissue at any developmental stage. However, the BmEnoY mRNA was expressed in the human cell line HepG2. Sequence analysis of BmEnoY showed that its amino acid sequence completely matched that of H. sapiens alpha enolase. Thus, BmEnoY might be a result of contamination by human alpha enolase that was incorrectly registered in the public database. In conclusion, our results suggest BmEno1, BmEno2, and BmEnoC are functional enolases in B. mori.
In this study, we performed a pipeline analysis using a combination of dry and wet bench processes. Using a molecular biological approach, we identified functional enolases in B. mori. BmEno1 was conserved across species. However, BmEno2 and BmEnoC appear to have a lepidopteran-specific function rather than a glycolytic enzyme function. Notably, enolase functions as a dimer. Therefore, research on how the enolases characterized in this study combine and function is needed. Future studies should compare the expression and enzymatic activities of the dimer proteins in different tissues and developmental stages. The key BmEno2 amino acid residues partially differed from those in BmEno1, and may play an important role in enzyme activity and metal binding.
We identified three B. mori enolases using a combination of dry and wet bench processes. These BmEnos have different functions within the tissues of B. mori. At some point, incomplete transcripts or uncorrected data were registered in public databases. If we can resolve these database errors using wet bench processes, then the usefulness of public databases will increase for all users. All public databases provide a wealth of information for future scientific research. Thus, we proposed a procedure for how to identify active genes from public databases in this study. It is important that public databases are regularly maintained by users. Our combination of dry and wet experiments is useful for the detection of minor genes and declared functional genes of non-model organisms in public databases.
The B. mori hybrid strain Kinshu × Showa used in this study was supplied by Ueda-Sha Co. Ltd. (Nagano, Japan). Silkworm larvae were reared on the artificial diet silk-mate 2S (Nosan, Tsukuba, Japan). Insects were maintained at 25 °C with a 12-h light/dark cycle. The B. mori strain o751 (wild-type) used in the RNA-seq analysis was obtained from the Institute of Genetic Resources, Faculty of Agriculture, Kyushu University (NBRP silkworm database: http://silkworm.nbrp.jp/index_en.html).
Identification of B. mori enolase sequences by HMM search and bioinformatics
The HMM search program in the HMMER package (version 3.1b1)  was used to detect enolase candidates. HMM profiles of the enolase N-terminal domain (Enolase_N, PF03952) and C-terminal domain (Enolase_C, PF00113) in the Pfam 27.0 database  were queried against deduced protein sequences in a B. mori Ensembl Gene dataset  and a cDNA dataset  with default parameters.
A search for enolase orthologs among the genes of the following species was conducted using BLAST methods: D. melanogaster, M. sexta, A. gambiae, A. mellifera, T. castaneum, and H. sapiens. Global homology searches were conducted using Genetyx ver. 10 (Genetyx Co. Ltd., Tokyo, Japan). A phylogenic analysis was conducted using MEGA ver. 7 . A protein motif search was conducted using SMART (http://smart.embl-heidelberg.de/). The alignment of the BmEno amino acid sequences and enolase orthologs from other species was conducted using CLC Sequence viewer 7.6.1 (CLC Bio Japan Inc. Tokyo, Japan). All analyses were performed with default parameters for the software.
Purification of total RNA and cDNA synthesis from different tissues and whole-body samples
Various tissues were dissected from day 3 fifth-instar larvae: brain, nerve ganglion, midgut, silk gland, fat body, Malpighian tubules, testis, and ovary. These tissues were stored at −80 °C until use. Larval, pupal, and adult whole bodies were also used for total RNA purification. Whole bodies were freeze-dried using a freeze drier (TAITECH Co. Ltd., Tokyo, Japan) for 12 h. Tissues and freeze-dried whole bodies were weighed and homogenized with lysis buffer from a PureLink® RNA extraction kit (Thermo Fisher Scientific Inc., Valencia, CA, USA) and then centrifuged at 13,000 × g for 10 min. Next, the supernatants were collected and processed for RNA purification according to the manufacturer’s instructions. Purified total RNA (1 μg) was processed for cDNA synthesis using a PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Co. Ltd., Tokyo, Japan).
cDNA cloning of B. mori enolases
Enolase homologs in species used for the phylogenic tree
Saccharomyces cerevisiae S288c
Oryza sativa Japonica
Primers used for Northern blot analysis (A), RT-PCR (B), and quantitative RT-PCR (C)
BmEno2 and BmEnoC
BmEno2 and BmEnoC
Tissue and developmental distribution analysis by RT-PCR
The tissue distribution of the BmEno genes was determined in the brain, nerve ganglion, suboesophageal ganglion, midgut, silk gland, fat body, Malpighian tubules, testis, and ovary of day 3 fifth-instar larvae. The distribution of the BmEno genes in the whole bodies of first instar to fifth instar larvae, pupae, and adults were determined. All samples were processed for extraction of total RNA and cDNA synthesis as previously described. Reverse transcriptase (RT)-PCR was performed with specific primers (Table 3) using AmpliTaq Gold® 360 Master Mix (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. B. mori actin (BmActin, Gene ID 187281813) was used as an endogenous control.
Northern blot analysis
Total RNA derived from the testis of day 3 fifth-instar larvae was used. Total RNA (12 μg) was separated on a 1.5% agarose and 6% formaldehyde gel and stained with ethidium bromide. Next, the gel was transferred to a nylon membrane. DIG-labeled probes were synthesized using a PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany) with specific primers (Table 3). After pre-hybridization, the membrane was hybridized with DIG-labeled probes at 50 °C overnight. The specific reaction was visualized on Kodak XOMAT AR X-ray film using a DIG chemiluminescence detection kit (Roche Diagnostics). 18S ribosomal RNA (rRNA) was used as a control. The mRNA size of BmEno genes was calculated using the image analysis software CS analyzer Ver. 3.0. A calibration curve was determined using the mobility of the DIG RNA ladder marker (Roche Diagnostics).
To quantify RNA expression levels, 1 μg of total RNA from pooled testis tissue dissected from day 0 fifth-instar larvae to day 0 adults (n = 3 each) was used for cDNA synthesis. qRT-PCR was performed in a 20 μl reaction volumes with 0.5 μl of the cDNA template and primers (Table 3) with a KAPA SYBR Fast qRT-PCR Kit (Nippon Genetics Co., Ltd., Tokyo, Japan) in accordance with the manufacturer’s instructions. qRT-PCR was performed on a Step One plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) following the Delta-Delta Ct method. Ribosome protein 49 (GeneID: 778453) was used as an endogenous reference for the standardization of RNA expression levels, and all data were calibrated against universal reference data. Relative quantification (RQ) values represent the relative expression level against a reference sample. All samples were assayed in triplicate as technical replications.
Total RNA was isolated from the testis of day 3 fifth-instar larvae of the B. mori o751 wild type strain using a PureLink® RNA extraction kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. The quality of RNA was assessed using an Agilent Bio-analyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing cDNA libraries were constructed with 4 μg of total RNA from o751 wild type testis samples (n = 3) with a Truseq RNA Sample Preparation Kit Set A (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol. RNA-seq was performed using a HiSeq 2500 system (Illumina Inc.). The data quality of the fastq files was verified with the FastQC tool (Babraham Bioinformatics, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The 44 M paired-end reads (2 × 150 bp) were mapped to the reference B. mori genomes available in the Ensembl Genome database [30, 32] using the Tophat program version 2.0.13 with default parameters . BAM formatted files generated by Tophat were sorted and indexed using SAMtools  and then converted to Wiggle track format (WIG) files using the bam2wig software (https://github.com/MikeAxtell/bam2wig). This allowed us to visualize the density of reads mapped to the specific region of interest. Histograms of mapped reads were generated using the Spotfire Cloud software with TIBCO Spotfire’s “Better World” program license (TIBCO Software, Inc., Palo Alto, CA, USA) (http://spotfire.tibco.com/better-world-donation-program/).
The wild type silkworm strain o751 was obtained from the National Bio Resource Project KAIKO (NBRP KAIKO) at the Center of Genetic Resources at Kyushu University. Computations were partially performed on the NIG supercomputer at the ROIS National Institute of Genetics. We thank Ms. Maaya Nishiko and Ms. Mai Sakamoto for the design of some pictures in Fig. 3.
This work was supported by JSPS KAKENHI grant number JP26450465 to HT, grant number JP15H02483 to KiI and HT, and grant number JP15H02837 to RS and HT.
This work was also supported by the NIG Collaborative Research Program (2014-A171 and 2015-A155).
Availability of data and materials
The nucleotide sequences for BmEno1, BmEno2, and BmEnoC were submitted to DDBJ/ENA/GenBank (Accession Nos. LC170036, LC170037, and LC170038, respectively). The RNA-seq reads supporting the conclusions of this article are available in the Sequence Read Archive (SRA) with accession ID DRA005094, https://www.ncbi.nlm.nih.gov/sra/.
Conceived and designed the experiments: AK and HT Performed the experiments: AK, YN, KaI, and AT Contributed reagents/materials/analysis tools: KiI, HB, TY, AF and RS Analyzed the data AK, TN, HB and HT Contributed to the writing of the paper AK, and HT All authors discussed the data and helped with manuscript preparation. HT supervised the project. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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