Novel gene encoding precursor protein consisting of possible several neuropeptides expressed in brain and frontal ganglion of the silkworm, Bombyx mori

Peptides 30 (2009) 1233–1240

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Novel gene encoding precursor protein consisting of possible several neuropeptides expressed in brain and frontal ganglion of the silkworm, Bombyx mori Kanako Mitsumasu a, Yoshiaki Tanaka b, Teruyuki Niimi a, Okitsugu Yamashita a, Toshinobu Yaginuma a,* a b

Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan Invertebrate Gene Function Research Unit, Division of Insect Sciences, National Institute of Agrobiological Sciences, Oowashi 1-2, Tsukuba 305-8634, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 September 2008 Received in revised form 4 March 2009 Accepted 9 March 2009 Available online 10 April 2009

A novel gene (BmK5) expressed in the central nervous system of the silkworm, Bombyx mori, was isolated using a cDNA subtraction method. BmK5 was first cloned as a candidate regulator of diapause hormone release from subesophageal ganglion via corpus cardiacum–corpus allatum into the hemolymph; however, subsequent analyses revealed that the gene expression patterns in brain–subesophageal ganglion complexes did not differ between diapause and nondiapause egg producers. The deduced amino acid sequence showed the characteristics of secretory protein precursor or nuclear localization protein. Immunohistochemical experiments with an anti-BmK5 antibody revealed that BmK5 precursor protein exists in the cytoplasm of specific cells of brain and frontal ganglion, but not in the nuclei. In addition, a peptide (GSGTKVGGAGAATKVVTKSGS-NH2) possibly processed from the BmK5 precursor protein was immunohistochemically detected in the axons connecting the anti-BmK5 antibody-positive cells to the neurohemal organ, corpus cardiacum–corpus allatum. These results suggest that BmK5 encodes a precursor of the novel neurosecretory protein and that several mature peptides are released into the hemolymph via the corpus cardiacum–corpus allatum, although the functions of these peptides are yet unclear. ß 2009 Elsevier Inc. All rights reserved.

Keywords: Bombyx mori BmK5 gene Novel neuropeptide Frontal ganglion Brain

1. Introduction Insects receive and integrate signals from environmental stimuli such as photoperiod and temperature through the brain and central nervous system (CNS), in order to synchronize growth and development in their population. Environmental signals are transduced into endogenous chemical messengers such as neurotransmitters, neuropeptides and hormones that are expressed in and released from the CNS to regulate physiological events in target organs [45]. A typical example is insect diapause [45]. In the silkworm, Bombyx mori, embryonic diapause is predetermined by a neuropeptide (diapause hormone, DH), which is synthesized in the mother’s subesophageal ganglion (SG), released via the neurohemal organ, corpus cardiacum–corpus allatum complex (CC–CA), into the hemolymph, and then targets developing ovaries during the pupal stage [15,16,21]. In B. mori bivoltine strains, whether the mother pupa releases DH into the hemolymph depends on her

* Corresponding author. Tel.: +81 52 789 4039; fax: +81 52 789 4036. E-mail address: [email protected] (T. Yaginuma). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.03.023

embryonic experience which is an incubation at 25 8C under light conditions or 15 8C under darkness [15–17,25,42]. In order to understand the molecular processes between the environmental signal during mother’s embryonic development and the release of DH, we must clarify the molecular mechanisms for releasing DH in diapause egg-producing pupae, or for blocking the DH release in nondiapause egg-producing pupae. Thus, we focused on the isolation of genes encoding such factors, which are expressed preferentially in the brain–SG of diapause or nondiapause egg producers. For this purpose, we adopted a cDNA subtraction method, selective amplification via biotin- and restriction-mediated enrichment (SABRE) [19], and then isolated several candidate genes. In this study, we describe the detail of analyses focused on one of them, the BmK5 gene. Unfortunately, as the gene was shown to be expressed at similar levels in both diapause and nondiapause egg-producing pupae, the relationship between the BmK5 gene and the release of DH was still unclear. Nevertheless, the mRNA was much more abundant in pupal brain–SG, while the translated product was localized in specific cells of the brain and frontal ganglion. As similar neurohormones or neuropeptides were not found in databases, the function of the gene product remains to be

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determined. However, this study suggests that the BmK5 gene encodes novel insect neuropeptides. 2. Materials and methods 2.1. Insects Larvae of the silkworm, B. mori, were reared on fresh mulberry leaves at 25–26 8C. Pupae were kept at 25 8C. For RNA extraction, various tissues were collected as follows: Eggs of no. 459 strain, which shows sex-specific pigmentation in serosa cells, were collected during embryonic development; samples of female brain–SG complex of the bivoltine Daizo strain were dissected each day from pupal ecdysis to adult eclosion; brain–SG, fat body, midgut and ovaries of 3-day-old female pupae of Daizo were collected. Samples were stored at 80 8C until use. For immunohistochemistry, female brain–SG–frontal ganglion (FG) complexes of Daizo, Shin-asagiri or a polyvoltine N4 strain were dissected at various developmental stages, and were treated as described below. 2.2. RNA extraction, first-strand cDNA synthesis and cloning of BmK5 cDNA RNA was prepared from about 25 pupal brain–SG complexes with TRIZOL reagent (Gibco BRL, Paisley, UK) according to the manufacturer’s protocol. First-strand cDNA was synthesized in 1 reverse transcriptase buffer (10 ml) containing 2 mM DTT, 1 mM dNTP, Superscript II (Gibco BRL), 1 mM modified oligo-dT and 1 mg of RNA. For cDNA subtraction, we adopted the SABRE method [19]. We used cDNAs from brain–SGs of 1- to 3-day-old diapause eggproducing pupae as a tester, and cDNAs from brain–SGs of 1- to 3day-old nondiapause egg-producing pupae as a driver, and vice versa, and we then selected tester homohybrid species by SABRE [19]. We selected several homohybrid species including BmK5 when cDNAs from diapause egg producers were used as a tester. Based on the partial nucleotide sequence of this BmK5, the fulllength cDNA was determined by 50 - and 30 -rapid amplification of cDNA ends (RACE) using the SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA). The nucleotide sequence of the open reading frame (ORF) was further confirmed by sequencing the nucleotides of PCR products amplified with two primer sets: 50 CTGTCCATCGACTATTCTGACCGACCGTGACTATG-30 (nucleotide nos. 155–189) and the antisense of 50 -CTTGTACACCCTTGCTGAGCTACTCCGCTCTG-30 (nucleotide nos. 1221–1252); and 50 -GAAGCTCAGGAACGCCGAAAGCAAG-30 (nucleotide nos. 853–877) and the antisense of 50 -CGCAGTCTGTAGCGGATACCTAACC-30 (nucleotide nos. 1501–1525) (GenBank accession no. AB162718) (Fig. 1). 2.3. Sequencing and sequence analysis PCR products were subcloned into the EcoRV site of a pBluescript KS+ vector (Stratagene, La Jolla, CA). Nucleotide sequence determination was performed by the dideoxy chaintermination method using an automatic DNA sequencer (ABI model 373A; PE Biosystems, Foster City, CA). Sequence analysis was performed using the DNASIS system (Hitachi Software Engineering, Yokohama, Japan). 2.4. Quantitative real-time PCR Quantitative real-time PCR was carried out using SYBR Green I doubled-stranded DNA binding dye chemistry on the GeneAmp 5700 Sequence Detection System (PE Biosystems). The following primers were used: 50 -CAGATCTTGCGTTCATACGGAAC-30 (nucleo-

tide nos. 1315–1339; two insertions of C at the positions of 1317 and 1325 were finally found after the sequence confirmation described in Section 2.2, see Fig. 1) and the antisense of 50 GCAGTCTGTAGCGGATACCTAACC-30 (nucleotide nos. 1502–1525) (GenBank accession no. AB162718) (Fig. 1). There was no problem with quantification of BmK5 mRNA using quantitative real-time PCR and this primer set (data not shown). For normalization, mRNA levels were divided by Bombyx ribosomal protein 49 (rp49) mRNA levels, which were quantified in the same manner [24]. 2.5. Northern blot analysis Eggs (no. 459 strain) were treated with HCl solution (specific gravity 1.075 at 15 8C) at 46 8C for 5 min to prevent diapause initiation at 21 h after oviposition. Eggs could be divided into male and female groups from 36 h after oviposition. Sex of embryos was judged based on pigmentation in serosa cells. Larvae hatched at 10 days after oviposition. Poly (A)+ RNAs were extracted from eggs in various stages. Ten micrograms of poly (A)+ RNAs were separated with 1% agarose gel, transferred to Hybond N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, UK) and probed with a-[P32]-labeled cDNA fragment containing 1250 bp from the 50 -end of the BmK5 gene. Blots were rinsed with 2 SSPE (20 mM NaH2PO4, 300 mM NaCl, 2 mM EDTA, pH 7.4) and analyzed by BAS2000 (Fuji Film, Tokyo, Japan). 2.6. Production of antibodies specific to BmK5 As antigens specific to the precursor form of BmK5 and an amidated peptide of BmK5, deduced from the amino acid sequence to be processed and modified through the protein maturation process, two peptides [C-VPTPSNNKDGSTISELPEN (BmK5) and CGSGTKVGGAGAATKVVTKSGS-NH2 (peptide 2)] corresponding to amino acid nos. 18–36 (box in Fig. 1) and to amino acid nos. 121– 141 (Figs. 1 and 7) of BmK5 (GenBank accession no. AB162718) were chemically synthesized. These peptides were respectively conjugated via an additional cysteine to keyhole limpet hemocyanin or bovine serum albumin as carriers, and were then injected into rabbits (BioGate, Yamagata, Japan). Using affinity columns, antibodies specific to the respective peptides were purified (BioGate). 2.7. Whole-mount immunohistochemistry All steps were performed as described previously [25,34]. FG– brain–SG complex fixed with 4% PFA/PBS (4% paraformaldehyde in 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4) was rinsed with PBS three times, dehydrated through a graded methanol series, and stored in 90% methanol/EGTA at 30 8C until use. Dehydrated tissue was then re-hydrated and equilibrated with PBS containing 0.2% of Tween 20 (PBT0.2). Specimens were bleached with 3% H2O2/PBS at 37 8C for 1 h, washed with PBT0.2, and permeabilized with PBS containing 2% of Tween 20 for 2 days at 4 8C. After washing with PBT0.2 twice, tissues were blocked with 2% BSA/PBT (B/PBT) and subsequently incubated with anti-BmK5 antibody (1:500 dilution) for 2 days at 4 8C. After rinsing with PBT0.2 four times and blocking with B/PBT, tissues were treated with Cy3-conjugated anti-rabbit IgG (Jackson Immuno Research, West Grove, PA; 1:500 dilution) overnight at 4 8C. Specimens were then washed with PBT0.2 five times and PBS once, and were mounted onto slide glasses with Fluoroguard (Bio-Rad, Hercules, CA). Detection was performed by confocal microscopy (Model LSM510, Carl Zeiss Japan, Tokyo) and purple staining indicated a positive reaction. In Fig. 7, CNS from Shin-asagiri was subjected to a whole-mount immunohistochemical procedure, as described by Roller et al. [32]. Anti-peptide 2 antibody was employed in this study at 1:4000

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Fig. 1. Nucleotide and deduced amino acid sequences of BmK5 cDNA (GenBank accession no. AB162718). *, stop codons. Putative polyadenylation signal is indicated by bold letters with dots. The ATTTA motifs are indicated by italic letters. Underlining and arrowheads indicate signal peptide and putative cleavage sites, respectively. Residues indicated by bold type with double-underline are putative protease cleavage sites. Bolded and italic letters represent amidation sites. Boxed amino acid residues are a peptides synthesized as an antigen. Underlined and double-underlined nucleotide sequences (nucleotide nos. 155–189 and 1221–1252; 853–877 and 1501–1525) indicate the primers used to amplify the region containing the ORF. Overlined nucleotide sequences (nucleotide nos. 1315–1339 and 1502–1525) indicate the primers used for quantitative real-time PCR.

dilution. As a second antibody, goat anti-rabbit IgG labeled with horseradish peroxidase (HRP) was used. HRP was stained with 0.05% 3,3-diaminobenidine tetrahydrochloride and 0.01% H2O2. To observe labeled preparations, we used the Leica MZ16 compound microscope and Leica DMLB microscope equipped with Leica DC300 photographic equipment (Leica Microsystems, Wetzlar, Germany). 3. Results and discussion 3.1. Cloning of full-length cDNA of BmK5 In the bivoltine Daizo strain of B. mori, embryonic diapause in the progeny occurs only when the mother experiences 25 8C under light conditions at the embryonic stage. DH is released only from the SG of diapause egg-producing pupas via CC–CA, although both diapause and nondiapause egg-producing pupae synthesize DH in the SG [15–17,25,42]. The brains of diapause and nondiapause egg producers are reported to regulate DH release in a stimulatory and inhibitory manner, respectively [10,20,37,38]. In this study, we attempted to identify the genes differentially expressed in the brain–SGs of diapause or nondiapause egg producers as candidate regulators of DH release.

We used the cDNA subtraction method, SABRE [19] with mRNAs extracted from brain–SG complexes of 1- to 3-day-old pupae in order to isolate genes expressed preferentially in diapause or nondiapause egg-producing pupae. We isolated several fragmented cDNAs for genes that are expressed abundantly in the brain–SG of diapause egg-producing pupae. One of these cDNAs was BmK5 (Bm and K5 represents B. mori and the isolated number, respectively). Subsequently, we determined the nucleotide sequence of a full-length cDNA (1927 nucleotides) of the BmK5 gene using 50 - and 30 -RACE (Fig. 1). As nucleotide nos. 172–174 is a stop codon (TGA), the downstream nucleotides 187–189 (ATG) were regarded as the translation initiation methionine. Another stop codon (TAA) was seen at nucleotide nos. 1303–1305; thus, the ORF consisted of 1116 nucleotides coding for 372 amino acids with a calculated molecular weight of 42,009. There were nine copies of the ATTTA motif, which is found in unstable mRNAs [36], at the 30 untranslated region and a polyadenylation signal (AATAAA) at nos. 1791–1796. A signal peptide leader was found at amino acid nos. 1–17 of the deduced amino acid sequence (Fig. 1). In addition, several consensus sequence were identified; the RRKR nuclear localization and cleavage site; KKR, RRK, KK, KR and RR cleavage sites; and GKK and GR amidation signals [5,18,41,43]. Although we

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compared this whole amino acid sequence with those in all DDBJpublished databases (http://www.ddbj.nig.ac.jp/index-e.html) by BLAST search, we could not find similar proteins. Thus, we tentatively designated this cDNA as BmK5 (GenBank accession no. AB162718). 3.2. Developmental profiles of BmK5 mRNA in brain–SG complex during pupal–adult development As the BmK5 gene was thought to be expressed more abundantly in the brain–SG of diapause egg-producing pupae than nondiapause egg-producing pupae, we followed the mRNA levels of BmK5 in brain–SGs of diapause and nondiapause egg producers during pupal–adult development. For measuring mRNA levels, we used a quantitative real-time PCR (ABI 5700 system). When diapause or nondiapause egg-producing pupae were incubated at 25 8C, adult emergence occurred 8 or 7 days later, respectively. Therefore, the duration from pupation to adult emergence was expressed as 100% in order to compare physiological events between diapause and nondiapause egg producers. In brain–SG complexes for diapause egg-producing pupae, BmK5 mRNA levels increased gradually with age (Fig. 2). The pattern of mRNA expression in nondiapause egg producers was similar to that of diapause egg producers. Surprisingly, there were no significant differences between the levels of BmK5 mRNA in diapause and nondiapause egg producers, indicating that BmK5 is a false-positive gene. Further investigation on BmK5 mRNA expression profiles demonstrated that the BmK5 gene exhibits a distinctive expression pattern: when the mRNA level in brain–SG from 3-day-old diapause egg producer was regarded as 100, those in fat body, midgut and ovary were respectively 0.020–0.051, 0.256–0.320 and 0.010–0.019 (Fig. 3). This tissue distribution analysis of BmK5 mRNA revealed the abundant expression of BmK5 mRNA in the brain–SG complex: BmK5 mRNA was expressed at higher levels in the brain–SG complex than in other tissues by two or three orders of magnitude (Fig. 3). As BmK5 mRNA expression was confirmed in brain–SG complex, we then investigated when mRNA expression began to occur during embryonic development. At 6 days after oviposition, mRNA clearly appeared in males on Northern blotting membranes (Fig. 4), as a major band at 2.1 kb, which corresponded to size of

Fig. 2. Profile of BmK5 mRNA levels in brain–SG complexes during pupal–adult development. Brain–SG complexes were dissected each day from both types of female pupae and adults of Daizo. Diapause and nondiapause egg-producing adults were eclosed at 8 and 7 days, respectively. Here, the duration from pupation to adult emergence was expressed as 100% to compare physiological events between diapause and nondiapause egg producers. Levels of BmK5 mRNA were quantified by real-time PCR and normalized against Bmrp49 mRNA. The results are shown as amounts relative to the value of 0-day-old diapause egg-producing pupae. Each point represents means  SEM (n = 2). Closed circles, diapause egg producers; Open circles, nondiapause egg producers.

Fig. 3. Tissue distribution of BmK5 mRNA. Tissues were dissected from 3-day-old female diapause and nondiapause egg-producing pupae of Daizo. Total RNA was extracted from the brain–SG, fat body, midgut and ovary, and first-strand cDNA was synthesized from 1 mg of total RNA. Quantification was performed by real-time PCR. Levels of BmK5 mRNA were normalized against Bmrp49 mRNA. mRNA levels were expressed in the logarithmic scale (mRNA level in brain–SGs from diapause egg producers was regarded as 1). Values shown are means  SEM (n = 2). Closed bars, diapause egg producers; Open bars, nondiapause egg producers.

BmK5 cDNA (1.9 kbp in Fig. 1). The BmK5 gene in males was expressed more rapidly than in females (Fig. 4). This expression probably reflects the faster development of males, as the similar expression pattern of the Sex-lethal gene was observed at the same time [29]. In fact, larval hatching was more rapid in males than in females. Thus, the pattern in mRNA levels corresponds with changes in morphology of the brain and SG [11,12,25], suggesting that the expression of BmK5 mRNA is induced in parallel with the development of the CNS. The specificity of BmK5 mRNA expression in the nervous system was thus interesting, despite the finding that BmK5 gene is probably not related to DH release; therefore, we decided to further characterize the BmK5 gene. 3.3. Localization of BmK5 protein in specific cells of CNS In order to define the localization of BmK5 gene products in the CNS, we first attempted in situ hybridization using BmK5 cDNA and brain–SG complex. However, we were unable to identify clear sites showing positive signals. Thereafter, we produced an antibody against a synthetic peptide corresponding to amino acid nos. 18– 36 of BmK5 protein (boxed region in Figs. 1 and 6). Several cells in the frontal ganglion and brain were immunostained with this antibody (Fig. 5). These staining intensities changed reverseproportionally in the antigen-dose dependent manner when the antibody was pre-incubated with the antigen peptide, while the pre-incubation with a peptide corresponding to amino acid nos. 216–238 of B. mori sorbitol dehydrogenase-2 (GenBank accession no. AB164059) never affected the staining (data not shown). Furthermore, when an antibody against the peptide for sorbitol dehydrogenase-2 was used, these specific cells in brain and FG

Fig. 4. Changes in BmK5 mRNA levels during embryonic development. Ten micrograms of poly (A)+ RNA was loaded in each lane, followed by separation and analysis by Northern blotting. When the same blot membrane was hybridized with the Bmrp49 cDNA as a control, levels of Bmrp49 mRNA were shown to be almost constant [29]. Eggs (no. 459 strain) with pigmented serosa cells were female. Male larvae hatched faster than females.

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Fig. 5. BmK5 immunoreactivity in the brain (Br) and frontal ganglion (FG). Tissues were dissected from various stages of N4, fixed with 4% PFA and stained with anti-BmK5 antibody against a peptide corresponding to amino acid nos. 18–36 of BmK5. Preparations were observed by confocal microscopy. Positive signals were detected in several specific cells of brain (arrowheads) and FG (arrows). A, 1st instar 0-day-old larva; B, 5th instar 4-day-old larva; C, 5th instar 6-day-old larva; D, 5th instar 7-day-old larva; E, 0day-old pupa; F, 3.5-day-old pupa; G, 6-day-old pupa; H, 0-day-old adult; SG, subesophageal ganglion. Reproducibility was poor when detecting positive signals was carried out in SG, although the reasons were unclear. Scale bars, 10 and 50 mm (left and right in A, respectively); 100 mm (in B–H).

were not stained (data not shown). Thus, the staining seems to result from the specific binding of this antibody to BmK5 protein. We followed the transition of staining patterns for these cells during the post-embryonic development of the silkworm. No differences were seen between diapause and nondiapause egg producers of Daizo, or between a commercial hybrid race (diapause egg producer) and the polyvoltine N4 strain (nondiapause egg producer) (data not shown). Further investigation was performed using the nervous tissues from N4 individuals. In newly hatched larvae, two cells in the FG and several medial cells in the brain were faintly immunostained (Fig. 5A). These cells in FG and brain were stained more clearly in the middle of the 5th instar larval stage (Fig. 5B–D), and lateral cells in the brain were also detected (Fig. 5B–D). In the early and middle pupal stages, four positive-stained cells were observed in the FG (Fig. 5E and F), and cells of the middle and lateral regions of the brain (Fig. 5E and F) were stained more intensely than in the larval stage. These signals became weak in 6-day-old pupal FG and brain (Fig. 5G). When adults emerged, immunostaining was weak, and the number of immunostained cells decreased; only four pairs of medial cells in the brain and two cells in the FG (Fig. 5H). Some cells in the SG were immunostained in some samples (broken line in Fig. 5F), but in other samples, positive signals were not observed, although the reasons remained unclear. No significant immunos-

taining was observed in the nuclear region of the anti-BmK5 antibody-positive cells or in the axons running from the anti-BmK5 antibody-positive cells to the CC–CA, where neurosecretion occurs. 3.4. Several neuropeptides may be processed from BmK5 precursor protein Although the BmK5 protein contains four copies of the RRKR motif (nuclear localization signal), it also has a signal peptide leader, and immunohistochemical analysis revealed that BmK5 protein is present in the cytoplasm of specific cells in the brain and FG, and was absent in the nuclei (Fig. 5). As described previously, the deduced amino acid sequence of BmK5 has several proteolytical cleavage sites [18,27,31,41,43] in addition to a signal peptide leader, which are the characteristics of secretory protein precursors (Fig. 1). These results suggest that the BmK5 protein is a type of neurosecretory protein and a precursor protein. In fact, the amino acid sequence of BmK5 with characteristic endoproteolytic cleavage sites (usually KR) seems to fit the structural criteria of neuropeptide preproproteins that were proposed based on the neuropeptide-like protein genes identified in Caenorhabditis elegans and other invertebrate species [27]. Various neurosecretory proteins were isolated from insect species and were localized in neurosecretory cells, while axons connected these cells to the CC–

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Fig. 6. Possible processed peptides of BmK5. The putative signal peptide is shaded. BmK5 has 11 putative cleavage sites, indicated by black. Peptides 2 and 7 have a putative amidation site. Peptides 2 and 4, and peptides 3 and 6 respectively have identical residues (double-underlined). Peptides 7 and 9 have identical residues (underlined). Boxed peptide was synthesized as an antigen. pE, pyroglutamate.

CA for release into the hemolymph [23,30,44]. However, the absence of BmK5 protein in the axons connecting the anti-BmK5 antibody-positive cells to CC–CA (Fig. 5) apparently conflicts with speculation that BmK5 is a neurosecretory precursor protein. The amino acid sequence of BmK5 shows that the protein has eleven putative prohormone convertase-recognition sites [18,27,31,41,43]. Several peptides could be processed based on the putative cleavage sites RRKR, KKR, RRK, KK, KR and RR, and amidation sites GKK and GR (Fig. 6). If these processed peptides pass through the axons, they would not be stained by antibody recognizing only the peptide corresponding to amino acid nos. 18– 36 shown in Fig. 1; therefore, we decided to prepare another antibody that recognizes peptides conceivably produced after processing and modification. Among candidate peptide sequences for the antigen, peptides 2 and 4 (GSGTKVGGAGAATKVVTKSGSamide and GSGTKVGGAAASAKTATKNSGGN, respectively; identical amino acid residues are italicized when they are aligned at the Nterminal side) shown in Fig. 6 are particularly interesting because neuropeptide precursors are typically known to include several homologous peptide fragments activated after processing and modification [7,36], and the sequences of peptides 2 and 4 are very similar among the peptide fragments possibly produced after BmK5 processing. Thus, we synthesized a peptide corresponding to peptide 2 with amidation at the C-terminus and used an antibody raised against peptide 2 to confirm whether BmK5 passes into the axons. Distinct staining in the axons [nervi corpora cardiaci (NCC) and nervi corpora allati (NCA)] joining to the CC–CA, as well as in the antiBmK5 antibody-positive cells in the brain and FG was observed (Fig. 7), indicating that BmK5 protein is processed into several neuropeptides, which are transported to the CC–CA and released into the hemolymph. In the present study, we isolated a novel gene expressed abundantly in the CNS of B. mori, although it is unclear whether the gene is involved in the regulation of the DH release. The function of BmK5 protein was initially unpredictable, as homologous sequences were not significantly found between BmK5 protein and known proteins; BmK5 protein localization, however, suggests that the protein is a precursor of neurosecretory peptides (Figs. 5 and 7). Generally, neurosecretory proteins act as neurohormones, neurotransmitters and neuromodulators through maturation steps to become active peptides and are localized in specific neurosecretory cells and neurons [26]. In B. mori and other lepidopteran insects, the detailed histological experiments on neurosecretory cells and neurons expressing several neuropeptides have been carried out [1–4,13,23,44]. Based on the location, size, and shape of such cells in nervous cells [1–4,13,23,44], the anti-BmK5 antibodypositive cells in brain and FG (Figs. 5 and 7) are thought to correspond to neurosecretory cells and neurons expressing neuropeptides. For examples, the four pairs of medial neurose-

cretory cells produce an insect insulin-like peptide, bombyxin, in B. mori [22] and tachykinin-related peptide in Spodoptera litura [14]. The lateral neurosecretory cells express prothoracicotropic hormone (PTTH) in B. mori [1,23] and Manduca sexta [44]. The FG neurons also express PTTH in M. sexta [44], and allatostatin and allatotropin in Helicoverpa armigera, Heliothis virescens, Lacanobia oleracea, S. frugiperda and M. sexta [2–4]. Bombyxin and other insect insulin-like peptides have been shown to act as regulators of carbohydrate metabolism, cell differentiation and lifespan [9,28,33,40]. Although PTTH is known to originally regulate biosynthesis of ecdysteroid in the prothoracic gland [8], PTTH is also thought to be involved in controlling feeding behavior [44]. Allatotropin and allatostatin are known to regulate the activity of JH biosynthesis in the CA [6,7,26,39], whereas the neuropeptides produced in the FG are involved in the modulation

Fig. 7. Immuno-localization of a peptide (GSGTKVGGAGAATKVVTKSGS-NH2 corresponding to amino acid nos. 121–141) possibly processed from the BmK5 precursor protein in the central nervous system. Tissues from Shin-asagiri were dissected from 4th instar 2-day-old larvae (A–C) or from 6-day-old pupae (D), fixed and stained with anti-peptide 2 antibody (see Fig. 6). Positive signals were detected in the anti-BmK5 antibody-positive cells (arrowheads) of the brain (Br) and the frontal ganglion (FG), and in axons (arrowheads) connected to the CC–CA. FC, frontal connective; NCC, nervi corpora cardiaci; NCA, nervi corpora allati. Scale bars, 100 mm.

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of foregut contraction [2–4]. Westbrook et al. [44] suggested that the targets and physiological functions of a neuropeptide vary depending on where the neuropeptide is produced and when it is released. Therefore, BmK5 could also have multiple roles in the developmental and physiological events, as BmK5 is present in neurosecretory cells and neurons of diverse regions. In addition, because BmK5 mRNA or protein expression was detected at embryonic, larval and pupal–adult developmental stages (Figs. 2, 4, 5 and 7), this gene is likely expressed at almost all developmental stages after differentiation of the CNS is completed, indicating that BmK5 has physiologically fundamental roles. BmK5 protein produced in the medial and lateral neurosecretory cells of the brain are likely released into the hemolymph via the CC–CA to act as a neurohormone to regulate certain biological events, and peptides in the FG are thought to participate in the modulation of foregut contraction. A remaining problem is that we were unable to elucidate the mature form, activity and function of BmK5 peptide. Neurosecretory cells and neurons producing neuropeptides also express prohormone convertases that are necessary to process the precursor proteins into mature forms [27,31,41,43]; therefore, to be qualified as a neuropeptide precursor protein, BmK5 protein is likely to be processed by these enzymes. Additionally, many active neuropeptides are known to have amidated C-termini [5]. The antibody raised against peptide 2, which has an amidated Cterminus, recognizes axons connecting the anti-BmK5 antibodypositive cells to the CC–CA (Fig. 7). This suggests that at least one of the mature forms of BmK5 includes amino acid residues corresponding to peptide 2 or may be amidated peptide 2 itself and that this peptide is transported into and released from the CC– CA. In addition, the amino acid sequence of peptide 4 is similar to that of peptide 2 (Fig. 6). Multiple homologous neuropeptides are occasionally encoded in a gene of the precursor protein [7,27,35]. To elucidate whether peptide 2 and peptide 4 are active neuropeptides, it is necessary to isolate the peptides from expressing tissues and hemolymph, and to confirm that the peptides show biological activity. At present, no information on the target of BmK5 peptide or its activity on the target is available. Because neuropeptides generally bind to specific receptors, which are mainly G-protein-coupled receptors or receptor tyrosine kinases, to elicit activity, the target of BmK5 would also have a receptor specific to this ligand. Detailed annotation on the genomic DNA sequence of B. mori is in progress, and neuropeptide receptor homologs encoded in the B. mori genome will eventually be clarified. Among these, several orphan receptors may be worth evaluating for binding ability with BmK5 peptide and activity in accelerating signaling pathways via appropriate second messengers. Information on the receptor for BmK5 will lead to elucidation of the role of BmK5 in the physiology of B. mori. The novel neuropeptide, BmK5, may provide new insights into the regulation of physiological events in B. mori and other insects. Acknowledgements We would like to thank Prof. Michihiro Kobayashi and Assoc. Prof. Motoko Ikeda of the Graduate School of Bioagricultural Sciences, Nagoya University, for helpful discussions. We are also grateful to Prof. Kunio Imai, Mie University, Prof. Kei-ichiro Maeda, Nagoya University, and Assoc. Prof. Kunihiro Shiomi, Shinshu University, for their advice concerning neuropeptides, and the National Institute of Agrobiological Sciences for providing silkworm strain no. 459. This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for the Promotion of

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