Archives of Insect Biochemistry and Physiology 67:87–96 (2008) Functional Analysis of Four Gloverin-like Genes in the Silkworm, Bombyx mori Shinpei Kawaoka,1 Susumu Katsuma,1 Takaaki Daimon,1 Ryoko Isono,1 Naoko Omuro,1 Kazuei Mita,2 and Toru Shimada1* To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1–4, in the Bombyx genome. Northern blot and RT– PCR analysis showed that BmGlov1–4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-κB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1–4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1–4 significantly inhibited the growth of E. coli. Arch. Insect Biochem. Physiol. 67:87–96, 2008. © 2007 Wiley-Liss, Inc. Keywords: Bombyx mori; EST; antibacterial protein; Gloverin INTRODUCTION In multicellular organisms, low-molecularweight, nonspecific antimicrobial proteins play an important role in the innate immunity to combat infectious microorganisms (Hoffman et al., 1999). These antimicrobial proteins are induced by invasion of microorganisms or an injury. In the study of the innate immunity of the silkworm Bombyx mori, at least four groups of antimicrobial proteins have been discovered: Cecropin, Attacin, Lebocin, and Moricin (Morishima et al., 1990; Sugiyama et al., 1995; Hara and Yamakawa, 1995a,b). These antimicrobial proteins might act with each other, and this cooperation may help in developing an effective defense mechanism against microorganisms. Many gene families encode antimicrobial proteins. Some of them, such as the Cecropin fam- ily (Ramos-Onsins and Aguade, 1998), are conserved beyond order or species, while others, such as the Drosomycin family (Jiggins and Kim, 2005), are specific among order or species. There are glycine-rich antibacterial proteins with molecular masses ranging from 8 to 30 kDa. Attacin is a representative of glycine-rich antibacterial proteins. These proteins inhibit the growth of gram-negative bacteria by interacting with lipopolysaccharides (LPS) and by specifically inhibiting the formation of outer membrane (Carlsson et al., 1991, 1998). The permeabilization of the bacterial outer membrane allows the large molecular antibacterial proteins to access the inner membrane easily, which results in setting a synergic interaction between these antibacterial proteins (Engström et al., 1984; Lazzaro and Clark, 2001). Gloverin, isolated from the giant silk moth Hyalophora gloveri, 1 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan 2 National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan Contract grant sponsor: Grants-in-Aid for Scientific Research (MEXT); Contract grant number: 17018007; Contract grant sponsor: Insect Technology–Agrigenome Program (MAFF-NIAS); Contract grant sponsor: National Bioresource Project (MEXT); Contract grant sponsor: Program for Agricultural Bioinformatics (JST). *Correspondence to: Dr. T. Shimada, Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: firstname.lastname@example.org Received 15 March 2007; Accepted 6 May 2007 © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20223 Published online in Wiley InterScience (www.interscience.wiley.com) 88 Kawaoka et al. also belongs to this group and seems to be a lepidopteran-specific protein (Axen et al., 1997). In this study, we describe the functional analysis of four Gloverin-like genes, BmGlov1–4, in B. mori, and show that recombinant BmGlov1–4 expressed by a baculovirus vector can cause the inhibition of bacterial growth. MATERIALS AND METHODS Insects, Cell Lines, and Viruses B. mori larvae (F1 hybrid Kinshu × Showa) were reared as described previously (Katsuma et al., 2005). The Sf-9 and High Five cell lines were cultured at 27°C in TC-100 medium supplemented with 10% fetal bovine serum and Express Five medium (Invitrogen, Carlsbad, CA), respectively. Autographa californica nucleopolyhedrovirus (AcMNPV) was propagated in the Sf-9 cells, and the virus titers were determined by a plaque assay on the Sf-9 cells. Alignment and Phylogenetic Analysis Amino acid sequences and nucleotide sequences of lepidopteran Gloverins were aligned using the CLUSTALW program (Thompson et al., 1994). Distances between the nucleotide sequences were calculated according to the F84 model with DNADIST in the PHYLIP 3.65 package (Felsenstein, 1989). A Neighbor-joining tree was constructed with the NEIGHBOR program in PHYLIP (Felsenstein, 1989). The reliability of the tree was tested by bootstrap analysis with 1,000 replications. Immune Challenge Escherichia coli (JCM5491; ATCC 25299) and Candida albicans (JCM1621; ATCC 10264) were grown overnight at 37°C in Luria-Bertani (LB) medium and potato-dextran medium, respectively. The cells were washed twice with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4), and the bacterial concentration was adjusted to an absorbance of 0.01 at 600 nm. Three-day-old fifth instar B. mori larvae were injected with 50 µl of E. coli or C. albicans suspensions. We collected fat body from the larvae at 0, 6, 12, 24, 36, and 48 h after the injection, and then isolated total RNA from these tissues using the Trizol reagent (Invitrogen). Construction of Expressed Sequence Tag (EST) Database From Fat Body of Immune-Challenged B. mori Larvae We constructed a cDNA library from the fat body RNA of E. coli- and C. albicans-challenged B. mori larvae (6 h and 12 h after the injection) using the method described previously (Kato et al., 2005). We sequenced 10,752 clones from the cDNA library and annotated these clones by the BLAST program against public protein databases. Reverse Transcription–Polymerase Chain Reaction (RT–PCR) The total RNA was reverse-transcribed, diluted, and used for PCR as described elsewhere (Katsuma et al., 2005). First-strand cDNA reaction was performed with TaKaRa RNA PCR Kit (AMV) version 3.0 (TaKaRa, Japan), using oligo dT-Adaptor primer. Primers for PCR are shown in Table 1. Northern Blot Analysis Total RNA of the B. mori fat body was prepared using the Trizol reagent (Invitrogen). Northern blot analysis was performed as described previously (Katsuma et al., 2005). Probes for B. mori Gloverin1, Cecropin B, and Actin3 were amplified by PCR with the primers listed in Table 1 using the B. mori cDNA clones as a template. Generation of Recombinant AcMNPVs Recombinant AcMNPVs were constructed using a Bac-to-Bac system (Invitrogen). The coding region of the BmGlov proteins (BmGlov1, BmGlov2, BmGlov3, and BmGlov4) with a His-tag sequence at the C-terminus was amplified by PCR with the Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. Characterization of B. mori Gloverin Genes 89 TABLE 1. PCR Primers Used in This Study Nucleotide sequence (5′–3′) Application CACGACTTTGTCACTTGG GCTTACGAGGCAAGAATG GTCTTGAGGAGCGAAACT GTCATAACAAAGCACGAG ACTAGCCAAACCACAAAC GATGGGATTGTGTTGACA ATTGGGAGGACGAAGAAG TGCAGAGTGAAAGTATCG AGATGAACCCAGATCATGTTCG GAGATCCACATCTGTTGGAAG AGCGCCGCTTGTGTCTTAA TGGAAATCCAGTCTTATACGAG CTAGGATCCAGAAATGTATTCCAAGGTGTTGTTATCC AGTGGATCCTCAATGGTGATGGTGATGATGACCCCACTCGTGAGTAATCTGGGCCTG CTAGGATCCAGAAATGAATACAAATCTGTTTTATATCTTCGC AGTGGATCCTCAATGGTGATGGTGATGATGACCCCAATCATGGCGGATCTCTGCTTG CTAGGATCCAGAAATGAATTCCAAATTGCTGTTTTTCATCGC AGTGGATCCTCAATGGTGATGGTGATGATGACCCCACTCATGCCGGATCTCTGCTTG CTAGGATCCAGAAATGAATTCCAAACTATTATATTTCTTCGCCAC AGTGGATCCTCAATGGTGATGGTGATGATGACCCCAATCATGACGGAACTCTGCCTG BmGlov1 (Forward): RT–PCR and Northern blot BmGlov1 (Reverse): RT–PCR and Northern blot BmGlov2 (Forward): RT–PCR BmGlov2 (Reverse): RT–PCR BmGlov3 (Forward): RT–PCR BmGlov3 (Reverse): RT–PCR BmGlov4 (Forward): RT–PCR BmGlov4 (Reverse): RT–PCR BmActin3 (Forward):RT–PCR and Northern blot BmActin3 (Reverse):RT–PCR and Northern blot BmCecB (Forward): Northern blot BmCecB (Reverse): Northern blot BmGlov1-His (Forward) BmGlov1-His (Reverse) BmGlov2-His (Forward) BmGlov2-His (Reverse) BmGlov3-His (Forward) BmGlov3-His (Reverse) BmGlov4-His (Forward) BmGlov4-His (Reverse) gene-specific primers (shown in Table 1) using the B. mori cDNA clones as a template. The PCR products were cloned into the cloning site (BamHI) of the pFastBac1 vector (Invitrogen). The nucleotide sequence was confirmed by DNA sequencing using the ABI Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) and the ABI Prism 3100 DNA sequencer (Applied Biosystems). The recombinant AcMNPVs were generated by transfection with bacmid DNAs into Sf-9 cells and propagated in the Sf-9 cells. HisGraviTrap column (GE Healthcare Bioscience, New York, NY). The eluted solution was concentrated by using AmiconUltra 10K (Millipore), then desalted by using a PD-10 column (GE Healthcare Bioscience). Elution was performed with 0.1 M phosphate buffer (pH 7.4). The protein concentrations were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), followed by staining with Coomassie brilliant blue. The BmGlov proteins were stored at –80°C until use. Expression and Purification of BmGlovs Antibacterial Assays The High Five cells cultured in a 150-mm dish were infected with the recombinant AcMNPVs at an MOI of 5. After 60 h, the cells were collected and the expressions of BmGlov1–4 were examined by Western blot analysis using anti-His antibody (Qiagen, Germany), as described previously (Katsuma et al., 2006). Purification of BmGlov1–4 was performed by using nickel chromatography. The collected cells were lysed with I-PER Reagent (Pierce, Rockford, IL), containing protease inhibitor cocktail (Roche, Switzerland). After centrifugation at 8,000g for 15 min, the resulting supernatants were filtered with a 0.45-µm pore filter (Millipore, Billerica, MA), and loaded onto a E. coli (JM109) were used in this antibacterial assay. Five or 20 µg of the purified BmGlov1–4 was added to LB medium in 96-well microtiter plates that contained log-phase bacteria in a total volume of 100 µl. The growth of the bacteria at 37°C was determined by recording optical density 595 nm. Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. Nucleotide Sequence Accession Number The nucleotide sequence reported in this article has been submitted to the DDBJ/EMBL/GenBank data bank under the accession number AB190863AB190866 (BmGlov1–4). 90 Kawaoka et al. RESULTS We constructed a cDNA library from the fat body RNA of E. coli- and C. albicans-challenged B. mori larvae. DNA sequencing yielded 10,752 ESTs from the cDNA library, and these ESTs were clustered into 858 contigs. From the EST data, we found four kinds of clone that were homologous to Gloverin from Hyalophora gloveri (Axen et al., 1997). Based on the whole genome shotgun sequence (Mita et al., 2004; Xia et al., 2004) and phylogenetic analysis, we identified four loci encoding Gloverin-like genes in the B. mori genome and termed them BmGlov1, BmGlov2, BmGlov3, and BmGlov4, respectively (Fig. 1A). A more detailed EST data analysis showed that each BmGlov gene had putative alleles. Nucleotide sequence similari- ties between the alleles were very high (>97%). We found that the BmGlov1–4 cDNAs were 828, 805, 809, and 840 bp long and potentially encode proteins composed of 178, 173, 173, and 171 amino acids, respectively (Fig. 1B). Hydropathy plot analysis showed that the N-terminus of each BmGlov protein had features consistent with a signal peptide, suggesting that BmGlovs might be secreted proteins. The signal sequence cleavage site was predicted to lie between amino acid positions 17 and 18 of BmGlov1 or 18 and 19 of BmGlov2, BmGlov3, and BmGlov4 (Fig. 1B). The deduced amino acid sequence of BmGlov1 showed 84%, 84%, and 82% identity to BmGlov2, BmGlov3, and BmGlov4, respectively, and 77% identity to Manduca sexta Gloverin (Fig. 1B). Gloverins from other insects are produced in a prepropeptide form (Lundström et al., 2002). After cleavage of the signal peptide, a cleavage at Fig. 1. Phylogenetic tree and multiple alignment of Gloverins from lepidopteran insects. A: Neighbor-joining tree generated from lepidopteran Gloverin gene sequences. Bootstrap values of 1,000 replicates are indicated. B: Mul- tiple alignment of BmGlovs with Gloverin from Hyalophora cecropia (P81048), Manduca sexta (AAO74639), and Trichoplusia ni (AAG44367). Putative signal peptidase (S) and endopeptidase (X) processing sites are indicated. Isolation of B. mori cDNAs Encoding Gloverin-like Genes Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. Characterization of B. mori Gloverin Genes TABLE 2. Molecular Weight (Mw) and Isoelectric Points (pI) of BmGlovs BmGlov1 BmGlov2 BmGlov3 BmGlov4 prepro pro mature prepro pro mature prepro pro mature prepro pro mature Mw (kDa) pI 19.2 17.4 14.4 18.9 16.4 14.1 19.0 17.1 14.1 18.8 16.8 14.1 6.97 6.57 5.49 6.41 6.48 7.03 6.91 6.49 6.32 6.30 5.93 6.94 RHPR present on propeptide, which is a typical motif (-Arg-X-X-Arg-) of the cleavage site for endopeptidase (Molloy et al., 1992), could result in the production of the mature form of BmGlovs. Theoretical molecular masses of BmGlov1, BmGlov2, BmGlov3, and BmGlov4 were 14.4, 14.1, 14.1, and 14.1 kDa, respectively (Table 2). Isoelectric points of BmGlovs were 5.49, 7.03, 6.32, and 6.94, respectively (Table 2). The putative mature form of BmGlov1 showed 92%, 93%, and 91% identity to that of BmGlov2, BmGlov3, and BmGlov4, respectively, and 80% identity to that of M. sexta Gloverin (Fig. 1B). The BmGlov proteins exhibit a typical amino acid composition of the Gloverin family: many glycine residues (14%) and no cysteine residues (Axen et al., 1997). The locations of glycine residues were conserved among BmGlovs. Transcriptional Analysis of BmGlov Genes Temporal expression profiles of the BmGlov genes after the challenge of E. coli or C. albicans 91 were examined by Northern blot analysis. As shown in Fig. 2, the injection of E. coli caused a strong induction of the BmGlov genes at 6 h after the immune challenge, and this induction continued for 48 h after the immune challenge. In contrast, the injection of C. albicans did not induce the expression of the BmGlov genes. The expression of the BmGlov genes reached its highest level at 12–24 h after the bacterial challenge. One distinct band with an approximate size of 800 bases was detected by Northern blot analysis. We could not separate the transcripts of BmGlov1–4 by Northern blot analysis because of the similarities of their sequences and sizes. In addition, the expression pattern of Cecropin B was examined by Northern blot analysis. Cecropin B is a well-known antibacterial gene of B. mori (Taniai et al., 2006). As shown in Figure 2, Cecropin B was significantly induced in the larval fat body after the bacterial challenge, but less induction was observed after the C. albicans injection, confirming that the immune-challenge experiments performed in this study worked well. To distinguish the expression profiles of each BmGlov, we next performed RT–PCR analysis using primers specific to BmGlov1–4. We found that the expressions of all BmGlov genes were significantly induced by the injection of E. coli (Fig. 3). Sequence Analysis of Promoter Regions of BmGlovs We searched the partial promoter sequences of BmGlovs using the B. mori whole-genome shotgun sequence (Mita et al., 2004; Xia et al., 2004). In the upstream regions of each BmGlov gene, a puta- Fig. 2. Effects of the bacterial challenge on the expression of BmGlov, BmCecB, and Actin3. Three-day-old fifth-instar larvae were immune challenged with the injection of either Escherichia coli or Candida albicans. Northern blot analysis was performed using total RNA of the larval fat body prepared at 0, 6, 12, 24, 36, and 48 h after the injection. Actin3 is shown as a control. Molecular sizes are shown to the right of the figure and time courses and experimental group are indicated above. Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. 92 Kawaoka et al. BmGlovs in both the cells and the medium, suggesting that both the propeptides and mature forms of BmGlovs exist in the cells and medium of the recombinant AcMNPV-infected cells. We then purified recombinant BmGlovs from recombinant AcMNPV-infected cells to near homogeneity by using nickel chromatography (Fig. 4B), and performed antibacterial assays using different concentrations of BmGlovs (5 or 20 µg in 100 µl E. coli culture medium). As shown Figure 5, we observed that all Fig. 3. RT–PCR analysis of BmGlov1–4. The total RNA samples used in Northern hybridization were used in this experiment. Actin3 was used as a control, and time courses and experimental groups are indicated above. tive TATA box was located 21 nt upstream from the transcription start site (data not shown). Moreover, a clear consensus sequence for the transcription factor NF-κB binding site (GGGAMTTYCC) was found 48 nt upstream from the transcription start site of BmGlov1, BmGlov2, BmGlov3, and BmGlov4, suggesting that the BmGlov genes may be induced by the NF-κB signaling cascade (Silverman and Maniatis, 2001; data not shown). Expression and Antibacterial Assays of Recombinant BmGlovs To examine the biochemical properties of the products of the BmGlov genes, we generated recombinant AcMNPVs expressing His-tagged BmGlov proteins. With Western blot analysis using anti-His antibody, we found that the recombinant Histagged BmGlovs were successfully expressed as approximately 15–20 kDa proteins in the High Five cells infected with the recombinant AcMNPVs (Fig. 4A). Moreover, we observed faint bands in the medium of AcMNPV-infected High Five cells (Fig. 4A), indicating that the recombinant BmGlovs were slightly secreted into the medium. Positive bands were not detected in the High Five cells infected with wild-type (wt) AcMNPV (Fig. 4A). In addition, Western blot analysis detected two forms of Fig. 4. Expression and purification of BmGlov1–4. A: Expression of BmGlov1–4. The High Five cells were infected with the wt AcMNPV or recombinant AcMNPVs expressing BmGlov1, BmGlov2, BmGlov3, or BmGlov4. Expression of BmGlov1–4 was examined by Western blot analysis with anti-His antibody. The molecular weights of the protein standards are shown on the left. B: Purification of BmGlov1–4. Purification was performed by using nickel chromatography. One µg of each purified BmGlov was electrophoresed, and the gel was stained with Coomassie brilliant blue. The molecular weights of the protein standards are shown on the left. Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. Characterization of B. mori Gloverin Genes 93 Fig. 5. Antibacterial assay of BmGlov1–4. The growth of bacteria at 37°C with or without BmGlovs was detected by recording optical density at 595 nm. 0.1 M phosphate buffer (pH 7.4) was used as a negative control. Five (A) or 20 µg (B) of each BmGlov was used in the experiments. The data show means ± the standard errors. recombinant BmGlovs had weak but significant antibacterial activities against E. coli in a dose-dependent manner. DISCUSSION To identify immune-related genes of B. mori, we obtained 10,752 ESTs from the cDNA library conArchives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. structed from the fat body RNA of E. coli- and C. albicans-challenged B. mori larvae. From the EST data, we identified four kinds of clone that were homologous to Hyalophora gloveri Gloverin (Axen et al., 1997). Phylogenetic analysis with whole genome shotgun data showed that there are four loci encoding Gloverin-like genes in the silkworm genome. In addition, we found that each BmGlov gene 94 Kawaoka et al. has putative alleles. These alleles might be derived from Kinshu or Showa, the parental strain of Kinshu × Showa used in this study. Cheng et al. (2006) reported there are at least seven Gloverin-like genes in the silkworm genome (Cheng et al., 2006). Thus, we compared sequences of the BmGlov genes obtained in this study to those of Gloverin-like genes they had reported. We found that none of the BmGlov genes showed 100% identity with Gloverinlike genes reported by Cheng et al. (2006). These differences may simply be caused by interstrain variations between F1 hybrid Kinshu × Showa used in this study and inbred strain Dazao used in Cheng et al. (2006). The present EST analysis showed that only four BmGlov genes were induced by bacterial challenge, suggesting that these BmGlov genes might mainly contribute to the innate immunity of this strain. To examine whether the BmGlov genes are induced by an immune challenge, we investigated their immune response by Northern blot analysis after the injection of a gram-negative bacteria, E. coli, or a yeast, C. albicans. The BmGlov genes were strongly induced by the challenge of E. coli, but not by the challenge of C. albicans. Moreover, RT– PCR analysis showed that some differences were present in the expression profiles of the BmGlov genes. Sequence analysis revealed that there were consensus NF-κB binding sites at 48 nucleotides upstream from the transcription start site of each BmGlov gene (data not shown). These results indicate that the BmGlov genes are likely to be regulated by the classical pathways through Toll (Lemaitre et al., 1996) and/or Imd (Georgel et al., 2001), which are well known as Rel/NF-κB-mediated immune responses in insects (Silverman and Maniatis, 2001; Tanaka et al., 2005). We next expressed recombinant BmGlovs using the baculovirus expression system. Sequence analysis and Western blot experiments showed that BmGlovs might be expressed as prepropeptides like most insect antibacterial peptides. Generally, the preproparts are cleaved sequentially to produce the mature form of antibacterial protein. Most often, the function of propart is to keep the antibacterial protein inactive (Lundström et al., 2002). Gunne et al. (1990) reported that a recombinant proattacin lost its antibacterial activity after introduction of a mutation blocks the correct processing of attacin (Gunne et al., 1990). In contrast, Lundström et al. (2002) showed that the expression of a recombinant T. ni Gloverin possessing its propart had similar, or actually somewhat higher, antibacterial activity compared with H. gloveri mature Gloverin (Lundström et al., 2002). They concluded that the propart cannot exert any major inhibitory effect on the activity of T. ni Gloverin (Lundström et al., 2002). We have now shown that all propeptides of recombinant BmGlovs could significantly inhibit the growth of E. coli (Fig. 5), indicating that the propart of the BmGlov proteins cannot exert any major inhibitory effect on their activities. In this study, we identified four functional Gloverin-like genes, BmGlov1, BmGlov2, BmGlov3, and BmGlov4. 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