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Multipeptide precursor structure of acaloleptin A isoforms, antibacterial peptides from the Udo longicorn beetle, Acalolepta luxuriosa
Developmental and Comparative Immunology 33 (2009) 1120–1127
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Multipeptide precursor structure of acaloleptin A isoforms, antibacterial peptides from the Udo longicorn beetle, Acalolepta luxuriosa Morikazu Imamura a,1, Sugino Wada a, Kenjiro Ueda b, Ayaka Saito b, Nobuo Koizumi a, Hidenori Iwahana a, Ryoichi Sato b,* a b
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho, Koganei, Tokyo 184-8588, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 April 2009 Received in revised form 5 June 2009 Accepted 6 June 2009 Available online 24 June 2009
We previously purified acaloleptin A1, A2, and A3, antibacterial peptides that are produced in the larval hemolymph of Acalolepta luxuriosa (Udo longicorn beetle). In this study, we performed cDNA cloning. The cDNA sequence showed a predicted acaloleptin A precursor that consisted of five acaloleptin A isoforms. Four (isoforms 1, 2, 3 and 4) of the five isoforms of the acaloleptin A precursor had high-level sequence identities with each other, but the N-terminal region of isoform 5 differed from those of the other acaloleptin A isoforms. Northern and Western blot analyses showed that acaloleptin A isoforms were mass-produced soon after bacterial inoculation. Finally, we purified isoform 5 from hemolymph of the immunized larvae. Isoform 5, unlike acaloleptin A1, A2 and A3, showed antimicrobial activities against a Gram-positive bacterium, Micrococcus luteus and a fungus, Magnaporthe grisea. These results suggest that the multipeptide structure of the acaloleptin A precursor allows A. luxuriosa high-level production of antibacterial peptides and resistance to a wide range of microorganisms. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Acaloleptin A Acalolepta luxuriosa Multipeptide precursor Antimicrobial peptide Insect
1. Introduction Insects produce a battery of antibacterial peptides to combat invading bacteria [1–3]. More than 170 antibacterial peptides have been found in lepidopteran, dipteran, hymenopteran, and coleopteran insects [4]. These peptides are classified into three main groups: (1) linear alpha-helical peptides lacking cysteine residues, (2) proline- and/or glycine-rich peptides, and (3) cysteine-rich peptides with cysteine-stabilized a-b (CSab) motifs [4–6]. In general, the antibacterial peptides produced by insects are of low molecular weight (2–25 kDa), and their expression is induced by bacterial infection [7]. Furthermore, these antibacterial peptides are capable of killing a broad spectrum of bacteria. However one antibacterial peptide is not effective against all infecting bacteria. Therefore, to survive bacterial infections, insects need to efficiently produce a range of antibacterial peptides with different specificities in a timely manner. We reported previously on the isolation of acaloleptin A1 cysteine-rich peptide (AlCRP), cecropin, defensin, and luxuriosin
Abbreviations: cDNA, DNA complementary to RNA; Arg R, arginine; K, lysine. * Corresponding author. Tel.: +81 423 88 7277; fax: +81 423 88 7277. E-mail address: [email protected] (R. Sato). 1 Present address: Research Center for Prion Disease, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8517, Japan. 0145-305X/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2009.06.004
from the larval hemolymph of the longicorn beetle, Acalolepta luxuriosa [8–12]. Acaloleptin A1 consists of 71 amino acid residues, shows antibacterial activity against Gram-negative bacteria only, and has sequence similarities with antibacterial peptides from other coleopteran insects, such as coleoptericin [13], holotricin 2 [14], rhinocerosin [15], and the Allomyrina dichotoma coleoptericin [16]. Acaloleptin A1 has two additional isoforms (acaloleptin A2 and 3) that show different retention times in reversed-phase highperformance liquid chromatography (HPLC). These isoforms have the same N-terminal sequence, and their antibacterial activity spectra are similar [8]. In the present study, we cloned and characterized the cDNA for the acaloleptin A precursor, and performed gene expression analyses. The acaloleptin A precursor turned out to be unusual in that it contained five acaloleptin A isoforms and an acidic peptide. Four (isoforms 1, 2, 3 and 4) of the five isoforms showed high-level sequence similarities to each other, but the fifth isoform (isoform 5) had a unique N-terminal region. Northern and Western blot analyses showed that large quantities of mRNA transcripts of uniform length appeared after immunization, and that high levels of the peptides which cross-react to the anti-acaloleptin A1 antiserum were present in the hemolymph up to 72 h later. Thus, multipeptide precursor structure seems to permit rapid and highlevel production of structurally similar peptides, and facilitates the simultaneous generation of peptides with different antimicrobial activity spectra.
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2. Materials and methods
2.5. Northern blot analysis
2.1. Insects and immunization
Total RNA was isolated from the fat bodies of immunized sixthinstar larvae 24 h post-induction. For the time-course experiment, the fat bodies were collected from three fourth-instar larvae that were maintained at 25 8C for the indicated time intervals following immunization. Total RNA (20 mg) was electrophoresed on a 1% agarose gel that contained 18% formaldehyde, and blotted onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). The membrane was then hybridized with the 30 -terminal 400-bp fragment. The hybridization and washing protocols were performed according to the supplier’s instructions.
Acalolepta luxuriosa larvae were reared as described previously [17]. In the immunization experiments, the larvae were injected with formaldehyde-fixed Escherichia coli K12 W3110 (2.5 107 cells/larva) suspended in an insect physiological saline (IPS; 150 mM NaCl, 5 mM KCl, and 1 mM CaCl2), and maintained at 25 8C until sampling. 2.2. Construction of the cDNA library Total RNA was extracted from the fat bodies of immunized fifthand sixth-instar larvae using the Quickprep Total RNA Extraction Kit (Amersham Pharmacia Biotech), and the mRNA transcripts were prepared using the mRNA Purification Kit (Amersham Pharmacia Biotech). First-strand cDNA was synthesized using the cDNA Synthesis Module Kit (Amersham Pharmacia Biotech), and the cDNA library was constructed in the lambda MOSElox vector using the Complete Rapid Cloning System (Amersham Pharmacia Biotech). 2.3. Probe preparation The probes used for screening the cDNA library were made from the degenerated PCR products. The design of the degenerated primers was based on the amino acid sequences of the mature acaloleptin A1 [8]. For the forward and reverse primers, the nucleotide sequences were deduced from Asn (11) to Gln (17) and Lys (57) to Trp (62), respectively, of the mature peptide. The following primers were used—forward primer: 50 -AA(C/T)AA(A/ G)GA(C/T)CA(A/G)CC(A/C/G/T)TGGCA-30 ; and reverse primer: 50 TGCCA(A/C/G/T)GT(A/C/G/T)GG(C/T)TT(A/C/G/T)GC(C/T)TT-30 . The phage DNA from the cDNA library was used as the template. The following PCR conditions were used: initial denaturation at 95 8C for 2 min, followed by 35 cycles of 95 8C for 1 min, 45 8C for 1 min, and 72 8C for 1 min. The amplified fragment was subcloned into the T-overhang vector p123T (MoBiTec). Dideoxy double-stranded sequencing of the inserted DNA using the ALFred DNA sequencer (Pharmacia) confirmed the identity of the amplified 161-bp fragment. This fragment was labeled with fluorescein using the Random Primer Labeling Kit (Amersham Pharmacia Biotech). Detection of the hybridized probe was carried out with an anti-fluorescein antibody that was conjugated to horseradish peroxidase (HRP), according to the manufacturer’s instructions. The probes used for Northern blotting and genomic Southern blotting were prepared from the acaloleptin A cDNA fragment. The plasmid that contained the full-length acaloleptin A cDNA was digested with EcoRI, thus generating fragments of approximately 1000 bp and 400 bp, since there was one EcoRI site in the cDNA (Fig. 1), and the vector plasmid had two EcoRI site flanking the insert site. The 30 -terminal 400-bp fragment (the region of nucleotides 1031–1404) was used as the probe. 2.4. Cloning of the acaloleptin A cDNA The cDNA library was screened with the fluoresceinlabeled probe. DNA was prepared from the positive phages, and the inserted cDNA was subcloned into pMOSElox by premediated in vivo excision of the plasmids in E. coli BM25.8, according to the supplier’s instructions. The plasmid that carried the longest insert was sequenced, as described above.
2.6. Southern blot analysis Genomic DNA was extracted from the whole body of a single fifth- or sixth-instar larva according to the method described by Sambrook et al. [18]. Samples (10 mg) of the genomic DNAs were digested with DraI, HindIII, or EcoRI. The resultant DNA fragments were separated by electrophoresis in a 0.9% agarose gel, and transferred onto the nylon membrane. The membrane was hybridized with 30 -terminal 400-bp fragment in 5 SSC, 0.5% blocking agent (Amersham), 0.1% SDS, 5% dextran sulphate at 60 8C for over night. After hybridization, the membrane was washed twice with 0.1 SSC, 0.1% SDS for 15 min at 60 8C. 2.7. Western blot analysis Tricine SDS–PAGE using 16.5% gel was carried out according to the method of Schagger and von Jagow [19]. The hemolymph was collected in an ice-cooled 1.5-ml tube from three fourth-instar larvae at the indicated time intervals following immunization. After centrifugation to remove the hemocytes, 1 ml of the clear supernatant was denatured by heating, and used as the sample for analysis. The proteins were separated by electrophoresis, and transferred electrophoretically from the gel onto a Hybond-P PVDF membrane (Amersham Pharmacia Biotech). The anti-acaloleptin A mouse antiserum, which was prepared by immunizing a mouse with HPLC-purified acaloleptin A, was used as the primary antibody at a 1:10,000 dilution in TBST that contained 1% BSA. The anti-acaloleptin A1 antiserum should detect with high sensitivity most of acaloleptin isoforms, since their amino acid sequences are almost identical. The goat anti-mouse IgG-HRP conjugate (BioRad) was used at a 1:3000 dilution as the secondary antibody. Immunoreactive signals were detected using the ECL Western Blotting System (Amersham Pharmacia Biotech). 2.8. Purification of the antibacterial peptide The hemolymph was collected 24 h after formalin-fixed E. coli cells injection to A. luxuriosa larvae as described previously [12], and centrifuged for 20 min at 1400 g at 4 8C to obtain a clear supernatant. A twofold diluted hemolymph sample was subjected to chromatography in a Sep-Pak C18 cartridge column (Sep-Pak Vac 6 cc, Waters) pre-equilibrated with 0.05% trifluoroacetic acid (TFA) and eluted with 30% acetonitrile acidified with 0.05% TFA. The eluted fraction was freeze-dried and a resulting pellet was solubilized in distilled water. The solubilized sample was subjected to reverse phase HPLC on a TSKgel ODS 120T (300 mm 7.8 mm, C18) column (TOSO), and fractions were isolated with a linear gradient of 10–40% acetonitrile with 0.05% TFA at a flow rate of 1 ml/min for 120 min, vacuum dried, and then dissolved in distilled water. The active fraction was dried, dissolved in distilled water, and subjected to a second HPLC step on a Synmetory1 C8 column (Waters). Fractions were isolated with a linear gradient of 10–40% acetonitrile with 0.05% TFA at a flow rate of 1 ml/min for
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Fig. 1. Nucleotide sequence, predicted amino acid sequence, and schematic structure of the acaloleptin A precursor. (A) Nucleotide sequence and putative amino acid sequence of the acaloleptin A cDNA. The numbers at the right indicate the nucleotide and amino acid positions. Four tandem repeats of the nucleotide sequence are indicated with double-headed arrows. The EcoRI site is boxed. The preprosequence is underlined, and the vertical arrow indicates its putative processing site to release signal peptide, as predicted by SignalP ver. 2.0 (http://www.cbs.dtu.dk/services/SignalP-2.0/). The putative mature peptides and basic dipeptides are indicated by open boxes and dotted boxes, respectively. The star symbol below the nucleotide sequence indicates the stop codon. The TTATTTAT, ATTTA, and polyadenylation signal (AATAGA) sequences are shown by the single line, double lines, and the dotted line, respectively. (B) Schematic representation of the acaloleptin A and holotricin 2 precursors. The shaded box represents the preprosequence. The open boxes and black boxes indicate the putative mature peptides and basic dipeptides, respectively. The acidic peptide and the Glu- and Asp-rich region at the N-terminus of isoform 5 are indicated as a crossed box and hatched box, respectively.
216 min, vacuum dried, and then dissolved in distilled water. The active fraction was dried, dissolved in distilled water, and subjected to a third HPLC step on a Synmetory1 C8 column. Fractions were isolated with a linear gradient of 25–30% acetonitrile with 0.05% TFA at a flow rate of 1 ml/min for 216 min, vacuum dried, and then dissolved in distilled water. The active fraction was subjected to Tricine SDS–PAGE [19]. The peptide concentration was determined by the Bradford method [20] using BSA as a standard. 2.9. Assay for the antimicrobial activity The bactericidal activity of hemolymph against E. coli was measured by the plate growth inhibition assay [8], in which the efficacy of bacterial killing correlated with the area of the inhibitory zone in the spot agar layer. Aliquots (1 ml) of the hemolymph mixture, which were collected from the three larvae at the indicated time intervals, were used in the assay. A liquid growth inhibition assay was performed to determine the antibacterial activity and spore germination and growth inhibitory activity against the conidia from rice blast fungus, Magnaporthe grisea was tested as previously described [12].
3. Results 3.1. Cloning and nucleotide sequencing of the acaloleptin A cDNA Approximately 2 104 clones from an immunized larval fat body cDNA library were screened using the 161-bp degenerated PCR product as the probe. Nine positive clones were recovered, two of which had long inserts (approximately 1400 bp). One of these clones was sequenced and found to contain a 1402-bp insert (GenBank accession no. AB094343; Fig. 1a). The putative translation initiation codon ATG was located 26-bp downstream of the 50 end of the cDNA. In the 30 -terminal region, poly(A) tails were identified, and the termination codon TGA was located at nucleotides 1295–1297. Although the typical polyadenylation signal (AATAAA) was lacking in the 30 -untranslated region, the related sequence (AATAGA) was located 36-bp downstream of the stop codon. In this region, there were two ATTTA motifs, which probably affect the stability of the mRNA [21], and one TTATTTAT motif, which represents the consensus sequence of mammalian acute phase protein genes [22]. These sequences were previously identified in other insect antibacterial peptides, such as sarcotoxin IIA and diptericin [23,24].
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Fig. 2. Alignment of the amino acid sequences of the five predicted acaloleptin A isoforms from the acaloleptin A precursor and acaloleptin A1. The boxed residues represent the minor amino acids among the corresponding residues of the isoform sequences.
The acaloleptin A cDNA was expected to encode a 423-amino acid residue precursor protein that contained a preprosequence (Fig. 1A). However, four tandem nucleotide repeats that encoded acaloleptin A-related mature peptides and the Arg-Arg dipeptide (double-headed arrows in Fig. 1) were found. The four deduced mature peptides consisted of 71 amino acid residues each, and shared high-level sequence similarities with acaloleptin A1 [8]. We designated these peptides as isoforms 1, 2, 3, and 4, in the order of their positions in the sequence (Fig. 1B). In the 105-aa region that followed the four tandem repeats of isoforms, 59 amino acid residues (from aa 365 in the putative precursor) showed significant sequence similarities with aa 13–71 of the acaloleptin A isoforms1, 2, 3, and 4. It was proposed that this C-terminal 69-aa region of the precursor is secreted as a peptide along with the other isoforms, as the acaloleptin A precursor appears to be processed at the presumed processing signal R-X-R/K-R (see Section 4) of the 351–354 region. We regarded this putative 69-aa peptide as isoform 5. Therefore, it appears that the acaloleptin A precursor forms a multipeptide structure that consists of five acaloleptin A isoforms.
3.3. Acaloleptin A gene expression in larval fat bodies Northern blot analysis of the larval fat bodies showed that acaloleptin A gene expression was induced only in the immunized larvae (Fig. 3A). Furthermore, only the 1.4-kbpositive band was detected, which suggests that the acaloleptin A gene(s) produces a single size of transcript. In contrast, the transcripts of apidaecin, which is a honeybee antibacterial peptide that is processed from a multipeptide precursor, vary in size from 650 bp to 1400 bp because of the existence of multiple genes [25].
3.2. Sequence variations among the acaloleptin A isoforms The primary structures of the five isoforms were aligned (Fig. 2) and shown to possess non-identical amino acid sequences, although they shared significant sequence similarities. Notably, high levels of sequence similarity were found among isoforms 1, 2, and 3 (1 and 2: 93.0%; 1 and 3: 95.8%; 2 and 3: 97.2%). The sequence of isoform 4 differed from those of the other three isoforms, with a sequence identity of approximately 90% (1 and 4: 90.1%; 2 and 4: 87.3%; 3 and 4: 90.1%). However, unlike the other isoforms, isoform 5 contained a unique N-terminal region that was rich in aspartic and glutamic acids. Furthermore, the region from positions 11–69 in isoform 5 contained many different amino acids compared to the corresponding regions in the other four isoforms. The sequence of isoform 5 was only 67.4% identical to isoform 4, which shared greater sequence similarity with isoform 5 than did isoforms 1, 2, or 3. The cloned cDNA did not encode any isoforms that were 100% identical to the amino acid sequence of acaloleptin A1, although there was only one amino acid difference between the sequences of isoform 3 and acaloleptin A1 (Fig. 2). This phenomenon is presumably due to allelic polymorphisms, as cDNA and hemolymph were prepared from larvae which enclosed from eggs laid by field-captured adult insects.
Fig. 3. Northern blot analysis of total RNA from naı¨ve and immunized larvae (A) and southern blot analysis for the acaloleptin A gene (B). Total RNA was extracted from naı¨ve and bacteria-challenged (immune) sixth-instar larvae. Samples (20 mg) were blotted from the denaturing gel onto a nylon membrane. The 30 -terminal 400-bp EcoRI fragment of the cDNA was used as the probe for Northern blotting. The ribosomal RNA band was used as the loading control (A). Genomic DNA (10 mg) was digested with DraI, HindIII, and EcoRI. The digested samples were electrophoresed on a 0.9% agarose gel, transferred to a nylon membrane, and hybridized with the 400-bp EcoRI cDNA probe (B).
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3.4. Southern blot analysis of genomic DNA Southern blot analysis was performed to estimate the number of copies of the acaloleptin A gene in the genome. Two HindIII fragments were detected, and two high-intensity bands and one weak band were detected in the DraI- and EcoRI-digested DNA, respectively, with a 400-bp C-terminal probe (nucleotides 10311404 of the cloned cDNA; Fig. 3B). Although no DraI or HindIII site was present, one EcoRI site was found in the cDNA at the 50 -end of the probe site. A faint band that was detected in the DraI- and EcoRI-digested DNAs may indicate the existence of these restriction enzyme sites within the probe region of the different alleles, which was not cloned in the cDNA. Genomic DNA was extracted from the whole body of a single fifth- or sixth-instar larva. Therefore, it is possible that the acaloleptin A gene exists as a single copy per haploid and that two different alleles exist in the genome. 3.5. Kinetics of acaloleptin A gene expression in the fat bodies The kinetics of acaloleptin A expression at the mRNA and peptide levels were investigated after inoculation with E. coli. Acaloleptin A transcripts were first recognized 2 h after injection, and expression peaked after 24 h. Subsequently, the expression level decreased gradually, although a considerable level of expression persisted after 72 h (Fig. 4A). On the other hand, the concentration of acaloleptin A isoforms in the hemolymph, as detected using the antiserum, increased 6 h after bacterial inoculation, and peaked after 48 h. High-level expression was maintained after 72 h, although this level was lower than that after 48 h (Fig. 4A). We also measured the antibacterial activity of hemolymph against E. coli at each time-point. Antibacterial activity appeared 12 h post-inoculation, peaked at 48 h, and was maintained at a high level after 72 h (Fig. 4B). Thus, the antibacterial activity levels correlated with the levels of acaloleptin A peptide in the hemolymph.
Fig. 4. Kinetics of acaloleptin A mRNA, peptide induction, and anti-E. coli activities of the larval hemolymph after immunization. The levels of mRNA expression and peptide synthesis were investigated by Northern and Western blotting, respectively, after inoculation with E. coli (A). The anti-E. coli activities of the larval hemolymph at each time point after inoculation are shown in (B). The activities of the hemolymph were inferred from the size of the inhibition zones.
3.6. Purification of a peptide with the same N-terminal amino acid sequence as isoform 5 from the hemolymph of A. luxuriosa larvae A fraction of the hemolymph of immune-challenged sixth instar larvae of A. luxuriosa, eluted from an ODS cartridge column (SepPak C18, Waters) with 30% acetonitrile were previously reported to possess potent antibacterial activity [8]. Multiple fractions were first separated by reverse phase HPLC on a TSKgel ODS 120T column (Toso), and fractions showing anti-Micrococcus luteus activity were identified as previously described [11,12]. We selected one of the fractions with activity against M. luteus (data not shown) and purified it in a stepwise manner via HPLC on a Symmetry1 C8 column (Fig. 5A). Purified peptide was obtained after elution with 25% acetonitrile at the final purification step. The purified product was observed as a single peptide band of 7.5 kDa by Tricine SDS–PAGE (Fig. 5B). The first 13 residues of the Nterminal amino acid sequence of the purified peptide were identified as SDDEDEEEEEDQP via automated Edman degradation. This sequence was identical to the N-terminal sequence of the putative acaloleptin A isoform 5, which was predicted in Section 3.1. These results indicated that isoform 5 was actually processed and secreted into the hemolymph. 3.7. Antimicrobial activity of the purified peptide Using a liquid growth inhibition assay, the antibacterial activity of the purified peptide was evaluated for four Gram-positive bacterial strains (M. luteus, B. subtilis, B. cereus, and S. aureus), five Gram-negative bacterial strains (E. coli, A. hydrophila, E. persicinus,
Fig. 5. Purification of an antimicrobial peptide from A. luxuriosa larvae. (A) Final step reverse-phase HPLC profile of the active fraction. The arrow indicates the fraction with antibacterial activity against M. luteus. (B) Tricine SDS–PAGE of the active fraction indicated in (A). The arrow indicates the purified peptide. The first 13 residues of the N-terminal amino acid sequence of the purified peptide obtained via Edman degradation was also indicated.
S. marcescens, and A. hydrophila), and one yeast strain (S. cerevisiae). The purified peptide inhibited more than 70% of the growth of M. luteus at 20 mg/ml (Fig. 6A), but did not inhibit the growth of other bacteria and yeast. The ability of the purified peptide to inhibit spore germination and growth was tested against the conidia of rice blast fungus, Magnaporthe grisea. The purified peptide at a concentration of 161.4 mg/ml inhibited the germination of conidia and/or the growth of mycelium (Fig. 6B). These results suggested that the purified peptide might possess antibacterial activity
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Fig. 6. Anti-bacterial and anti-fungal activities of the purified peptide. (A) Growth inhibitory activity of the purified peptide against M. luteus. Growth inhibitory activities against four bacteria and one yeast were estimated as described in Section 2. *, M. luteus; *, B. subtilis; , S. aureus; &, B. cereus. (B and C) Germination- and growth-inhibitory activity of the purified peptide against conidia from the rice blast fungus, M. grisea. Conidia of M. grisea were incubated with (B) or without (C) the purified peptide at 20 8C for 24 h and then spore germination and the growth of mycelia were observed via phase-contrast microscopy. Arrowheads indicate the conidia of M. grisea.
against some Gram-positive bacteria and antifungal activity, and has different antimicrobial spectrum from that of Acaloleptin A1, 2 and 3, which show antibacterial activity against only Gramnegative bacteria [8].
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a single precursor (Fig. 1). It was reported that apidaecin and the putative cysteine-rich antibacterial peptides from the hemipteran insect Riptortus clavatus are processed from multipeptide precursors [25,26]. In other organisms except for insects, only the magainins from Xenopus, the cysteine-rich antibacterial peptides from the seeds of Impatiens balsamina (Ib-AMPs) and naegleriapore B from amoeboflagellate are derived from multipeptide precursors [27–30]. Bearing in mind that numerous antibacterial peptides have been isolated from a wide range of organisms, it seems likely that the occurrence of multipeptide precursor structures in antibacterial peptides is rare. Nevertheless, antibacterial peptides with multipeptide precursors have been identified in diverse organisms, including invertebrates, vertebrates, protozoa and plants. These antibacterial peptides do not share sequence similarities, with the exception of the apidaecin and cysteine-rich antibacterial peptides from hemiptera. This suggests that multipeptide precursors of antibacterial peptides did not evolve from a single ancestor, but instead, each antibacterial peptide acquired independently the multipeptide precursor structure during evolution of the organism. The precursors of many secreted proteins and membrane proteins in non-endocrine cells contain an R-X-K/R-R signal tetrapeptide that is recognized and processed by a furin-like protease [31]. The cDNAs of six antibacterial peptides have been cloned from coleopteran insects, and shown to contain this tetrapeptide, which is used for the processing of the precursors [14–16,32,33]. The C-terminal tetrapeptide in the preprosequence of the acaloleptin A precursor is R-W-K-R, which suggests that this tetrapeptide is also used for processing. The C-terminal dipeptides of isoforms 14 are R-W, and R-R dipeptides link each isoform (Fig. 1A). Therefore, it seems likely that the acaloleptin A precursor is processed by the furin-like protease not only at the C-terminus of the preprosequence, but also at the C-terminus of isoforms 1–4. In fact, three different isoforms (acaloleptin A1, 2, 3), which share a SLQPGA sequence at the N-terminus but differ in terms of retention times in the ODS column, were confirmed to be secreted into the hemolymph as mature peptides [8]. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis has shown that peptides with molecular weights similar to those of the four acaloleptin A isoforms exist in immunized larval hemolymph (data not shown). An acidic sequence between two R-X-K/R-R motives at positions 315–318 and 351–354 have no homology with the acaloleptin A mature peptides, and no proteins with homology to this region were detected using BLAST. Because the acaloleptin A precursor is presumably processed at this tetrapeptide motif, we expect the precursor to be processed at the C-terminus of amino acid 354. Assuming that this is true, four 71-aa isoforms, one 69-aa isoform (isoform 5), which has a different N-terminal region than the other isoforms, and a 34-aa acidic peptide should be generated from the acaloleptin A precursor. Indeed, a peptide with the same N-terminal amino acid sequence as isoform 5 was purified from larval hemolymph in this study (Fig. 5). However, we have no information regarding the presence or function of the acidic peptide in the hemolymph. In the case of magainin, acidic 6-aa peptides are secreted with the mature peptides after precursor processing; the function of these acidic peptides has not yet been clarified [28]. 4.2. Origin of the precursor structure of acaloleptin A
4. Discussion 4.1. Structure of the acaloleptin A precursor and its mature peptides Based on the nucleotide sequence of the acaloleptin A cDNA, it appeared that five isoforms were generated through processing of
The precursors of holotricin 2, rhinocerosin, and the A. dichotoma coleoptericin, which are thought to be orthologs of acaloleptin A, produce single mature peptides and are unlike the acaloleptin A precursor. As far as we know, there are no reports that the precursor structures among the orthologs are completely
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maintained until 72 h post-inoculation (Fig. 4B). The antibacterial activity levels mirrored the acaloleptin A concentrations in the hemolymph (Fig. 4A). This result suggests that acaloleptin A is an important inhibitor of the proliferation of Gram-negative bacteria in the hemocoel. 4.4. Biological significance of multiple acaloleptin A genes
Fig. 7. Phylogenetic analysis of the coleoptericin family of peptides. The tree was constructed based on the complete amino acid sequence of the mature coleoptericin peptides using the Clustal X program. The numbers indicate the values that were obtained from 1000 bootstrap replicates. The accession numbers of the peptide sequences are as follows: acaloleptin A1, P81592; coleoptericin, A41711; holotricin 2, Q25054; rhinocerosin, O76145; A. dichotoma coleoptericin A, BAB40436; A. dichotoma coleoptericin B, BAB40437; A. dichotoma coleoptericin C, BAB40438; diptericin, 1403303A.
different, which is interesting from both a phylogenetic and evolutionary perspective. A phylogenetic tree was constructed based on the amino acid sequences of the five mature isoforms of the acaloleptin A precursor, coleoptericin, holotricin 2, rhinocerosin, and the A. dichotoma coleoptericin (Fig. 7). The five acaloleptin A isoforms formed a cluster with coleoptericin, whereas rhinocerosin, holotricin 2, and the A. dichotoma coleoptericin formed another cluster. According to the morphological classification of the coleopteran insects, A. luxuriosa and Zophobas atratus, from which coleoptericin has been isolated, belong to the Chrysomeloidea and Tenebrionoidea superfamilies, respectively. Because both of these superfamilies belong to Cucujiformia, it appears that these two species are relatively closely related. On the other hand, Holotrichia idiomphalia, Oryctes rhinoceros, and A. dichotoma, from which holotricin 2, rhinocerosin, and the A. dichotoma coleoptericin have been isolated, respectively, are classified into superfamily Scarabaeoidea of the infraorder Scarabaeiformia. Therefore, the peptide sequence-based phylogenetic tree is consistent with the tree based on morphological characters. Taken together, these findings suggest that the multipeptide precursor in the coleoptericin family emerged after the divergence of Cucujiformia and Scarabaeiformia, during the evolution of coleopteran insects. However, the timing of multipeptide acquisition is unknown. 4.3. Alterations in the levels of acaloleptin A mRNA and peptide after bacterial inoculation In Drosophila, various antibacterial peptide gene expression patterns have been observed after bacterial inoculation [34]. With respect to cecropin, attacin, and defensin, the peak mRNA levels were observed 6–12 h after immune-stimulation, whereas the levels of diptericin, metchnikowin, and drosocin transcripts peaked 12–24 h later. In the case of acaloleptin A, gene expression increased for up to 48 h after inoculation with E. coli, and remained high for an additional 72 h (Fig. 4A). Therefore, the expression pattern of the acaloleptin A gene is similar to those of the diptericin, metchnikowin, and drosocin genes, which have antibacterial activities against Gram-negative bacteria. This suggests that the regulation of acaloleptin A gene expression may be similar to that of Drosophila antibacterial peptide genes. The antibacterial activity of the immunized A. luxuriosa larval hemolymph against E. coli peaked 48 h after inoculation, and was
In this study, we showed that the acaloleptin A precursor had a multipeptide structure. It is possible that the multipeptide structure of the precursor ensures rapid mass production of acaloleptin A isoforms following bacterial invasion, thereby raising an effective immune response. By comparing the signal intensity between immunized hemolymph and graduated dilutions of purified acaloleptin A in Western blots, the concentration of acaloleptin A isoforms in the hemolymph was determined to exceed 100 mg/ml at 48 h after bacterial inoculation (data not shown). No significant difference was observed regarding the activities of acaloleptin A1, A2, or A3 [8], which supports the idea that redundancy of the isoforms quantitatively amplifies the immune response. The N-terminal sequence of isoform 5 was completely different from the sequences of isoforms 1, 2, 3, and 4, and the orthologs of various insects (Fig. 2), although this region was strictly conserved in the coleoptericin family of peptides. A peptide that has an identical N-terminal sequence to acaloleptin A isoform 5 was isolated from larval hemolymph and shown to have antimicrobial activity against a Gram-positive bacterium, M. luteus, and a fungus, M. grisea (Fig. 6). These results suggest that the multipeptide precursor structure confers upon the acaloleptin A isoforms a broader range of antimicrobial activity. Acaloleptin A is unique among the antibacterial peptides in having a precursor that contains several related peptides with different activities, although a similar phenomenon has been described for neuropeptide precursors [35]. References [1] Lemaitre B, Kromer-Metzger E, Michaut L, Nicolas E, Meister M, Georgel P, et al. A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense. Proc Natl Acad Sci USA 1995;92: 9465–9. [2] Engstro¨m Y. Induction and regulation of antimicrobial peptides in Drosophila. Dev Comp Immunol 1999;23:345–58. [3] Hedengren M, Asling B, Dushay MS, Ando I, Ekengren S, Wihlborg M, et al. Relish, a central factor in the control of humoral but not cellular immunity in Drosophila. Mol Cell 1999;4:827–37. [4] Bulet P, Hetru C, Dimarcq JL, Hoffmann D. Antimicrobial peptides in insects; structure and function. Dev Comp Immunol 1999;23:329–44. [5] Meister M, Lemaitre B, Hoffmann JA. Antimicrobial peptide defense in Drosophila. Bioessays 1997;19:1019–26. [6] Dimarcq JL, Bulet P, Hetru C, Hoffmann J. Cysteine-rich antimicrobial peptides in invertebrates. Biopolymers 1998;47:465–77. [7] Hoffmann JA, Reichhart JM, Hetru C. Innate immunity in higher insects. Curr Opin Immunol 1996;8:8–13. [8] Imamura M, Wada S, Koizumi N, Kadotani T, Yaoi K, Sato R, et al. Inducible antibacterial peptides from larvae of the beetle, Acalolepta luxuriosa. Arch Insect Biochem Physiol 1999;40:88–98. [9] Saito A, Ueda K, Imamura M, Atsumi S, Tabunoki H, Miura N, et al. Purification and cDNA cloning of a cecropin from the longicorn beetle, Acalolepta luxuriosa. Comp Biochem Physiol B Biochem Mol Biol 2005;142:317–23. [10] Saito A, Ueda K, Imamura M, Miura N, Atsumi S, Tabunoki H, et al. Purification and cDNA cloning of a novel antibacterial peptide with a cysteine-stabilized alphabeta motif from the longicorn beetle, Acalolepta luxuriosa. Dev Comp Immunol 2004;28:1–7. [11] Ueda K, Imamura M, Saito A, Sato R. Purification and cDNA cloning of a novel insect defensin from larvae of the longicorn beetle, Acalolepta luxuriosa. Appl Entomol Zool 2005;40:335–45. [12] Ueda K, Saito A, Imamura M, Miura N, Atsumi S, Tabunoki H, et al. Purification and cDNA cloning of luxuriosin, a novel antibacterial peptide with Kunitz domain from the longicorn beetle, Acalolepta luxuriosa. Biochim Biophys Acta 2005;1722:36–42. [13] Bulet P, Cociancich S, Dimarcq JL, Lambert J, Reichhart JM, Hoffmann D, et al. Insect immunity. Isolation from a coleopteran insect of a novel inducible
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[25] Casteels-Josson K, Capaci T, Casteels P, Tempst P. Apidaecin multipeptide precursor structure: a putative mechanism for amplification of the insect antibacterial response. EMBO J 1993;12:1569–78. [26] Miura K, Ueno S, Kamiya K, Kobayashi J, Matsuoka H, Ando K, et al. Cloning of mRNA sequences for two antibacterial peptides in a Hemipteran insects, Riptortus clavatus. Zoolog Sci 1996;13:111–7. [27] Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA 1987;84:5449–53. [28] Terry AS, Poulter L, Williams DH, Nutkins JC, Giovannini MG, Moore CH, et al. The cDNA sequence coding for prepro-PGS (prepro-magainins) and aspects of the processing of this prepro-polypeptide. J Biol Chem 1988;263:5745–51. [29] Tailor RH, Acland DP, Attenborough S, Cammue BP, Evans IJ, Osborn RW, et al. A novel family of small cysteine-rich antimicrobial peptides from seed of Impatiens balsamina is derived from a single precursor protein. J Biol Chem 1997;272:24480–7. [30] Herbst R, Marciano-Cabral F, Leippe M. Antimicrobial and pore-forming peptides of free-living and potentially highly pathogenic Naegleria fowleri are released from the same precursor molecule. J Biol Chem 2004;279:25955–8. [31] Hosaka M, Nagahama M, Kim WS, Watanabe T, Hatsuzawa K, Ikemizu J, et al. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J Biol Chem 1991;266:12127– 21230. [32] Moon HJ, Lee SY, Kurata S, Natori S, Lee BL. Purification and molecular cloning of cDNA for an inducible antibacterial protein from larvae of the coleopteran, Tenebrio molitor. J Biochem 1994;116:53–8. [33] Ishibashi J, Saido-Sakanaka H, Yang J, Sagisaka A, Yamakawa M. Purification, cDNA cloning and modification of a defensin from the coconut rhinoceros beetle, Oryctes rhinoceros. Eur J Biochem 1999;266:616–23. [34] Rutschmann S, Jung AC, Hetru C, Reichhart JM, Hoffmann JA, Ferrandon D. The Rel protein DIF mediates the antifungal but not the antibacterial host defense in Drosophila. Immunity 2000;12:569–80. [35] Douglass J, Civelli O, Herbert E. Polyprotein gene expression: generation of diversity of neuroendocrine peptides. Annu Rev Biochem 1984;53:665–715.
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Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
Multipeptide precursor structure of acaloleptin A isoforms, antibacterial peptides from the Udo longicorn beetle, Acalolepta luxuriosa Morikazu Imamura a,1, Sugino Wada a, Kenjiro Ueda b, Ayaka Saito b, Nobuo Koizumi a, Hidenori Iwahana a, Ryoichi Sato b,* a b
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho, Koganei, Tokyo 184-8588, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 April 2009 Received in revised form 5 June 2009 Accepted 6 June 2009 Available online 24 June 2009
We previously purified acaloleptin A1, A2, and A3, antibacterial peptides that are produced in the larval hemolymph of Acalolepta luxuriosa (Udo longicorn beetle). In this study, we performed cDNA cloning. The cDNA sequence showed a predicted acaloleptin A precursor that consisted of five acaloleptin A isoforms. Four (isoforms 1, 2, 3 and 4) of the five isoforms of the acaloleptin A precursor had high-level sequence identities with each other, but the N-terminal region of isoform 5 differed from those of the other acaloleptin A isoforms. Northern and Western blot analyses showed that acaloleptin A isoforms were mass-produced soon after bacterial inoculation. Finally, we purified isoform 5 from hemolymph of the immunized larvae. Isoform 5, unlike acaloleptin A1, A2 and A3, showed antimicrobial activities against a Gram-positive bacterium, Micrococcus luteus and a fungus, Magnaporthe grisea. These results suggest that the multipeptide structure of the acaloleptin A precursor allows A. luxuriosa high-level production of antibacterial peptides and resistance to a wide range of microorganisms. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Acaloleptin A Acalolepta luxuriosa Multipeptide precursor Antimicrobial peptide Insect
1. Introduction Insects produce a battery of antibacterial peptides to combat invading bacteria [1–3]. More than 170 antibacterial peptides have been found in lepidopteran, dipteran, hymenopteran, and coleopteran insects [4]. These peptides are classified into three main groups: (1) linear alpha-helical peptides lacking cysteine residues, (2) proline- and/or glycine-rich peptides, and (3) cysteine-rich peptides with cysteine-stabilized a-b (CSab) motifs [4–6]. In general, the antibacterial peptides produced by insects are of low molecular weight (2–25 kDa), and their expression is induced by bacterial infection [7]. Furthermore, these antibacterial peptides are capable of killing a broad spectrum of bacteria. However one antibacterial peptide is not effective against all infecting bacteria. Therefore, to survive bacterial infections, insects need to efficiently produce a range of antibacterial peptides with different specificities in a timely manner. We reported previously on the isolation of acaloleptin A1 cysteine-rich peptide (AlCRP), cecropin, defensin, and luxuriosin
Abbreviations: cDNA, DNA complementary to RNA; Arg R, arginine; K, lysine. * Corresponding author. Tel.: +81 423 88 7277; fax: +81 423 88 7277. E-mail address: [email protected] (R. Sato). 1 Present address: Research Center for Prion Disease, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8517, Japan. 0145-305X/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2009.06.004
from the larval hemolymph of the longicorn beetle, Acalolepta luxuriosa [8–12]. Acaloleptin A1 consists of 71 amino acid residues, shows antibacterial activity against Gram-negative bacteria only, and has sequence similarities with antibacterial peptides from other coleopteran insects, such as coleoptericin [13], holotricin 2 [14], rhinocerosin [15], and the Allomyrina dichotoma coleoptericin [16]. Acaloleptin A1 has two additional isoforms (acaloleptin A2 and 3) that show different retention times in reversed-phase highperformance liquid chromatography (HPLC). These isoforms have the same N-terminal sequence, and their antibacterial activity spectra are similar [8]. In the present study, we cloned and characterized the cDNA for the acaloleptin A precursor, and performed gene expression analyses. The acaloleptin A precursor turned out to be unusual in that it contained five acaloleptin A isoforms and an acidic peptide. Four (isoforms 1, 2, 3 and 4) of the five isoforms showed high-level sequence similarities to each other, but the fifth isoform (isoform 5) had a unique N-terminal region. Northern and Western blot analyses showed that large quantities of mRNA transcripts of uniform length appeared after immunization, and that high levels of the peptides which cross-react to the anti-acaloleptin A1 antiserum were present in the hemolymph up to 72 h later. Thus, multipeptide precursor structure seems to permit rapid and highlevel production of structurally similar peptides, and facilitates the simultaneous generation of peptides with different antimicrobial activity spectra.
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2. Materials and methods
2.5. Northern blot analysis
2.1. Insects and immunization
Total RNA was isolated from the fat bodies of immunized sixthinstar larvae 24 h post-induction. For the time-course experiment, the fat bodies were collected from three fourth-instar larvae that were maintained at 25 8C for the indicated time intervals following immunization. Total RNA (20 mg) was electrophoresed on a 1% agarose gel that contained 18% formaldehyde, and blotted onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). The membrane was then hybridized with the 30 -terminal 400-bp fragment. The hybridization and washing protocols were performed according to the supplier’s instructions.
Acalolepta luxuriosa larvae were reared as described previously [17]. In the immunization experiments, the larvae were injected with formaldehyde-fixed Escherichia coli K12 W3110 (2.5 107 cells/larva) suspended in an insect physiological saline (IPS; 150 mM NaCl, 5 mM KCl, and 1 mM CaCl2), and maintained at 25 8C until sampling. 2.2. Construction of the cDNA library Total RNA was extracted from the fat bodies of immunized fifthand sixth-instar larvae using the Quickprep Total RNA Extraction Kit (Amersham Pharmacia Biotech), and the mRNA transcripts were prepared using the mRNA Purification Kit (Amersham Pharmacia Biotech). First-strand cDNA was synthesized using the cDNA Synthesis Module Kit (Amersham Pharmacia Biotech), and the cDNA library was constructed in the lambda MOSElox vector using the Complete Rapid Cloning System (Amersham Pharmacia Biotech). 2.3. Probe preparation The probes used for screening the cDNA library were made from the degenerated PCR products. The design of the degenerated primers was based on the amino acid sequences of the mature acaloleptin A1 [8]. For the forward and reverse primers, the nucleotide sequences were deduced from Asn (11) to Gln (17) and Lys (57) to Trp (62), respectively, of the mature peptide. The following primers were used—forward primer: 50 -AA(C/T)AA(A/ G)GA(C/T)CA(A/G)CC(A/C/G/T)TGGCA-30 ; and reverse primer: 50 TGCCA(A/C/G/T)GT(A/C/G/T)GG(C/T)TT(A/C/G/T)GC(C/T)TT-30 . The phage DNA from the cDNA library was used as the template. The following PCR conditions were used: initial denaturation at 95 8C for 2 min, followed by 35 cycles of 95 8C for 1 min, 45 8C for 1 min, and 72 8C for 1 min. The amplified fragment was subcloned into the T-overhang vector p123T (MoBiTec). Dideoxy double-stranded sequencing of the inserted DNA using the ALFred DNA sequencer (Pharmacia) confirmed the identity of the amplified 161-bp fragment. This fragment was labeled with fluorescein using the Random Primer Labeling Kit (Amersham Pharmacia Biotech). Detection of the hybridized probe was carried out with an anti-fluorescein antibody that was conjugated to horseradish peroxidase (HRP), according to the manufacturer’s instructions. The probes used for Northern blotting and genomic Southern blotting were prepared from the acaloleptin A cDNA fragment. The plasmid that contained the full-length acaloleptin A cDNA was digested with EcoRI, thus generating fragments of approximately 1000 bp and 400 bp, since there was one EcoRI site in the cDNA (Fig. 1), and the vector plasmid had two EcoRI site flanking the insert site. The 30 -terminal 400-bp fragment (the region of nucleotides 1031–1404) was used as the probe. 2.4. Cloning of the acaloleptin A cDNA The cDNA library was screened with the fluoresceinlabeled probe. DNA was prepared from the positive phages, and the inserted cDNA was subcloned into pMOSElox by premediated in vivo excision of the plasmids in E. coli BM25.8, according to the supplier’s instructions. The plasmid that carried the longest insert was sequenced, as described above.
2.6. Southern blot analysis Genomic DNA was extracted from the whole body of a single fifth- or sixth-instar larva according to the method described by Sambrook et al. [18]. Samples (10 mg) of the genomic DNAs were digested with DraI, HindIII, or EcoRI. The resultant DNA fragments were separated by electrophoresis in a 0.9% agarose gel, and transferred onto the nylon membrane. The membrane was hybridized with 30 -terminal 400-bp fragment in 5 SSC, 0.5% blocking agent (Amersham), 0.1% SDS, 5% dextran sulphate at 60 8C for over night. After hybridization, the membrane was washed twice with 0.1 SSC, 0.1% SDS for 15 min at 60 8C. 2.7. Western blot analysis Tricine SDS–PAGE using 16.5% gel was carried out according to the method of Schagger and von Jagow [19]. The hemolymph was collected in an ice-cooled 1.5-ml tube from three fourth-instar larvae at the indicated time intervals following immunization. After centrifugation to remove the hemocytes, 1 ml of the clear supernatant was denatured by heating, and used as the sample for analysis. The proteins were separated by electrophoresis, and transferred electrophoretically from the gel onto a Hybond-P PVDF membrane (Amersham Pharmacia Biotech). The anti-acaloleptin A mouse antiserum, which was prepared by immunizing a mouse with HPLC-purified acaloleptin A, was used as the primary antibody at a 1:10,000 dilution in TBST that contained 1% BSA. The anti-acaloleptin A1 antiserum should detect with high sensitivity most of acaloleptin isoforms, since their amino acid sequences are almost identical. The goat anti-mouse IgG-HRP conjugate (BioRad) was used at a 1:3000 dilution as the secondary antibody. Immunoreactive signals were detected using the ECL Western Blotting System (Amersham Pharmacia Biotech). 2.8. Purification of the antibacterial peptide The hemolymph was collected 24 h after formalin-fixed E. coli cells injection to A. luxuriosa larvae as described previously [12], and centrifuged for 20 min at 1400 g at 4 8C to obtain a clear supernatant. A twofold diluted hemolymph sample was subjected to chromatography in a Sep-Pak C18 cartridge column (Sep-Pak Vac 6 cc, Waters) pre-equilibrated with 0.05% trifluoroacetic acid (TFA) and eluted with 30% acetonitrile acidified with 0.05% TFA. The eluted fraction was freeze-dried and a resulting pellet was solubilized in distilled water. The solubilized sample was subjected to reverse phase HPLC on a TSKgel ODS 120T (300 mm 7.8 mm, C18) column (TOSO), and fractions were isolated with a linear gradient of 10–40% acetonitrile with 0.05% TFA at a flow rate of 1 ml/min for 120 min, vacuum dried, and then dissolved in distilled water. The active fraction was dried, dissolved in distilled water, and subjected to a second HPLC step on a Synmetory1 C8 column (Waters). Fractions were isolated with a linear gradient of 10–40% acetonitrile with 0.05% TFA at a flow rate of 1 ml/min for
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Fig. 1. Nucleotide sequence, predicted amino acid sequence, and schematic structure of the acaloleptin A precursor. (A) Nucleotide sequence and putative amino acid sequence of the acaloleptin A cDNA. The numbers at the right indicate the nucleotide and amino acid positions. Four tandem repeats of the nucleotide sequence are indicated with double-headed arrows. The EcoRI site is boxed. The preprosequence is underlined, and the vertical arrow indicates its putative processing site to release signal peptide, as predicted by SignalP ver. 2.0 (http://www.cbs.dtu.dk/services/SignalP-2.0/). The putative mature peptides and basic dipeptides are indicated by open boxes and dotted boxes, respectively. The star symbol below the nucleotide sequence indicates the stop codon. The TTATTTAT, ATTTA, and polyadenylation signal (AATAGA) sequences are shown by the single line, double lines, and the dotted line, respectively. (B) Schematic representation of the acaloleptin A and holotricin 2 precursors. The shaded box represents the preprosequence. The open boxes and black boxes indicate the putative mature peptides and basic dipeptides, respectively. The acidic peptide and the Glu- and Asp-rich region at the N-terminus of isoform 5 are indicated as a crossed box and hatched box, respectively.
216 min, vacuum dried, and then dissolved in distilled water. The active fraction was dried, dissolved in distilled water, and subjected to a third HPLC step on a Synmetory1 C8 column. Fractions were isolated with a linear gradient of 25–30% acetonitrile with 0.05% TFA at a flow rate of 1 ml/min for 216 min, vacuum dried, and then dissolved in distilled water. The active fraction was subjected to Tricine SDS–PAGE [19]. The peptide concentration was determined by the Bradford method [20] using BSA as a standard. 2.9. Assay for the antimicrobial activity The bactericidal activity of hemolymph against E. coli was measured by the plate growth inhibition assay [8], in which the efficacy of bacterial killing correlated with the area of the inhibitory zone in the spot agar layer. Aliquots (1 ml) of the hemolymph mixture, which were collected from the three larvae at the indicated time intervals, were used in the assay. A liquid growth inhibition assay was performed to determine the antibacterial activity and spore germination and growth inhibitory activity against the conidia from rice blast fungus, Magnaporthe grisea was tested as previously described [12].
3. Results 3.1. Cloning and nucleotide sequencing of the acaloleptin A cDNA Approximately 2 104 clones from an immunized larval fat body cDNA library were screened using the 161-bp degenerated PCR product as the probe. Nine positive clones were recovered, two of which had long inserts (approximately 1400 bp). One of these clones was sequenced and found to contain a 1402-bp insert (GenBank accession no. AB094343; Fig. 1a). The putative translation initiation codon ATG was located 26-bp downstream of the 50 end of the cDNA. In the 30 -terminal region, poly(A) tails were identified, and the termination codon TGA was located at nucleotides 1295–1297. Although the typical polyadenylation signal (AATAAA) was lacking in the 30 -untranslated region, the related sequence (AATAGA) was located 36-bp downstream of the stop codon. In this region, there were two ATTTA motifs, which probably affect the stability of the mRNA [21], and one TTATTTAT motif, which represents the consensus sequence of mammalian acute phase protein genes [22]. These sequences were previously identified in other insect antibacterial peptides, such as sarcotoxin IIA and diptericin [23,24].
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Fig. 2. Alignment of the amino acid sequences of the five predicted acaloleptin A isoforms from the acaloleptin A precursor and acaloleptin A1. The boxed residues represent the minor amino acids among the corresponding residues of the isoform sequences.
The acaloleptin A cDNA was expected to encode a 423-amino acid residue precursor protein that contained a preprosequence (Fig. 1A). However, four tandem nucleotide repeats that encoded acaloleptin A-related mature peptides and the Arg-Arg dipeptide (double-headed arrows in Fig. 1) were found. The four deduced mature peptides consisted of 71 amino acid residues each, and shared high-level sequence similarities with acaloleptin A1 [8]. We designated these peptides as isoforms 1, 2, 3, and 4, in the order of their positions in the sequence (Fig. 1B). In the 105-aa region that followed the four tandem repeats of isoforms, 59 amino acid residues (from aa 365 in the putative precursor) showed significant sequence similarities with aa 13–71 of the acaloleptin A isoforms1, 2, 3, and 4. It was proposed that this C-terminal 69-aa region of the precursor is secreted as a peptide along with the other isoforms, as the acaloleptin A precursor appears to be processed at the presumed processing signal R-X-R/K-R (see Section 4) of the 351–354 region. We regarded this putative 69-aa peptide as isoform 5. Therefore, it appears that the acaloleptin A precursor forms a multipeptide structure that consists of five acaloleptin A isoforms.
3.3. Acaloleptin A gene expression in larval fat bodies Northern blot analysis of the larval fat bodies showed that acaloleptin A gene expression was induced only in the immunized larvae (Fig. 3A). Furthermore, only the 1.4-kbpositive band was detected, which suggests that the acaloleptin A gene(s) produces a single size of transcript. In contrast, the transcripts of apidaecin, which is a honeybee antibacterial peptide that is processed from a multipeptide precursor, vary in size from 650 bp to 1400 bp because of the existence of multiple genes [25].
3.2. Sequence variations among the acaloleptin A isoforms The primary structures of the five isoforms were aligned (Fig. 2) and shown to possess non-identical amino acid sequences, although they shared significant sequence similarities. Notably, high levels of sequence similarity were found among isoforms 1, 2, and 3 (1 and 2: 93.0%; 1 and 3: 95.8%; 2 and 3: 97.2%). The sequence of isoform 4 differed from those of the other three isoforms, with a sequence identity of approximately 90% (1 and 4: 90.1%; 2 and 4: 87.3%; 3 and 4: 90.1%). However, unlike the other isoforms, isoform 5 contained a unique N-terminal region that was rich in aspartic and glutamic acids. Furthermore, the region from positions 11–69 in isoform 5 contained many different amino acids compared to the corresponding regions in the other four isoforms. The sequence of isoform 5 was only 67.4% identical to isoform 4, which shared greater sequence similarity with isoform 5 than did isoforms 1, 2, or 3. The cloned cDNA did not encode any isoforms that were 100% identical to the amino acid sequence of acaloleptin A1, although there was only one amino acid difference between the sequences of isoform 3 and acaloleptin A1 (Fig. 2). This phenomenon is presumably due to allelic polymorphisms, as cDNA and hemolymph were prepared from larvae which enclosed from eggs laid by field-captured adult insects.
Fig. 3. Northern blot analysis of total RNA from naı¨ve and immunized larvae (A) and southern blot analysis for the acaloleptin A gene (B). Total RNA was extracted from naı¨ve and bacteria-challenged (immune) sixth-instar larvae. Samples (20 mg) were blotted from the denaturing gel onto a nylon membrane. The 30 -terminal 400-bp EcoRI fragment of the cDNA was used as the probe for Northern blotting. The ribosomal RNA band was used as the loading control (A). Genomic DNA (10 mg) was digested with DraI, HindIII, and EcoRI. The digested samples were electrophoresed on a 0.9% agarose gel, transferred to a nylon membrane, and hybridized with the 400-bp EcoRI cDNA probe (B).
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3.4. Southern blot analysis of genomic DNA Southern blot analysis was performed to estimate the number of copies of the acaloleptin A gene in the genome. Two HindIII fragments were detected, and two high-intensity bands and one weak band were detected in the DraI- and EcoRI-digested DNA, respectively, with a 400-bp C-terminal probe (nucleotides 10311404 of the cloned cDNA; Fig. 3B). Although no DraI or HindIII site was present, one EcoRI site was found in the cDNA at the 50 -end of the probe site. A faint band that was detected in the DraI- and EcoRI-digested DNAs may indicate the existence of these restriction enzyme sites within the probe region of the different alleles, which was not cloned in the cDNA. Genomic DNA was extracted from the whole body of a single fifth- or sixth-instar larva. Therefore, it is possible that the acaloleptin A gene exists as a single copy per haploid and that two different alleles exist in the genome. 3.5. Kinetics of acaloleptin A gene expression in the fat bodies The kinetics of acaloleptin A expression at the mRNA and peptide levels were investigated after inoculation with E. coli. Acaloleptin A transcripts were first recognized 2 h after injection, and expression peaked after 24 h. Subsequently, the expression level decreased gradually, although a considerable level of expression persisted after 72 h (Fig. 4A). On the other hand, the concentration of acaloleptin A isoforms in the hemolymph, as detected using the antiserum, increased 6 h after bacterial inoculation, and peaked after 48 h. High-level expression was maintained after 72 h, although this level was lower than that after 48 h (Fig. 4A). We also measured the antibacterial activity of hemolymph against E. coli at each time-point. Antibacterial activity appeared 12 h post-inoculation, peaked at 48 h, and was maintained at a high level after 72 h (Fig. 4B). Thus, the antibacterial activity levels correlated with the levels of acaloleptin A peptide in the hemolymph.
Fig. 4. Kinetics of acaloleptin A mRNA, peptide induction, and anti-E. coli activities of the larval hemolymph after immunization. The levels of mRNA expression and peptide synthesis were investigated by Northern and Western blotting, respectively, after inoculation with E. coli (A). The anti-E. coli activities of the larval hemolymph at each time point after inoculation are shown in (B). The activities of the hemolymph were inferred from the size of the inhibition zones.
3.6. Purification of a peptide with the same N-terminal amino acid sequence as isoform 5 from the hemolymph of A. luxuriosa larvae A fraction of the hemolymph of immune-challenged sixth instar larvae of A. luxuriosa, eluted from an ODS cartridge column (SepPak C18, Waters) with 30% acetonitrile were previously reported to possess potent antibacterial activity [8]. Multiple fractions were first separated by reverse phase HPLC on a TSKgel ODS 120T column (Toso), and fractions showing anti-Micrococcus luteus activity were identified as previously described [11,12]. We selected one of the fractions with activity against M. luteus (data not shown) and purified it in a stepwise manner via HPLC on a Symmetry1 C8 column (Fig. 5A). Purified peptide was obtained after elution with 25% acetonitrile at the final purification step. The purified product was observed as a single peptide band of 7.5 kDa by Tricine SDS–PAGE (Fig. 5B). The first 13 residues of the Nterminal amino acid sequence of the purified peptide were identified as SDDEDEEEEEDQP via automated Edman degradation. This sequence was identical to the N-terminal sequence of the putative acaloleptin A isoform 5, which was predicted in Section 3.1. These results indicated that isoform 5 was actually processed and secreted into the hemolymph. 3.7. Antimicrobial activity of the purified peptide Using a liquid growth inhibition assay, the antibacterial activity of the purified peptide was evaluated for four Gram-positive bacterial strains (M. luteus, B. subtilis, B. cereus, and S. aureus), five Gram-negative bacterial strains (E. coli, A. hydrophila, E. persicinus,
Fig. 5. Purification of an antimicrobial peptide from A. luxuriosa larvae. (A) Final step reverse-phase HPLC profile of the active fraction. The arrow indicates the fraction with antibacterial activity against M. luteus. (B) Tricine SDS–PAGE of the active fraction indicated in (A). The arrow indicates the purified peptide. The first 13 residues of the N-terminal amino acid sequence of the purified peptide obtained via Edman degradation was also indicated.
S. marcescens, and A. hydrophila), and one yeast strain (S. cerevisiae). The purified peptide inhibited more than 70% of the growth of M. luteus at 20 mg/ml (Fig. 6A), but did not inhibit the growth of other bacteria and yeast. The ability of the purified peptide to inhibit spore germination and growth was tested against the conidia of rice blast fungus, Magnaporthe grisea. The purified peptide at a concentration of 161.4 mg/ml inhibited the germination of conidia and/or the growth of mycelium (Fig. 6B). These results suggested that the purified peptide might possess antibacterial activity
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Fig. 6. Anti-bacterial and anti-fungal activities of the purified peptide. (A) Growth inhibitory activity of the purified peptide against M. luteus. Growth inhibitory activities against four bacteria and one yeast were estimated as described in Section 2. *, M. luteus; *, B. subtilis; , S. aureus; &, B. cereus. (B and C) Germination- and growth-inhibitory activity of the purified peptide against conidia from the rice blast fungus, M. grisea. Conidia of M. grisea were incubated with (B) or without (C) the purified peptide at 20 8C for 24 h and then spore germination and the growth of mycelia were observed via phase-contrast microscopy. Arrowheads indicate the conidia of M. grisea.
against some Gram-positive bacteria and antifungal activity, and has different antimicrobial spectrum from that of Acaloleptin A1, 2 and 3, which show antibacterial activity against only Gramnegative bacteria [8].
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a single precursor (Fig. 1). It was reported that apidaecin and the putative cysteine-rich antibacterial peptides from the hemipteran insect Riptortus clavatus are processed from multipeptide precursors [25,26]. In other organisms except for insects, only the magainins from Xenopus, the cysteine-rich antibacterial peptides from the seeds of Impatiens balsamina (Ib-AMPs) and naegleriapore B from amoeboflagellate are derived from multipeptide precursors [27–30]. Bearing in mind that numerous antibacterial peptides have been isolated from a wide range of organisms, it seems likely that the occurrence of multipeptide precursor structures in antibacterial peptides is rare. Nevertheless, antibacterial peptides with multipeptide precursors have been identified in diverse organisms, including invertebrates, vertebrates, protozoa and plants. These antibacterial peptides do not share sequence similarities, with the exception of the apidaecin and cysteine-rich antibacterial peptides from hemiptera. This suggests that multipeptide precursors of antibacterial peptides did not evolve from a single ancestor, but instead, each antibacterial peptide acquired independently the multipeptide precursor structure during evolution of the organism. The precursors of many secreted proteins and membrane proteins in non-endocrine cells contain an R-X-K/R-R signal tetrapeptide that is recognized and processed by a furin-like protease [31]. The cDNAs of six antibacterial peptides have been cloned from coleopteran insects, and shown to contain this tetrapeptide, which is used for the processing of the precursors [14–16,32,33]. The C-terminal tetrapeptide in the preprosequence of the acaloleptin A precursor is R-W-K-R, which suggests that this tetrapeptide is also used for processing. The C-terminal dipeptides of isoforms 14 are R-W, and R-R dipeptides link each isoform (Fig. 1A). Therefore, it seems likely that the acaloleptin A precursor is processed by the furin-like protease not only at the C-terminus of the preprosequence, but also at the C-terminus of isoforms 1–4. In fact, three different isoforms (acaloleptin A1, 2, 3), which share a SLQPGA sequence at the N-terminus but differ in terms of retention times in the ODS column, were confirmed to be secreted into the hemolymph as mature peptides [8]. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis has shown that peptides with molecular weights similar to those of the four acaloleptin A isoforms exist in immunized larval hemolymph (data not shown). An acidic sequence between two R-X-K/R-R motives at positions 315–318 and 351–354 have no homology with the acaloleptin A mature peptides, and no proteins with homology to this region were detected using BLAST. Because the acaloleptin A precursor is presumably processed at this tetrapeptide motif, we expect the precursor to be processed at the C-terminus of amino acid 354. Assuming that this is true, four 71-aa isoforms, one 69-aa isoform (isoform 5), which has a different N-terminal region than the other isoforms, and a 34-aa acidic peptide should be generated from the acaloleptin A precursor. Indeed, a peptide with the same N-terminal amino acid sequence as isoform 5 was purified from larval hemolymph in this study (Fig. 5). However, we have no information regarding the presence or function of the acidic peptide in the hemolymph. In the case of magainin, acidic 6-aa peptides are secreted with the mature peptides after precursor processing; the function of these acidic peptides has not yet been clarified [28]. 4.2. Origin of the precursor structure of acaloleptin A
4. Discussion 4.1. Structure of the acaloleptin A precursor and its mature peptides Based on the nucleotide sequence of the acaloleptin A cDNA, it appeared that five isoforms were generated through processing of
The precursors of holotricin 2, rhinocerosin, and the A. dichotoma coleoptericin, which are thought to be orthologs of acaloleptin A, produce single mature peptides and are unlike the acaloleptin A precursor. As far as we know, there are no reports that the precursor structures among the orthologs are completely
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maintained until 72 h post-inoculation (Fig. 4B). The antibacterial activity levels mirrored the acaloleptin A concentrations in the hemolymph (Fig. 4A). This result suggests that acaloleptin A is an important inhibitor of the proliferation of Gram-negative bacteria in the hemocoel. 4.4. Biological significance of multiple acaloleptin A genes
Fig. 7. Phylogenetic analysis of the coleoptericin family of peptides. The tree was constructed based on the complete amino acid sequence of the mature coleoptericin peptides using the Clustal X program. The numbers indicate the values that were obtained from 1000 bootstrap replicates. The accession numbers of the peptide sequences are as follows: acaloleptin A1, P81592; coleoptericin, A41711; holotricin 2, Q25054; rhinocerosin, O76145; A. dichotoma coleoptericin A, BAB40436; A. dichotoma coleoptericin B, BAB40437; A. dichotoma coleoptericin C, BAB40438; diptericin, 1403303A.
different, which is interesting from both a phylogenetic and evolutionary perspective. A phylogenetic tree was constructed based on the amino acid sequences of the five mature isoforms of the acaloleptin A precursor, coleoptericin, holotricin 2, rhinocerosin, and the A. dichotoma coleoptericin (Fig. 7). The five acaloleptin A isoforms formed a cluster with coleoptericin, whereas rhinocerosin, holotricin 2, and the A. dichotoma coleoptericin formed another cluster. According to the morphological classification of the coleopteran insects, A. luxuriosa and Zophobas atratus, from which coleoptericin has been isolated, belong to the Chrysomeloidea and Tenebrionoidea superfamilies, respectively. Because both of these superfamilies belong to Cucujiformia, it appears that these two species are relatively closely related. On the other hand, Holotrichia idiomphalia, Oryctes rhinoceros, and A. dichotoma, from which holotricin 2, rhinocerosin, and the A. dichotoma coleoptericin have been isolated, respectively, are classified into superfamily Scarabaeoidea of the infraorder Scarabaeiformia. Therefore, the peptide sequence-based phylogenetic tree is consistent with the tree based on morphological characters. Taken together, these findings suggest that the multipeptide precursor in the coleoptericin family emerged after the divergence of Cucujiformia and Scarabaeiformia, during the evolution of coleopteran insects. However, the timing of multipeptide acquisition is unknown. 4.3. Alterations in the levels of acaloleptin A mRNA and peptide after bacterial inoculation In Drosophila, various antibacterial peptide gene expression patterns have been observed after bacterial inoculation [34]. With respect to cecropin, attacin, and defensin, the peak mRNA levels were observed 6–12 h after immune-stimulation, whereas the levels of diptericin, metchnikowin, and drosocin transcripts peaked 12–24 h later. In the case of acaloleptin A, gene expression increased for up to 48 h after inoculation with E. coli, and remained high for an additional 72 h (Fig. 4A). Therefore, the expression pattern of the acaloleptin A gene is similar to those of the diptericin, metchnikowin, and drosocin genes, which have antibacterial activities against Gram-negative bacteria. This suggests that the regulation of acaloleptin A gene expression may be similar to that of Drosophila antibacterial peptide genes. The antibacterial activity of the immunized A. luxuriosa larval hemolymph against E. coli peaked 48 h after inoculation, and was
In this study, we showed that the acaloleptin A precursor had a multipeptide structure. It is possible that the multipeptide structure of the precursor ensures rapid mass production of acaloleptin A isoforms following bacterial invasion, thereby raising an effective immune response. By comparing the signal intensity between immunized hemolymph and graduated dilutions of purified acaloleptin A in Western blots, the concentration of acaloleptin A isoforms in the hemolymph was determined to exceed 100 mg/ml at 48 h after bacterial inoculation (data not shown). No significant difference was observed regarding the activities of acaloleptin A1, A2, or A3 [8], which supports the idea that redundancy of the isoforms quantitatively amplifies the immune response. The N-terminal sequence of isoform 5 was completely different from the sequences of isoforms 1, 2, 3, and 4, and the orthologs of various insects (Fig. 2), although this region was strictly conserved in the coleoptericin family of peptides. A peptide that has an identical N-terminal sequence to acaloleptin A isoform 5 was isolated from larval hemolymph and shown to have antimicrobial activity against a Gram-positive bacterium, M. luteus, and a fungus, M. grisea (Fig. 6). These results suggest that the multipeptide precursor structure confers upon the acaloleptin A isoforms a broader range of antimicrobial activity. Acaloleptin A is unique among the antibacterial peptides in having a precursor that contains several related peptides with different activities, although a similar phenomenon has been described for neuropeptide precursors [35]. References [1] Lemaitre B, Kromer-Metzger E, Michaut L, Nicolas E, Meister M, Georgel P, et al. A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense. Proc Natl Acad Sci USA 1995;92: 9465–9. [2] Engstro¨m Y. Induction and regulation of antimicrobial peptides in Drosophila. Dev Comp Immunol 1999;23:345–58. [3] Hedengren M, Asling B, Dushay MS, Ando I, Ekengren S, Wihlborg M, et al. Relish, a central factor in the control of humoral but not cellular immunity in Drosophila. Mol Cell 1999;4:827–37. [4] Bulet P, Hetru C, Dimarcq JL, Hoffmann D. Antimicrobial peptides in insects; structure and function. Dev Comp Immunol 1999;23:329–44. [5] Meister M, Lemaitre B, Hoffmann JA. Antimicrobial peptide defense in Drosophila. Bioessays 1997;19:1019–26. [6] Dimarcq JL, Bulet P, Hetru C, Hoffmann J. Cysteine-rich antimicrobial peptides in invertebrates. Biopolymers 1998;47:465–77. [7] Hoffmann JA, Reichhart JM, Hetru C. Innate immunity in higher insects. Curr Opin Immunol 1996;8:8–13. [8] Imamura M, Wada S, Koizumi N, Kadotani T, Yaoi K, Sato R, et al. Inducible antibacterial peptides from larvae of the beetle, Acalolepta luxuriosa. Arch Insect Biochem Physiol 1999;40:88–98. [9] Saito A, Ueda K, Imamura M, Atsumi S, Tabunoki H, Miura N, et al. Purification and cDNA cloning of a cecropin from the longicorn beetle, Acalolepta luxuriosa. Comp Biochem Physiol B Biochem Mol Biol 2005;142:317–23. [10] Saito A, Ueda K, Imamura M, Miura N, Atsumi S, Tabunoki H, et al. Purification and cDNA cloning of a novel antibacterial peptide with a cysteine-stabilized alphabeta motif from the longicorn beetle, Acalolepta luxuriosa. Dev Comp Immunol 2004;28:1–7. [11] Ueda K, Imamura M, Saito A, Sato R. Purification and cDNA cloning of a novel insect defensin from larvae of the longicorn beetle, Acalolepta luxuriosa. Appl Entomol Zool 2005;40:335–45. [12] Ueda K, Saito A, Imamura M, Miura N, Atsumi S, Tabunoki H, et al. Purification and cDNA cloning of luxuriosin, a novel antibacterial peptide with Kunitz domain from the longicorn beetle, Acalolepta luxuriosa. Biochim Biophys Acta 2005;1722:36–42. [13] Bulet P, Cociancich S, Dimarcq JL, Lambert J, Reichhart JM, Hoffmann D, et al. Insect immunity. Isolation from a coleopteran insect of a novel inducible
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