Purification and cDNA cloning of a cecropin from the longicorn beetle, Acalolepta luxuriosa

Comparative Biochemistry and Physiology, Part B 142 (2005) 317 – 323 www.elsevier.com/locate/cbpb

Purification and cDNA cloning of a cecropin from the longicorn beetle, Acalolepta luxuriosa B Ayaka Saito a, Kenjiro Ueda a, Morikazu Imamura b, Shogo Atsumi a, Hiroko Tabunoki a, Nami Miura a, Ayako Watanabe a, Madoka Kitami a, Ryoichi Sato a,* a

b

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan Prion Disease Research Center, National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan Received 7 April 2005; received in revised form 4 August 2005; accepted 8 August 2005 Available online 13 September 2005

Abstract We have cloned and characterized a novel antibacterial peptide from the hemolymph of the coleopteran insect Acalolepta luxuriosa, of the superfamily Cerambyocidea. This peptide is active against Micrococcus luteus and Escherichia coli, and the amino acid sequence deduced by cloning of the cDNA identifies it as a coleopteran cecropin. Sequence comparisons and phylogenetic analyses performed using Clustal X suggest that this cecropin is evolutionarily intermediate between dipteran and lepidopteran cecropins. The results of MALDI-TOF mass spectrometry indicate that the mature form of this antibacterial peptide is 35 amino acid residues in length and has an amidated C-terminal isoleucine. This report is the first description of a cecropin from a coleopteran insect. D 2005 Elsevier Inc. All rights reserved. Keywords: Cecropin; Antibacterial peptide; Purification; Coleoptera; Insect immunity; Acalolepta luxuriosa; Hemolymph; MALDI-TOF

1. Introduction Insects employ both cellular and humoral defense systems. Much research has shown that antibacterial peptides comprise a very important aspect of humoral defense systems in insects. Today, more than 200 such peptides have been identified in insects. Although these peptides exhibit great structural diversity, certain common structural patterns are apparent, and the peptides are often classified into three groups. These are 1) linear alpha-helical peptides devoid of cysteine residues, 2) peptides in which proline and/or glycine residues are over-represented, and 3) cysteine-rich peptides with cysteine-stabilized a-h (CSah) motifs (Bulet et al., 1999). Cecropin, an inducible antibacterial peptide that is found in the hemolymph of the pupae of Hyalophora cecropia, was i

DDBJ accession number of the cDNA: AB188500. * Corresponding author. Tel./fax: +81 42 388 7277. E-mail address: [email protected] (R. Sato).

1096-4959/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2005.08.001

the first insect antimicrobial peptide discovered (Steiner et al., 1981). Subsequently, cecropins from tunicate (Zhao et al., 1977) and Ascaris nematodes (Andersson et al., 2003) have also been identified, suggesting that cecropins are widespread throughout the animal kingdom. In insects, however, cecropins have been described only in the orders Diptera and Lepidoptera, and it was uncertain whether cecropins had a broader distribution among the insects. Mature cecropin peptides lack cysteine residues, are 35 to 39 amino acids in length (Cociancich et al., 1994), and form two linear ahelices connected by a hinge (Holak et al., 1988). The Cterminal amino acid of the H. cecropia cecropin B is amidated (van Hofsten et al., 1985), and many other cecropins are also expected to be amidated (Cociancich et al., 1994). Insects are the most diverse animal group, accounting for more than 70% of the species in the animal kingdom. Insects have diversified remarkably since they appeared 300 million years ago. Consequently, some antibacterial peptides are found only in restricted groups of insects. For instance, coleoptericins are found only in coleopterans (Bulet et al.,

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1991; Lee et al., 1994; Yang et al., 1998; Imamura et al., 1999; Sagisaki et al., 2001), and lebocin is found only in lepidopterans (Hara and Yamakawa, 1995). Since exploration of antimicrobial peptides in coleopteran insects had thus far been limited to only six insect species, all of which are in the Scarabaeoidea or Tenebrionidea superfamilies, we sought to discover new groups of antibacterial peptides by examining insects of other superfamilies. In previous studies, we described the purification of three novel antibacterial peptides from the hemolymph of the unusual coleopteran insect Acalolepta luxuriosa, of the superfamily Cerambyocidea. These peptides are the coleopteran-specific antibacterial peptide Acaloleptin A (Imamura et al., 1999), a CSah motif-containing peptide that is only distantly related to insect defensins (Saito et al., 2004) and Luxuriosin, a novel antibacterial peptide with Kunitz domain (Ueda et al., 2005). In this paper we describe the purification, cDNA cloning, and deduced amino acid sequence of a cecropin from A. luxuriosa. Using MALDITOF mass spectrometry (MS), we found that the mature form of this antibacterial peptide appears to have 35 amino acid residues, and that the C-terminal isoleucine residue is amidated. This report constitutes the first description of a cecropin from a coleopteran insect.

E. coli K12 W3110 and M. luteus IAM1056 were used. A plate growth inhibition assay was performed as described previously (Saito et al., 2004). Briefly, 5 ml of LB 0.5% agar medium containing 1 107 logarithmic-phase bacterial cells were poured on 1.5% agar LB medium in a 9-cm Petri dish. After cooling, 5 Al of the sample containing 1 Ag of peptide were dropped on the gel and the plates were incubated at 37 or 30 -C for E. coli and M. luteus, respectively.

2. Materials and methods

2.5. Measuring molecular mass and partial amino acid sequence analysis

2.1. Insects A. luxuriosa were reared as described previously by Akutsu (Akutsu, 1985) and hemolymph was collected from 6th instar larvae. 2.2. Immunization and collection of hemolymph In our experience, formalin-fixed bacteria are most effective for inducing overall antimicrobial activity in A. luxuriosa (data not shown). Formalin-fixed Escherichia coli, washed and suspended in physiological saline (150 mM NaCl, 5 mM KCl, pH 6.8) at 8.5  108 cells/ml, were prepared as the inoculum. A. luxuriosa larvae were injected with 30 Al of formalin-fixed E. coli. The hemolymph was collected 24 h later, and centrifuged for 20 min at 1400 g at 4 -C to obtain a clear supernatant. 2.3. Purification of the antibacterial peptide A two-fold diluted hemolymph sample was subjected to chromatography in a Sep-Pak C18 cartridge column (Waters) pre-equilibrated with 0.05% trifluoroacetic acid (TFA) and eluted with 0%, 20%, 30%, 50%, 80%, and 100% acetonitrile acidified with 0.05% TFA. The 30% fraction was subjected to reverse phase HPLC on a TSKgel ODS 120T (300  7.8 mm, C18) column (TOSO) since it displayed the most potent anti-E. coli and anti-M. luteus

activity. Fractions were isolated with a linear gradient of 20– 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 TSKgel ODS 120T column. Fractions were isolated with a linear gradient of 28 –40% 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 (Schagger and von Jagow, 1987). The peptide concentration was determined by the Bradford method (Bradford, 1976) using BSA as a standard. 2.4. Assay for antibacterial activity

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was conducted on a Voyager Elitei Biospectrometer (PerSeptive Biosystems) operating in reflector-delayed extraction positive ion mode at a resolution of 10,000 using a-cyano-4-hydroxycinnamic acid (a-CHCA) (Aldrich) as the matrix. a-CHCA was dissolved in 70% acetonitrile at a concentration of 10 mg/ ml and a 1 Al portion of the matrix solution was deposited onto a sample target plate and allowed to dry in air at room temperature. One microliter of the peptide sample solution was deposited onto dried matrix and allowed to dry in air at room temperature and then a 1 Al portion of the matrix solution was deposited onto it and allowed to dry in air at room temperature. Mass spectra were internally calibrated with bacitracin (1422.75) and oxidized insulin B chain (3494.65), the SDS-PAGE molecular weight standards (BIO RAD). The active fraction was subjected to Tricine SDS-PAGE, transferred to PVDF membrane, and then the N-terminal amino acid sequence of the antibacterial peptide was determined by automated Edman degradation performed with a model HP241 amino acid sequencer (Hewlett & Packard). 2.6. Cloning the cDNA and nucleotide sequencing Total RNA was extracted from the sixth instar larval fat body using a QuickPrep\ Total RNA Extraction Kit

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(Pharmacia), 12 h after injecting 1  06 formaldehyde-fixed E. coli cells as described previously (Saito et al., 2004). The first-strand cDNA was synthesized with 12 to 18 oligo dT primers associated with M13M4 nucleotide sequences ( M 1 3 M 4 o l i g o d ( 1 2 – 1 8 m i x ) T p r i m e r : 5 VGTTTTCCCAGTCACGACdT(12 – 18)-3V) (Takara) using Rever Tra Ace (TOYOBO) as the reverse transcriptase. For 5V RACE (5V Rapid Amplification of cDNA Ends) firststrand cDNA was synthesized with an anti-sense-1 primer (5V-TGTTTTGCAACTCCTG-3V) designed from the A. luxuriosa cecropin cDNA and then polyadenylated with terminal deoxynucleotidyl transferase (Amersham Pharmacia). Double-stranded cDNA fragments were amplified by PCR using the primer sets i) sense-1 (5V-GAYAAYTTYTTYAARMG-3V) or sense-2 (5V-GARAARGTNGGN A A R A A - 3 V) a n d M 1 3 M 4 ( 5 V- G T T T T C C C A GTCACGAC-3V) for 3V RACE, and for 5V RACE ii) M13M4 oligo d(12 – 18 mix)T primer or M13M4 and antisense-2 (5V-AACTCCTGCATAGCCTAC-3V) or anti-sense-3 (5V-CATAGCCTACCACGGTT-3V). cDNA fragment spanning the entire coding region of the peptide was amplified with the primer sets of sense-3 (5V-GTCTCTAGCAGTCTTCAG-3V) or sense-4 (5V-CATCACTACACATCA -3V) and M13M4. The PCR cycling conditions were as follows: initial denaturation at 94 -C for 1 min and 37 cycles of 94 -C for 1 min, 45 -C for 1 min, and 72 -C for 1 min. The amplified fragment was cloned into T-overhang vector p123T (Mo Bi Tec). The cDNA was sequenced using a Long Read Tower DNA sequencer (Amersham Pharmacia). A computer-assisted homology search was performed using the Internet BLAST and FASTA searches of the Bioinformatics Center of Kyoto University. The sequences were aligned with CLUSTAL X (Thompson et al., 1997) and DRAWTREE of PHYLIP ver.3.5 C was used to generate an unrooted bootstrap tree.

3. Results and discussion 3.1. Purification of an antibacterial peptide from A. luxuriosa hemolymph Cell-free hemolymph collected from immunized larvae was loaded onto a Sep-Pak C18 cartridge column (Waters) and eluted stepwise with 0%, 20%, 30%, 50%, 80%, and 100% acetonitrile in 0.05% trifluoroacetic acid (TFA). When aliquots of the resulting fractions were tested using a plate growth inhibition assay, the 30% fraction displayed the most potent anti-E. coli and anti-M. luteus activity (data not shown). To further purify the antibacterial peptide, the Sep-Pak C18 30% eluate was subjected to reverse phase HPLC on a TSKgel ODS column (TOSO). The fraction eluted at 36% acetonitrile displayed activity against E. coli and M. luteus (Fig. 1A), and was subjected to a second reverse phase HPLC step on a TSKgel ODS column (Fig. 1B). A single peak eluted at 30% acetonitrile showed

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antibacterial activity, producing clear zones on E. coli and M. luteus culture plates (Fig. 1C and D). Tricine SDS-PAGE showed that the final, 30% acetonitrile peak contained a 2.3-kDa peptide (data not shown). Analysis of the peptide by MALDI-TOF MS yielded five isotopic ions ([MH+]) at a resolution of 10,000. The peak of the monoisotopic ion was observed at m / z = 3901.78 (Fig. 1E). 3.2. cDNA cloning and amino acid sequence determination Twenty-one residues of the N-terminal amino acid sequence of the purified peptide were determined using automated Edman degradation (Fig. 2A). Homology searches using the BLAST and FASTA programs indicated that the peptide sequence is highly similar to that of insect cecropins. The complete amino acid sequence of the peptide was deduced via cDNA cloning. The first-strand cDNA was synthesized with 12 to 18 oligo(dT) primers associated with M13M4 nucleotide sequences. We designed degenerate primers (sense-1 and sense-2) and amplified a doublestranded cDNA fragment by two-step PCR using these primers and an M13M4 adapter primer with the singlestranded cDNA from the sixth instar larval fat body as the template. The resulting 215-bp cDNA fragment contained the region from the degenerate primer (sense-2) to the polyA sequence and was sequenced. Next, we designed three anti-sense primers (anti-sense-1, -2, and -3) from this sequence, performed reverse transcription with the antisense-1 primer using total RNA as the template, and polyadenylated the product. We then performed two-step PCR with the anti-sense-2, anti-sense-3, and oligo d(12 – 18 mix)T primer associated with M13M4 nucleotide sequences and M13M4 adapter primers to clone the region upstream from the cloned cDNA fragment. Consequently, a 211-bp cDNA fragment containing the 5V-untranslated region was cloned and sequenced. Finally, we designed primers sense-3 and sense-4 for the 5V-untranslated regions, performed twostep PCR with these primers and the M13M4 adapter primer, and cloned and sequenced a cDNA fragment encoding the entire peptide. 3.3. Analysis of A. luxuriosa cecropin processing Using NetStart software (Pedersen and Neilsen, 1997), we estimated the site of translation initiation to be at the adenine residue 79 bases downstream from the 5V terminus of the cDNA fragment (Fig. 2B). The Kozak consensus sequence AAAATGAAG (Kozak, 1984) is present at this site. Using the Signal P program (Nielsen et al., 1997) the cleavage site for the signal peptide was predicted to be between Ser23 and Lys24 (Fig. 2B). The Nterminal amino acid sequence obtained for the purified peptide was equivalent to residues 24 to 44 (Fig. 2B). The 60th amino sequences (ATAAA) were present 81 and 87

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Fig. 1. Purification of an antibacterial peptide from A. luxuriosa. (A) Initial purification of the antibacterial peptide. The fraction eluted at 30% TFA from a SepPak C18 cartridge column was loaded onto a TSKgel ODS-120T column. The elution profile of the second column is shown here. The arrow indicates the fraction with antibacterial activity against E. coli and M. luteus. (B) Final purification of the antibacterial peptide. The active fraction indicated in A was loaded onto a second TSKgel ODS-120T column, which was eluted over 100 min with a linear gradient of 23 – 33% acetonitrile. The arrow indicates the fraction with antibacterial activity against M. luteus and E. coli. (C) and (D) Growth inhibition of M. luteus (C) and E. coli (D) by the active fraction indicated in B. Arrows indicate the bacterial growth inhibition zones caused by the active fraction. Arrowheads indicate sites at which control samples were placed. (E) MALDI-TOF MS spectrum of the purified antibacterial peptide. The peaks at m / z = 3901.78, 3902.82, 3903.81, 3904.78, and 3905.84 indicate the mono-, di-, tri-, tetra- and penta-isotopic ions for [MH]+, respectively.

Fig. 2. Amino acid and nucleotide sequences of the A. luxuriosa antibacterial peptide. (A) Partial amino acid sequence of the purified antibacterial peptide as determined by automated Edman degradation. (B) Nucleotide sequence of the cDNA and deduced amino acid sequence of the antibacterial peptide. The putative mature peptide is boxed and an asterisk indicates the amino acid that is postulated to be amidated. The vertical arrow indicates the putative processing site for the signal peptide as predicted by Signal P vs 1.1. The solid and dashed lines indicate the Kozak consensus sequence (AAAATGAAG) and the polyadenylation signal-like sequences (ATAAA), respectively.

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residues following the terminal codon (Fig. 2B). From these analyses, we postulated that the peptide precursor consists of a 23-residue signal sequence and a 37-residue mature peptide. However, the theoretical monoisotopic

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molecular weight for the mature peptide of 37 amino acid residues is 4087.34, which differs greatly from that of the monoisotopic ion obtained by MALDI-TOF MS (3901.78) (Fig. 1F).

Fig. 3. Multiple sequence alignment and phylogenetic tree of mature cecropin peptides. (A) Multiple sequence alignment performed using CLUSTAL X. AgCec, cecropin form A. gambiae (Vizioli et al., 2000); BactericidinB2, bactericidin B-2 from Manduca sexta (Dickinson et al., 1988); HcCecA, HcCecB, and HcCecD, cecropins A, B, and D, respectively, from H. cecropia (Gudmundsson et al., 1991; Xanthopoulos et al., 1988); BmCecA, BmCecB, BmCecD, and Enbocin, cecropins A, B, and D and Enbocin, respectively, from Bombyx mori (Yamano et al., 1994; Taniai et al., 1995; Taniai et al., 1999; Kim et al., 1998); DmCecA1, DmCecB, and DmCecC, cecropins A1, A2, B, and C, respectively, from Drosophila melanogaster (Date et al., 1998; Samakovlis et al., 1990; Tryselius et al., 1992); DsiCecB, cecropin B from Drosophila simulans (Ramos-Onsins and Aguade, 1998); DvCec2, Cecropin 2 from Drosophila virilis (Zhou et al., 1997); DyCecC, cecropin C from Drosophila yakuba (Ito et al., 2002); DseCecC, cecropin C from (Ito et al., 2002); Sarcotoxin IA and Sarcotoxin IB, sarcotoxin IA and sarcotoxin IB, respectively, from Sarcophaga peregrine (Matsumoto et al., 1986; Kanai and Natori, 1989); CcCec1 and CcCec2, Cec 1 and 2, respectively, from Ceratitis capitata (Rosetto et al., 1993); GmCec, cecropin from Glossina morsitans (Boulanger et al., 2002); AaCecA1, AaCecB, and AaCecC, cecropins A1, B, and C, respectively, from Aedes albopictus (Sun et al., 1998, 1999); CecropinP1, cecropin P1 from Ascaris suum (Andersson et al., 2003); StyelinC, styelin C from Styela clava (Zhao et al., 1977); TnCecA, cecropin A from Trichoplusia ni (Kang et al., 1996); AlCec, A. luxuriosa cecropin (this work). (B) Phylogenetic tree drawn with PHYLIP vs 3.5C. The numbers are the bootstrap values using 1000 replicates with CLUSTAL X.

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Cecropin B from H. cecropia has an amidated Cterminal amino acid (van Hofsten et al., 1985), as is expected for many insect cecropins (Cociancich et al., 1994). Amide groups which are involved in amidation are believed to derive from C-terminal Gly or second Cterminal Gly (Lidholm et al., 1997). The calculated monoisotopic mass of the mature A. luxuriosa cecropin peptide with amidation at Ile35 is 3901.23, which is in close agreement with the MS result. Therefore, we conclude that the mature peptide of A. luxuriosa cecropin consists of 35 amino acid residues and has an amidated Cterminus (Fig. 2B). 3.4. Cecropin sequence alignment and phylogenetic analysis of A. luxuriosa cecropin Cecropins have been reported not only in insects but also in a tunicate (Zhao et al., 1977) and ascarid nematodes (Andersson et al., 2003). However, cecropins have never before been reported in insects other than dipterans and lepidopterans. This report constitutes the first demonstration that coleopteran insects also produce cecropins. The evolutionary relationship between the cecropin from A. luxuriosa and cecropins from dipteran and lepidopteran insects was examined via a multiple sequence alignment performed using CLUSTAL X (Thompson et al., 1997). Some amino acid residues conserved for both dipteran and lepidopteran insects were also conserved for A. luxuriosa, and some amino acid residues conserved only within the dipteran or lepidopteran sequences were also conserved for A. luxuriosa (Fig. 3A). This result suggests that the A. luxuriosa cecropin is of an intermediate type. The presence of a tryptophan at the 1st or 2nd position of the N-terminal of the molecule was reported to be a common feature of almost all the insects (Hetru et al., 1998) and important for the activity of Hyalophora cecropin A (Andreu et al., 1985). However, broad activity especially toward yeasts and Grampositive bacteria was reported in Anopheles gambiae cecropin (AngCecA) which doesn’t have N-terminal tryptophan (Vizioli et al., 2000). It is interesting to search the activity of A. luxuriosa cecropin since it doesn’t have tryptophan residue (Fig. 3A). In further analysis, an unrooted bootstrap tree was created using CLUSTAL X and DRAWTREE of PHYLIP with 1000 bootstrap replicates. By this analysis, the A. luxuriosa cecropin is outside all groups composed of dipteran or lepidopteran cecropins (Fig. 3B), consistent with the evolutionary relationship of these three insect orders. References Akutsu, K., 1985. PhD thesis, University of Tokyo,Tokyo. Andersson, M., Bonan, A., Bonan, H.G., 2003. Ascaris nematodes from pig and human make three antibacterial peptides: isolation of cecropin P1 and two ASABF peptides. Cell Mol. Life Sci. 60, 599 – 606.

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