Purification and cDNA cloning of inducible antibacterial peptides from Protaetia brevitarsis (Coleoptera).код для вставкиСкачать
92 Yoon et al. Archives of Insect Biochemistry and Physiology 52:92–103 (2003) Purification and cDNA Cloning of Inducible Antibacterial Peptides From Protaetia brevitarsis (Coleoptera) Hye Suk Yoon,1 Chang Seok Lee,1 Sang Yong Lee,1 Chung Sik Choi,1 In Hee Lee,2 Sung Moon Yeo,3 and Hak R. Kim1* Three antibacterial peptides, named protaetins 1, 2, and 3, were purified and characterized from immunized larval hemolymph of Protaetia brevitarsis, a fruit tree pest in Korea. Also, protaetin 1 was cloned. Acid extraction, gel filtration, preparative acidurea PAGE, and reversed-phase FPLC were used for purification of peptides. Protaetins 1 and 3 had molecular masses of 7.5 and 12 kDa on Tricine SDS-PAGE, respectively, and the molecular mass of protaetin 2 was 9,283.95 Da as determined by MALDI-TOF mass spectrometry. In an antibacterial assay, protaetins showed antibacterial activities against a panel of Grampositive and -negative bacteria. For the RT-PCR (reverse transcription polymerase chain reaction) to obtain the complete primary sequence, the primer was designed according to the N-terminal amino acid sequence of protaetin 1. Amino acid sequence homology of protaetin 1 with holotricin 2, an antibacterial peptide from Holotrichia diomphalia, showed 99% identity. Northern blot analysis showed that the protaetin 1 gene was strongly expressed in the fat body after Escherichia coli injection, but not in normal fat body. Also, it was expressed in the gut, but was much weaker after immunization. Arch. Insect Biochem. Physiol. 52:92–103, 2003. © 2003 Wiley-Liss, Inc. KEYWORDS: Protaetia brevitarsis; antibacterial peptide; Protaetin; MALDI-TOF mass spectrometry; RT-PCR; N-terminal amino acid sequence; Northern blot analysis INTRODUCTION Insects produce a variety of antimicrobial proteins when they are infected. These proteins or peptides play a crucial role in protecting them from invading microorganisms. The major antimicrobial proteins found so far in insects include the cecropins, insect defensins, attacin-like proteins, proline-rich peptides, and lysozyme (Powning and Davidson, 1973; Hultmark et al, 1983; Casteels et al, 1989; Boman, 1995). These antimicrobial pro- 1 Graduate School of Biotechnology, Korea University, Seoul, Korea 2 Department of Life Science, Hoseo University, Choongnam, Korea 3 Department of Biology, Dankook University, Chunan, Korea teins have been mostly identified from the Hymenoptera, the Diptera, and the Lepidoptera. Antimicrobial proteins from the Coleoptera have been studied only in relatively very few insect species such as Zophobas atratus (Bulet et al., 1991), Tenebrio molitor (Moon et al., 1994), H. diomphalia (Lee et al., 1994), Allomyrina dichotoma (Miyanoshita et al., 1996), Acalolepta luxuriosa (Morikazu et al., 1999). So far, six different groups of antibacterial proteins have been identified. These groups contain the insect difensins (4 kDa) and *Correspondence to: Hak R. Kim, Ph.D., Graduate School of Biotechnology, Korea University, 5-1 Anam-Dong, Seoul 136-701, Korea. E-mail : firstname.lastname@example.org Received 2 February 2002; Accepted 19 October 2002 © 2003 Wiley-Liss, Inc. DOI: 10.1002/arch.10072 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology Antibacterial Peptides From P. brevitarsis glycine-rich family (Dimarcq et al., 1988; Lambert et al., 1989). Holotricin 2 and coleoptericin have been purified and identified as a glycine-rich family. It is surmised that the finding of antimicrobial peptides in other coleopteran species will be helpful in understanding the molecular mechanism of insect immune responses as a whole. Moreover, all studies related to antimicrobial peptides might give insight into the innate immunity of invertebrates and produce templates for designing novel broadspectrum antibiotics that might function in humans (Boman, 1995; Lee et al., 1997; Tossi et al., 1997). Genes for various defense proteins are activated simultaneously in the fat body when their integument is injured (Takahashi et al., 1986). The insect immune genes and their related molecules have been isolated using N-terminal amino acid sequence of purified antibacterial proteins and characterized. In this study, we identified three types of antimicrobial peptides named protaetin-1, 2, and 3 from the larval hemolymph of immunized P. brevitarsis (Coleoptera), fruit tree pests in Korea, and cloned cDNA encoding protaetin-1. Also, the expression pattern of protaetin-1 was examined in normal and immunized states. MATERIALS AND METHODS Immunization and Hemolymph Collection Last instar larvae of P. brevitarsis were obtained from the insect rearing company, “Choganongsan,” Kyoungki-Province, Korea. For immunization, 5 ml containing 1 ´107 viable log phase bacteria E. coli K112 were injected into the last instar larvae of P. brevitarsis. After 24 h, hemolymph was collected through an incision in the front of the prothorax, and put into a chilled tube containing anticoagulation buffer (93 mM NaCl, 0.1 mM glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM EDTA, pH 4.6) and small amounts of phenylthiourea. To remove hemocytes and cell debris, immunized hemolymph was centrifuged at 10,000g for 20 min. The clear supernatant was stored at –70°C until use. February 2003 93 Purification Procedure of the Antibacterial Peptides To purify antibacterial peptides, a series of general biophysico-chemical procedures were adapted including gel permeation chromatography, preparative acid-urea PAGE, and reversed-phase FPLC. The cell-free hemolymph (50 ml) from immunized insects (50 larvae) was mixed with 10% acetic acid (1:1 v/v) and stirred overnight at 4°C. To remove acid-insoluble material, acid-extracted hemolymph was centrifuged at 25,000g for 30 min. The supernatant containing acid-soluble protein (6.68 mg/ ml) was subjected to a column (1.9 ´ 120 cm) of Sephadex G-50 equilibrated with 5% acetic acid. The flow rate was 20 ml/h and 3-ml fractions were collected. The fractions showing antibacterial activity were pooled and then freeze-dried for the next purification step. The antibacterial activities of fractions against E. coli K112 were monitored by radial diffusion assay. Freeze-dried fractions were resuspended in 5% acetic acid and then subjected to Prep cell (Preparative acid-urea PAGE). Prep cell was pre-run at 35 mA for 20 min, and then was run with 5% acetic acid at a flow rate of 1 ml/min at 25 mA and monitored at 280 nm. The antibacterial activity of each fraction was monitored by overlay assay. Antibacterial peptides were finally purified by reversed-phase FPLC (fast performance liquid chromatography) (PepRPC HR 5/5 column, Pharmacia). Fractions showed antibacterial activity were acidified by adding trifluoroacetic acid (TFA). The sample was eluted with various linear gradients of acetonitrile containing 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min and monitored at 214 nm. Aliquots of eluted fractions were dried in a vacuum drier and used for antibacterial assay. Antibacterial activity was monitored during the purification procedure using ultrasensitive radial diffusion assay and overlay assay. Ultrasensitive Radial Diffusion Assay The assay conditions were those essentially described by Lambert et al. (1989). Six milliliters of underlay agar (9 mM sodium phosphate, 1 mM 94 Yoon et al. sodium citrate, 1% agarose [w/v], pH 7.4, and 0.3 mg/ml tryptic soy broth), containing 5 ´ 107 log phase cells of the E. coli K112 strain was poured into sterile petri dishes. Wells 3 mm in diameter were cut on the underlay agar and 5 ml aliquots of purified protaetins resuspended in 0.01% acetic acid were added to the wells. After 3 h incubation for diffusion, a nutrient-rich TSB overlay agar was poured and incubated overnight at 37°C. The diameters of the clear zone around the 3-mm holes were measured and determined as antibacterial activity. The diameter of the clear zone indicating antibacterial activity was graphed against the log10 of the peptide concentration. Overlay Assay The AU-PAGE gel was washed in the 10 mM sodium phosphate buffer (pH 7.4) and put on the solidified underlay agar containing 5 ´ 107 cells/ ml of microbes. After 3-h incubation at 37°C, the AU-PAGE gel was removed and nutrient-rich TSB overlay agar was poured onto the underlay agar. The antibacterial regions were monitored as clearing zones on the plate after overnight incubation at 37°C. Measurement of Minimal Inhibitory Concentration (MIC) MIC of the protaetin 2 against bacteria was determined by ultrasensitive radial diffusion assay. Bacteria were plated on an agar medium and twofold diluted proteatin 2 (from 6 to 200 mg/ml) was applied to each well. After 3-h incubation for diffusion of the added samples, a nutrient-rich TSB agar was poured and incubated overnight at 37°C. The concentration of protein was determined by the Bradford method using BSA (Sigma, St. Louis, MO) as a standard. MIC of the peptides was estimated as x intercept calculated by a least-meansquare formula in the semilog graph (using log10 transformed peptide concentration). Acid Urea (AU)-PAGE AU-PAGE was conducted as described by Ganz et al. (1985). The gel was prepared using a 5% ace- tic acid containing 12.5% acrylamide, and pre-electrophoresed at 150V for 1 h with 5% acetic acid. The gel was stained with Coomassie blue G-50 and the duplicated gel was tested by the overlay assay (Lehrer et al., 1991) to visualize the antibacterial activity. Tricine SDS-PAGE The antibacterial peptide was analyzed by 16.5% Tricine SDS-PAGE according to the method of Schägger and von Jagow (1987). After electrophoresis, the gel was stained in Coomassie brilliant blue G-250 and destained with a 30% methanol aqueous solution containing 3.5% acetic acid. N-terminal Amino Acid Sequence and MALDI-TOF/Mass Spectrometry The amino acid sequencing analysis was conducted by the Korea Basic Science Institute. For N-terminal amino acid sequencing, purified protaetins 1 and 3 were transferred to a polyvinylidine difluoride membrane after Tricine SDSPAGE according to Towbin et al. (1979). Also, the purified protaetin 2 was dried and directly analyzed. Amino acid sequence was determined with automated protein sequencer (Perkin Elmer Procise Model). The molecular mass of the purified protaetin 2 was measured by MALDI-TOF/mass spectrometry (Kratos Kompact MAL DI 2, Manchester, UK) and protaetins 1 and 3 were measured by 16.5% Tricine SDS-PAGE. Protaetin 1 Gene cDNA Cloning Total RNA was prepared from immunized last instar larval fat bodies after immunization using the SDS/Phenol RNA extraction method. The RNA was reverse transcribed using oligo (dT)20-M4 adaptor primer (TaKaRa, M13 primer M4; 5¢GTTTTCCCAGTCACGAC-3¢) and double-stranded cDNA were synthesized using RT-PCR kit (TaKaRa). The degenerate oligonucleotide primer (PB1) (5¢TCNCTNCARCCNGGNGCNCC-3¢ N-A, C, G, T; RArchives of Insect Biochemistry and Physiology Antibacterial Peptides From P. brevitarsis 95 A,G) was designed using an N-terminal amino acid sequence of purified protaetin 1. The thermal cycle involved 40 cycles of 1 min at 95°C, 1 min at 60°C, and 1 min at 72°C using the designed 5¢-degenerate primer and the M13 primer M4. The products were electrophoresed in 1.3% agarose gel (Goldberg, 1980) and a gel fraction containing DNA with an expected size was excised. The DNA was extracted and purified. The extracted DNA was cloned into pGEM-T easy vector (Promega, Madison, WI) and transformed to E. coli DH10B. The nucleotide sequence of the insert DNA of clones was determined using 372 DNA automatic sequencing system (Perkin-Elmer, Oak Brook, IL). The 5¢-region of protaetin 1 DNA was analyzed using Marathon™ cDNA Amplification kit (Clontech, Palo Alto, CA) and a gene specific primer (PB 4) (5¢-ACTTCCAG GAATAGGAAAAGAGG-3¢). Fig. 1. The profile of gel permeation chromatography and 16.5% Tricine SDS-PAGE of gel permeation chromatography. Acid extracts of immunized larval hemolymph were applied to Sephadex G-50 column equilibrated with 5% acetic acid (A). Each vertical bar under the I (No. 1-4) and II (No. 5-8) on A was electrophoresed on B. Vertical bars indicate the antibacterial activities in fraction. Fractions (II: 31–45) were pooled and freeze-dried for the next purification step. The right-hand axis indicates antibacterial activity as clear zone units through ultrasensitive radial diffusion assay (1 mm = 10 Units). February 2003 96 Yoon et al. Total RNA Isolation and Northern Blot Analysis For the accumulation of transcripts after immunization, larvae were prepared at 4 h following the injection of bacterial suspension. Total RNA was isolated from the larval fat body and gut using the method of Chomczynski and Sacchi (1987). Twenty micrograms of total RNA per sample were electrophoresed on 1.3% agarose/2.2 M formaldehyde gel. The RNA was transferred to a Hybond N membrane (Amersham, Arlington Heights, IL) using a capillary method (Sambrook et al., 1989) and then the blot was cross-linked by UV irradiation. The blot was prehybridized in a prehybridization solution (16 mM Na2HPO4, 33 mM NaH2PO4, 0.5% NaCl, 1 mM EDTA, 1% SDS, 0.5 mg/ml BSA, 0.5 mg/ml Ficoll, 0.5 mg/ml Polyvinyl pyrolidine, 0.1 mg/ml salmon sperm DNA, 50% Formamide) for 16 h at 42°C. Hybridization was performed for 18 h at 42°C in the Fig. 2. Profile of reversed-phase FPLC chromatograms (RPC). After gel permeation chromatography, peak II was subjected to RPC. Peaks 1, 2, and 3 were eluted at 33.09, 26.68, and 35.55% of acetonitrile concentration, respec- tively (A), and these peaks were electrophoresed on the 16.5% Tricine SDS gel (B). M, molecular mass marker; numbers 1, 2, and 3 are peaks 1 (protaetin 2), 2 (protaetin 1), and 3 (protaetin 1 and 3), respectively. Archives of Insect Biochemistry and Physiology Antibacterial Peptides From P. brevitarsis 97 Fig. 3. Profiles of the second RPC and Tricine SDS-PAGE. To finally purify protaetins 2 (A) and 3 (B), RPC was performed. Chromatographic conditions are as follows; column, PepRPC HR 5/5 connected to Pharmacia FPLC system; linear gradient of (0– 22% for 10 min/22–35% for 60 min, protaetin 2; 1–25% for 10 min/25– 35% for 60 min, protaetin 3) acetonitrile. C: The purity of protaetins (a, protaetin 2; b, protaetin 3) was confirmed on the Tricine SDS-PAGE. prehybridizaton solution containing a [a-32P]-labeled probe which incorporated the entire coding region of protaetin 1 cDNA, prepared by using Klenow fragment of DNA polymerase (Sambrook et al., 1989). After hybridization, the filter was washed at 42°C in 2 ´ SSC, 0.1% SDS. For the detection of transcripts, the filter was exposed to X-ray film for autoradiography at –80°C in the presence of an intensifying screen. RESULTS Purification of Antibacterial Peptides Three types of antibacterial peptides named protaetins 1, 2, and 3 were identified from the immunized larvae of P. brevitarsis against E. coli K112. February 2003 To purify the peptides, a series of general biophysicochemical procedures were conducted, including gel permeation chromatography, preparative acid-urea PAGE, and Reversed-Phase FPLC. The antibacterial peptides were extracted in an acidic medium in order to maximize the solubility of the peptides. The extracts were subjected to gel permeation chromatography. As seen in Figure 1, peaks I and II have antibacterial activity, and peak II shows major bands with small molecular masses between 6.5 and 14 kDa (Fig. 1B). Peak II containing antibacterial activity was further fractionated by gel permeation chromatography (data not shown). The reversed-phase FPLC was carried out for further purification of the proteins fractionated above. The three peaks (peaks 1, 2, and 3) were eluted at 98 Yoon et al. in various acetonitrile concentrations for final purification (Fig. 3A and B). But, the peak including protaetin 1 was not reanalyzed because it had only one peptide in the first FPLC program. Finally purified protaetins 2 and 3 were confirmed by Tricine SDS-PAGE (Fig. 3C). At the end, antibacterial activities of the ultimate protaetins were observed by acid urea PAGE (Fig. 4). The finally purified fractions were used for the estimation of molecular weight and the N-terminal amino acid sequencing. Characterization of Protaetins Fig. 4. Acid-urea PAGE and overlay assay of finally purified protaetins. Protaetins 1, 2, and 3 were electrophoresed on the acid-urea PAGE (A) and overlay assay was conducted with the duplicate gel to confirm the antibacterial activities (B). 26.28, 33.09, and 35.55% acetonitrile concentration, respectively (Fig. 2A). The first, second, and the third antibacterial peaks contained protaetins 2, 1, and 1+3, respectively (Fig. 2B). These peaks showed strong antibacterial activity against E. coli K112. Among them, peaks 1 and 3 were collected, concentrated, and reanalyzed on the same column Fig. 5. N-terminal amino acid sequences of protaetins 1, 2, and 3 and comparison of amino acid sequence of protaetin 1 with other antibacterial peptides. The N-terminal amino acid sequences of protaetins were determined by gas-phase Edman degradation (A) and, among them, Molecular masses of the antibacterial peptides purified by reversed phase FPLC were estimated by MALDI-TOF/mass spectrometry and protaetins 1, 2, and 3 were named based on their molecular weights. The masses of protaetins 1, 2, and 3 were determined to be about 7.5, 9, and 12 kDa by 16.5% Tricine SDS-PAGE, respectively, and the following MALDI-TOF/mass spectrometry analysis revealed the mass of protaetin 2 as 9,283.5 Da. The N-Terminal amino acid sequences of protaetins 1, 2, and 3 were determined to 15, 15, and 7 residues, respectively (Fig. 5A). The N-terminal amino acid sequences of protaetins 2 and 3 did N-terminal amino acid sequence of protaetin 1 was compared with other antibacterial peptides (coleoptericin from Z. atratus; holotricin 2 from H. diomphalia) from coleopteran insects (B). Archives of Insect Biochemistry and Physiology Antibacterial Peptides From P. brevitarsis Figure 6. Figure and legend continues on overleaf. February 2003 99 100 Yoon et al. Fig. 6. cDNA encoding P. brevitarsis protaetin 1. The diagram of PCR and sequencing strategy. The coding region and UTR regions were generated by RT-PCR using total RNA from the fat body. PB1 primer was designed from Nterminal amino acid sequence of purified protaetin 1. AP 1 and AP 2 primers were adaptor primers supplied on the Marathon™ cDNA Amplification Kit (Clontech). The PCR products were cloned into pGEM T-Easy Vector. The horizontal lines and arrows indicate the length and direction of the sequenced fragments and nucleotide numbers are shown on each arrow and box (A). Nucleotide sequence and deduced amino acid sequence of protaetin 1 cDNA. The italic deduced amino acid sequence absolutely matches that of the N-terminal amino acid sequence obtained from the purified protaetin 1. The termination codon is indicated by an asterisk and the putative polyadenylation signal (AATAAA) and the potential processing signal (Arg-Glu-Arg-Arg) are underlined. The numbers in the left-hand margin denote nucleotide and amino acid number (B). Alignment of amino acid sequences of protaetin 1 (from P. brevitarsis), holotricin 2 (from H. diomphalia, Lee et al., 1994), and coleoptericin (from Z. atratus, Bulet et al., 1991). Sequence identities of protaetin 1 and holotricin 2, and protaetin 1 and coleoptericin are about 99 and 39%, respectively. N-terminal amino acid sequence of protaetin 1 is underlined and identical residues in protaetin 1 and holotricin 2 are indicated by shading. Signal peptides of holotricin 2 and protaetin 1 are shown by dark shading and perfectly matched amino acid residues in all proteins are indicated by asterisks (C). TABLE 1. Antibacterial Activities of Protaetin 2* sequence homology among protaetin 1, holotricin 2, and coleoptericin and only one amino acid residue is different between protaetin 1 and holotricin 2 in the N-terminal amino acid sequence. It showed that the primary structure of protaetin 1 was quite homologous to holotricin and coleoptericin. Therefore, it was surmised that these amino terminal residues are important to their antibacterial activities. Bacteria Staphylococcus aureus Micrococcus luteus Bacillus subtilus Bacillus thuringiensis Escherichia coli Escherichia coli Salmonella typhimurium Pseudomonas acidovorans Salmonella typhimurium Pseudomonas putida Serraia marcescens Strain KCTC EK 132 EK 112 UK-1 LT-2 KOT KU9 Gram +/– Minimal inhibitory concentration (MIC/ug/ml) + + + + – – – – – – – No inhibition 4.52 2.09 7.37 19.25 11.12 29.41 18.32 33.24 57.43 3.66 *Antibacterial activities of protaetin 2 against a panel of bacteria were determined by radial diffusion assay. Bacteria was plated on agar medium and antibacterial peptide diluted 2-fold (from 6 to 200 mg/ml) was applied to each well, the diameter of growth inhibition was recorded after 24-h incubation at 37°C. not have sequence homology to any other antibacterial proteins, but protaetin 1 has a sequence homology with the known antibacterial proteins in Coleoptera. Figure 5B shows that the amino acid Antibacterial Activity of the Isolated Peptides and Synthetic Peptide The antibacterial activity of protaetin 2 was tested on several different microorganisms including Gram-negative (Escherichia coli K132, Escherichia coli K112, Salmonella typhimurium UK-1, Pseudomonas acidovorans, Salmonella typhimurium LT-2, Pseudomonas putida KTT, Serraia marcescens KU9) and Grampositive bacteria (Staphylococcus aureus, Micrococcus luteus KCTC, Bacillus subtilus, Bacillus thuringiensis). Archives of Insect Biochemistry and Physiology Antibacterial Peptides From P. brevitarsis 101 As shown in Table 1, proaetin 2 displayed strong antibacterial activities against a broad spectrum of bacteria and was more effective against Gram-positive bacteria. However, no appreciable hemolytic activities were observed for protaetin 2 when tested against rabbit red blood cells (data not shown). Also, we could confirm that the antibacterial activities of protaetins 1 and 3 were effective against Gram-negative and Gram-positive bacteria using ultra-sensitive radial diffusion assay. Protaetin 1 cDNA Cloning and Northern Blot Analysis The N-terminal sequence data were also used to design the degenerate primer for RT-PCR (as indicated in Materials and Methods). Through RTPCR, the cDNA of protaetin 1 was amplified and cloned in the pGEM-T Easy vector. Deduced amino acid sequences of the clone containing the cDNA insert exactly matched the N-terminal amino acid sequence of purified protaetin 1. But this clone did not contain 5¢-untranslated region (5¢-UTR). Therefore, we performed a 5¢-RACE (Rapid Amplification of cDNA Ends) PCR to identify 5¢-UTR (Fig. 6A). This result confirmed that the insert encoding protaetin 1 had a 549-bp-long DNA, which had an open reading frame of 351 nucleotides encoding 117 amino acids (Fig. 6B). Structural analysis of protaetin 1 indicated that it has a recognition sequence for the cleavage site within the constitutive secretory pathway (Arg-Xa.a-Lys/Arg-Arg; Hosaka et al., 1992) suggesting that a matured peptide (72 amino acid residues residues; predicted molecular mass of 7.8 kDa) is produced by cleavage of the signal peptide and propeptide from the 117 amino acid precursor protein. The amino acid sequence analysis of protaetin 1 deduced from cDNA sequence revealed a 99% identity with holotricin 2 except for only one amino acid residue through the entire amino acid sequence and also showed 39% identity with coleoptericin (Fig. 6C). On the other hand, the mRNA sizes of protaetin 1 and holotricin 2 are 549 bp and 519 bp, respectively, and 5¢ and 3¢-untranslated regions are quite different (data not shown). By Northern blot analysis using a [a-32P]-laFebruary 2003 Fig. 7. Northern blot analysis of mRNA from fat body and gut before and after the bacterial injections. Twenty micrograms of total RNA per sample were electrophoresed on 1.3% agarose/2.2 M formaldehyde gel. A: Fat body. B: Gut (N, not injected; I, injected). Top, bottom: Northern blot image of mRNA and agarose gel electrophoretic pattern of rRNA, respectively. beled probe cDNA of protaetins, the transcript of protaetin 1 was detected in the lane of total RNA from immunized fat body and gut. In the fat body, mRNA was strongly expressed following the bacterial injection and the same signal was detected in the gut after bacterial injection, but expression in the gut was much weaker and took more time than the fat body after injection (Fig. 7). This result is perfectly in accord with the fact that antibacterial peptides are induced by bacterial invasion. DISCUSSION We found antibacterial substances in the hemolymph of P. brevitarsis larvae that were induced by the injection of E. coli. Three antibacterial peptides named protaetins 1, 2, and 3 were purified and characterized. Furthermore, the complete cDNA sequence of one (protaetin 1) of these was determined. There are remarkable similarities between cloned protaetin 1 and other coleopteran antibacterial peptides such as holotricin 2, coleoptericin, and acaloleptin A. Interestingly, protaetin 1 has the 102 Yoon et al. same structure as holotricin 2 from H. diomphalia. For this reason, we carefully examined the relationship between these antibacterial peptides. But no significant similarity was found between protaetin 2 and 3 and other antibacterial peptides. It is surmised that these protaetins are notable antibacterial peptides involved in efficient insect defense mechanism. Coleoptericin, holotricin 2, and acaloleptin A are 8-kDa antibacterial peptides. Also, the molecular mass of protaetin 1 was determined to be 7.5 kDa on Tricine SDS-PAGE (Figs. 1B and 2B). The deduced amino acid sequence of protaetin 1 cDNA revealed that protaetin 1 belongs to glycine-rich antibacterial peptides such as coleoptericin, holotricin, and acaloleptin A. Like other antibacterial peptides, protaetin 1 is basic and the basicity tends to correlate with the higher antibacterial activities (Fink et al., 1989; Cociancich et al., 1994). The first six Nterminal amino acid residues (SLQPGA) are highly conserved in coleoptericin, holoticn 2, acaloleptin A, and protaetin 1. Therefore, the conserved N-terminal amino acid sequence may play an important role in the antibacterial activity of coleopteran insects. We have identified only 15 N-terminal amino acid residues of protaetin 1, but we could obtain a complete structure through the cloning of these peptides. Pairwise sequence alignment of protaetin 1 and holotricin 2 revealed that their sequences are almost identical except for only one amino acid residue (M in holotricin 2 was replaced by I in protaetin 1). These findings reveal that they have a common ancestry to both coleopteran insects. The cDNA sequence of protaetin 1 showed a 98% identity with that of holotricin 2 but their 5¢ and 3¢ untranslated regions differ in several base pairs. The cDNA of holotricin 2 and protaetin 1 contained about 519 and 549 bp, respectively, and had similar structures. By Northern-blot analysis with a [a-32P]-labeled probe encoding protaetin 1, it was clarified that protaetin 1 gene was expressed in the fat body from immunized larvae, but not in normal fat body. Also, the same transcript was detected in the gut following bacterial injection. The transcription of the most antibacterial peptide gene rapidly starts after injection of bacteria, lasts for 24 h, and then decreases. In summary, the protaetins are remarkable antibacterial peptides in P. brevitarsis and have an efficient self-defense response against invading bacteria. Furthermore, more intensive study about protaetins is necessary to clarify the relationship among them in P. brevitarsis. LITERATURE CITED Boman HG. 1995. Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13:61–92. Bulet P, Cociancich S, Dimarq JL, Lambert J, Reichhart JM, Hoffmann D, Hetru C, Hoffmann J. 1991. 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