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A novel lebocin-like gene from eri-silkworm, Samia cynthia ricini, that does not encode the antibacterial peptide lebocin
Comparative Biochemistry and Physiology, Part B 140 (2005) 127 – 131 www.elsevier.com/locate/cbpb
A novel lebocin-like gene from eri-silkworm, Samia cynthia ricini, that does not encode the antibacterial peptide lebocin Yanyuan Bao, Yoshiaki Yamano, Isao Morishima* Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan Received 5 August 2004; received in revised form 28 September 2004; accepted 28 September 2004
Abstract A cDNA clone with homology to lebocin gene was isolated from fat body of immunized Samia cynthia ricini larvae. The cDNA has an open reading frame encoding 162 amino acid residues. The deduced amino acid sequence shows significant homology to lebocin precursor proteins from Bombyx mori and Trichoplusia ni only in the bprosegmentQ region, but no homology to mature lebocin, a proline-rich antibacterial peptide, indicating the protein is not a precursor for lebocin antibacterial peptide. Northern analysis indicates that transcript of the lebocin-like gene is not detected in any tissues of naive larvae, but induced mainly in fat body after injection of the larvae with bacterial cells or cell wall components, such as peptidoglycan. D 2004 Elsevier Inc. All rights reserved. Keywords: Insect immunity; Immune-induced gene; Lebocin-like gene; Eri-silkworm; Samia cynthia ricini
1. Introduction In insects, inducible humoral responses play important roles to combat infectious microorganisms by producing a wide range of immune molecules at different levels (Hoffmann et al., 1999). Although many immune-related proteins have been isolated and characterized from various insects (Hetru et al., 1998), the mechanisms leading to the expression of immune molecules and how they participate in the humoral response against bacteria are still not well understood. For more complete understanding of the insect immune responses, we have studied the genes that are differentially expressed in the fat body of Samia cynthia ricini after bacterial challenge using suppression subtractive hybridization method (Diatchenko et al., 1996) and identified several immune-related genes (Bao et al., 2003). One of the cDNA clones from the subtracted cDNA library had significant homology to lebocin precursor genes from Bombyx mori (Chowdhury et al., 1995;
* Corresponding author. Tel.: +81 857 31 5359; fax: +81 857 31 5360. E-mail address: [email protected] (I. Morishima). 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.09.022
Furukawa et al., 1997). Lebocin is a proline-rich antibacterial peptide so far characterized only in B. mori (Hara and Yamakawa, 1995). The lebocin has been isolated in three isoforms from the immunized larval hemolymph. All lebocins with 32 amino acid residues contain seven or eight proline residues and O-glycosylated threonine. They are active against Gram-negative bacteria, and O-glycosylation has been shown to be necessary for antibacterial activity. The lebocin cDNA encoding a precursor protein with 179 amino acid residues has subsequently been isolated from B. mori (Chowdhury et al., 1995). The deduced protein contains a presumed signal peptide, a putative bprosegmentQ of 104 amino acid residues and a mature lebocin peptide of 32 amino acid residues followed by additional 27 amino acid residues. The role of unusually long bprosegmentQ is not known. Another lebocin cDNA has been cloned from Trichoplusia ni and shown to encode a similar precursor protein with 143 amino acid residues containing putative bprosegmentQ and mature lebocin peptide (Liu et al., 2000). The lebocin gene expression was inducible upon bacterial challenge in fat body of the lepidopteran insects (Chowdhury et al., 1995; Liu et al., 2000).
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2. Materials and methods
Uncross-linked linear PGN from Micrococcus luteus was prepared as described (Iketani et al., 1999). Lipopolysaccharide (LPS) from Escherichia coli K235, laminarin from Laminaria digitata, and zymosan from Saccharomyces cerevisiae were purchased from Sigma. Oligo chitin (hexa-N-acetylchitohexaose) was purchased from Seikagaku Kogyo, and Curdlan (h-1,3-glucan) was from Wako.
2.1. Insects and bacteria
2.3. Injection of insects and collection of tissues
Eri-silkworms (S. cynthia ricini) were reared on an artificial diet (Silkmate L4M, Nihon Nosan Kogyo, Japan) at 27 8C under aseptic conditions as previously described (Fujimoto et al., 2001). The larvae on the third day of the fifth instar were used for the experiments. Bacteria were as previously described (Fujimoto et al., 2001).
The larvae were injected with either UV-killed bacteria (5107 cells per larva) suspended in 10 Al of phosphatebuffered saline (PBS) or samples to be tested (20 Ag in 10 Al PBS per larva). The larvae were dissected, and the tissues were isolated, immediately frozen on dry ice, and stored at 80 8C.
2.2. Chemicals
2.4. cDNA cloning and nucleotide sequencing
Peptidoglycan (PGN) was prepared from bacterial cell wall and solubilized by hydrolysis with hen egg lysozyme as previously described (Morishima, 1998).
cDNA clones were randomly selected from subtracted cDNA library enriched with differentially expressed genes (Bao et al., 2003), and the plasmid DNA was purified using
In this paper, we describe a novel inducible lebocin-like gene from S. ricini larva that encodes a protein with significant homology to the bprosegmentQ region of lebocin precursor proteins from B. mori and T. ni, but does not encode the antibacterial peptide.
Fig. 1. Nucleotide and deduced amino acid sequences of Samia lebocin-like cDNA. Nucleotides are numbered on the right of each line. Deduced amino acid sequence is shown below the nucleotide sequence and numbered from the first methionine. Initiation and termination codons are shown in bold face.
Y. Bao et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 127–131
Quantum Prep Plasmid Miniprep Kit (Bio-Rad Lab). A cDNA library was constructed from the larval fat body taken 6 h after the mixed injection of UV-killed E. coli and Bacillus licheniformis cells using Creator SMART cDNA Library Construction Kit (Clontech). A cDNA fragment (232 bp) with high homology to T. ni lebocin cDNA (Liu et al., 2000) was used as a probe to screen the cDNA library. The nucleotide sequences were determined using Big Dye Terminator Cycle Sequencing Kit and ABI DNA sequencer (Applied Biosystems). 2.5. Northern blot analysis Total RNA was extracted from tissues with guanidine thiocyanate, and 10 Ag of the RNA was denatured and electrophoresed through a 1% agarose-formaldehyde gel. After transferring the RNA to Hybond-N nylon membrane, the membrane was hybridized with 32P-labeled probe as previously described (Bao et al., 2003). In some experiments, the membrane was washed with 0.1% SDS in a boiling water bath for several minutes, then used for rehybridization with 32 P-labeled attacin cDNA (Kishimoto et al., 2002). The membrane was exposed to imaging plate (Fuji Film) for 3 h, and the image was visualized and analyzed with a fluoroimage analyzer (Fuji Film FLA-5000).
3. Results
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a probe after injecting the larvae with solubilized PGN (Fig. 2A). The expression of attacin, an antibacterial protein, gene was also examined for comparison. No transcript of lebocinlike gene was detected in naive larvae (time 0), but was detectable 6 h after injection and increased to reach the highest level at 12 h, then gradually decreased until 60 h after injection. Injection of PBS alone caused no detectable expression of the gene. The induction pattern was similar to that of attacin. The expression was mainly in fat body and weak in other tissues tested (Fig. 2B). The very weak expression in hemocytes was a remarkable contrast to the expression of attacin gene, which was similar in both fat body and hemocytes. In the tissues from naive larvae, no expression of both genes was detected in any tissues tested (data not shown). 3.3. Elicitor specificity for induction of lebocin-like gene The elicitor specificity was tested by injecting Samia larvae with UV-killed bacterial cells, bacterial cell wall components, or structurally related glucans. The expression levels of lebocin-like gene in the fat body were analyzed by Northern blotting 9 h after the injection (Fig. 3). Strong expression was induced by UV-killed Gramnegative and -positive bacteria, B. licheniformis cell wall PGN, and M. luteus uncross-linked PGN, whereas M. luteus cell wall PGN was a weak elicitor. LPS, oligo chitin, and h-1,3 glucans, such as zymosan, laminarin, and curdlan, induced no detectable levels of the expression.
3.1. Isolation of cDNA clones encoding lebocin-like protein A 232-bp cDNA fragment with homology to T. ni lebocin cDNA was used as a probe to screen a cDNA library of immunized S. ricini larval fat body. Four independent positive clones were isolated for sequencing. All of the clones had a 955 bp insert of identical sequence, which contained a single open reading frame encoding 162 amino acid residues. The entire nucleotide sequence and deduced amino acid sequence are shown in Fig. 1 (DDBJ/GenBank/EMBL accession no.AB182995). The N-terminal 127-amino acid sequence of the deduced protein is 44.7% and 41.4% identical to B. mori lebocin 1 bprosegementQ and the corresponding region of T. ni lebocin precursor protein, respectively. The Samia protein, however, contains no region with similarity to Bombyx mature lebocin. The first 21 amino acid residues of Samia protein constitute a predicted signal peptide (Bendtsen et al., 2004). The protein without signal sequence is cationic with a calculated isoelectric point of 11.0 and has a molecular mass of 15.7 kd. 3.2. Time course and tissue specificity of lebocin-like gene expression Time course of lebocin-like gene expression in the fat body was analyzed by Northern blotting using the cDNA as
Fig. 2. Time course and tissue-specific expression of lebocin-like gene. (A) Total RNA was extracted from the fat body at the indicated time after injection of solubilized PGN from B. licheniformis cell wall (20 Ag in 20 Al PBS) or PBS (20 Al) and subjected to Northern blot hybridization using 32Plabeled lebocin-like cDNA as a probe (top: Leb). The membrane was washed with 0.1% SDS and rehybridized with 32P-labeled attacin cDNA (middle: Att). Ethidium bromide staining of 18S rRNA is shown in the bottom panel. (B) Total RNA was extracted from fat body (Fb), midgut (Mg), silkgland (Sg), Malpighian tube (Mt), and hemocyte (Hc) of the larvae 9 h after injection of UV-killed E. coli K12 cells (5107 cells/larva).
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Fig. 3. Induction of lebocin-like gene by bacterial cells and cell wall components. Fifth instar larvae were injected with PBS (lane 2), solubilized cell wall PGNs from B. licheniformis (lane 3) and M. luteus (lane 4), uncross-linked linear PGN from M. luteus (lane 5), LPS from E. coli K235 (lane 6), UV-killed cells of B. licheniformis (lane 7), M. luteus (lane 8) and E. coli K12 (lane 9), oligo chitin (lane 10), zymosan (lane 11), laminarin (lane 12), and curdlan (lane 13), respectively. The amount of injection was 20 Ag or 5107 cells in 20 Al PBS per larva. Noninjected control is shown in lane 1. Total RNA was extracted from the fat body 9 h after the injection, and 10 Ag each of the RNA was analyzed by Northern blot hybridization as described in legend to Fig. 2.
The elicitor specificity was essentially the same as that for attacin.
4. Discussion A cDNA clone with significant homology to lebocin gene was isolated from the fat body of immunized S. ricini
larvae. Amino acid sequence of the deduced protein is compared with lebocin precursor proteins so far available on the data base (Fig. 4). Three isoforms of lebocin, 1, 2, and 3, have been isolated from immunized hemolymph of B. mori (Hara and Yamakawa, 1995). Lebocin 1 and 2 are identical in amino acid sequence but differ only in sugar moiety, and lebocin 3 differs from lebocin 2 in one amino acid. A cDNA clone encoding lebocin 1 precursor protein and genomic clones encoding precursors for lebocin 3 and another isoform, lebocin 4, have subsequently been isolated (Chowdhury et al., 1995; Furukawa et al., 1997). The precursor proteins are 179-amino acid long consisting of signal peptide, "prosegment" (104 amino acids), mature lebocin, and "postsegment" (27 amino acids). The authors suggest that the mature lebocin peptides are excised out from the precursor proteins by an unknown mechanism. The Samia protein has significant homology to Bombyx, Trichoplusia (Liu et al., 2000), and Pseudoplusia includens (only available on data base,AY533675) lebocin precursor proteins in the "prosegment" region, but the C-terminal region has no homology to mature lebocin. Mature lebocins from Bombyx and putative mature lebocins from Trichoplusia and Pseudoplusia have 7 to 10 proline residues and O-glycosylated threonine. The corresponding C-terminal
Fig. 4. Comparison of lebocin-like proteins from lepidopteran insects. (A) Samia lebocin-like protein is compared to precursor protein for lebocin 1, 3, and 4 from B. mori (S79612, AB003035, AB003036; Chowdhury et al., 1995; Furukawa et al., 1997) and lebocin-like proteins from T. ni (AF233589; Liu et al., 2000) and P. includens (AY533675). A CLUSTALW program (DDBJ) was used for alignments. Residues identical in Samia, Trichoplusia, and Pseudoplusia proteins are shown in boldface. A cleavage site for predicted signal peptide (Bendtsen et al., 2004) is shown by arrowhead. Mature lebocins from Bombyx are double underlined, and putative mature lebocin from Trichoplusia is underlined. O-glycosylated threonine in Bombyx lebocins is boxed. Homologous protein segments, which are compared in panel B, are dotted-underlined. The number of amino acids are given at the right side of each line. (B) A sequence alignment of the Samia C-terminal 31 amino acid segment with Samia protein segment from 96 to 127, Trichoplusia segment from 80 to 111, and Pseudoplusia segment from 82 to 113. Residues identical in all segments are shown in boldface.
Y. Bao et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 127–131
region of the Samia protein, however, has been replaced by a modified copy of the preceding segment (96 to 127; Fig. 4A) and has only two proline residues and no possible Oglycosylation site (Christlet and Veluraja, 2001). The sequence of this region is also homologous to the corresponding region of Trichoplusia (80 to 111) and Pseudoplusia (82 to 113) proteins, but totally different from Bombyx proteins. All of the segments have an identical sequence of THPGYNRR (Fig. 4B), whereas Bombyx protein lacks this sequence. The fact that Samia protein has no region corresponding to mature lebocin indicates that this protein is not a precursor for the proline-rich antibacterial peptide but plays some other unknown role in immune reaction in this insect. The function of the protein is difficult to speculate, because a database search gave only lepidopteran lebocin precursor proteins but no other proteins with significant homology. The physiological function of the lebocin-like protein may be shared by the lebocin precursor proteins from other lepidopteran insects. The Bombyx proteins may have some original function other than as precursor for antibacterial peptide and deliver lebocin peptides after enzymatic hydrolysis of the protein. Many antibacterial peptides have been known to be generated through proteolytical digestion of milk or egg proteins, such as lactoferrin, lysozyme, and ovotransferrin (Pellegrini, 2003). The expression of lebocin-like gene was not detected in naive Samia larvae but was induced after injection of the larvae with bacteria or PGN (Figs. 2 and 3). The expression was mainly in fat body and very weak in hemocytes (Fig. 2B), in contrast to attacin and other antibacterial protein genes, which are expressed similarly in both fat body and hemocytes (Kishimoto et al., 2002; Fujimoto et al., 2001; Yamano et al., 1994). In B. mori and T. ni, the lebocin precursor genes were also mainly expressed in fat body (Chowdhury et al., 1995; Liu et al., 2000). The PGN from M. luteus cell wall induced very weak expression of the gene in contrast to the PGN from B. licheniformis, but uncross-linked linear PGN from the same bacteria was a strong elicitor (Fig. 3). M. luteus cell wall PGN has a charged bulky pentapeptide bridge between adjacent peptide side chains, which lacks in Bacillus PGN and has been shown to be a poor elicitor for the induction of antibacterial proteins in B. mori (Iketani et al., 1999). Chitin oligomer and h-1,3 glucans were not effective as elicitors. The elicitor specificities for the induction of lebocin-like gene are essentially the same as those for attacin. These findings strongly suggest that the lebocin-like protein has some important role in immune reaction.
Acknowledgements We thank Kazuhiko Hashimoto for constructing the cDNA library, and Dr. Osamu Shimizu, Gunma Sericultural
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Station, for supplying the eggs of S. ricini. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References Bao, Y., Mega, K., Yamano, Y., Morishima, I., 2003. cDNA cloning and expression of bacteria-induced Hdd11 gene from eri-silkworm, Samia cynthia ricini. Comp. Biochem. Physiol., C 136, 337 – 342. Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: signalP 3.0. J. Mol. Biol. 340, 783 – 795. Chowdhury, S., Taniai, K., Hara, S., Kadono-Okuda, K., Kato, Y., Yamamoto, M., Xu, J., Choi, S.K., Debnath, N.C., Choi, H.K., Miyanoshita, A., Sugiyama, M., Asaoka, A., Yamakawa, M., 1995. cDNA cloning and gene expression of lebocin, a novel member of antibacterial peptides from the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 214, 271 – 278. Christlet, T.H.T., Veluraja, K., 2001. Database analysis of O-glycosylation sites in proteins. Biophys. J. 80, 952 – 960. Diatchenko, L., Lau, Y.F.C, Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U. S. A. 93, 6025 – 6030. Fujimoto, S., Toshimori-Tsuda, I., Kishimoto, K., Yamano, Y., Morishima, I., 2001. Protein purification, cDNA cloning and gene expression of lysozyme from eri-silkworm, Samia cynthia ricini. Comp. Biochem. Physiol., B 128, 709 – 718. Furukawa, S., Taniai, K., Ishibashi, J., Hara, S., Shono, T., Yamakawa, M., 1997. A novel member of lebocin gene family from the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 238, 769 – 774. Hara, S., Yamakawa, M., 1995. A novel antibacterial peptide family isolated from the silkworm, Bombyx mori. Biochem. J. 310, 651 – 656. Hetru, C., Hoffmann, D., Bulet, P., 1998. Antimicrobial peptides from insects. In: Brey, P.T., Hultmark, D. (Eds.), Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall, London, pp. 41 – 66. Hoffmann, J.A., Kafatos, F.C., Janeway Jr., C.A., Ezekowitz, R.A.B., 1999. Phylogenetic perspectives in innate immunity. Science 284, 1313 – 1318. Iketani, M., Nishimura, H., Akayama, K., Yamano, Y., Morishima, I., 1999. Minimum structure of peptidoglycan required for induction of antibacterial protein synthesis in the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 29, 19 – 24. Kishimoto, K., Fujimoto, S., Matsumoto, K., Yamano, Y., Morishima, I., 2002. Protein purification, cDNA cloning and gene expression of attacin, an antibacterial protein, from eri-silkworm, Samia cynthia ricini. Insect Biochem. Mol. Biol. 32, 881 – 887. Liu, G., Kang, D., Steiner, H., 2000. Trichoplusia ni lebocin, an inducible immune gene with a downstream insertion element. Biochem. Biophys. Res. Commun. 269, 803 – 807. Morishima, I., 1998. Cecropin and lysozyme induction by peptidoglycan fragments in cultured fat body from Bombyx mori. In: Wiesner, A., Dunphy, G.B., Marmaras, V.J., Morishima, I., Sugumaran, M., Yamakawa, M. (Eds.), Techniques in Insect Immunology. SOS Publications, New Jersey, pp. 59 – 64. Pellegrini, A., 2003. Antimicrobial peptides from food proteins. Curr. Pharm. Des. 9, 1225 – 1238. Yamano, Y., Matsumoto, M., Inoue, K., Kawabata, T., Morishima, I., 1994. Cloning of cDNAs for cecropin A and B, and expression of the genes in the silkworm, Bombyx mori. Biosci. Biotech. Biochem. 58, 1476 – 1478.
A novel lebocin-like gene from eri-silkworm, Samia cynthia ricini, that does not encode the antibacterial peptide lebocin Yanyuan Bao, Yoshiaki Yamano, Isao Morishima* Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan Received 5 August 2004; received in revised form 28 September 2004; accepted 28 September 2004
Abstract A cDNA clone with homology to lebocin gene was isolated from fat body of immunized Samia cynthia ricini larvae. The cDNA has an open reading frame encoding 162 amino acid residues. The deduced amino acid sequence shows significant homology to lebocin precursor proteins from Bombyx mori and Trichoplusia ni only in the bprosegmentQ region, but no homology to mature lebocin, a proline-rich antibacterial peptide, indicating the protein is not a precursor for lebocin antibacterial peptide. Northern analysis indicates that transcript of the lebocin-like gene is not detected in any tissues of naive larvae, but induced mainly in fat body after injection of the larvae with bacterial cells or cell wall components, such as peptidoglycan. D 2004 Elsevier Inc. All rights reserved. Keywords: Insect immunity; Immune-induced gene; Lebocin-like gene; Eri-silkworm; Samia cynthia ricini
1. Introduction In insects, inducible humoral responses play important roles to combat infectious microorganisms by producing a wide range of immune molecules at different levels (Hoffmann et al., 1999). Although many immune-related proteins have been isolated and characterized from various insects (Hetru et al., 1998), the mechanisms leading to the expression of immune molecules and how they participate in the humoral response against bacteria are still not well understood. For more complete understanding of the insect immune responses, we have studied the genes that are differentially expressed in the fat body of Samia cynthia ricini after bacterial challenge using suppression subtractive hybridization method (Diatchenko et al., 1996) and identified several immune-related genes (Bao et al., 2003). One of the cDNA clones from the subtracted cDNA library had significant homology to lebocin precursor genes from Bombyx mori (Chowdhury et al., 1995;
* Corresponding author. Tel.: +81 857 31 5359; fax: +81 857 31 5360. E-mail address: [email protected] (I. Morishima). 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.09.022
Furukawa et al., 1997). Lebocin is a proline-rich antibacterial peptide so far characterized only in B. mori (Hara and Yamakawa, 1995). The lebocin has been isolated in three isoforms from the immunized larval hemolymph. All lebocins with 32 amino acid residues contain seven or eight proline residues and O-glycosylated threonine. They are active against Gram-negative bacteria, and O-glycosylation has been shown to be necessary for antibacterial activity. The lebocin cDNA encoding a precursor protein with 179 amino acid residues has subsequently been isolated from B. mori (Chowdhury et al., 1995). The deduced protein contains a presumed signal peptide, a putative bprosegmentQ of 104 amino acid residues and a mature lebocin peptide of 32 amino acid residues followed by additional 27 amino acid residues. The role of unusually long bprosegmentQ is not known. Another lebocin cDNA has been cloned from Trichoplusia ni and shown to encode a similar precursor protein with 143 amino acid residues containing putative bprosegmentQ and mature lebocin peptide (Liu et al., 2000). The lebocin gene expression was inducible upon bacterial challenge in fat body of the lepidopteran insects (Chowdhury et al., 1995; Liu et al., 2000).
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2. Materials and methods
Uncross-linked linear PGN from Micrococcus luteus was prepared as described (Iketani et al., 1999). Lipopolysaccharide (LPS) from Escherichia coli K235, laminarin from Laminaria digitata, and zymosan from Saccharomyces cerevisiae were purchased from Sigma. Oligo chitin (hexa-N-acetylchitohexaose) was purchased from Seikagaku Kogyo, and Curdlan (h-1,3-glucan) was from Wako.
2.1. Insects and bacteria
2.3. Injection of insects and collection of tissues
Eri-silkworms (S. cynthia ricini) were reared on an artificial diet (Silkmate L4M, Nihon Nosan Kogyo, Japan) at 27 8C under aseptic conditions as previously described (Fujimoto et al., 2001). The larvae on the third day of the fifth instar were used for the experiments. Bacteria were as previously described (Fujimoto et al., 2001).
The larvae were injected with either UV-killed bacteria (5107 cells per larva) suspended in 10 Al of phosphatebuffered saline (PBS) or samples to be tested (20 Ag in 10 Al PBS per larva). The larvae were dissected, and the tissues were isolated, immediately frozen on dry ice, and stored at 80 8C.
2.2. Chemicals
2.4. cDNA cloning and nucleotide sequencing
Peptidoglycan (PGN) was prepared from bacterial cell wall and solubilized by hydrolysis with hen egg lysozyme as previously described (Morishima, 1998).
cDNA clones were randomly selected from subtracted cDNA library enriched with differentially expressed genes (Bao et al., 2003), and the plasmid DNA was purified using
In this paper, we describe a novel inducible lebocin-like gene from S. ricini larva that encodes a protein with significant homology to the bprosegmentQ region of lebocin precursor proteins from B. mori and T. ni, but does not encode the antibacterial peptide.
Fig. 1. Nucleotide and deduced amino acid sequences of Samia lebocin-like cDNA. Nucleotides are numbered on the right of each line. Deduced amino acid sequence is shown below the nucleotide sequence and numbered from the first methionine. Initiation and termination codons are shown in bold face.
Y. Bao et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 127–131
Quantum Prep Plasmid Miniprep Kit (Bio-Rad Lab). A cDNA library was constructed from the larval fat body taken 6 h after the mixed injection of UV-killed E. coli and Bacillus licheniformis cells using Creator SMART cDNA Library Construction Kit (Clontech). A cDNA fragment (232 bp) with high homology to T. ni lebocin cDNA (Liu et al., 2000) was used as a probe to screen the cDNA library. The nucleotide sequences were determined using Big Dye Terminator Cycle Sequencing Kit and ABI DNA sequencer (Applied Biosystems). 2.5. Northern blot analysis Total RNA was extracted from tissues with guanidine thiocyanate, and 10 Ag of the RNA was denatured and electrophoresed through a 1% agarose-formaldehyde gel. After transferring the RNA to Hybond-N nylon membrane, the membrane was hybridized with 32P-labeled probe as previously described (Bao et al., 2003). In some experiments, the membrane was washed with 0.1% SDS in a boiling water bath for several minutes, then used for rehybridization with 32 P-labeled attacin cDNA (Kishimoto et al., 2002). The membrane was exposed to imaging plate (Fuji Film) for 3 h, and the image was visualized and analyzed with a fluoroimage analyzer (Fuji Film FLA-5000).
3. Results
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a probe after injecting the larvae with solubilized PGN (Fig. 2A). The expression of attacin, an antibacterial protein, gene was also examined for comparison. No transcript of lebocinlike gene was detected in naive larvae (time 0), but was detectable 6 h after injection and increased to reach the highest level at 12 h, then gradually decreased until 60 h after injection. Injection of PBS alone caused no detectable expression of the gene. The induction pattern was similar to that of attacin. The expression was mainly in fat body and weak in other tissues tested (Fig. 2B). The very weak expression in hemocytes was a remarkable contrast to the expression of attacin gene, which was similar in both fat body and hemocytes. In the tissues from naive larvae, no expression of both genes was detected in any tissues tested (data not shown). 3.3. Elicitor specificity for induction of lebocin-like gene The elicitor specificity was tested by injecting Samia larvae with UV-killed bacterial cells, bacterial cell wall components, or structurally related glucans. The expression levels of lebocin-like gene in the fat body were analyzed by Northern blotting 9 h after the injection (Fig. 3). Strong expression was induced by UV-killed Gramnegative and -positive bacteria, B. licheniformis cell wall PGN, and M. luteus uncross-linked PGN, whereas M. luteus cell wall PGN was a weak elicitor. LPS, oligo chitin, and h-1,3 glucans, such as zymosan, laminarin, and curdlan, induced no detectable levels of the expression.
3.1. Isolation of cDNA clones encoding lebocin-like protein A 232-bp cDNA fragment with homology to T. ni lebocin cDNA was used as a probe to screen a cDNA library of immunized S. ricini larval fat body. Four independent positive clones were isolated for sequencing. All of the clones had a 955 bp insert of identical sequence, which contained a single open reading frame encoding 162 amino acid residues. The entire nucleotide sequence and deduced amino acid sequence are shown in Fig. 1 (DDBJ/GenBank/EMBL accession no.AB182995). The N-terminal 127-amino acid sequence of the deduced protein is 44.7% and 41.4% identical to B. mori lebocin 1 bprosegementQ and the corresponding region of T. ni lebocin precursor protein, respectively. The Samia protein, however, contains no region with similarity to Bombyx mature lebocin. The first 21 amino acid residues of Samia protein constitute a predicted signal peptide (Bendtsen et al., 2004). The protein without signal sequence is cationic with a calculated isoelectric point of 11.0 and has a molecular mass of 15.7 kd. 3.2. Time course and tissue specificity of lebocin-like gene expression Time course of lebocin-like gene expression in the fat body was analyzed by Northern blotting using the cDNA as
Fig. 2. Time course and tissue-specific expression of lebocin-like gene. (A) Total RNA was extracted from the fat body at the indicated time after injection of solubilized PGN from B. licheniformis cell wall (20 Ag in 20 Al PBS) or PBS (20 Al) and subjected to Northern blot hybridization using 32Plabeled lebocin-like cDNA as a probe (top: Leb). The membrane was washed with 0.1% SDS and rehybridized with 32P-labeled attacin cDNA (middle: Att). Ethidium bromide staining of 18S rRNA is shown in the bottom panel. (B) Total RNA was extracted from fat body (Fb), midgut (Mg), silkgland (Sg), Malpighian tube (Mt), and hemocyte (Hc) of the larvae 9 h after injection of UV-killed E. coli K12 cells (5107 cells/larva).
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Fig. 3. Induction of lebocin-like gene by bacterial cells and cell wall components. Fifth instar larvae were injected with PBS (lane 2), solubilized cell wall PGNs from B. licheniformis (lane 3) and M. luteus (lane 4), uncross-linked linear PGN from M. luteus (lane 5), LPS from E. coli K235 (lane 6), UV-killed cells of B. licheniformis (lane 7), M. luteus (lane 8) and E. coli K12 (lane 9), oligo chitin (lane 10), zymosan (lane 11), laminarin (lane 12), and curdlan (lane 13), respectively. The amount of injection was 20 Ag or 5107 cells in 20 Al PBS per larva. Noninjected control is shown in lane 1. Total RNA was extracted from the fat body 9 h after the injection, and 10 Ag each of the RNA was analyzed by Northern blot hybridization as described in legend to Fig. 2.
The elicitor specificity was essentially the same as that for attacin.
4. Discussion A cDNA clone with significant homology to lebocin gene was isolated from the fat body of immunized S. ricini
larvae. Amino acid sequence of the deduced protein is compared with lebocin precursor proteins so far available on the data base (Fig. 4). Three isoforms of lebocin, 1, 2, and 3, have been isolated from immunized hemolymph of B. mori (Hara and Yamakawa, 1995). Lebocin 1 and 2 are identical in amino acid sequence but differ only in sugar moiety, and lebocin 3 differs from lebocin 2 in one amino acid. A cDNA clone encoding lebocin 1 precursor protein and genomic clones encoding precursors for lebocin 3 and another isoform, lebocin 4, have subsequently been isolated (Chowdhury et al., 1995; Furukawa et al., 1997). The precursor proteins are 179-amino acid long consisting of signal peptide, "prosegment" (104 amino acids), mature lebocin, and "postsegment" (27 amino acids). The authors suggest that the mature lebocin peptides are excised out from the precursor proteins by an unknown mechanism. The Samia protein has significant homology to Bombyx, Trichoplusia (Liu et al., 2000), and Pseudoplusia includens (only available on data base,AY533675) lebocin precursor proteins in the "prosegment" region, but the C-terminal region has no homology to mature lebocin. Mature lebocins from Bombyx and putative mature lebocins from Trichoplusia and Pseudoplusia have 7 to 10 proline residues and O-glycosylated threonine. The corresponding C-terminal
Fig. 4. Comparison of lebocin-like proteins from lepidopteran insects. (A) Samia lebocin-like protein is compared to precursor protein for lebocin 1, 3, and 4 from B. mori (S79612, AB003035, AB003036; Chowdhury et al., 1995; Furukawa et al., 1997) and lebocin-like proteins from T. ni (AF233589; Liu et al., 2000) and P. includens (AY533675). A CLUSTALW program (DDBJ) was used for alignments. Residues identical in Samia, Trichoplusia, and Pseudoplusia proteins are shown in boldface. A cleavage site for predicted signal peptide (Bendtsen et al., 2004) is shown by arrowhead. Mature lebocins from Bombyx are double underlined, and putative mature lebocin from Trichoplusia is underlined. O-glycosylated threonine in Bombyx lebocins is boxed. Homologous protein segments, which are compared in panel B, are dotted-underlined. The number of amino acids are given at the right side of each line. (B) A sequence alignment of the Samia C-terminal 31 amino acid segment with Samia protein segment from 96 to 127, Trichoplusia segment from 80 to 111, and Pseudoplusia segment from 82 to 113. Residues identical in all segments are shown in boldface.
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region of the Samia protein, however, has been replaced by a modified copy of the preceding segment (96 to 127; Fig. 4A) and has only two proline residues and no possible Oglycosylation site (Christlet and Veluraja, 2001). The sequence of this region is also homologous to the corresponding region of Trichoplusia (80 to 111) and Pseudoplusia (82 to 113) proteins, but totally different from Bombyx proteins. All of the segments have an identical sequence of THPGYNRR (Fig. 4B), whereas Bombyx protein lacks this sequence. The fact that Samia protein has no region corresponding to mature lebocin indicates that this protein is not a precursor for the proline-rich antibacterial peptide but plays some other unknown role in immune reaction in this insect. The function of the protein is difficult to speculate, because a database search gave only lepidopteran lebocin precursor proteins but no other proteins with significant homology. The physiological function of the lebocin-like protein may be shared by the lebocin precursor proteins from other lepidopteran insects. The Bombyx proteins may have some original function other than as precursor for antibacterial peptide and deliver lebocin peptides after enzymatic hydrolysis of the protein. Many antibacterial peptides have been known to be generated through proteolytical digestion of milk or egg proteins, such as lactoferrin, lysozyme, and ovotransferrin (Pellegrini, 2003). The expression of lebocin-like gene was not detected in naive Samia larvae but was induced after injection of the larvae with bacteria or PGN (Figs. 2 and 3). The expression was mainly in fat body and very weak in hemocytes (Fig. 2B), in contrast to attacin and other antibacterial protein genes, which are expressed similarly in both fat body and hemocytes (Kishimoto et al., 2002; Fujimoto et al., 2001; Yamano et al., 1994). In B. mori and T. ni, the lebocin precursor genes were also mainly expressed in fat body (Chowdhury et al., 1995; Liu et al., 2000). The PGN from M. luteus cell wall induced very weak expression of the gene in contrast to the PGN from B. licheniformis, but uncross-linked linear PGN from the same bacteria was a strong elicitor (Fig. 3). M. luteus cell wall PGN has a charged bulky pentapeptide bridge between adjacent peptide side chains, which lacks in Bacillus PGN and has been shown to be a poor elicitor for the induction of antibacterial proteins in B. mori (Iketani et al., 1999). Chitin oligomer and h-1,3 glucans were not effective as elicitors. The elicitor specificities for the induction of lebocin-like gene are essentially the same as those for attacin. These findings strongly suggest that the lebocin-like protein has some important role in immune reaction.
Acknowledgements We thank Kazuhiko Hashimoto for constructing the cDNA library, and Dr. Osamu Shimizu, Gunma Sericultural
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Station, for supplying the eggs of S. ricini. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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