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Identification of up-regulated proteins in the hemolymph of immunized Bombyx mori larvae
Comparative Biochemistry and Physiology, Part D 1 (2006) 260 – 266 www.elsevier.com/locate/cbpd
Identification of up-regulated proteins in the hemolymph of immunized Bombyx mori larvae Kyung Han Song a , Su Jin Jung a , Young R. Seo c , Seok Woo Kang d , Sung Sik Han a,b,⁎ a
c
School of Life Science and Biotechnology, Korea University, Seoul 136-701, South Korea b Korean Entomological Institute, Korea University, Seoul, 136-701, South Korea Department of Pharmacology, Medical Research Center, College of Medicine, Kyung Hee University, Seoul 130-701, South Korea d Department of Agricultural Biology, NIAST, RDA, Suwon 441-100, South Korea Received 4 April 2005; received in revised form 12 January 2006; accepted 16 January 2006 Available online 21 April 2006
Abstract Insects defend themselves against foreign invaders via both a cellular response and a humoral response. The objective of this study was to identify proteins which were differently regulated in the immunized Bombyx mori larvae. Heat-inactivated bacteria (Bacillus megaterium) were injected into B. mori larvae, 4 days after final ecdysis. After 6 h, we identified the immune proteins in the hemolymph which had been differentially regulated in the immune-challenged larvae, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS). After the bacterial injection, more than 30 spots determined to have been up-regulated, and 11 spots were down-regulated. The heat shock 70 kDa protein cognate was one of the up-regulated hemocytic proteins, and peptidoglycan recognition protein, antichymotrypsin precursor, and gloverin-like protein 1∼4 were newly synthesized in the plasma. Antennal binding protein 7 was up-regulated in the plasma. Our results indicated that these immune response proteins were involved with the carrying out of innate immune responses. © 2006 Elsevier Inc. All rights reserved. Keywords: Proteomic analysis; Immune response proteins; Heat shock 70 kDa protein (Hsp70); Antichymotrypsin; Gloverin; Peptidoglycan recognition protein (PGRP); Antennal binding protein 7; Bombyx mori; Innate immunity
1. Introduction Unlike the immune system of vertebrates, which is composed of both innate and adaptive immune defenses, insects defend themselves solely by means of an innate immune system (Tzou et al., 2002). This, of course, means that the innate immune system of insects must necessarily be efficient; therefore, insects are a good model to investigate innate immunity. Drosophila melanogaster is a particularly favored model system to study innate immunity through its fully sequenced genome and convenient genomics (Brennan and Anderson, 2004). Innate immunity involves both cellular (hemocytic) and humoral reactions, including phagocytosis and nodulation by ⁎ Corresponding author. Cell Engineering and 3-D structure Laboratory, School of Life Science and Biotechnology, Korea University, Seoul, 136-701, South Korea. Tel.: +82 2 3290 3424; fax: +82 2 3290 3924. E-mail address: [email protected] (S.S. Han). 1744-117X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2006.01.001
hemocytes, the activation of proteolytic cascades leading to localized melanization and coagulation, and the synthesis of potent antimicrobial peptides (AMPs) (Levy et al., 2004a,b; Miller and Stanley, 2004). Recent studies have generally focused on the expression of these antimicrobial molecules, and two major pathways involved in these immune responses (Bae and Kim, 2003; Trepod and Mott, 2004). The Toll pathway is known to modulate defenses against fungal or Gram-positive bacterial molecules. The features of this pathway are similar to the cytokine-induced activation of NF-κB in mammals (Mead et al., 1986). The immune deficiency pathway (Imd) is involved in the expression of most of the antibacterial peptide genes related to defense against Gram-negative infections (De Gregorio et al., 2002). Although many studies have previously revealed a large number of differentially expressed genes, mRNA-based approaches measure message abundances and thus are somewhat removed from the mediators of physiological
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functions (Vierstraete et al., 2004). In addition, there can be a poor correlation between mRNA level and protein abundance in the cell (Ideker et al., 2001). Therefore, a proteomic approach, which could characterize both the proteins and their post-translational modifications, would clearly be a more appropriate method for protein/functional profiling. Many AMPs and pattern recognition proteins are synthesized within the fat bodies or hemocytes and then secreted into the plasma. Therefore, hemolymph, composed of hemocytes and plasma, contains a lot of AMPs, recognition-related proteins and hemocytes, and is also the space for cellular responses. Even though important, genomics cannot be used for fluids, such as the hemolymph. Unlike the fruit fly, the proteomic knowledge of silkworm innate immunity is still limited, despite sufficient genomic databases and relatively convenient handling. Here, we have identified the immune response proteins synthesized in hemolymph of Bombyx mori larvae, through two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with Q-TOF MS/MS, in order to acquire proteomic information regarding the components of the innate immune system of insects. 2. Materials and methods 2.1. Insects and immunization The fourth-day final instar larvae (L5D4) of B. mori (Baekokjam, Jam 123 × Jam 124) were used in this study. The larvae were reared on an artificial diet at a temperature of 25 ± 1 °C, 60% humidity, and a 16 / 8 h light / dark photoperiod. Seven larvae were injected with Bacillus megaterium and then compared with the naïve larvae after six h. The B. megaterium was incubated for 12 h, heat-treated for 1 h at 60 °C, and then, 8.4 × 106 cells in 12 μL sterile silkworm saline (Taniai et al., 1997) were injected into the experimental larvae.
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of isoelectric points. Isoelectric focusing (IEF) was performed at a total of 80,000 V h, using the IPG-phor system (Amersham Biosciences). After the IEF, the strips were equilibrated in equilibration buffer (6 M urea, 50 mM Tris–HCl, 2% SDS and 30% glycerol) containing 13 mM DTT for 20 min, then alkylated for 20 min in an equilibration buffer containing 2.5% iodoacetamide. The proteins were then re-separated according to their molecular masses, through a 12% non-gradient gel, using standard SDS-polyacrylamide gel electrophoresis (Pasquali et al., 1997). After the electrophoresis, the proteins were visualized by silver-staining the gels (Mortz et al., 2001). Protein spots differentially regulated in the immunized larvae were excised from the 2-D gels and then processed for ESI-MS/ MS. 2-D PAGE was repeated at least five times of three separate experiments. 2.4. In-gel digestion and Q-TOF MS/MS The excised spots were destained in order to remove any remaining silver ions, and incubated with 200 mM ammonium bicarbonate. The gel pieces were dehydrated with 100 μL of acetonitrile and dried in a vacuum centrifuge. The dried gel pieces were then rehydrated with 20 μL of 50 mM ammonium bicarbonate, containing 0.2 μg of modified trypsin (Promega). After 45 min, the solution was removed, and 30 μL of 50 mM ammonium bicarbonate was added. Digestion was performed overnight at 37 °C. The peptide solution was desalted using a C18 nano-column. The MS/MS of the peptides generated by in-gel digestion was performed by nano-ESI on a Q-TOF2 mass spectrometer (Micromass, Manchester) (Shevchenko et al., 1996). In order to identify the proteins, all of the MS/MS spectra recorded on the tryptic peptides derived from the spots were searched against the protein sequences from the NCBInr databases, using the MASCOT search program (www.matrixscience.com, Matrix Science, UK) and SEQUEST with MS BLAST (Shevchenko et al., 2001).
2.2. Protein preparation 3. Results The larvae were sterilized in 70% alcohol, and bled 6 h after injection. The hemolymph was collected in anticoagulant buffer (Yamashita and Iwabuchi, 2001) and centrifuged for 4 min at 1160 g. The separated hemocytes were then stored in lysis buffer (30 mM Tris–HCl pH 8.0, 1% Triton X-100, 65 mM DTT) containing protease inhibitors (Complete mini, Roche) for 1.5 h (4 °C), and finally sonicated (Ultrasonifier, Branson). The sonicated hemocytes and separated plasma were subjected to 20 min of centrifugation at 19,320 g (4 °C), and then each of the supernatants was dissolved in 6 M urea/2 M thiourea, and subjected to 2-D PAGE.
3.1. 2-D PAGE of hemocyte proteins About 300 protein spots were released in the naïve L5D4 (Fig. 1A), and the black-boxed section shows a map of the spots which were identical between the naïve and immunized L5D4. A total of 11 spots were determined to have been differentially released in the immunized L5D4 (Fig. 1), and the up-regulated spots are shown in the white box (Fig. 1). Three spots had disappeared, three spots were down-regulated in the bacteriainjected hemocytes (Fig. 1A), and five spots (H1, H2, H3, H4, H5) were up-regulated (Fig. 2B).
2.3. 2-D PAGE 3.2. Identification of hemocyte protein Three hundred micrograms of each protein was applied to an IPG strip (18 cm, linear pH 3–10) (Immobiline DryStrip, Amersham Biosciences) with rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer), and initially separated on the basis
According to the result of the nano-ESI on a Q-TOF MS/MS, H2 was identified as the heat shock 70 kDa protein cognate (Hsp70), a type of molecular chaperone. Three sub-peptides
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Fig. 1. 2-D PAGE analysis of proteins in hemocytes. Proteins were separated according to their isoelectric points on immobilized 3–10 pH-gradients, then separated by 12% SDS-PAGE. More than 300 spots were counted, and the white boxes indicate the areas in which up-regulated spots were observed (enlarged in Fig. 2). Spots in the black box were released at identical levels in the immunized and naïve L5D4. A: hemocyte proteins in naïve larvae. B: hemocyte proteins in immunized larvae. Five spots were up-regulated (black circles), three spots disappeared (thin arrows), and three spots were down-regulated (arrow heads) in the activated larvae.
were sequenced, and all of the sequences matched the Hsp70 from B. mori, according to our MASCOT database searches (Table 1). The B. mori Hsp70 was composed of 659 amino acids, and 45 of these amino acids matched the database records. The matched regions consisted of the conserved domains of the heat shock 70 kDa protein (NCBI PSSM-id: 4036 in the conserved domain database). 3.3. 2-D PAGE of plasma proteins Among 250 total spots, 21 were differentially regulated in the plasma. Five of these spots were down-regulated (Fig. 3A), 11 spots (P1∼P11) had been newly synthesized (Fig. 4B), and five spots (P12∼P16) were up-regulated (Fig. 4B,D). 3.4. Identification of plasma proteins Seven spots were identified in the plasma (Table 1) and P1 was determined to be the peptidoglycan recognition protein (PGRP) expressed in B. mori. This protein was composed of 195 amino acids, and is known to recognize the peptidoglycan
constituting part of the bacterial cell wall. This protein has also been reported that play a major role in process of self/non-self discrimination (Yoshida et al., 1996). The antichymotrypsin (ACT) precursor, a member of the serine protease inhibitor (serpin) family, was released at elevated levels in the immune-activated B. mori larvae (Fig. 4B, spot P5). The antichymotrypsin of B. mori larvae was first characterized in 1984, and its cDNA sequence was published in 1993 (Eguchi et al., 1984; Narumi et al., 1993). In this study, we determined that antichymotrypsin is actually up-regulated in immunized B. mori larvae. Gloverin-like proteins were newly synthesized in the experimental silkworms after bacterial injection (Fig. 4B, spot P4, P6, P8, P9). The gloverin-like protein 2 (P9) was the protein which was synthesized most abundantly among all 21 of the upregulated proteins. Gloverin was first reported in 1997, as a novel antibacterial protein isolated from Hyalophora gloveri and homologue of that protein was discovered in Helicoverpa armigera. Gloverins are glycine-rich proteins, and exhibit potent activity against Gram-negative bacteria (Axen et al., 1997; Mackintosh et al., 1998). Four gloverin-like genes in the
Fig. 2. A: hemocyte proteins in naïve larvae. B: hemocyte proteins in immunized larvae. Among 300 total spots, five spots (H1∼H5) were up-regulated in the immunized silkworm hemocytes. H2 was identified as the 70 kDa heat shock protein.
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Fig. 3. 2-D PAGE analysis of proteins in plasma. A: plasma proteins in naïve larvae. B: plasma proteins in immunized larvae. About 250 spots were detected using 12% acrylamide gels, ranging in pI from 3 to 10. White boxes indicate selected spot sections (enlarged Fig. 4). Spots in the black box were released at identical levels in the immunized and naïve larvae. Fifteen spots were up-regulated (black circles), and five spots (arrow heads) were down-regulated in the activated plasma.
GenBank (GenBank No. AB190863, AB190864, AB190865, AB190866) were cloned with the microbe-challenged silkworms in 2004, and in this study, we discovered that the four gloverin-like proteins were apparently induced from the immunized B. mori larvae. The antennal binding protein 7 (ABP7) was determined to have been up-regulated in immune-challenged larvae (Fig. 4B, spot P12). This protein is a member of the insect odorantbinding protein (OBP) family, and is involved with the relaying of sensory communication. Recently, pherokines, members of the OBP family found in Drosophila, were induced by both viral and bacterial infections, indicating that these proteins serve a primary defensive function by recognizing and/or neutralizing invading microorganisms (Sabatier et al., 2003).
4. Discussion Using 2-D PAGE with Q-TOF MS/MS, a fairly powerful technique, we have identified several differently regulated proteins in the hemolymph of immunized silkworms. We observed 5 spots in the hemocytes, and 16 spots in the plasma, which had been up-regulated during the carrying out immune responses (Table 2). Among these protein spots, we identified the antichymotrypsin (ACT) precursor, a member of the serpin superfamily. This protein has many previously characterized homologues, including silkworm antitrypsin, tobacco hornworm alaserpin, human alpha-1-antitrypsin and human alpha-1-antichymotrypsin (Narumi et al., 1993). The insect serpins are well known to
Fig. 4. A: plasma proteins in naïve larvae. B: plasma proteins in immunized larvae. Among 250 total spots, 11 spots (P1∼P11) were newly released (white arrows), and four spots (P12∼P15) were up-regulated (black arrows) in the immunized plasma. P1, P5, and P12 were identified as peptidoglycan recognition protein (PGRP), antichymotrypsin (ACT) precursor, and antennal binding protein 7, respectively. P4, 6, 8, and 9 were all identified as gloverin-like proteins.
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Table 1 The identification of immune response proteins up-regulated in the hemolymph of immunized Bombyx mori larvae Function
Spot no.
Identity
Location of matched peptides a
Accession no. b
Score c
Chaperon Signal cascade Antibacterial protein
H2 P5 P4 P6 P8 P9
Heat shock 70 kDa protein cognate (Hsp70) Antichymotrypsin precursor (ACT) Gloverin-like protein 1 Gloverin-like protein 3 Gloverin-like protein 4 Gloverin-like protein 2
65∼77 85∼99 200∼216 272∼285 298∼308 60∼74 128∼147 55∼69 75∼92 53∼67 91∼104 126∼140 55∼69 93∼106
BAA32395 BAA02995 BAD51473 BAD51475 BAD51476 BAD51474
125 123 212 112 191 125
Protein identification was accomplished by amino acid sequencing, using ESI Q-TOF MS/MS and MASCOT. Each spot from the 2-D gels was digested by trypsin into several sub-peptides and these were divided into single amino acid by ion energy. Each amino acid was sequenced by time of flight and identified using MASCOT (www.matrixscience.com) for searches of the Bombyx mori nr and EST database. a Matched regions of digested peptides in total amino acid sequences. b Accession number in NCBI database. c Individual score in MASCOT. This score is >50 indicate identity or extensive homology (p < 0.05) in MASCOT search results.
regulate the levels of proteinases involved in hemolymph coagulation, antimicrobial peptide synthesis and melanization of pathogen surfaces (Gorman and Paskewitz, 2001). Members of the serpin-1 family are released from the fat bodies and include signal peptides. Serpin-1 K is found in the plasma of Manduca sexta, and appears to function as an inhibitor of the chymotrypsin-like enzymes expressed constitutively in the fat bodies (Kanost et al., 2004). The ACT from B. mori harbored 16 amino acid signal peptides (Narumi et al., 1993) and was isolated in the hemolymph. These results indicate that the ACT from B. mori might be a member of the serpin-1 family. We also identified a chaperone protein (Hsp70) in the hemocytes of the immunized samples. This protein is evolutionarily conserved, with little variations between prokaryotes and eukaryotes. It is induced on exposure to many kind of stress, including heat, depletion of oxygen, nutrients, in order to promote refolding of proteins after cell injury and recruit another immune cells (Abbas and Lichtman, 2003). Therefore, in cases in which B. mori larvae were infected with microorganisms (for example, in the case of Bacillus infiltration), the observed up-regulation of Hsp70 may have been involved with the carrying out of these functions. Hsp70 has a molecular mass of 70kDa, but we detected Hsp70 at 32 kDa in the 2-D gel. However, the Hsp70 protein is comprised of two domains, an ATPase domain on the amino terminus, and a substrate-binding region on the carboxyl terminus. Therefore, we assumed that H2 was simply one of the Hsp70 subunits. The peptidoglycan recognition protein (PGRP) is a newly entered protein which appears to function during immune response. This protein binds specifically to peptidoglycan, a component of the bacterial cell wall, and result in the triggering of responses such as phagocytosis, nodule formation, the synthesis of antimicrobial peptides, and the activation of proteinase/prophenoloxidase cascades. It is also believed to be one of the pattern recognition proteins involved with the innate immunity of insects (Ochiai and Ashida, 1999). PGRP also contributes to the regulation of the two major signal pathways, Toll and Imd (Kanost et al., 2004). Twelve PGRPs have been identified in Drosophila (Werner et al., 2000) and three PGRPs; PGRP-SA, PGRP-LC, and PGRP-LE, have been shown to be involved with the carrying
out of immune responses. PGRP-SA activates the Toll pathway, and PGRP-LC (transmembrane protein) and PGRP-LE (secreted PGRP) have been implicated in the activation of the Imd pathway (Choe et al., 2005; Takehana et al., 2002). In this study, we found PGRP in the plasma, and thus propose that the newly synthesized PGRP in the plasma of our immunized silkworms was a PGRP secreted for the activation of the Imd pathway, PGRP-LE. Gloverin has been identified as an inducible glycine-rich antibacterial protein, isolated from H. gloveri (Axen et al., 1997), and was also discovered in H. armigera (Mackintosh et al., 1998). Gloverin cDNA was reported from Trichoplusia ni and Galleria mellonella (Lundstrom et al., 2002; Seitz et al., 2003), and four gloverin-like genes (GenBank accession nos. AB190863, AB190864, AB190865, AB190866) have been reported in the fat bodies of the immune-challenged silkworms. In our study, we observed four gloverin-like proteins are actually induced in immunized silkworms. These proteins may have been synthesized in the fat bodies and then secreted into the plasma. Gloverins exhibit potent activity against Gramnegative bacteria and have no effect against Gram-positive bacteria (Mackintosh et al., 1998). Recent studies have shown that the lysine-type and diaminopimelic acid (DAP)-type peptidoglycan function as Gram-positive and Gram-negative pathogens, respectively (Leulier et al., 2003). Therefore, the DAP-type of peptidoglycan found in Bacillus, although it belongs to Gram-positive bacteria, could activate the immune
Table 2 The identification of immune response proteins using ESI Q-TOF MS and SEQUEST Fuction
Spot no.
Recognition P1 Olfaction
P12
Species
Identity
Accession no. a
Bombyx mori Manduca sexta
Peptidoglycan AAL32058 recognition protein Antennal binding AAL60425 protein 7
Score b 84 76
Protein identification by amino acid sequencing using ESI Q-TOF MS/MS and SEQUEST for searches of the insect nr and EST database including Bombyx mori and Manduca sexta. a Accession number in NCBI database. b Score in MS BLAST.
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system of B. mori, and thereby inducing the four gloverin-like proteins observed in our study. Antennal binding protein 7 is a member of the insect odorant binding protein (OBP) family. These proteins are believed to participate in the sensing of odors and pheromones (Krieger et al., 1996). However, Pherokine-2 (Phk-2) and -3 (Phk-3) were observed to be up-regulated by viral and bacterial infection, and suggesting that they may also serve a primary defense function, possibly via the recognition and/or neutralization of invading microorganisms (Sabatier et al., 2003). Recently, another member of the OBP family that was induced during immune responses was isolated in Drosophila (Vierstraete et al., 2004; Levy et al., 2004a,b). Our results also indicated that another odorant binding protein from the silkworm was induced during immune responses, which supports the aforementioned hypothesis. The difference between the pI values of the 2-D gel and those calculated from the database-stored sequence is mainly to be attributable to posttranslational modifications. Molecular mass values shown on the 2-D gel occasionally do not match those calculated from the database-stored sequence. For example, the visualized masses of Hsp70 and ACT on the 2-D gel were lower than their computed masses. It is possible that these two proteins contain only a fragment of the identified protein, because the identification of these spots was based on tryptic peptides originating from regions between residues 65∼77, 85∼99, 200∼216, and 272∼285, 298∼308, respectively (Table 1). We ignore whether this fragmentation may have been induced by protease activity during sample preparation or in vivo biological processing. In this study, we demonstrated that antichymotrypsin (ACT), Hsp70, PGRP, gloverin-like proteins and antennal binding protein 7 are all proteins which are up-regulated in the hemolymph of B. mori larvae in which immune reactions have been induced. Taken it together, our results will be valuable to further study on insect innate immunity. Acknowlegdements This study was supported by a grant (H0453002) from the Bio-Green 21 Program (Rural Development Administration, Republic of Korea), and the Brain Korea 21 (Korea Ministry of Education) in 2005. References Abbas, A.K., Lichtman, A.H., 2003. Cellular and Molecular Immunology, 5th ed. Saunders press, pp. 345–366. Axen, A., Carlsson, A., Engstrom, A., Bennich, H., 1997. Gloverin, an antibacterial protein from the immune hemolymph of Hyalophora pupae. Eur. J. Biochem. 247, 614–619. Bae, S., Kim, Y., 2003. Lysozyme of the beet armyworm, Spodoptera exigua: activity induction and cDNA structure. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 135, 511–519. Brennan, C.A., Anderson, K.V., 2004. Drosophila: the genetics of innate immune recognition and response. Annu. Rev. Immunol. 22, 457–483. Choe, K.M., Lee, H., Anderson, K.V., 2005. Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor. Proc. Natl. Acad. Sci. U. S. A. 102, 1122–1126.
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Identification of up-regulated proteins in the hemolymph of immunized Bombyx mori larvae Kyung Han Song a , Su Jin Jung a , Young R. Seo c , Seok Woo Kang d , Sung Sik Han a,b,⁎ a
c
School of Life Science and Biotechnology, Korea University, Seoul 136-701, South Korea b Korean Entomological Institute, Korea University, Seoul, 136-701, South Korea Department of Pharmacology, Medical Research Center, College of Medicine, Kyung Hee University, Seoul 130-701, South Korea d Department of Agricultural Biology, NIAST, RDA, Suwon 441-100, South Korea Received 4 April 2005; received in revised form 12 January 2006; accepted 16 January 2006 Available online 21 April 2006
Abstract Insects defend themselves against foreign invaders via both a cellular response and a humoral response. The objective of this study was to identify proteins which were differently regulated in the immunized Bombyx mori larvae. Heat-inactivated bacteria (Bacillus megaterium) were injected into B. mori larvae, 4 days after final ecdysis. After 6 h, we identified the immune proteins in the hemolymph which had been differentially regulated in the immune-challenged larvae, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS). After the bacterial injection, more than 30 spots determined to have been up-regulated, and 11 spots were down-regulated. The heat shock 70 kDa protein cognate was one of the up-regulated hemocytic proteins, and peptidoglycan recognition protein, antichymotrypsin precursor, and gloverin-like protein 1∼4 were newly synthesized in the plasma. Antennal binding protein 7 was up-regulated in the plasma. Our results indicated that these immune response proteins were involved with the carrying out of innate immune responses. © 2006 Elsevier Inc. All rights reserved. Keywords: Proteomic analysis; Immune response proteins; Heat shock 70 kDa protein (Hsp70); Antichymotrypsin; Gloverin; Peptidoglycan recognition protein (PGRP); Antennal binding protein 7; Bombyx mori; Innate immunity
1. Introduction Unlike the immune system of vertebrates, which is composed of both innate and adaptive immune defenses, insects defend themselves solely by means of an innate immune system (Tzou et al., 2002). This, of course, means that the innate immune system of insects must necessarily be efficient; therefore, insects are a good model to investigate innate immunity. Drosophila melanogaster is a particularly favored model system to study innate immunity through its fully sequenced genome and convenient genomics (Brennan and Anderson, 2004). Innate immunity involves both cellular (hemocytic) and humoral reactions, including phagocytosis and nodulation by ⁎ Corresponding author. Cell Engineering and 3-D structure Laboratory, School of Life Science and Biotechnology, Korea University, Seoul, 136-701, South Korea. Tel.: +82 2 3290 3424; fax: +82 2 3290 3924. E-mail address: [email protected] (S.S. Han). 1744-117X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2006.01.001
hemocytes, the activation of proteolytic cascades leading to localized melanization and coagulation, and the synthesis of potent antimicrobial peptides (AMPs) (Levy et al., 2004a,b; Miller and Stanley, 2004). Recent studies have generally focused on the expression of these antimicrobial molecules, and two major pathways involved in these immune responses (Bae and Kim, 2003; Trepod and Mott, 2004). The Toll pathway is known to modulate defenses against fungal or Gram-positive bacterial molecules. The features of this pathway are similar to the cytokine-induced activation of NF-κB in mammals (Mead et al., 1986). The immune deficiency pathway (Imd) is involved in the expression of most of the antibacterial peptide genes related to defense against Gram-negative infections (De Gregorio et al., 2002). Although many studies have previously revealed a large number of differentially expressed genes, mRNA-based approaches measure message abundances and thus are somewhat removed from the mediators of physiological
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functions (Vierstraete et al., 2004). In addition, there can be a poor correlation between mRNA level and protein abundance in the cell (Ideker et al., 2001). Therefore, a proteomic approach, which could characterize both the proteins and their post-translational modifications, would clearly be a more appropriate method for protein/functional profiling. Many AMPs and pattern recognition proteins are synthesized within the fat bodies or hemocytes and then secreted into the plasma. Therefore, hemolymph, composed of hemocytes and plasma, contains a lot of AMPs, recognition-related proteins and hemocytes, and is also the space for cellular responses. Even though important, genomics cannot be used for fluids, such as the hemolymph. Unlike the fruit fly, the proteomic knowledge of silkworm innate immunity is still limited, despite sufficient genomic databases and relatively convenient handling. Here, we have identified the immune response proteins synthesized in hemolymph of Bombyx mori larvae, through two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with Q-TOF MS/MS, in order to acquire proteomic information regarding the components of the innate immune system of insects. 2. Materials and methods 2.1. Insects and immunization The fourth-day final instar larvae (L5D4) of B. mori (Baekokjam, Jam 123 × Jam 124) were used in this study. The larvae were reared on an artificial diet at a temperature of 25 ± 1 °C, 60% humidity, and a 16 / 8 h light / dark photoperiod. Seven larvae were injected with Bacillus megaterium and then compared with the naïve larvae after six h. The B. megaterium was incubated for 12 h, heat-treated for 1 h at 60 °C, and then, 8.4 × 106 cells in 12 μL sterile silkworm saline (Taniai et al., 1997) were injected into the experimental larvae.
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of isoelectric points. Isoelectric focusing (IEF) was performed at a total of 80,000 V h, using the IPG-phor system (Amersham Biosciences). After the IEF, the strips were equilibrated in equilibration buffer (6 M urea, 50 mM Tris–HCl, 2% SDS and 30% glycerol) containing 13 mM DTT for 20 min, then alkylated for 20 min in an equilibration buffer containing 2.5% iodoacetamide. The proteins were then re-separated according to their molecular masses, through a 12% non-gradient gel, using standard SDS-polyacrylamide gel electrophoresis (Pasquali et al., 1997). After the electrophoresis, the proteins were visualized by silver-staining the gels (Mortz et al., 2001). Protein spots differentially regulated in the immunized larvae were excised from the 2-D gels and then processed for ESI-MS/ MS. 2-D PAGE was repeated at least five times of three separate experiments. 2.4. In-gel digestion and Q-TOF MS/MS The excised spots were destained in order to remove any remaining silver ions, and incubated with 200 mM ammonium bicarbonate. The gel pieces were dehydrated with 100 μL of acetonitrile and dried in a vacuum centrifuge. The dried gel pieces were then rehydrated with 20 μL of 50 mM ammonium bicarbonate, containing 0.2 μg of modified trypsin (Promega). After 45 min, the solution was removed, and 30 μL of 50 mM ammonium bicarbonate was added. Digestion was performed overnight at 37 °C. The peptide solution was desalted using a C18 nano-column. The MS/MS of the peptides generated by in-gel digestion was performed by nano-ESI on a Q-TOF2 mass spectrometer (Micromass, Manchester) (Shevchenko et al., 1996). In order to identify the proteins, all of the MS/MS spectra recorded on the tryptic peptides derived from the spots were searched against the protein sequences from the NCBInr databases, using the MASCOT search program (www.matrixscience.com, Matrix Science, UK) and SEQUEST with MS BLAST (Shevchenko et al., 2001).
2.2. Protein preparation 3. Results The larvae were sterilized in 70% alcohol, and bled 6 h after injection. The hemolymph was collected in anticoagulant buffer (Yamashita and Iwabuchi, 2001) and centrifuged for 4 min at 1160 g. The separated hemocytes were then stored in lysis buffer (30 mM Tris–HCl pH 8.0, 1% Triton X-100, 65 mM DTT) containing protease inhibitors (Complete mini, Roche) for 1.5 h (4 °C), and finally sonicated (Ultrasonifier, Branson). The sonicated hemocytes and separated plasma were subjected to 20 min of centrifugation at 19,320 g (4 °C), and then each of the supernatants was dissolved in 6 M urea/2 M thiourea, and subjected to 2-D PAGE.
3.1. 2-D PAGE of hemocyte proteins About 300 protein spots were released in the naïve L5D4 (Fig. 1A), and the black-boxed section shows a map of the spots which were identical between the naïve and immunized L5D4. A total of 11 spots were determined to have been differentially released in the immunized L5D4 (Fig. 1), and the up-regulated spots are shown in the white box (Fig. 1). Three spots had disappeared, three spots were down-regulated in the bacteriainjected hemocytes (Fig. 1A), and five spots (H1, H2, H3, H4, H5) were up-regulated (Fig. 2B).
2.3. 2-D PAGE 3.2. Identification of hemocyte protein Three hundred micrograms of each protein was applied to an IPG strip (18 cm, linear pH 3–10) (Immobiline DryStrip, Amersham Biosciences) with rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer), and initially separated on the basis
According to the result of the nano-ESI on a Q-TOF MS/MS, H2 was identified as the heat shock 70 kDa protein cognate (Hsp70), a type of molecular chaperone. Three sub-peptides
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Fig. 1. 2-D PAGE analysis of proteins in hemocytes. Proteins were separated according to their isoelectric points on immobilized 3–10 pH-gradients, then separated by 12% SDS-PAGE. More than 300 spots were counted, and the white boxes indicate the areas in which up-regulated spots were observed (enlarged in Fig. 2). Spots in the black box were released at identical levels in the immunized and naïve L5D4. A: hemocyte proteins in naïve larvae. B: hemocyte proteins in immunized larvae. Five spots were up-regulated (black circles), three spots disappeared (thin arrows), and three spots were down-regulated (arrow heads) in the activated larvae.
were sequenced, and all of the sequences matched the Hsp70 from B. mori, according to our MASCOT database searches (Table 1). The B. mori Hsp70 was composed of 659 amino acids, and 45 of these amino acids matched the database records. The matched regions consisted of the conserved domains of the heat shock 70 kDa protein (NCBI PSSM-id: 4036 in the conserved domain database). 3.3. 2-D PAGE of plasma proteins Among 250 total spots, 21 were differentially regulated in the plasma. Five of these spots were down-regulated (Fig. 3A), 11 spots (P1∼P11) had been newly synthesized (Fig. 4B), and five spots (P12∼P16) were up-regulated (Fig. 4B,D). 3.4. Identification of plasma proteins Seven spots were identified in the plasma (Table 1) and P1 was determined to be the peptidoglycan recognition protein (PGRP) expressed in B. mori. This protein was composed of 195 amino acids, and is known to recognize the peptidoglycan
constituting part of the bacterial cell wall. This protein has also been reported that play a major role in process of self/non-self discrimination (Yoshida et al., 1996). The antichymotrypsin (ACT) precursor, a member of the serine protease inhibitor (serpin) family, was released at elevated levels in the immune-activated B. mori larvae (Fig. 4B, spot P5). The antichymotrypsin of B. mori larvae was first characterized in 1984, and its cDNA sequence was published in 1993 (Eguchi et al., 1984; Narumi et al., 1993). In this study, we determined that antichymotrypsin is actually up-regulated in immunized B. mori larvae. Gloverin-like proteins were newly synthesized in the experimental silkworms after bacterial injection (Fig. 4B, spot P4, P6, P8, P9). The gloverin-like protein 2 (P9) was the protein which was synthesized most abundantly among all 21 of the upregulated proteins. Gloverin was first reported in 1997, as a novel antibacterial protein isolated from Hyalophora gloveri and homologue of that protein was discovered in Helicoverpa armigera. Gloverins are glycine-rich proteins, and exhibit potent activity against Gram-negative bacteria (Axen et al., 1997; Mackintosh et al., 1998). Four gloverin-like genes in the
Fig. 2. A: hemocyte proteins in naïve larvae. B: hemocyte proteins in immunized larvae. Among 300 total spots, five spots (H1∼H5) were up-regulated in the immunized silkworm hemocytes. H2 was identified as the 70 kDa heat shock protein.
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Fig. 3. 2-D PAGE analysis of proteins in plasma. A: plasma proteins in naïve larvae. B: plasma proteins in immunized larvae. About 250 spots were detected using 12% acrylamide gels, ranging in pI from 3 to 10. White boxes indicate selected spot sections (enlarged Fig. 4). Spots in the black box were released at identical levels in the immunized and naïve larvae. Fifteen spots were up-regulated (black circles), and five spots (arrow heads) were down-regulated in the activated plasma.
GenBank (GenBank No. AB190863, AB190864, AB190865, AB190866) were cloned with the microbe-challenged silkworms in 2004, and in this study, we discovered that the four gloverin-like proteins were apparently induced from the immunized B. mori larvae. The antennal binding protein 7 (ABP7) was determined to have been up-regulated in immune-challenged larvae (Fig. 4B, spot P12). This protein is a member of the insect odorantbinding protein (OBP) family, and is involved with the relaying of sensory communication. Recently, pherokines, members of the OBP family found in Drosophila, were induced by both viral and bacterial infections, indicating that these proteins serve a primary defensive function by recognizing and/or neutralizing invading microorganisms (Sabatier et al., 2003).
4. Discussion Using 2-D PAGE with Q-TOF MS/MS, a fairly powerful technique, we have identified several differently regulated proteins in the hemolymph of immunized silkworms. We observed 5 spots in the hemocytes, and 16 spots in the plasma, which had been up-regulated during the carrying out immune responses (Table 2). Among these protein spots, we identified the antichymotrypsin (ACT) precursor, a member of the serpin superfamily. This protein has many previously characterized homologues, including silkworm antitrypsin, tobacco hornworm alaserpin, human alpha-1-antitrypsin and human alpha-1-antichymotrypsin (Narumi et al., 1993). The insect serpins are well known to
Fig. 4. A: plasma proteins in naïve larvae. B: plasma proteins in immunized larvae. Among 250 total spots, 11 spots (P1∼P11) were newly released (white arrows), and four spots (P12∼P15) were up-regulated (black arrows) in the immunized plasma. P1, P5, and P12 were identified as peptidoglycan recognition protein (PGRP), antichymotrypsin (ACT) precursor, and antennal binding protein 7, respectively. P4, 6, 8, and 9 were all identified as gloverin-like proteins.
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Table 1 The identification of immune response proteins up-regulated in the hemolymph of immunized Bombyx mori larvae Function
Spot no.
Identity
Location of matched peptides a
Accession no. b
Score c
Chaperon Signal cascade Antibacterial protein
H2 P5 P4 P6 P8 P9
Heat shock 70 kDa protein cognate (Hsp70) Antichymotrypsin precursor (ACT) Gloverin-like protein 1 Gloverin-like protein 3 Gloverin-like protein 4 Gloverin-like protein 2
65∼77 85∼99 200∼216 272∼285 298∼308 60∼74 128∼147 55∼69 75∼92 53∼67 91∼104 126∼140 55∼69 93∼106
BAA32395 BAA02995 BAD51473 BAD51475 BAD51476 BAD51474
125 123 212 112 191 125
Protein identification was accomplished by amino acid sequencing, using ESI Q-TOF MS/MS and MASCOT. Each spot from the 2-D gels was digested by trypsin into several sub-peptides and these were divided into single amino acid by ion energy. Each amino acid was sequenced by time of flight and identified using MASCOT (www.matrixscience.com) for searches of the Bombyx mori nr and EST database. a Matched regions of digested peptides in total amino acid sequences. b Accession number in NCBI database. c Individual score in MASCOT. This score is >50 indicate identity or extensive homology (p < 0.05) in MASCOT search results.
regulate the levels of proteinases involved in hemolymph coagulation, antimicrobial peptide synthesis and melanization of pathogen surfaces (Gorman and Paskewitz, 2001). Members of the serpin-1 family are released from the fat bodies and include signal peptides. Serpin-1 K is found in the plasma of Manduca sexta, and appears to function as an inhibitor of the chymotrypsin-like enzymes expressed constitutively in the fat bodies (Kanost et al., 2004). The ACT from B. mori harbored 16 amino acid signal peptides (Narumi et al., 1993) and was isolated in the hemolymph. These results indicate that the ACT from B. mori might be a member of the serpin-1 family. We also identified a chaperone protein (Hsp70) in the hemocytes of the immunized samples. This protein is evolutionarily conserved, with little variations between prokaryotes and eukaryotes. It is induced on exposure to many kind of stress, including heat, depletion of oxygen, nutrients, in order to promote refolding of proteins after cell injury and recruit another immune cells (Abbas and Lichtman, 2003). Therefore, in cases in which B. mori larvae were infected with microorganisms (for example, in the case of Bacillus infiltration), the observed up-regulation of Hsp70 may have been involved with the carrying out of these functions. Hsp70 has a molecular mass of 70kDa, but we detected Hsp70 at 32 kDa in the 2-D gel. However, the Hsp70 protein is comprised of two domains, an ATPase domain on the amino terminus, and a substrate-binding region on the carboxyl terminus. Therefore, we assumed that H2 was simply one of the Hsp70 subunits. The peptidoglycan recognition protein (PGRP) is a newly entered protein which appears to function during immune response. This protein binds specifically to peptidoglycan, a component of the bacterial cell wall, and result in the triggering of responses such as phagocytosis, nodule formation, the synthesis of antimicrobial peptides, and the activation of proteinase/prophenoloxidase cascades. It is also believed to be one of the pattern recognition proteins involved with the innate immunity of insects (Ochiai and Ashida, 1999). PGRP also contributes to the regulation of the two major signal pathways, Toll and Imd (Kanost et al., 2004). Twelve PGRPs have been identified in Drosophila (Werner et al., 2000) and three PGRPs; PGRP-SA, PGRP-LC, and PGRP-LE, have been shown to be involved with the carrying
out of immune responses. PGRP-SA activates the Toll pathway, and PGRP-LC (transmembrane protein) and PGRP-LE (secreted PGRP) have been implicated in the activation of the Imd pathway (Choe et al., 2005; Takehana et al., 2002). In this study, we found PGRP in the plasma, and thus propose that the newly synthesized PGRP in the plasma of our immunized silkworms was a PGRP secreted for the activation of the Imd pathway, PGRP-LE. Gloverin has been identified as an inducible glycine-rich antibacterial protein, isolated from H. gloveri (Axen et al., 1997), and was also discovered in H. armigera (Mackintosh et al., 1998). Gloverin cDNA was reported from Trichoplusia ni and Galleria mellonella (Lundstrom et al., 2002; Seitz et al., 2003), and four gloverin-like genes (GenBank accession nos. AB190863, AB190864, AB190865, AB190866) have been reported in the fat bodies of the immune-challenged silkworms. In our study, we observed four gloverin-like proteins are actually induced in immunized silkworms. These proteins may have been synthesized in the fat bodies and then secreted into the plasma. Gloverins exhibit potent activity against Gramnegative bacteria and have no effect against Gram-positive bacteria (Mackintosh et al., 1998). Recent studies have shown that the lysine-type and diaminopimelic acid (DAP)-type peptidoglycan function as Gram-positive and Gram-negative pathogens, respectively (Leulier et al., 2003). Therefore, the DAP-type of peptidoglycan found in Bacillus, although it belongs to Gram-positive bacteria, could activate the immune
Table 2 The identification of immune response proteins using ESI Q-TOF MS and SEQUEST Fuction
Spot no.
Recognition P1 Olfaction
P12
Species
Identity
Accession no. a
Bombyx mori Manduca sexta
Peptidoglycan AAL32058 recognition protein Antennal binding AAL60425 protein 7
Score b 84 76
Protein identification by amino acid sequencing using ESI Q-TOF MS/MS and SEQUEST for searches of the insect nr and EST database including Bombyx mori and Manduca sexta. a Accession number in NCBI database. b Score in MS BLAST.
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system of B. mori, and thereby inducing the four gloverin-like proteins observed in our study. Antennal binding protein 7 is a member of the insect odorant binding protein (OBP) family. These proteins are believed to participate in the sensing of odors and pheromones (Krieger et al., 1996). However, Pherokine-2 (Phk-2) and -3 (Phk-3) were observed to be up-regulated by viral and bacterial infection, and suggesting that they may also serve a primary defense function, possibly via the recognition and/or neutralization of invading microorganisms (Sabatier et al., 2003). Recently, another member of the OBP family that was induced during immune responses was isolated in Drosophila (Vierstraete et al., 2004; Levy et al., 2004a,b). Our results also indicated that another odorant binding protein from the silkworm was induced during immune responses, which supports the aforementioned hypothesis. The difference between the pI values of the 2-D gel and those calculated from the database-stored sequence is mainly to be attributable to posttranslational modifications. Molecular mass values shown on the 2-D gel occasionally do not match those calculated from the database-stored sequence. For example, the visualized masses of Hsp70 and ACT on the 2-D gel were lower than their computed masses. It is possible that these two proteins contain only a fragment of the identified protein, because the identification of these spots was based on tryptic peptides originating from regions between residues 65∼77, 85∼99, 200∼216, and 272∼285, 298∼308, respectively (Table 1). We ignore whether this fragmentation may have been induced by protease activity during sample preparation or in vivo biological processing. In this study, we demonstrated that antichymotrypsin (ACT), Hsp70, PGRP, gloverin-like proteins and antennal binding protein 7 are all proteins which are up-regulated in the hemolymph of B. mori larvae in which immune reactions have been induced. Taken it together, our results will be valuable to further study on insect innate immunity. Acknowlegdements This study was supported by a grant (H0453002) from the Bio-Green 21 Program (Rural Development Administration, Republic of Korea), and the Brain Korea 21 (Korea Ministry of Education) in 2005. References Abbas, A.K., Lichtman, A.H., 2003. Cellular and Molecular Immunology, 5th ed. Saunders press, pp. 345–366. Axen, A., Carlsson, A., Engstrom, A., Bennich, H., 1997. Gloverin, an antibacterial protein from the immune hemolymph of Hyalophora pupae. Eur. J. Biochem. 247, 614–619. Bae, S., Kim, Y., 2003. Lysozyme of the beet armyworm, Spodoptera exigua: activity induction and cDNA structure. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 135, 511–519. Brennan, C.A., Anderson, K.V., 2004. Drosophila: the genetics of innate immune recognition and response. Annu. Rev. Immunol. 22, 457–483. Choe, K.M., Lee, H., Anderson, K.V., 2005. Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor. Proc. Natl. Acad. Sci. U. S. A. 102, 1122–1126.
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