Differential protein expression in the honey bee head after a bacterial challenge.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 65:223–237 (2007) Differential Protein Expression in the Honey Bee Head After a Bacterial Challenge Bieke Scharlaken,1 Dirk C. de Graaf,1* Samy Memmi,2 Bart Devreese,2 Jozef Van Beeumen,2 and Frans J. Jacobs1 Insect immune proteins and peptides induced during bacterial infection are predominantly synthesized by the fat body or by haemocytes and released into the hemolymph. However, tissues other than the “immune-related” ones are thought to play a role in bacteria-induced responses. Here we report a proteomic study of honey bee heads designed to identify the proteins that are differentially expressed after bacterial challenge in a major body segment not directly involved in insect immunity. The list of identified proteins includes structural proteins, an olfactory protein, proteins involved in signal transduction, energy housekeeping, and stress responses, and also two major royal jelly proteins. This study revealed a number of bacteria-induced responses in insect head tissue directly related to typical functions of the head, such as exocrine secretion, memory, and senses in general. Arch. Insect Biochem. Physiol. 65:223–237, 2007. © 2007 Wiley-Liss, Inc. KEYWORDS: Apis mellifera; honey bee; proteomics; head; insect immunity; bacterial infection INTRODUCTION Insects are provided with an extraordinary ability to resist infection. Infection or introduction of foreign material in the hemolymph of insects triggers cellular and humoral immune responses that function to eliminate the invading agent (Gillespie and Kanost, 1997). Cellular immunity includes phagocytosis, nodule formation (Bedick et al., 2001), and encapsulation. The different types of haemocytes (de Graaf et al., 2002) that react towards a foreign invading agent undergo changes in morphology, behavior, and population composition. Humoral immunity includes activation of proteolytic cascades (Zufelato et al., 2004; Lourenço et al., 2005) leading to localized melanization and coagulation, and also to synthesis of several pep- 1 Laboratory of Zoophysiology, Ghent University, Ghent, Belgium 2 Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, Ghent, Belgium tides by the fat body resulting in a broad spectrum of antimicrobial activity (Casteels, 1997). Many proteomic studies on insect immunity have identified differentially expressed proteins in “immune related tissues” (such as the fat body) or in hemolymph (Vierstraete et al., 2004a,b; Levy et al., 2004; Guedes et al., 2005; Song et al., 2006). Transcriptomic studies also identified differentially expressed genes with putative immune function in epithelial layers of the integument (Brey et al., 1993) and the gut (Lehane et al., 2003), and both involved in the insects’ first line of defense. This raises the question whether we could identify proteins that are differentially expressed after bacterial challenge in a major body segment not directly involved in insect immunity, for instance heads. The head mainly consists of the brains and associ- Abbreviations used: ESI: electrospray ionization; HPLC: high pressure liquid chromatography; IEF: iso-electric focusing; MALDI: matrix-assisted laser desorption ionization; PAGE: polyacrylamide gelelectrophoresis; TOF: time of flight; 2D: two-dimensional. *Correspondence to: Dirk C. de Graaf, Laboratory of Zoophysiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. E-mail: Dirk.deGraaf@UGent.be Received 20 January 2007; Accepted 26 January 2007 © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20179 Published online in Wiley InterScience (www.interscience.wiley.com) 224 Scharlaken et al. ated ganglia, hypopharyngeal glands, mandibular glands, salivary glands, and antennae, all contributing to neural, endocrine and/or exocrine functions. Here we describe a differential proteomic study of the head of the honey bee worker. WI). Acetonitrile was from Biosolve (Volkenswaard, The Netherlands). Trifluoroacetic acid was from Pierce (Rockford, IL) and α-cyano-4-hydroxy-cinnamic acid was from Sigma (St. Louis, MO). Water was obtained using a Milli-Q System (Millipore, Billerica, MA). MATERIALS AND METHODS Preparation of Protein Samples Honey Bee Workers and Microbial Challenge Newly emerged (up to 1-day-old) Carniolan honeybee workers (Apis mellifera carnica) were collected from hives of the experimental apiary in Ghent. A set of 50 worker bees were pricked in the abdomen (between second and third tergite) with a sterile needle dipped in a bacterial suspension of Escherichia coli NCTC 9001 (three fresh colonies, grown overnight on nutrient-agar plate, suspended in 500 µl sterile physiological solution containing 15 mM NaCl, 75 mM KCl, 3 mM CaCl2, 10 mM MgCl2, 55.5 mM glucose, 15 mM sucrose, and 55.5 mM fructose). A control group of 50 worker bees was pricked in the same way with a sterile needle dipped in the described physiological solution to distinguish between immune-induced and injury- or stress-induced proteins. After pricking, bees were put in laboratory cages and incubated at 34°C and 70 % RH, with ad libitum water and sugar water. Reagents and Chemicals Magnesium chloride, calcium chloride, glucose, sodium dodecyl sulphate, Coomassie Brilliant Blue G-250, and formic acid were purchased from Merck (Darmstadt, Germany). Sodium chloride, potassium chloride, sucrose, fructose, and Tris (hydroxymethyl) aminomethane were from Acros Organics (Geel, Belgium). Urea, CHAPS, and ammonium bicarbonate were from Fluka/Riedel-de Haën (Seelze, Germany). Acrylamide:N,N’-methylenbisacrylamide solution, 37.5:1 (electrophoresis purity reagent) for preparing gels was from Bio-Rad (Hercules, CA). Sequencing grade modified trypsin was purchased from Promega Corporation (Madison, At different time points (8, 24, and 48 h) after bacterial challenge, animals were anesthetized by chilling. The whole head of each worker was separated from the body by cutting precisely at the end of the thoracal tagmatum using a pair of scissors. Pooled samples, five heads each, were suspended in a 500-µl lysis solution (8 M urea, 4% CHAPS, 40 mM Tris) and immediately frozen at –20°C to avoid loss of protein content. Thawed samples were homogenised manually with an eppendorf pestle, sonicated three times for 15 sec with an interface of 30 sec at 100 W (Labsonic 1510) and centrifuged for 5 min at 9,660g. The supernatant was collected and the total protein content was determined by the Protein Assay Kit (Bio-Rad, Hercules, CA). 2-D Gel Electrophoresis Approximately 875-µg proteins were loaded on 17-cm immobilized pH gradient (IPG) strips, pH range 3–10 (Bio-Rad) and iso-electric focusing (IEF) was performed as described elsewhere (Vanrobaeys et al., 2003). The strips were then placed on lab-cast SDS–PAGE gels (13.5% acrylamide). The second dimension was carried out in a Protean Plus Dodeca Cell system (Bio-Rad) using five replicate gels for each experimental situation (infected/control). Electrophoresis of these ten gels was performed for 15 min at 10 mA/gel, followed by a 10-h run at 200 V until the bromophenol blue front reached the bottom of the gel. Gel Imaging and Image Analysis Staining was performed using Coomassie Brilliant Blue G-250 according to Anderson et al. Archives of Insect Biochemistry and Physiology August 2007 doi: 10.1002/arch. Protein Expression in the Honey Bee Head (1989). The gel images were digitized with a 12bit GS-710 calibrated densitometer (Bio-Rad) and analyzed with the PDQuest 7.3.1 software (BioRad). After spot detection, the 2-D maps were automatically aligned, followed by manual spot editing to increase the correlation between the different 2-D maps. Two replicate groups were created. This allows us to group the five duplicate gels for each experimental situation (infected/control) and determine the average quantities of their protein spots. Next, a master gel was constructed that summarized all the observed spots. Normalization in PDQuest is performed using the following formula: [normalized spot quantity = raw spot quantity * scaling factor * pipetting error compensation factor (= 1 in this study) / normalization factor]. Statistical analysis of the relative abundance of each matched protein spot was accomplished by using a two-tailed t-test (P < 0.05). The confidence threshold for the up- and down-regulation of protein spots was set at 3-fold above and 2-fold below the spot intensity seen in controls. In-Gel Digestion Differential protein spots were excised from the gel: each spot was excised twice from different gels and pooled. Tryptic digestion of the protein spots was performed according to Rosenfeld et al. (1992), with minor modifications. Briefly, excised protein spots were washed twice with 150 µl of 200 mM ammonium bicarbonate in 50% acetonitrile/water (20 min at 30°C). After drying at room temperature, for 10 min, the tubes were chilled on ice. Twelve microliters of digestion buffer (50 mM ammonium bicarbonate, pH 7.8), containing 0.002 µg/ µl trypsin, was added and samples were kept on ice for 45 min. Then 30 µl of digestion buffer was added. After overnight incubation (37°C), the supernatant was recovered. The remaining peptides were extracted from the gel piece using 60% acetonitrile/0.1% formic acid in water and pooled with the supernatant. For mass spectrometric analysis, the pooled samples were dried and redissolved in 12 µl 0.1 % formic acid. Archives of Insect Biochemistry and Physiology August 2007 doi: 10.1002/arch. 225 Mass Spectrometric Analyses and Protein Identification Tryptic peptide mixtures were analyzed on a MALDI TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Framingham, CA). The peptide mixture (1 µl) was co-crystallized with an equal volume of matrix solution (100 mM α-cyano-4-hydroxy-cinnamic acid dissolved in 50% v/v acetonitrile/0.1% trifluoroacetic acid in water) and 1 µl of the resultant mix was applied to the MALDI target plate. When the peptide mass fingerprint analysis was not conclusive, several peptides were subjected to further MS/MS analysis using collision-induced dissociation. A genome database for Apis mellifera was downloaded from http://www.ncbi.nlm.nih.gov and the GLEAN3 database with genome-based protein predictions was provided by the Honeybee Genome Sequencing Consortium (2006). Both databases were formatted to make them accessible for the database searching program MASCOT (http:// www.matrixscience.com). If the identification, based on MALDI TOF/TOF mass spectral data, was uncertain, the peptide mixture was separated by nano-HPLC and detected on-line by an ESI-QTRAP mass spectrometer (Applied Biosystems). RESULTS AND DISCUSSION This work is a first study on the proteome change in the head of an insect after bacterial challenge. Five samples, each containing five pooled whole heads from newly emerged honey bees, were collected after 8, 24, and 48 h for both the infected and control situation. These time points were chosen because the induction of immune effector molecules in hemolymph of honey bees becomes clear between 6 to 9 h post-infection and the highest levels of expression in hemolymph are obtained 48 h post-infection (Casteels,1997). Approximately 250–350 proteins were detected on each replicate gel. Differential analysis of the head elucidated the up-regulation of 18, 2, and 6 spots and the downregulation of 11, 10, and 9 spots, respectively, at 8, 24, and 48 h after challenge. Of all the differen- 226 Scharlaken et al. Figure 1 Archives of Insect Biochemistry and Physiology August 2007 doi: 10.1002/arch. Protein Expression in the Honey Bee Head tially regulated spots, 33 were identified (see Table 1). Most of the identified proteins have not yet been isolated or characterized. Their molecular function and biological role were predicted based on specific homology domains shared with proteins from other insect species (Table 2). Differentially expressed spots that were not identified are not listed in Table 1. Several identified proteins were present as multiple spots. This can be due to post-translational modifications that change pI or molecular weight of the protein. But also cleavage or protease activity during sample preparation could be the cause of degradative products of proteins. For example “jelleines” (antimicrobial peptides found in Royal Jelly) are suggested to be the result of degradation or maturation of precursor protein MRJP1 (Fontana et al., 2004). Structural Function An actin-binding protein (CG5023-PA; spot 1) was identified as a down-regulated target of the anti-bacterial response in head. CG5023-PA is a calponin homolog (Scp1-like) (Goodman et al., 2003). The calponin homology domain is found in actin-associated proteins that cross-link actin filaments, link actin to other cytoskeletal systems, and form signaling scaffolds (Galkin et al., 2006). Although at least two myosin proteins (spots 7, 8, and 30) are down-regulated after bacterial challenge, paramyosin (spot 10, 11) is up-regulated. The same observation was made in immune-challenged larval hemolymph of Drosophila (Guedes et al., 2005). Two differentially regulated proteins in the head were identified as cuticle domain-containing proteins Fig. 1. Two-dimensional gel maps (13.5% SDS-PAGE) of honey bee head tissue obtained in pH 3–10 and stained with Coomassie Brilliant Blue G-250. A,C,E: Head tissue of control honey bees 8, 24, and 48 h, respectively. B,D,F: Head tissue of immunized honey bees 8, 24, and 48 h, respectively, after bacterial challenge. Circles indicate those spots detected to be differentially expressed between control and immunized bees. Numerically indicated spots were identified by mass spectrometry and are listed in Table 1. Archives of Insect Biochemistry and Physiology August 2007 doi: 10.1002/arch. 227 (spots 12, 18 up-regulated; spot 27a down-regulated). The cuticular epithelial cells of Hyalophora cecropia have also been shown to actively participate in defence (Brey et al., 1993). A global gene expression analysis of whole mosquitoes in responses to microbial challenge showed the induction of five transcripts encoding cuticle domain–containing proteins (Aguilar et al., 2005). Spots 12 and 18 show homology with pupal cuticle protein C1B of Tenebrio molitor (Andersen et al., 1997). Signal Transduction Spot 2 is similar to the 38-kDa subunit (NURF38) of the nucleosome remodeling factor (NURF) complex. NURF acts as a negative regulator within the Drosophila JAK/STAT pathway (Badenhorst et al., 2002). This signaling pathway is involved in Drosophila immune responses (Agaisse and Perrimon, 2004). The recent sequencing of the honey bee genome (Honeybee Genome Sequencing Consortium, 2006) showed the presence of the JAK/ STAT cytokine receptor domeless and all other members of this pathway, except for the ligand unpaired, which suggests that the JAK/STAT pathway is functional in honey bees (Evans et al., 2006). Downregulation of this honey bee NURF-38 suggests that this signaling pathway is activated in head tissue after bacterial challenge. Spot 22 was identified as similar to the 14-3-3like protein. It is located in a ladder of spots possibly representing a series of post-translational modifications of a protein similar to myosin regulatory light chain 2 (spot 7 and 8). The 14-3-3 proteins were first discovered as abundant proteins in the brain. They have the ability to bind to phosphoserine residues in a sequence-specific manner, by which they can mediate signal transduction cascades (Aitken, 2006). The Drosophila 14-3-3 zeta homolog (Leonardo) is highly expressed in the central nervous system and preferentially enriched in the mushroom bodies (MBs) of the adult brain (Skoulakis and Davis,1996), which are believed to be involved in olfactory learning and are important structures for memory formation in the insect brain. It is tempting to suggest that bacterial challenge can Density ratiob Accession no.c 0.25 (⇓) <0.05 (⇓) 0.39 (⇓) 0.37 (⇓) <0.05 (⇓) <0.05 (⇓) 0.33 (⇓) 2 3 4 5 6 7 8 gi 66555437 gi 66555437 gi 110761968 gi 66546657 gi 58585170 gi 94158711 GB18261-PA 8 hours after bacterial challenge 1 0.14 (⇓) gi 66525458 Spot no.a Similar to myosin regulatory light chain 2 (MLC-2) Similar to myosin regulatory light chain 2 (MLC-2) Similar to enolase CG17654, isoform A, partial Similar to ERp60 CG8983-PA, isoform A isoform 2 Major royal jelly protein 4 Predicted: similar to nucleosome remodeling factor, 38kDa CG4634-PA Odorant binding protein 17 Similar to CG5023-PA Protein Idd 4.78 4.78 5.66 5.57 5.89 4.54 6.57 8.49 pIe 23545.21 23545.21 40305.27 55856.54 52915.46 15031.43 37661.34 19096.88 Massf 452.11 478.82 597.31 770.28 1806.94 504.75 540.99 597.34 689.94 732.87 740.34 755.44 473.15 498.83 557.81 590.37 613.85 617.37 859.36 519.88 614.93 618.48 673.90 714.33 798.97 923.95 954.94 1498.82 500.23 1498.82 1824 494.95 653.39 912.23 1135.63 600.39 681.89 1426.76 511.31 726.82 Observed peptide massg TABLE 1. Identification of Differentially Displayed Proteins in Honey Bee Head Tissue After Bacterial Challenge 35 80 71 34 97 189 231 30 230 156 51 ILNM1VVEIPR TTIETAEEVLEK TINQILNS ILEC2FFK YKTINQILNS FNVVDENANFNEK NTLIIYQNADDSFHR IAIDEYER YLDYDFDNDERR NEYLLALSDR VQDVFDSQLTVK LLQPYPDWSFAK YNGVPSSLNVVSDK LFAFDLNTSQLLK ILQNKFEANIVK NYFGIAGVR YSVSGYPTLK GFPTLYWAPK GDFVSDYNGPR LAPVYDELGEK AAEM1LLDNDPSITLAK NLYLQFIK EALNLIIDSIK SEVPSGASTGIHEALELR IGM1DVAASEFYK SDPSTYLDSDSLK AISNINNTIGPELIK GNPTVEVDLVTDDGLFR SNLDVTSQSDIDNFLLK HALM1TYGDKFTAK HALM1TYGDKFTAK HALM1TYGDKFTAK EAFQLM1DQDKDGIIGK HALMTYGDKFTAK AGSSVFSM1FTQK EAFQLM1DQDKDGIIGK LVTDKELDDOLNE 22 58 Mowse Scorei ITQKHPEYTGPR LFTEDQLR GTNFQLM1ENVQR Sequenceh LC LC LC LC MS/MS LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC MS/MS LC MS/MS MS/MS LC LC LC LC LC LC MS/MS LC LC Methodj Blastp Myosin 3 light chain [Lonomia obliqua] Myosin 3 light chain [Lonomia obliqua] Enolase, CG17654PA, isoform E [Drosophila melanogaster] ERp60 [Drosop hila melanogaster] Inorganic pyrophosphatase [Aedes aegypti] Calponin/transgelin [Aedes aegypti] Protein Id (continued) 5e –48 5e –48 5e –132 2e –166 1e –117 7e –76 E-value 6.25 (⇑) >20 (⇑) >20 (⇑) 6.7 (⇑) 3.6 (⇑) 5.6 (⇑) >20 (⇑) 6.12 (⇑) 3.16 (⇑) >20 (⇑) 10 11 12 13 14 15 16 17 18 19 gi 58585214 GB10628-PA GB10347-PA gi 48100966 gi 66506786 gi 66550890 GB15046-PA GB10628-PA gi 66510482 gi 66510482 gi 58585142 0.2 (⇓) <0.05 (⇓) >20 (⇑) 3.33 (⇑) 21 22 23 24 GB10347-PA GB10347-PA GB10347-PA gi 48097086 24 hours after bacterial challenge 20 <0.05 (⇓) gi 48097100 >20 (⇑) 9 Predicted: hypothetical protein Predicted: hypothetical protein Predicted: hypothetical protein Similar to 14-3-3-like protein (Leonardo protein) (14-3-3 zeta) isoform 1 Similar to yippee interacting protein 2 CG4600-PA FABP-like protein Predicted: similar to CG8541-PA, partial Predicted: hypothetical protein Similar to bellwether CG3612-PA, isoform 1 Similar to phosphoglyceromutase CG1721-PA, isoform A Similar to CG5362-PA, isoform 1 Predicted: hypothetical protein Similar to paramyosin CG5939-PA, isoform A Similar to paramyosin CG5939-PA, isoform A Predicted: similar to CG8541-PA, partial Major royal jelly protein 3 6.07 6.07 6.07 4.79 8.89 5.46 10.2 6.07 9 6.25 9.36 5.65 10.2 5.33 5.33 6.47 28901.02 28901.02 28901.02 28076.46 42503.97 15549.35 16696.67 28901.02 59511.61 36158.58 35360.77 63942.52 16696.67 102054.96 102054.96 61661.79 571.17 765.69 564.17 571.2 722.63 765.81 571.25 605.14 1889.0 736.36 515.31 571.44 581.84 583.27 503.35 641.28 654.31 546.27 666.88 666.94 680.28 571.27 1553.77 1624.92 500.83 513.85 617.33 812.84 554.88 617.9 617.83 515.78 576.31 616.80 515.76 VLDTPEVAVAK SADVLAVAGPALAYGR VVDTPEVAVAK VLDTPEVAVAK SGGGGPSPSQATVTLK SADVLAVAGPALAYGR VLDTPEVAVAK YLAEVATGETR M1NVPFEETLPSLPDRK FQTVTSIEGNTFK INLAQETLK VVDTPEVALAK IVPAAPLGPDGR LVPGAPIGLDGR FTFDIAHTSLLSR YYEIIVNDPR YGEEQVQIWR LLDIPVM1M1K NVAAAFLVGAM1PR IVQGLSINDFAR EVVPTADPSVAFK VLDTPEVAVAK EAYPGDVFYLHSR TGAIVDVPVGEELLGR VLSIGDGIAR AVDSLVPIGR SAEISSILEER TGAIVDVPVGEELLGR SIDTPFSSVR GIGNLGAISAYAK GIGNLGAISAYAK LSAELTQLR LTVAGESFTVK ILGANVDDLM1R LSAELTQLR 191 78 58 53 56 11 80 124 37 48 30 173 98 157 213 57 37 44 79 LC LC LC LC MS MS/MS LC LC LC LC LC LC LC LC LC LC LC LC LC MS/MS MS/MS LC LC LC LC LC LC LC LC LC LC LC Tyrosine 3monooxygenase protein zeta polypeptide [Bombyx mori] 3-ketoacyl-coa thiolase, mitochondrial [Aedes aegypti] Pupal cuticle protein C1B (TM-C1B) (TMPCP C1B) Lipid binding protein protein 9, Isoform a [Caenorhabditis elegans] Mitochondrial ATP Synthase alpha subunit [Aedes aegypti] Cytosolic malate dehydrogenase [Lysiphlebus testaceipes] Phosphoglyceromutase [Apis cerana] Paramyosin [Drosophila melanogaster] Paramyosin [Drosophila melanogaster] Pupal cuticle protein C1B (TM-C1B) (TM-PCP C1B) (continued) 2e –116 1e –124 9e –08 3e –08 0.0 4e –140 1e –135 3e –08 0.0 0.0 Density ratiob Accession no.c 0.39 (⇓) 0.33 (⇓) <0.05 (⇓) <0.05 (⇓) >20 (⇑) 6.66 (⇑) >20 (⇑) 27 28 29 30 31 32 33 gi 48100966 gi 66504546 gi 48138568 gi 110768236 gi 110757651 gi 58585146 GB10347-PA GB13208-PA GB10347-PA Similar to protein lethal (2) essential for life, isoform 1 Similar to bellwether CG3612-PA, isoform 1 Similar to CG14207-PB, isoform B isoform 1 Similar to myosin alkali light chain 1 CG5596-PA, isoform A isoform 4 Similar to Uev1A CG10640-PA, isoform A isoform 1 Arginine kinase Predicted: similar to CG15884-PA Predicted: hypothetical protein Predicted: hypothetical protein Predicted: hypothetical protein Protein Idd 9 5.85 6.58 4.47 5.48 5.66 6.07 5.22 6.07 6.07 pIe 59511.61 21975.63 16359.71 16980.47 21252.87 40008.36 28901.02 28225.88 28901.02 28901.02 Massf 500.66 617.18 592.74 700.29 561.34 609.28 619.34 660.2 850.14 564.19 571.2 766.01 571.14 765.8 628.31 717 571.26 765.93 Observed peptide massg VLSIGDGIAR SAEISSILEER 39 IDC2GQRYPDDAPNVR 63 54 52 119 147 152 122 141 68 160 Mowse Scorei ALNLNPTNATIEK LGLTEYQAVK GTFYPLTGM1SK IISM1QM1GGDLGQVYRR VSSTLSGLEGELK IISM1QM1GGDLGQVYR VVDTPEVAVAK VLDTPEVAVAK SADVLAVAGPALAYGR VLDTPEVAVAK SADVLAVAGPALAYGR YGFVDDTGNIR YGFVDDTGNIREVEYGASR VLDTPEVAVAK SADVLAVAGPALAYGR Sequenceh LC MS LC LC MS MS LC LC LC LC LC Methodj Blastp 4e –42 5e –62 6e –177 1e –29 E-value Ubiquitin-conjugating 6e –64 enzyme E2 [Aedes aegypti] Small heat shock protein 3e –57 [Venturia canescens] Mitochondrial ATP synthase 0.0 alpha subunit [Aedes aegypti] Heat shock protein hsp21.4 [Bombyx mori] Myosin 1 light chain, putative [Aedes aegypti] Putative arginine kinase [Homalodisca coagulata] RE05963p [Drosophila melanogaster] Protein Id b Numbers refer to the spot numbers given in Fig 1. The relative averaged integrated density values from five replicate 2D-PAGE gels were compared by determining the ratio of protein abundance after bacterial challenge to that of control situations. Only proteins with at least P < 0.05 were considered to differ significantly in abundance. ⇓ = down-regulated; ⇑ = up-regulated. c Prefix “gi” refers to protein entry code of the National Center for Biotechnology Information (NCBI): http://ncbi.nlm.nih.gov; prefix “GB” refers to protein entry code of Glean3 database. d Protein identification corresponding to the given accession number in NCBI. Glean3 data were BLAST searched and the predicted ID is given. e Theoretical iso-electric point of the protein. f Theoretical molecular mass of the protein. g Mass of the observed peptide. h Peptide sequence corresponding to peptide mass. 1refers to oxidation, 2refers to carbamidomethyl modification. i Probability-based MOWSE (molecular weight search) score of the Mascot search program, representing the total score of a protein match. j Protein spots analyzed and identified using either MALDI-TOF/TOF (MS or MS/MS) or nano-HPLC Q-TRAP (LC) mass spectrometer. a 0.38 (⇓) 26 48 hours after bacterial challenge 25 0.49 (⇓) GB10347-PA Spot no.a TABLE 1. Identification of Differentially Displayed Proteins in Honey Bee Head Tissue After Bacterial Challenge (continued) After 8 h After 8 h After 48 h After 8 h After 48 h After 8 h After 24 h After 48 h After 8 h ⇓ ⇑ ⇓ ⇑ ⇓ ⇑ ⇓ 12, 18 27a ⇓ ⇓ 30 31 3 After 8 h After 8 h After 8 h ⇓ ⇑ ⇑ 6 14 15 22 2 10, 11 7, 8 After 8 h 1 ⇑/⇓ ⇓ Spot n° Ener gy housekeeping Energy Carbohydrate metabolism Similar to enolase CG17654, isoform A, partial Similar to phosphoglyceromutase CG1721-PA, isoform A Similar to CG5362-PA, isoform 1 Similar to Uev1A CG10640PA, isoform A, isoform 1 Olfactory function Odorant binding protein 17 Similar to 14-3-3-like protein (Leonardo protein) (14-3-3 zeta) isoform 1 Similar to Myosin alkali light chain 1 CG5596-PA, isoform A isoform 4 Predicted: similar to CG8541-PA, partial Predicted: similar to CG15884-PA Signal transduction Predicted:similar to nucleosome remodeling factor - 38kDa CG4634-PA Similar to Paramyosin CG5939-PA, isoform A Similar to Myosin regulatory light chain 2 (MLC-2) Structural function Similar to CG5023-PA TABLE 2. Classification of the Differentially Displayed Proteins Malate dehydrogenases (MDH) cytoplasmic and cytosolic domain Phosphoglycerate mutase 1 Enolase domain Ubiquitin-conjugating enzyme E2, catalytic (UBCc) domain Inorganic pyrophosphatase; SPARC_EC extracellular Ca2+ binding domain KAZAL type serine protease inhibitors and follistatin-like domains; thyroglobulin type I repeats 14-3-3 homologues EF-hand, calcium binding motif EF-hand, calcium binding motif; Ca2+ binding protein (EF-hand superfamily), myosin tail 1 Eucaryotic SMC2 proteins; Chromosome segregation ATPases; myosin tail 1 Ca2+ binding protein (EF-hand superfamily) Calponin homology domain Conserved domain(s) L-malate dehydrogenase activity Phosphoglycerate mutase activity Phosphopyruvate hydratase activity Odorant binding (diacylglycerol-activated phospholipid-dependent) protein kinase C inhibitory activity; protein (domain specific) binding; protein kinase C inhibitor activity; transcription regulator activity; tryptophan hydroxylase activator activity Ligase activity; ubiquitin conjugating enzyme activity Protease inhibitor activity Inorganic diphosphatase activity; calcium ion binding Structural constituent of cuticle Structural constituent of cuticle (sensu Insecta) Microfilament motor activity; calcium ion binding; ATPase activity, coupled Cytoskeletal protein binding; motor activity; structural constituent of cytoskeleton Actin binding; structural constituent of cytoskeleton Microfilament motor activity; calcium ion binding; calmodulin binding; ATPase activity, coupled Molecular function (continued) Krebs cyclus; malate metabolism Glycolysis Glycolysis Sensory perception of chemical stimulus; transport Protein modification; proteolysis; ubiquitin cycle Signal transduction pathways; cell cycle Cell organization and biogenesis; negative regulator of JAK/STAT pathway Cytoskeleton organization and biogenesis; phosphorylation Cytoskeleton organization and biogenesis Cytoskeleton organization and biogenesis; calcium-mediated signaling; phosphorylation Cytoskeleton organization and biogenesis Biological proces After 8 h/ After 48 h After 48 h ⇑ ⇓ 17, 33 After 8 h After 48 h After 48 h After 8 h After 8 h After 8 h After 8 h After 24 h After 24 h After 48 h ⇓ ⇓ ⇑ ⇓ ⇑ ⇑ ⇑ ⇓ ⇑ ⇓ 29 32 4 9 13 16 21 23, 24 25, 26, 27b 5 28 After 8 h After 24 h ⇑ ⇓ ⇑/⇓ 19 20 Spot n° *⇓, down-regulated; ⇑, up-regulated. Similar to CG14207-PB, isoform B, isoform 1 Similar to protein lethal (2) essential for life, isoform 1 Others MRJP-4 MRJP-3 GB15046-PA GB10347-PA GB10347-PA GB10347-PA GB10347-PA Str ess rresponse esponse Stress Similar to ERp60 CG8983-PA, isoform A, isoform 2 Similar to arginine kinase Lipid metabolism FABP-like protein Similar to yippee interacting protein 2 CG4600-PA ATP production and transfer Similar to bellwether CG3612-PA, isoform 1 Major royal jelly protein Major royal jelly protein Alpha-crystallin-Hsps; IbpA PDIa family, C-terminal TRX domain (a’) subfamily; PDIa family, PDIR subfamily; PDIb family ERp57 subfamily, first redox inactive TRX-like domain b; PDIb’ family, ERp72 and ERp57 subfamily, second redox inactive TRX-like domain b’ Alpha-crystallin-Hsps F1 ATP synthase alpha, central domain; ATP synthase alpha/beta family, beta-barrel domain; ATP synthase alpha/beta chain, C-terminal domain ATP:guanido phosphotransfer, N-terminal domain; ATP:guanido phosphotransfer, C-terminal catalytic domain Thiolase domain Conserved domain(s) TABLE 2. Classification of the Differentially Displayed Proteins (continued) Protein disulfide isomerase activity Arginine kinase activity ATP binding; hydrogen-exporting ATPase activity, phosphorylative mechanism/rotational mechanism; hydrogen-transporting ATP synthase activity, rotational mechanism Fatty acid binding; transporter activity Acetyl-CoA C-acyltransferase activity Molecular function Honeybee nutrition Honeybee nutrition Heat shock response; defense response Heat shock response Electron transport; protein folding; protein modification Phosphorylation ATP biosynthesis Transport Fatty acid beta-oxidation Biological proces 232 Scharlaken et al. Archives of Insect Biochemistry and Physiology August 2007 doi: 10.1002/arch. Protein Expression in the Honey Bee Head impair mushroom body–mediated learning and long-term memory formation by down-regulation of the 14-3-3 zeta homologue in the head of the honey bee. A protein similar to Uev1A (spot 31) is up-regulated in the honey bee head after bacterial challenge. Uevs (ubiquitin-conjugating E2 enzyme variants) are defined as proteins similar in sequence and structure to the E2 ubiquitin-conjugating enzymes, but are predicted to be catalytically inactive, since they lack a critical Cys residue essential for the conjugation and transfer of ubiquitin to substrates (Sancho et al., 1998). It has been proposed that Uev 1A in humans has a role in NF-κB activation by interacting with an active ubiquitinating enzyme, ubiquitin-conjugating enzyme 13 (Ubc13) (Andersen et al., 2005). Similarly, the homologs of Ubc13 and Uev1A in Drosophila, Bendless and dUev1a, respectively, also associate with each other in vivo. It was shown that the BendlessdUev1a E2 complex is required for signaling by the IMD pathway in innate immunity (Zhou et al., 2005). The IMD signaling pathway is highly conserved in the honey bee, with plausible orthologues for all components (Evans et al., 2006). Olfactory Function Odorant binding protein 17 (OBP 17; spot 3) is down-regulated following bacterial challenge. Such a response suggests a reduced odor-sensing in challenged bees. OBPd-1, an OBP domain-containing protein of the mosquito’s olfactory system, is also known to be down-regulated upon microbial challenge (Aguilar et al., 2005). Obp99c was found to be an immune-responsive protein in Drosophila after fungal infection but is repressed after bacterial infection (Levy et al., 2004). Energy Housekeeping Carbohydrate metabolism. Several enzymes involved in carbohydrate metabolism are affected in the head by bacterial challenge. With respect to the glycolysis, phosphoglyceromutase (spot 14) is upregulated whereas enolase (spot 6) is down-reguArchives of Insect Biochemistry and Physiology August 2007 doi: 10.1002/arch. 233 lated. Up-regulation of phosphoglyceromutase is also observed in hemolymph of immune-challenged Drosophila larvae (Guedes et al., 2005). The majority of genes encoding carbohydrate-metabolizing enzymes found in the bee genome have 1:1:1 orthology (Apis: Drosophila: Anopheles), enolase has 2:1:1 orthology (Kunieda et al, 2006). Spot 15 (up-regulated) contains a malate dehydrogenase domain, possibly involved in the final reaction of the Krebs cycle. Up-regulation of this protein has also been reported in immunechallenged hemolymph of Drosophila larvae (Vierstraete et al., 2004b; Guedes et al., 2005). Lipid metabolism. In general, there is a high degree of homology in lipid-metabolizing genes between bee and dipteran species (typically >50% identity). The number of genes for lipid metabolism in honey bees is more evolutionarily stabile than for carbohydrate metabolism (Kunieda et al., 2006). Spot 19 was identified as an FABP-like protein. The primary role of all the FABP family members is the regulation of fatty acid uptake and intracellular transport. Participation in signal transduction and regulation of gene expression in lipid transport and metabolism has also been proposed (Esteves and Ehrlich, 2006). A retinoid and fattyacid-binding protein (RfaBp) is also up-regulated in infected Drosophila larval hemolymph (Vierstraete et al., 2004a), as well as a lipocalin-related FABP in Drosophila blood cells following LPS exposure (Loseva and Engström, 2004). A protein similar to yippee interacting protein (2) (yip2) is down-regulated (spot 20) in the head after bacterial challenge. Blastp indicates homology with the mitochondrial 3-ketoacyl-coA thiolase of Aedes aegypti. This mitochondrial thiolase can cleave longer fatty acid molecules and plays an important role in the beta-oxidative degradation of fatty acids. Down-regulation of this protein indicates reduced degradation of fatty acids in head tissue. ATP production and transfer. Up-regulation of a protein similar to bellwether, which encodes the alpha subunit of the mitochondrial ATP synthase (spots 17, 33) (Jacobs et al., 1998), points to an increase in ATP synthesis in head tissue. In addition, the up-regulation of enzymes in head tissue 234 Scharlaken et al. involved in glycolysis and the Krebs cycle (spot 14, 15) indicates that these pathways are used for ATP production. Spot 28 was identified as arginine kinase, an important component of the energy-releasing mechanism, belonging to the conserved family of ATP:guanidino phospho-transferases. Arginine kinase mRNA is relatively abundant in the central nervous system and the antennae of the honey bee, but the highest expression is found in its compound eye (Kucharski and Maleszka, 1998). It can thus be suggested that the function of arginine kinase, in the visual system, as an energy shuttle that delivers mitochondrial produced ATP to high energy-requiring processes, such as membrane turnover and pigment regeneration in the retina, is down-regulated by bacterial infection. Stress Response Spot 5 was identified as similar to ERp60, an endoplasmic reticulum (ER)-resident eukaryotic protein involved in oxidative protein folding. Among others, it contains a domain that belongs to the protein disulfide isomerase a (PDIa) family, subfamily PDI related (PDIR). It exhibits isomerase and chaperone activities. PDIR-proteins are preferentially expressed in cells actively secreting proteins, and its expression is induced by stress (Ferrari and Söling, 1999). Although heads contain actively secreting gland cells (hypopharyngeal and mandibular gland) and immunological stress was imposed, this putative honey bee ERp60 is down-regulated in head tissue. This suggests that another type of stress is necessary to induce this protein and it is also an indication of a possible reduced gland secretion during bacterial infection. Spots 29 and 32 belong to the alpha-crystallintype heat shock proteins (α-Hsps), a family of small stress-induced proteins that are believed to be ATP-independent chaperones that prevent aggregation and are important in protein refolding in combination with other Hsps (Narberhaus, 2002). These two α-Hsps are affected by immunological stress. One protein (spot 32, similar to “protein lethal (2) essential for life”) is induced, another (spot 29) is down-regulated. A function other than chaperoning may be implicated in the regulation of these α-Hsps. For example, in vertebrates an interaction between α-Hsps and nucleic acids has been demonstrated (Pietrowski et al., 1994). Others Major royal jelly protein 3 (MRJP3) and MRJP4 are members of the MRJP family. MRJPs occur in the hypopharyngeal glands as the major component of the larval bee queen food, also known as royal jelly. MRJP 3 (spot 9) becomes up-regulated and MRJP 4 (spot 4) becomes down-regulated 8 h after challenge. Our hypothesis that gland secreting is reduced during bacterial infection is in accordance with the down-regulation of MRJP4. However, Schmitzova et al. (1998) suggest that the nutritional function of MRJP3 and MRJP4 does not exclude other roles, especially because these two proteins contain lower amounts of essential amino acids. CONCLUSION This study shows that several proteins in the honey bee head, belonging to different functional classes, become differentially expressed after a bacterial challenge. None of them are immune effector proteins of the gene families implicated in honey bee immunity, as described in Evans et al. (2006). However, when compared to the immune-related tissues of other insects, several biological processes occurring in the honey bee head are affected in the same way. ATP synthesis is increased and the upregulated enzymes involved in glycolysis and Krebs cycle suggest also the involvement of these pathways in energy production in the head. An FABPlike protein is up-regulated whereas beta-oxidative degradation of fatty acids is reduced. Two α-crystallin-type Hsps are affected by bacterial challenge and ubiquitination of proteins is up-regulated. One major difference was found when compared to the immune-related tissues: most structural proteins are down-regulated. In hemolymph of immune-challenged insects, the up-regulation Archives of Insect Biochemistry and Physiology August 2007 doi: 10.1002/arch. Protein Expression in the Honey Bee Head of structural proteins reflects hemocyte migration and/or activated phagocytosis (Pearson et al., 2003). We believe that due to the small quantities of hemolymph flowing through the head, this phenomenon is overruled by the down-regulation of other biological processes, for instance exocytosis in the exocrine glands (confirmed by down-regulation of MRJP4 and ERp60). Moreover, this study revealed a number of bacteria-induced responses in insect heads directly related to the typical functions of the head. (1) The exocrine secretion of a nutritional MRJP family member is down-regulated. (2) A 14-3-3 zeta homologue involved in signal transduction is down-regulated, possibly impairing MB-mediated learning and long-term memory formation of the honey bee. In addition, (3) a reduction in odor sensing seems to occur, and (4) a component involved in processes in the visual system seems to be down-regulated. Thus, the senses in particular, appear to be affected by bacterial infection of the honey bee. LITERATURE CITED Badenhorst P, Voas M, Rebay I, Wu C. 2002. 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