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Differential protein expression in the honey bee head after a bacterial challenge.

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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.
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