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Contents lists available at ScienceDirect
Journal of Insect Physiology
journal homepage: www.elsevier.com/locate/jinsphys
Identification of major royal jelly proteins in the brain of the honeybee
Apis mellifera
Leonardo Gomes Peixoto a, Luciana Karen Calábria a, Liudy Garcia b,c, Fausto Emı́lio Capparelli a,
Luiz Ricardo Goulart a, Marcelo Valle de Sousa c, Foued Salmen Espindola a,*
a
b
c
Instituto de Genética e Bioquı´mica, Universidade Federal de Uberlândia, 38400-902 Uberlândia/MG, Brazil
Centro de Aplicaciones Tecnológicas y Desarrollo Nuclear, Ciudad Habana, Cuba
Instituto de Ciências Biológicas, Universidade de Brası´lia, 70910-900 Brası´lia/DF, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 17 September 2007
Received in revised form 5 May 2009
Accepted 7 May 2009
The consumption of royal jelly (RJ) determines the differences between castes and behavioral
development in the honeybee Apis mellifera. However, it is not known whether the proteins of RJ are
related to these differences, or which proteins are responsible for the changes. To understand the
functions of RJ proteins that are present in other tissues of the bee, in addition to hypopharyngeal gland,
we used a polyclonal antibody anti-MRJP1 to investigate the presence of this protein in nervous system
of honeybee. This study showed the presence of three polypeptides (p57, p70 and p128) in specific
tissues of bee brain. Mushroom body, optic lobe and antennal lobe neuropils all contained proteins
recognized by anti-MRJP1. Proteomic analysis showed that the three polypeptides are correlated with
proteins of the MRJP family. p57 is correlated with MRJP1, p70 with MRJP3, while p128 may be an
oligomeric form or a new polypeptide. Immunostaining of the brain and hypopharyngeal gland revealed
differential expression of MRJPs in various brain regions and in different honeybee castes and subcastes.
The identification and localization of these MRJPs contribute to the elucidation of the biological roles of
this protein family.
ß 2009 Elsevier Ltd. All rights reserved.
Keywords:
Major royal jelly protein
Honeybee
Brain
Hypopharyngeal gland
Royal jelly
1. Introduction
The fate of an adult honeybee is determined at the larval stage,
as its caste, development, phenotypical plasticity and behavior are
related to an early consumption of royal jelly (O’Shea and Schaffer,
1985; Tublitz et al., 1991; Nässel, 1993; Schmitzová et al., 1998).
Royal jelly (RJ) is secreted by hypopharyngeal glands of nurse bees
and constitutes a nutritious complex, in which the major royal jelly
proteins (MRJPs) are the most abundant proteins (Lensky and
Rakover, 1983; Knecht and Kaatz, 1990). The MRJP genes express
evolutionarily unique proteins, wich present homology with genes
preserved in dipterans (Drosophila melanogaster and Anopheles
gambiae), known as the Yellow–MRJP protein family. Several
studies have been published using proteomic analyses of royal jelly
as well as of the MRJPs (Li et al., 2007; Furusawa et al., 2008; Qu
et al., 2008; Li et al., 2008). Besides the expression these proteins in
hypopharyngeal glands, MRJP1 (mRNA) (Kucharski et al., 1998)
and MRJP2 (cDNA) (Kucharski and Maleszka, 2002) have also been
found in the brain of worker bees (Garcia et al., 2009).
* Corresponding author. Tel.: +55 34 3218 2477; fax: +55 34 3218 2203.
E-mail address: [email protected] (F.S. Espindola).
The molecular and functional features of MRJPs in honeybee
tissues outside the hypopharyngeal gland are almost unknown.
However, studies on the transcriptome of Apis mellifera L.
(Whitfield et al., 2002; Nunes et al., 2004) revealed contigs of
MRJPs, principally MRJP1, in libraries of the brain of this honeybee.
The complete sequencing of the honeybee (A. mellifera) genome
has revealed additional genes in the MRJP family. Nine of these
genes have apparently evowed from one ancestral yellow gene, and
now are functionally related to queen and brood nursing
suggesting achievement of new functions during the evolution
of sociality (The Honeybee Genome Sequencing Consortium,
2006). Moreover, microarray studies that compared young adult
functionally sterile (wild-type), bees with a stain in wich workers
were reproductivelly active, found that two genes (MRJP2 and 7)
were expressed at significantly higher levels in the heads of wildtype bees (Thompson et al., 2006). Given the assumed role RJ plays
in modulating caste hierarchy, the possibility that RJ proteins
might be endogenous participants in brain activities seems quite
interesting (Garcia et al., 2009).
The aim of this study was to apply immunopurified antibodies
against MRJP1 to investigate the distribution of MRJP1 in the head
and brain of the A. mellifera castes and subcastes, comparing with
three others species of hymenopterans. This approach allowed the
identification of 57 and 70 kDa polypeptides, which are related to
0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2009.05.005
Please cite this article in press as: Peixoto, L.G., et al., Identification of major royal jelly proteins in the brain of the honeybee Apis
mellifera. J. Insect Physiol. (2009), doi:10.1016/j.jinsphys.2009.05.005
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MRJP1 and MRJP3, respectively, as well as an additional 128 kDa
polypeptide which may correspond to an oligomeric form, or a not
yet described MRJP. We also present immunohistochemistry data
showing MRJP1 localization in different areas of the brain of the
nurse and worker honeybees.
2. Materials and methods
containing 0.1% sodium azide, quantified (Bradford’s method)
and stored at 20 8C. The specificity of the antibodies was
determined by ELISA.
2.4. Enzyme-linked immunosorbent assay (ELISA)
Micro-titration plates were coated overnight at 4 8C with 50
mL/well of WSP at a 10 mg/mL dilution in phosphate-buffered
2.1. Biological specimens
Bees (A. mellifera, Mellipona scutellaris, Scaptotrigona postica)
and ants (Camponotus atriceps) used in the biochemical assay were
collected at the University’s experimental garden. To distinguish
between honeybee nurse and forager workers anatomical features,
i.e., coat condition, damage to the wings, and development of the
hypopharyngeal gland were considered. Rabbits and mice used in
the production of the polyclonal antibodies for this study were
kept at the University’s animal facilities under supervision of the
Animal Experiments Review Board at our University.
2.2. Preparation of the samples
Head homogenates were prepared with aid of a hand blender in
cold homogenization buffer (40 mM Hepes at pH 7.7 containing
10 mM EDTA, 2 mM EGTA, 5 mM ATP, 2 mM DTT, 1 mM
benzamidine, 0.1 mM aprotinin and 0.5 mM PMSF). The same
buffer was also used to prepare brain and hypopharyngeal gland
homogenates (n = 30). The supernatant fraction was obtained by
centrifugation at 40,000 g for 40 min at 4 8C. Total protein
concentration (Bradford, 1976) of these samples was determined
prior to SDS-PAGE and Western blotting analysis as described
below. Alternatively, A. mellifera brain regions were dissected
(n = 30) to obtain the optic lobe, antennal lobe, peduncle, and
mushroom body neuropils. Each sample of these neuropils was
also prepared using same buffer and centrifugation as described
above.
saline (PBS) and then blocked with 1% bovine serum albumin (BSA)
in PBS. Subsequently, 50 mL of anti-MRJP1, serially diluted (1/100
to 1/512,000) with 0.1% BSA in PBS were incubated in the coated
wells for 2 h at room temperature. The binding of anti-MRJP1 with
antigen was visualized by using 50 mL of an anti-rabbit IgG
conjugated to peroxidase at 0.4 mg/mL. The color reaction was
initiated by adding 50 mL/well of orthophenylene diamine
chromogen at 0.4 mg/mL in substrate buffer (citric acid 0.1 M,
sodium acetate 0.1 M, pH 5.4, in H2O2 at 0.33% final concentration).
The reaction was stopped after 15 min by adding 25 mL of 4 N
H2SO4 to the wells. Optical density was measured at 492 nm with
an ELISA plate reader. Two rinses with PBS were performed
between each step described above.
2.5. Immunoprecipitation of MRJPs from nervous system
Protein extracts were incubated overnight at 4 8C with
20 mg/mL of anti-MRJP1 or non-immune rabbit IgG and
150 mM NaCl 0.2% Triton X-100. Protein A Sepharose beads
(2 mg/mL) were added and the suspensions were gently mixed
on a rocking platform for 30 min at 4 8C. The immunoprecipitation fractions were then collected by centrifugation at 14,000 g for 10 min. Supernatants were kept for further analysis, and
pellets were washed once with TBS supplemented with 0.5 M
NaCl and then six times in TBS without salt addition. Finally, the
pellets and the initial supernatants were diluted in SDS sample
buffer, boiled at 100 8C for 5 min and analyzed for MRJP1
content by immunoblot using rabbit anti-MRJP1 and mouse
anti-MRJP1 (negative control).
2.3. Production and purification of polyclonal antibodies
2.6. Immunohistochemical analysis of MRJPs in the bee brain
Rabbits and mice were inoculated with water soluble proteins
(WSP), isolated from RJ homogenates. The RJ was provided by
Cláudio Franco Lemos (Apiários Girassol Ltda of Uberlândia/MG,
Brazil). To prepare WSP, the royal jelly sample was homogenized in
PBS pH 8.0 containing 20 mM EDTA, centrifuged at 10,000 g,
10 min at 4 8C, supernatant fraction was dialyzed in Tris–HCl
buffer pH 8.0 containing 0.5 mM EDTA, after dialysis the final
supernatant was obtained by another centrifugation step. To
challenge animals with WSP antigen samples of 500 mg/mL
(rabbit) or 100 mg/mL (mouse) of a mixture of WSP and Freund’s
complete adjuvant (1:1), and subsequent 15-day reinforcement of
250 mg/mL (rabbit) or 25 mg/mL (mouse) were employed for
generation of polyclonal antibodies. Animals were bled three days
after the last inoculation. Serum was obtained by centrifugation at
3000 rpm for 3 min and stored at 80 8C. For purification of
polyclonal antibodies for MRJP1 (anti-MRJP1), 2 mg/mL WSP
fraction was separated in SDS-PAGE 5–22% (300 V, 2 h) and
stained (Coomassie brilliant blue—R250, at 1 h). The bands of
interest were excised and electrotransferred onto a nitrocellulose
membrane (300 V, overnight). Membranes were incubated in TBST (Tris–HCl pH 7.4, 0.5% Tween 20) containing 5% of dried milk
overnight at room temperature, prior to incubation with diluted
serum at TBS (1:1) at 2 h. Subsequently membranes were washed
three times for 5 min with TBS-T and bound antibodies (antiMRJP1) were eluted with 1.4% triethylamine (1 min) and
transferred to vials containing 1 M Tris–HCl pH 8.5 at room
temperature. After elution antibodies were dialyzed in TBS
Brains were dissected and fixed in 4% paraformaldehyde, as
described by McLean and Nakane (1974). Fixed tissues were
dehydrated in ethanol, cleared in xylene and embedded in
paraffin. Five micrometer sections were pretreated with 4 mM
citrate buffer (pH 6.0), containing 0.025% Tween 20, in a
microwave for 5 min. Thereafter, sections were incubated with
MRJP1 antibody for 16 h followed by incubation with the
NovoLinkTM Max Polymer Detection System-Post Primary Block
(Novocastra Laboratories Ltd, Newcastle Upon Tyne, UK). After
three washes with Tris buffer with Tween, the sections were
incubated with the NovoLink polymer for 30 min at 37 8C.
Chromogen development was performed with 3,30 -diaminobenzidine, and the material was counterstained with Harris
hematoxylin, dehydrated, and mounted with Permount and
analyzed using a light microscope (Zeiss Axiolab, MC80
911258, Germany). Negative controls consisted of the omission
of the primary antibody in the reaction.
2.7. MALDI-TOF MS and database search
Protein fragments corresponding to the previously immunoprecipitated MRJP1 were excised and digested for further
identification by peptide mass fingerprinting (PMF). The method
employed was based on the reduction of proteins with DTT,
alkylation with iodoacetamide and digestion with trypsin. The
yielded fragments were submitted to microchromatography using
Please cite this article in press as: Peixoto, L.G., et al., Identification of major royal jelly proteins in the brain of the honeybee Apis
mellifera. J. Insect Physiol. (2009), doi:10.1016/j.jinsphys.2009.05.005
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C18 Zip Tips (Millipore, Billerica, USA), followed by matrix assisted
laser desorption ionization-time of flight (MALDI-TOF) in a Bruker
Reflex IV mass spectrometer. Known trypsin autolysis peaks and
keratin contaminants were removed. Mascot software, assuming
p < 0.05 (Perkins et al., 1999) was used to search the PMFidentified proteins in the NCBI nr database. No restrictions were
made with respect to the molecular mass or the taxonomy of the
proteins. The fragment mass tolerance was <0.2 Da for MH+
monoisotopic data. The protein sequence identified by MASCOT
was aligned with other MRJPs sequences using CLUSTALW (http://
www.ebi.ac.uk/Tools/clustalw2/index.html).
2.8. Analysis of MRJP1 secondary structure
A 96-well microtitter plate was coated with 100 mg/mL of antiMRJP1 in bicarbonate–carbonate buffer at pH 8.6 (BCB) overnight
at 4 8C. After coating solution was poured off each well was filled
with BCB and 6.5 mg/mL bovine serum albumin (BSA) for 1 h at
4 8C. Subsequently, 100 mL of TBS-T with 4 1010 phage were
added (Ph, D – 12 mer – New England Biolabs) and incubated at
room temperature for 1 h. Bound phages were eluted at room
temperature for 10 min with 100 mL of 200 mM glycine–HCl pH
2.2, 1 mg/mL BSA, and immediately neutralized with 1 M Tris–HCl,
pH 9.1. E. coli cells (ER2738 strain) were then infected with 100 mL
of eluted during the rounds of phage amplification (input
inoculum).
Colonies deriving from the fourth cycle of biopanning (nonamplified eluted) were used for the amplification process in deepwell plates. After amplification, clones were tested by ELISA for
their ability to bind to serum-purified antibodies. Positive clones
were sequenced according to a cut-off value of 0.127 (average
readings of the negative control).
The nucleic acid sequences of positive clones were translated
using
DNA2PRO12
software (http://relic.bio.anl.gov/dna2pro12.aspx). Amino acid frequency was calculated with AAFREQS
software (http://relic.bio.anl.gov/aafreqs.aspx). To further investigate the importance of the identified residues or motifs, a
secondary structure prediction of the MRJP1 protein was made
using the PredictProtein server (http://www.predictprotein.org).
For selected peptides, biochemical properties, such as antigenicity,
surface exposure, and hydrophilicity, were assessed through
DnaStar-Lasergene software (subprogram Protean). Every peptide
homology with MRJP1 was tested with MATCH (http://relic.
3
bio.anl.gov/match.aspx) and CLUSTALW (18.1) (http://services.bioasp.nl/blast/cgi-bin/clustal.cg) softwares.
3. Results
3.1. Identification of brain MRJP1
Immunoprecipitation of A. mellifera brain homogenate with
rabbit anti-MRJP1 allowed partial purification of a p57 polypeptide
(Fig. 1a). Controls were carried out in presence of non-immune IgG.
Unspecific bands failed to cross-react with mouse anti-MRJP1 and
were discarded from our analysis. These results confirm the
specificity of p57 immunoprecipitation (Fig. 1b). For further
identification the p57 band was excised and processed for MALDITOF mass spectrometry measurements. Analysis revealed fourteen
peptides with mass m/z ranging from 582.3367 to 2854.6471, in
accordance to the predicted mass of in silico digested MRJP1
(Fig. 2a). Searches using these peptides mass in the MASCOT
protein database covered 39% of the A. mellifera MRJP1 sequence
(Fig. 2b). The MRJP1 identified in this search has accession number
58585098, estimated molecular mass (mw) of 49.311 kDa and
isoelectric point (pI) 5.1 (Fig. 2c). Protein multi alignment studies
of the MRJP1 with the MRJP2 and MRJP3 expressed in the A.
mellifera nervous system presented 44% of identity. Furthermore,
MRJP1 has more than 44% identity with all other major RJ proteins
(Fig. 3a).
Phage display and bioinformatic analyses showed that the
linear sequence of the brain MRJP1 exhibit seven hydrophilic
regions with high identity to other MRJPs. Although hydrophilic
regions in the linear sequences of MRJP proteins (as showed by
the phage display data) were recognized by anti-MRJP1 (Fig. 3b
and c), only the MRJP1 native form was immunoprecipitated by
this antibody. MRJP multiple alignments showed similarity with
Yellow proteins of the D. melanogaster. In order to predict the
functions of the honeybee brain MRJP1 we compared its
sequence with D. melanogaster paralogues using FatiGO analysis
of the MRJP1 sequence based on cellular content, molecular
function and biological process (Al-Shahrour et al., 2004, http://
fatigo.bioinfo.cnio.es/). This ontology analysis suggests that
MRJP1 is probably located in intracellular organelles, potentially
binds nucleic acids, and is potentially involved in morphogenesis,
embryonic development, metabolism, cell differentiation and sex
determination.
Fig. 1. Immunoprecipitation of p57 polypeptide from worker bee brain homogenate using anti-MRJP1 antibody. Immunoprecipitated proteins (IP) were analyzed by SDSPAGE (a) and Western blotting (b) using anti-MRJP1 produced at mouse as positive control, in supernatant (S) and pellet (P) fractions. The IgG non-immune was loaded 10fold more concentrated than IgG immune (anti-MRJP1). Numbers at left indicate the standard molecular mass.
Please cite this article in press as: Peixoto, L.G., et al., Identification of major royal jelly proteins in the brain of the honeybee Apis
mellifera. J. Insect Physiol. (2009), doi:10.1016/j.jinsphys.2009.05.005
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Fig. 2. MALDI-TOF MS spectrum of the MRJP1 immunoprecipitated. (a) Spectrum of the peptide masses generated from the digested immunoprecipitated band (p57) showed
the m/z – mass to charge ratio – (x-axis) and the intensity of the molecular ions (y-axis). From the results of MS/MS analysis, peaks with asterisk were identified as MRJP1. The
peaks with k/t indicate keratin/trypsin. (b) MRJP1 sequence with access number 58585098 indicates the fourteen peptides of the spectrum (underlane/bold). (c) Table of the
main characteristics of the MRJP1 identified in database.
3.2. Analysis of the expression of MRJPs in honeybee tissues
SDS-PAGE revealed differential protein content in head
homogenates of A. mellifera, M. scutellaris, S. postica and C.
atriceps (Fig. 4a). Remarkably, Western blots showed that only A.
mellifera homogenates contain bands reactive to MRJP1, as
shown in Fig. 4. Honeybee workers presented three main
polypeptides bands, termed p57, p70 and p128 while in
honeybee drones the p70 band was absent. We observed a
predominance of p70 in the honeybee subcaste of nurse worker
(Fig. 4b). The expression levels of each one of the immunoreactive bands also seemed to be caste-specific, especially in
nurses, whose p57 levels were higher than in foragers (Fig. 4c).
The anti-MRJP1 antibody also recognized all three polypeptides
in hypopharyngeal gland homogenates, regardless of caste.
However, p70 was clearly more expressed in brain samples
and p128 in hypopharyngeal glands samples (Fig. 4d). As the
remaining insects failed to display any immunoreactions,
regardless of antibody titration or span of incubation, further
localization experiments were carried out only for A. mellifera.
Supernatant fractions of the homogenates from different
regions of A. mellifera brain such as optic lobe, antennal lobe,
peduncles and mushroom body showed variable cross-reaction to
the three polypeptides described above with a predominance of
p57 in all regions (Fig. 4e). Immunolocalization of MRJP1 indicated
that the protein is localized similarly in specific regions of the
nurse and forager worker brain (Fig. 5). Kenyon cells were strongly
immunoreactive to anti-MRJP1, represented by a brown coloring,
as well as the calyx. On the other hand, anti-MRJP1 strongly stained
the non-compacted Kenyon cells (Fig. 5b and c). In the optic lobe,
Fig. 3. Phage display and bioinformatics analysis of the MRJPs immunodetected in the honeybee brain. (a) The MRJP1, MRJP2 and MRJP3 genes showed five introns (lane) and
six exons (rectangle), respectively. (b) Indices of antigenicity predicted for the recognized regions of the MRJP1 with the antibody anti-MRJP1 in accordance with the Protean
program (Lasergene softwares). (c) Alignment and identity of the linear sequences of these MRJPs. Boxes indicate the regions in the linear sequence of these proteins that are
recognized by anti-MRJP1.
Please cite this article in press as: Peixoto, L.G., et al., Identification of major royal jelly proteins in the brain of the honeybee Apis
mellifera. J. Insect Physiol. (2009), doi:10.1016/j.jinsphys.2009.05.005
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5
Fig. 4. Immunodetection of major polypeptides related with MRJPs in head, brain and hypopharyngeal gland of A. mellifera. Supernatant fractions of head, brain and
hypopharyngeal gland were analyzed by SDS-PAGE (left panel) and Western blotting (right panel) with immune detection of p57, p70 and p128 by anti-MRJP1. (a)
Supernatant fractions of head: C. atriceps (Ca), S. postica (Sp), M. scutellaris (Ms), A. mellifera (Am). (b and c) Supernatant fractions of A. mellifera head: drone (D), forager (F),
nurse (N). (d) Supernatant fractions from worker A. mellifera brain (br) and hypopharyngeal gland (hg). (e) Supernatant from different regions of A. mellifera brain: optic lobe
(ol), antennal lobe (al), peduncles (ped), mushroom body (mb). Numbers at left indicate the standard molecular mass.
anti-MRJP1 diffusely stained cells in the retina, monopolar neurons
in the fenestrated layer and fibers in the lamina (Fig. 5f and g). This
could be distinguished from the pigmented cells of the retina that
we seen in control section (Fig. 5h). In the antennal lobe, anti-
MRJP1 showed considerable staining among interneurons and in
the glomerulus. In detail was possible to observe anti-MRJP1
staining in fibers and pericellular regions of the interneurons
(Fig. 5j and k).
Fig. 5. Immunolocalization of MRJP1 in honeybee brain section. (b, c, f, g, j and k) labeled with anti-MRJP1, bar: 55 mm; (a, d, e, h, i and l) control, bar: 55 mm. (b, f and j) nurse
worker; (c, g and k) forager worker. (a–d) mushroom body; (e–h) optic lobe; (i–l) antennal lobe. (b and c) anti-MRJP1 immunodetection in internal compacted (ikc) and noncompacted Kenyon cells (nkc) and in the mushroom body calyx (cx). (f and g) labeling of anti-MRJP1 in retina (re), fenestrated layer (fl) and lamina fibers (la). In detail, the
photomicrograph shows the pigmented layer (pc). (j–k) densely stained of anti-MRJP1 in antennal lobe interneurons (in) and glomerulus (gl). Detail of the non-compacted
Kenyon cells, fenestrated layer and interneurons (inset box) show the MRJP1 localization in fibers and pericellular regions, bar: 22 mm.
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4. Discussion
The major royal jelly proteins (MRJP1-5) of honeybee (A.
mellifera) are highly expressed and secreted by the hypopharyngeal gland of nurse bees to feed the queen and the growing
larvae. We described in this study the immunodetection and
immunolocalization of MRJPs with affinity-purified polyclonal
antibodies. These antibodies specifically recognized protein bands
in A. mellifera that were absent in S. postica, M. scutellaris and C.
atriceps. A caste-specific pattern of staining was observed for the
MRJP1 antibody. Proteomic analysis revealed that the main
polypeptides recognized by anti-MRJP1 in the A. mellifera head
homogenates are well correlated with the MRJPs.
The hypopharyngeal gland is localized in the bee head and
produces most of the royal jelly proteins (Patel et al., 1960;
Klaudiny et al., 1994; Kubo et al., 1996). The protein profile of this
gland from Melipona and Apis bees reported by Silva de Moraes
et al. (1996) showed four polypeptides with similar molecular
mass. Also morphological analysis failed to clarify whether there is
hypopharyngeal gland secretion in the larval food by S. postica bee
(Costa and Cruz-Landim, 2002a,b). Considering the cross-reactivity
of the anti-MRJP1 and the phylogenetic profile of this family, our
results suggest that the proteins expressed in the head of the A.
mellifera differ considerably from those expressed in the heads of
M. scutellaris and S. postica.
We detected in the honeybee brain homogenate three
polypeptides, p51, p70 and p128, wich cross-reacted to antiMRJP1 with some particularities regarding the caste and the
subcastes. The p57 polypeptide was immunoprecipitated from bee
brain and identified as MRJP1. MRJP1 and MRJP3 were detected
and localized in different honeybee brain neuropils. In addition, a
p128 detected polypeptide is suggested were to be a potential
MRJP1 dimer, or a new not yet reported MRJP-like protein.
Remarkably of the p70 polypeptide was differentially expressed,
the amount of this protein being considerably greater in nurse
brain homogenates than in queen, drone or forager brain
homogenates. Nurse bee brains also presented higher expression
of p57 polypeptide than the others. This protein was immunoprecipitated suggesting that the anti-MRJP1 antibody recognized
the native form of this brain protein since immunoprecipitation
assay works with soluble protein instead of immobilized polypeptides in the nitrocellulose filter. Mass spectrometry analysis
identified the immunoprecipitated protein as A. mellifera MRJP1,
with a molecular mass of 49 kDa. Other studies have also reported
that MRJP1 may present variant forms of 49–60 kDa (Santos et al.,
2005).
Immunoreactive p70 in the nervous system and hypopharyngeal gland of nurse and forager worker bees co-migrated in the
same range as MRJP3 in SDS-PAGE. The polypeptide p70 has also
been differentially detected in brain supernatants of queen,
drone, nurse and forager worker bees. This finding indicates that
p70 is a good marker to distinguish forager from nurse worker
bees. Furthermore, Albert et al. (1999) have previously shown
that MRJP3 exhibits length polymorphism. In fact, our observations of p70 identified in distinct samples of honeybee brain
validate this finding. Strikingly, our results suggest the existence
of a queen/nurse-specific isoform of MRJP3 suggesting a length
polymorphism of p70 in caste and subcaste.
The p128 band was immunodetected in the honeybee’s head,
brain and gland tissues. Compared to the two polypeptides
described earlier, p128 was less immunoreactive, except in head of
drones and head of nurse worker. There were variations in its
presence in the brain for different castes and brain regions. This
polypeptide seems to be more expressed in the hypopharyngeal
gland than in brain tissue; although we did not compare the gland
tissues from different castes and subcastes, the data from the head
homogenates suggest that nurse workers express more p128 in the
hypopharyngeal gland. These MRJPs are secreted solely by the
hypopharyngeal gland of A. mellifera and are evolutionarily
conserved, presenting a high identity among each other and,
together with the Yellow proteins of D. melanogaster, they
constitute a new protein family as described by Albert and
Klaudiny (2004). The polypeptide p128 could be an oligomeric
form or an unknown MRJP, since this polypeptide was ignored in a
previous proteomic study of the hypopharyngeal gland of
Africanized A. mellifera (Santos et al., 2005). In their study, Santos
et al. (2005) identified 27 isoforms of MRJPs in hypopharyngeal
gland homogenates, which presented relative molecular masses
from 49 to 87 kDa. Because p128 has a molecular mass unlike the
ones reported above and is immunoreactive with anti-MRJP1, even
under the denaturated conditions of the immunoblot analysis, we
suggest that p128 is a new member of the A. mellifera MRJP family
and in future studies should be immunoprecipitated and
sequenced.
Analogous antigenic regions among MRJPs found in phage
display and bioinformatic analyses show that MRJP family proteins
contain seven antigenic conserved regions which probably
correspond to the epitopes for anti-MRJP1. The identities among
MRJP sequences have been suggested by hybridization assay and
sequence alignment (Klaudiny et al., 1994; Schmitzová et al., 1998;
Albert et al., 1999; Drapeau et al., 2006). This antibody recognizes
members of the MRJP family in contiguous sequences in the
denatured protein. In addition, sequence analysis agree with
available data showing that all of the MRJP family members have
an N-terminal hydrophobic sequence that would function as a
cleavable signal peptide as well as a putative N-linked glycosylation site, suggesting that these proteins are also secreted by the cell
(Klaudiny et al., 1994; Drapeau et al., 2006).
Immunolocalization data showed intense staining of MRJP1 in
different regions of the worker’s brain. In the mushroom bodies,
MRJP1 was localized in both internal compacted and noncompacted Kenyon cells, and in the calyx. Western blots revealed
the presence of polypeptides corresponding to MRJP1 and MRJP3,
as well as p128 in this region. In situ hybridization data on
mushroom bodies indicate the presence of this MRJP in specific
neurons, the intrinsic Kenyon cells, whose function is unknown
(Kucharski et al., 1998). It is known that the mushroom bodies are
central for visual and mechanosensory (primary), and olfactory
(secondary) integration. They are constituted of densely packed
parallel neurons called internal and external compact Kenyon cells.
These neuropils constitute the brain centers for processing of
information and memory in the bee’s brain (Hammer and Menzel,
1995; Capaldi et al., 1999).
Immunohistochemical analysis of the optic lobe showed
MRJP1 to be present in the retina, fenestrated layer and fibers
of the lamina. These neuropils are formed by axonal, pigment
cells, photoreceptors, and monopolar and laminar neurons
(Nässel et al., 1985). The localization of MRJP1 in the optic lobe
suggests multiple functions of these proteins in the bee nervous
system. In the antennal lobe both antibodies localized MRJPs in
the interneurons and glomeruli. These primary neuropils receive
impulses from chemosensory axons, and transmit this information to terminal olfactory receptors, which finally carry it to the
mushroom bodies and the alpha and beta lobes (Nässel et al.,
1986; Kloppenburg, 1995; Galizia and Menzel, 2000; Menzel and
Giurfa, 2001). Other studies reporting MRJP transcripts in the
brain consider that their function remains unknown (Kucharski
et al., 1998; Albert and Klaudiny, 2004). Notably, anti-MRJP1
stained the fibers and pericellular regions of interneurons more
intensely than other regions in the honeybee brain, and the
immunolocalization was coincident between the workers, nurse
and forager.
Please cite this article in press as: Peixoto, L.G., et al., Identification of major royal jelly proteins in the brain of the honeybee Apis
mellifera. J. Insect Physiol. (2009), doi:10.1016/j.jinsphys.2009.05.005
G Model
IP-2270; No of Pages 7
L.G. Peixoto et al. / Journal of Insect Physiology xxx (2009) xxx–xxx
Other studies have shown the presence of MRJP1 mRNA and
MRJP2 cDNA in the honeybee brain (Kucharski et al., 1998;
Kucharski and Maleszka, 2002; Albert and Klaudiny, 2004). Brain
histological sections stained with the anti-MRJP1 revealed specific
distributions of MRJP protein, confirming the results of immunoblotting experiments and eliminating the possibility of crosscontamination by hypopharyngeal gland tissue in the brain
samples. These data indicate that the honeybee brain expresses
MRJP family members, although their functions remain open to
speculation. FatiGO analysis also suggests their involvement in
developmental processes in the A. mellifera nervous system.
In conclusion, the affinity-purified antibodies for the MRJPs in
the brain and hypopharyngeal gland of A. mellifera revealed
differential expression of MRJPs regarding brain regions and
different castes and subcastes. The identification and localization
of these MRJPs in various honeybee brain regions may contribute
to elucidate the biological role of this protein family.
Acknowledgements
This work was supported by grants from CNPq and FAPEMIG to
FSE, LRG and MVS, CAPES fellowship to LGP and LKC, CNPq
fellowship to FEC, and TWAS to LG. Also, we thank Pablo Marco
Veras Peixoto (New York University) for critically reading the
manuscript.
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