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Insect Biochemistry and Molecular Biology 35 (2005) 85–91

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Short communication

Profiling the proteome complement of the secretion fromhypopharyngeal gland of Africanized nurse-honeybees

(Apis mellifera L.)

Keity Souza Santosa,b, Lucilene Delazari dos Santosa,b, Maria Anita Mendesa,b,Bibiana Monson de Souzaa,b, Osmar Malaspinaa,b, Mario Sergio Palmaa,b,�

aCenter of Study of Social Insects (CEIS) Department of Biology, Institute of Biosciences, Sao Paulo State University (UNESP), 13506900 Rio Claro,

SP, BrazilbInstitute of Immunological Investigations (CNPq/MCT);CAT/CEPID-FAPESP

Received 27 July 2004; received in revised form 5 October 2004; accepted 7 October 2004

Abstract

The protein complement of the secretion from hypopharyngeal gland of nurse-bees (Apis mellifera L.) was partially identified by

using a combination of 2D-PAGE, peptide sequencing by MALDI-PSD/MS and a protein engine identification tool applied to the

honeybee genome. The proteins identified were compared to those proteins already identified in the proteome complement of the

royal jelly of the honey bees. The 2-D gel electrophoresis demonstrated this protein complement is constituted of 61 different

polypepides, from which 34 were identified as follows: 27 proteins belonged to MRJPs family, 5 proteins were related to the

metabolism of carbohydrates and to the oxido-reduction metabolism of energetic substrates, 1 protein was related to the

accumulation of iron in honeybee bodies and 1 protein may be a regulator of MRJP-1 oligomerization. The proteins directly

involved with the carbohydrates and energetic metabolisms were: alpha glucosidase, glucose oxidase and alpha amylase, whose are

members of the same family of enzymes, catalyzing the hydrolysis of the glucosidic linkages of starch; alcohol dehydrogenase and

aldehyde dehydrogenase, whose are constituents of the energetic metabolism. The results of the present manuscript support the

hypothesis that the most of these proteins are produced in the hypoharyngeal gland of nurse-bees and secreted into the RJ.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Africanized Apis mellifera; Royal jelly; Peptide mass fingerprint; Proteome; MALDI-TOF

1. Introduction

The honeybee (Apis mellifera L.) is a social insect,living in colonies constituted of different castes: a queen,workers and drones (Lercker et al., 1982; Palma, 1992).The age-dependent role is one of the most notablefeatures of the workers in these colonies (Ohashi et al.,

e front matter r 2004 Elsevier Ltd. All rights reserved.

mb.2004.10.003

ing author. Center of Study of Social Insects (CEIS)

Biology, Institute of Biosciences, Sao Paulo State

NESP), 13506900 Rio Claro, SP, Brazil. Tel.:

63; fax: +55 19 3534 8523.

ess: mspalma@rc.unesp.br (M.S. Palma).

1999). Young workers, known as nurse-bees take care ofthe brood, by synthesizing and secreting many compo-nents of the royal jelly (RJ), while the older workersusually forage for nectar, converting it into honey(Robinson, 1987). This process is biologically regulatedand known as age polyethism, which is paralleled byphysiological changes in certain organs of the workerhoneybees (Ohashi et al., 1997).The RJ is believed to be synthesized both by the

mandibular and hypopharyngeal glands of nurse-hon-eybees (Knecht and Kaatz, 1990; Lensky and Rakover,1983). The hypoharyngeal gland is well developed in thenurse-bees, but shrinks in the older workers, to adapt

ARTICLE IN PRESSK.S. Santos et al. / Insect Biochemistry and Molecular Biology 35 (2005) 85–9186

the insect to forage and honey production (Ohashi et al.,1999). Thus, it may be assumed that the glandsynthesizes different proteins according to age polyeth-ism (Kubo et al., 1996).Taking into account that the secretion of this gland

constitutes an important part of the RJ composition, itis necessary to identify the biochemical contributions ofthe hypopharyngeal gland to the composition of the RJ,in order to get a better understanding the role of thisgland for the brood care.The RJ contains proteins, free amino acids, fatty

acids, sugars, vitamins, some minerals (Palma, 1992)and constitutes the principal food of the queenhoneybees; also it is part of the initial diet of honeybeelarvae (Moritz and Southwick, 1992). The proteincontent represent up to 12% (m/m) of freshly harvestedRJ and the major components seem to constitutemembers of the most known family of proteins of thissecretion, the major royal jelly proteins (MRJP) (Knechtand Kaatz, 1990; Lensky and Rakover, 1983); thisfamily of proteins represent about 82% of the totalprotein content of RJ (Schimitzova et al., 1998).The complexity of the protein complement composi-

tion of the RJ and the absence of fundamentalsequential data about the most of genes involved withthe expression of these proteins, make their biochemicaland physiological characterizations difficult, which inturn makes the understanding of their functions notfully known. In addition to this, there has been fewsubstantial biochemical analysis of the proteins synthe-sized by the hypopharyngeal gland.Four major proteins (50, 56, 57 and 64 kDa) were

demonstrated to be selectively synthesized by thehypopharyngeal gland and secreted as RJ proteins(Hanes and Simuth, 1992; Kubo et al., 1996). Theseresults constitute strong evidences that the someproteins found in RJ are in fact products of thehypopharyngeal gland secretion.Recently, the basic composition of the RJ was

analyzed by a proteomic approach, which identifiedthe presence of several different forms of MRJP-1 toMRJP-5 and glucose oxidase, presenting heterogeneitiesin terms of molecular weights and isoelectric points(Sato et al., 2004). This study offered a wide view of thecomposition of the proteome complement of the RJ,opening the possibility to develop a similar investigationwith the hypopharyngeal gland, in order to compareboth compositions considering a large number ofproteins. Thus, the aim of the present study was toobtain a proteome profile from the secretion ofhypopharyngeal gland of nurse-bees (A. mellifera) byusing the honeybee (HB) genome databank as referencefor proteins identification and to compare the proteinsidentified with those already described in RJ, in order toget a better understanding about the contribution of thisgland to the composition of the RJ.

2. Material and methods

2.1. Insects and the collection of the secretion from

hypopharyngeal-gland

Africanized honeybees (A. mellifera L.) kept in theapiary of the Institute of Biosciences of Sao PauloState University, at Rio Claro, southeast Brazil,were used. Newly emerged workers were marked (day-0) on their thorax using paint and introduced into anormal queenright colony; marked nurse beeswere collected 7 days later when they were feedingbrood. In order to extract proteins of the hypophar-yngeal gland secretion the bees were anesthetized byusing carbonic gas and the hypopharyngeal glands weredissected under a binocular microscope. The glands(500 glands/ml) were washed in buffered saline solution(10mM Tris-HCl pH 7.4, containing 13 mM NaCl,5mM KCl and 1mM CaCl2), containing a mixture ofprotease inhibitors: 1mM phenylmethylsulfonyl fluor-ide, 0.1 mg/ml pepstatin and 100 mg/ml leupeptin asdescribed elsewhere (Kubo et al., 1996). The washingextracts were centrifuged at 2000� g during 15minat 4 1C.The supernatant was collected and the debris dis-

carded; the supernatant was then filtered through aMillex membrane (0.45 mm pore diameter) at 4 1C,lyophilized and dissolved in 1ml urea/thiourea buffer(2M thiourea, 7M urea, 4 w/v DTT, 2% v/v carrierampholytes pH 3–10). Protein content was determinedby using the modified method of Bradford (Sedmak andGroosber, 1977).

2.2. D-polyacrylamide gel electrophoresis (PAGE)

2-D PAGE was performed by using 13 cm gels andIPG strips with immobilized pH gradients (IPG_Dalt);for the first dimension (IPG-IEF) IPG gels werecast with pH gradients of 3–10 (Gorg et al., 1999,2000). Samples (25 ml) were applied by cup-loadingclose to the anode and the focusings were performed byusing the following voltage gradient: 500V/1 h, 1000V/1 h and 8000V/2 h permitting the accumulation of16 000Vh. The runnings were performed at 20 1C,under a current of 0.05mA with a maximal power of5.0W. After equilibration of the IPG strips withSDS buffer were applied to vertical SDS-PAGEgels [12.5% (w/v) polyacrylamide and 0.8%(w/v)Bis(N,N0-methylenebisacrylamide)]. The second dimen-sion runnings were performed according to thefollowing program: 5W/gel during 30min and 17W/gel during 5 h, at 10 1C. After the electrophoreticseparation the gels were stained with CoomassieBrilliant Blue (CBB) and stored at room temperature(21 1C).

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2.3. Image acquisition

The 2-D gels were scanned using a transparency modescanner, connected to a PC system, at 24-bit red-green-blue colors and 300 dpi resolution for documentation.Images were analyzed using Image Master 2D Databasesoftware version 4.02 (Amersham Biosciences, Uppsala,Sweden).

2.4. Tryptic digestion

The CBB stained spots were excised and destained for30min using 100 ml acetonitrile (50%) and 25mM(NH4)HCO3 pH 8 (50%); dried for 20min withacetonitrile and incubated with 30 ml of 5mM DTT[in 25mM (NH4)HCO3] for 30min at 60 1C. The gelpieces were incubated for 30min with 30 ml of 55mMiodoacetamide [in 25mM (NH4)HCO3] for 30min atroom temperature, washed two times with 100 ml of25mM (NH4)HCO3 during 20min, followed by dehy-dration with acetonitrile. Finally the spots were dried at401C for 30min using a Speed-Vac system.Trypsin solution was prepared by dissolving 20 mg of

the enzyme in 100 ml 1mM HCl. Before use, 150 ml of5mM (NH4)HCO3 were added to the trypsin solution(final concentration 12.5 ng/ml); 10 ml of this solutionwas pipetted on each dried protein spot and incubatedfor 45min at 0 1C, the supernatant was discarded tominimize auto digestion of trypsin. Then 20 ml of 5mM(NH4)HCO3 were added and the sample was incubatedfor 18 h at 37 1C under soft shaking. To extract thepeptide fragments from the tryptic digests 50 ml of 50%(v/v) acetonitrile [containing 0.5% (v/v) TFA] wereadded and incubated for 30min at room temperature.Thereafter, 50 ml of 50% (v/v) formic acid were addedand incubated for 20min at room temperature. Aftereach step the supernatants were pooled together anddried using a Speed-Vac system.

2.5. Sample preparation

The digested sample was mixed with a matrix solution[10mg/ml a-cyano-4-hydroxycinnamic acid in 50% (v/v)acetonitrile+0.1% (v/v) TFA]; 0.4 ml of this solutionwere pipetted on the MALDI slide sampler and air-dried.

2.6. Mass spectrometry

The masses of the tryptic peptides were determined byusing MALDI-TOF mass spectrometry. Ettan z2 MAL-DI-TOF mass spectrometer (Amersham Biosciences,Uppsala, Sweden) equipped with UV nitrogen laser(334 nm) and harmonic reflectron, was used in thepositive ion mode at 20 kV, adjusted to perform delayedextraction and low mass rejection. Calibration was

performed with peptide samples (angiotensin II, ACTH1-39). Spectra were acquired by accumulation of 600single shots.A switch to fragmentation analysis in MALDI

instrument allowed the sequencing of some trypticpeptides. Tryptic peptides were derivatized with theCAF-kit (Amersham Biosciences) according to themanufacturer instructions. Peaks were selected fromcomplex spectra using the timed ion gate; each pulse ofthe laser provided a Post Source Decay (PSD) spectrumover the entire m/z range, without the need for datastitching. The spectra were acquired using an accelerat-ing potential of 20 kV, and adjusting the pulsed laser to1 shot per second; 300 shots per spectrum wereaccumulated

2.7. Database queries

The sequences of some tryptic peptides from eachdigested protein were used to interrogate the proteinengine search ‘‘Blast of Honeybee Genome’’ at theNational Center for Biotechnology Information (NCBI)home page in Internet (URL:http://www.ncbi.nlm.nih.-gov/genome/seq/AmeBlast.html). The search para-meters were carried out by using the ‘‘Build proteins’’databases and the ‘‘blastp’’ program. A protein wasregarded as identified when the E-value calculated bythe search algorithm was lower than 1� 10�3.

3. Results and discussion

The preliminary protein sequencing by MALDI-TOF-PSD/MS and the access to the honeybee genomedatabase through the protein engine search used in thepresent study revealed suitable for the identification ofseveral proteins from the secretion of the hypophar-yngeal gland of nurse-bees. Fig. 1 shows the 2-Delectrophoresis pattern of proteins CBB stained fromthe hypopharyngeal gland. A total of 61 proteins weredetected after background subtraction, from which 34were identified and represented in the Table 1. Amongthe identified proteins, 27 belong to the MRJPs family, 5proteins were related to the metabolism of carbohy-drates and energy production, 1 protein was related toiron accumulation and 1 protein was identified as aregulator of MRJP-1 oligomerization (Table 1). About27 protein spots were detected but not identified sincetheir analysis resulted in poor spectral quality due to anoise background and/or low protein amount to getreliable sequencing data .Workers perform the most of tasks in the honeybee

hive, except egg laying. The tasks performed byindividual workers are susceptible to age-dependentchanges, known as age polyethism (Moritz andSouthwick, 1992). Thus, nurse bees take care of the

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Fig. 1. 2-D PAGE of the proteins complement of the hypopharyngeal

gland of nurse-bee, IPG 3-10, 12.5% T, showing all the CBB stained

proteins.

K.S. Santos et al. / Insect Biochemistry and Molecular Biology 35 (2005) 85–9188

brood by producing the RJ, while the forager bees(generally older than 10 days post-eclosion) forage fornectar processing it into honey (Ohashi et al., 1997).Apparently, the hypopharyngeal glands seems to exist intwo distinct differentiation states, characterized bydifferent patterns of protein expression as function ofthe age polyethism (Ohashi et al., 1997). Thus, there is aperiod of time during which there is a transition betweenthe roles of nursing and foraging for the workers,causing an overlapping of functions for the organ. Asconsequence, in this period of time the hypopharyngealgland may present an overlapped pattern of proteinsexpression.It was previously demonstrated that the nurse-bee

hypopharyngeal gland is involved with the productionof three major groups of RJ proteins, while thehypopharyngeal gland of the forager bees seem to beinvolved with the production of an a-glucosidase(70 kDa) (Kubo et al., 1996) and selectively it expressesmRNAs for amylase (57 kDa) and glucose oxidase(85 kDa) (Ohashi et al., 1997; Ohashi et al., 1999).Among the identified proteins in the present investi-

gation some aspects are specially important and must beemphasized:Six different forms of MRJP-1 were identified with

MW values changing from 48,810 to 59,995Da and withpI values from 4.23 to 5.50 (spots 21, 23, 24, 36, 38 and41; Fig. 1), as also reported in a previous publicationwhich demonstrated that MRJP-1 may presents differ-ent variant forms with MW around 55,000Da in the pIrange from 4.50 to 5.20 (Hanes and Simuth, 1992).

However, the NCBI protein databank (http://www.ncbi.nlm.nih.gov/) reveals three hypothetical gly-cosylation sites for this protein, which may be used toexplain the observed MW heterogeneity.In the present investigation were identified six

different forms of MRJP-1 in the proteome complementof the hypopharyngeal gland of nurse-bees, while Sato etal. (2004) identified only two different forms of thisprotein in the RJ of Africanized honeybees and oneform in European honeybees. The experimental ap-proach used in the present investigation did not permitto know if the most forms of MRJP-1 are retained,degraded or even metabolically used by the nurse-bees.Eight different forms of MRJP-2 were identified

(spots 25, 26, 27, 28, 29, 31, 32 and 37; Fig. 1) withMW from 50,673 to 59,995Da in the pI range from 4.92to 7.02 in the proteome complement of the secretionfrom the hypopharyngeal gland of nurse-bees, while 15and 12 different forms of this protein were observed inthe RJ from Africanized and European honeybees,respectively (Sato et al., 2004). Bilikova et al, (1999)reported eight isoforms of this protein with MW valuesaround 49 kDa in pI range from 7.5 to 8.5 in RJ fromEuropean honeybees. Exploiting the NCBI proteindatabank (http://www.ncbi.nlm.nih.gov/) is possible toidentify two potential glycosylation sites, suggesting thepossible existence of different forms of MRJP-2,presenting different degrees of glycosylation. It mustbe emphasized that all the forms of this protein observedin the secretion of hypopharyngeal gland were alsopresent in the RJ as described by Sato et al, (2004).Thus, the higher number of forms observed in RJ maybe due to structural changes suffered by this protein,such as proteolysis, glycosylation/deglycosylation oc-curred during RJ storage.Five different forms of MRJP-3 were identified in the

proteome complement of the hypopharyngeal glands ofnurse-bees (spots 4,5,6,7 and 8; Fig. 1), in the MW rangefrom 80,590 to 87,000Da and pI values from 7.05 to8.04 (Fig. 1 and Table 1), while 10 and 24 differentforms of this protein were reported in RJ fromAfricanized and European honeybees, respectively (Satoet al., 2004). Thus, taking into account that all formsidentified in the hypopharyngeal glands were alsopresent in the RJ and considering that the MRJP-3may suffer partial proteolysis during the RJ storage(Kimura et al., 1995), it may be speculated that thisprotein is probably produced by the hypopharyngealgland, secreted into the RJ, in which it may suffer partialdegradation during the storage, originating the exceed-ing number of forms observed in RJ.No form of MRJP-4 was identified in the proteomic

analysis of the secretion of the hypopharyngeal gland,by using the honeybee genome as reference for proteinidentification; however the common sequence of atryptic peptide obtained from the spots 33 and 39 was

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Table 1

Proteins identified in the hypopharyngeal gland of nurse-bee

Spot number Sequences of tryptic fragments used in protein ID Protein identification in HB genome database Accession number E-value

21 VGDGGPLLQPYPDWSFAK Major royal jelly protein 1 XP_393380.1 2� 10�6

23 VGDGGPLLQPYPDWSFAK Major royal jelly protein 1 XP_393380.1 2� 10�6

24 VGDGGPLLQPYPDWSFAK Major royal jelly protein 1 XP_393380.1 2� 10�6

36 VGDGGPLLQPYPDWSFAK Major royal jelly protein 1 XP_393380.1 2� 10�6

38 VGDGGPLLQPYPDWSFAK Major royal jelly protein 1 XP_393380.1 2� 10�6

41 VGDGGPLLQPYPDWSFAK Major royal jelly protein 1 XP_393380.1 2� 10�6

25 SLNVIHEWKYFDYDFGSEER Major royal jelly protein 2 XP_393061.1 2� 10�7

26 SLNVIHEWKYFDYDFGSEER Major royal jelly protein 2 XP_393061.1 2� 10�7

27 SLNVIHEWKYFDYDFGSEER Major royal jelly protein 2 XP_393061.1 2� 10�7

28 SLNVIHEWKYFDYDFGSEER Major royal jelly protein 2 XP_393061.1 2� 10�7

29 SLNVIHEWKYFDYDFGSEER Major royal jelly protein 2 XP_393061.1 2� 10�7

31 SLNVIHEWKYFDYDFGSEER Major royal jelly protein 2 XP_393061.1 2� 10�7

32 SLNVIHEWKYFDYDFGSEER Major royal jelly protein 2 XP_393061.1 2� 10�7

37 SLNVIHEWKYFDYDFGSEER Major royal jelly protein 2 XP_393061.1 2� 10�7

4 WLLLVVCLGIACQDVTSAAVNHQRK Major royal jelly protein 3 XP_391893.1 2� 10�9

5 WLLLVVCLGIACQDVTSAAVNHQRK Major royal jelly protein 3 XP_391893.1 2� 10�9

6 WLLLVVCLGIACQDVTSAAVNHQRK Major royal jelly protein 3 XP_391893.1 2� 10�9

7 WLLLVVCLGIACQDVTSAAVNHQRK Major royal jelly protein 3 XP_391893.1 2� 10�9

8 WLLLVVCLGIACQDVTSAAVNHQRK Major royal jelly protein 3 XP_391893.1 2� 10�9

33 LSSHTLNHNSDKMSDQQENLTLK n.i.a — —

39 LSSHTLNHNSDKMSDQQENLTLK n.i.a — —

9 LANSMNVIHEWKYLDYDFGSDER Major royal jelly protein 5 XP_396121.1 5� 10�9

10 LANSMNVIHEWKYLDYDFGSDER Major royal jelly protein 5 XP_396121.1 5� 10�9

11 LANSMNVIHEWKYLDYDFGSDER Major royal jelly protein 5 XP_396121.1 5� 10�9

40 SSKNLEHSMNVIHEWK Major royal jelly protein 6 XP_393001.1 1� 10�4

35 GDGLIVYQNSDDSFHR Major royal jelly protein 7 XP_393063.1 2� 10�4

42 LWVLDNGISGETSVCPSQIVVFDLK Major royal jelly protein 8 XP_393049.1 2� 10�9

20 PLPENLKEDL IVYQVYPRSF alpha- glucosidase XP_392790.1 1� 10�6

17 GKNLGGTSSHNGMMYTR glucose oxidase XP_392386.1 4� 10�5

30 ANTYNFDYPQVPYTVKNFHPR alpha-amylase XP_391983.1 2� 10�8

50 HFVLAFSRSLARYYNNTGIR alcohol dehydrogenase XP_392596.1 2� 10�6

16 RKNLKPLGQLVSLEMG aldehyde dehydrogenase XP_394614.1 2� 10�3

53 AINDQINFELHASYIYLSMAYYFDR ferritin-like XP_392201.1 2� 10�9

60 NISKIQIEPSFLKAIPNSTKIKMSK apisimin XP_393206.1 3� 10�8

aIn spite this sequence did not permit the identification of the proteins in HB genome database, these sequences were observed in within the

primary sequence of the protein RJP57-2 (in NCBI protein database; accession number Q17061), which is equivalent to MRJP-4.

K.S. Santos et al. / Insect Biochemistry and Molecular Biology 35 (2005) 85–91 89

observed in the C-terminal region of the protein RJP57-2 (in NCBI protein database; accession numberQ17061), which is equivalent to MRJP-4. In the RJ ofAfricanized and European honeybees were observed 5and 2 different forms of this protein, respectively (Satoet al., 2004).Three different forms of MRJP-5 were identified in

the protein complement of the secretion from thehypopharyngeal gland of nurse-bees, presenting MWfrom 79,075 to 79,471Da, and pI values from 6.34 to6.80 (spots 9, 10 and 11; Fig. 1 and Table 1). In the RJfrom Africanized and European honeybees were identi-fied respectively, 7 and 4 different forms of this protein(Sato et al., 2004). All the spots corresponding toMRJP-5 were also detected in the RJ of Africanized

honeybees with similar MW and pI values. In spite tothese multiple forms the honeybee genome has a singlecopy of the gene of MRJP-5, which suggest that thisprotein may be susceptible to post-translational mod-ifications.A single form of the proteins MRJP-6, -7 and -8

(spots 40, 35 and 42, respectively; Fig. 1 and Table 1)were identified in the present proteomic analysis in asingle form. According to Sato et al, (2004) the MRJP-6,-7 and -8 were not observed in the proteome comple-ment of the RJ; however, there are a series of proteinspots still not identified in RJ proteome, which couldcorrespond to these proteins.The second largest group of the identified components

in RJ protein complement are those related to the

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metabolism of carbohydrates. Thus, the proteins identi-fied in this group are:Glucose-oxidase, alpha-glucosidase and alpha-amy-

lase were identified in the spots 17, 20 and 30,respectively (Fig. 1 and Table 1). Sato et al, (2004)identified five different forms of glucose oxidase enzymein the RJ from Africanized honeybees, with MW and pIvalues higher than those observed in the presentinvestigation.Aldehyde dehydrogenase and alcohol dehydrogenase

were identified in the HB genome the spots 16 and 50(Fig. 1 and Table 1). These enzymes are related to theenergetic metabolism, promoting the formation ofreduced coenzymes from their oxidated forms (NAD+

or NADP+).At first these results suggest that the secretion from

hypoharyngeal gland of seven days old nurse-bees maybe starting to produce proteins related to the processingof nectar into honey and also proteins related to thecatabolism of glucose to produce energy—a behaviortypical of workers bees, reflecting the first molecularsigns of the age-dependent role changes. However, stillmust be considered that these enzymes may haveoriginated from the disruption of some cells ofhypoharyngeal glands during the manipulation of thismaterial, representing intracellular proteins (not exo-crine products) which contaminated the samples, beingdetected and identified together the secretion of thegland.A protein ferritin-like was identified in the spot 53

(Fig. 1), presenting pI 4.21 and MW: 24,767Da (Table1). Ferritin is an intracellular molecule that stores iron ina soluble and non-toxic form, readily available for use.The peptide component apisimin was identified in the

spot number 60 (Fig. 1 and Table 1) presenting a MWvalue higher than the expected value from the HBgenome suggesting that it may be produced in aprecursor form. This peptide seems to be involved withthe oligomerization of MRJP-1 (Bilikova et al., 2002).Apparently, this polypeptide may be produced by thehypopharyngeal gland of nurse-bees and secreted intothe RJ, in spite it was not detected in the RJ by Sato etal, (2004); however, these authors also observed a wideheterogeneity of composition from one sample toanother one.Among the 27 not identified proteins, some spots

seem to be characteristic of the hypopharyngeal gland,since apparently they were not detected in the RJ bySato et al, (2004); these proteins correspond to the spots1,2,3, 12,14, 15,18,19 and 22. The proteins of all thesespots generated a series spectral data which apparentlydid not permit their identification, because the amountof material was very reduced, resulting poor MALDI-PSD/MS spectra.In summary, the CBB-stained 2-D gel electrophoresis

demonstrated that the proteome complement of the

secretion from the hypopharyngeal gland of 7 days oldnurse-bees contains at least 61 different proteins, fromwhich 34 were identified through the use of peptidesequencing by MALDI-PSD/MS combined with Blasttool to search for the protein identification in thehoneybee genome. When the identified proteins werecompared to those previously identified in RJ, itbecomes clear that the MRJPs are produced in thehypoharyngeal gland and secreted into the RJ, wheresome of these proteins may suffer glycosylation and/orpartial proteolysis, generating new isoforms of theseproteins in stored RJ. It was also demonstrated thatsome proteins characteristic of the hypopharyngealgland of worker bees are starting to be synthesized atthe seventh day of age, probably as consequence of age-controlled biochemical and physiological changes toprepare the transition from nurse to worker role of thehoneybees.Although the most likeliest candidates in the database

to match the spots were found, these identifications needto be physiologically confirmed in order to clearlyunderstand the functional role of these proteins in queenhoneybee development.

Acknowledgments

This work was supported by the grants from FAPESPand CNPq. M.A.M is Post-Doc fellow from FAPESP(proc. 01/05060-4), KSS was fellow from CNPq. M.S.P.(proc., 300337/2003-50) and O.M. .are researchers of theBrazilian Council for Scientific and TechnologicalDevelopment.

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