ARTICLE IN PRESS Insect Biochemistry and Molecular Biology Insect Biochemistry and Molecular Biology 35 (2005) 85–91 www.elsevier.com/locate/ibmb

Short communication

Profiling the proteome complement of the secretion from hypopharyngeal 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, a

Center of Study of Social Insects (CEIS) Department of Biology, Institute of Biosciences, Sa˜o Paulo State University (UNESP), 13506900 Rio Claro, SP, Brazil b Institute 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 notable features of the workers in these colonies (Ohashi et al., Corresponding author. Center of Study of Social Insects (CEIS) Department of Biology, Institute of Biosciences, Sa˜o Paulo State University (UNESP), 13506900 Rio Claro, SP, Brazil. Tel.: +55 19 3526 4163; fax: +55 19 3534 8523. E-mail address: [email protected] (M.S. Palma).

0965-1748/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.10.003

1999). Young workers, known as nurse-bees take care of the brood, by synthesizing and secreting many components of the royal jelly (RJ), while the older workers usually forage for nectar, converting it into honey (Robinson, 1987). This process is biologically regulated and known as age polyethism, which is paralleled by physiological changes in certain organs of the worker honeybees (Ohashi et al., 1997). The RJ is believed to be synthesized both by the mandibular and hypopharyngeal glands of nurse-honeybees (Knecht and Kaatz, 1990; Lensky and Rakover, 1983). The hypoharyngeal gland is well developed in the nurse-bees, but shrinks in the older workers, to adapt

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the insect to forage and honey production (Ohashi et al., 1999). Thus, it may be assumed that the gland synthesizes different proteins according to age polyethism (Kubo et al., 1996). Taking into account that the secretion of this gland constitutes an important part of the RJ composition, it is necessary to identify the biochemical contributions of the hypopharyngeal gland to the composition of the RJ, in order to get a better understanding the role of this gland 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 queen honeybees; also it is part of the initial diet of honeybee larvae (Moritz and Southwick, 1992). The protein content represent up to 12% (m/m) of freshly harvested RJ and the major components seem to constitute members of the most known family of proteins of this secretion, the major royal jelly proteins (MRJP) (Knecht and Kaatz, 1990; Lensky and Rakover, 1983); this family of proteins represent about 82% of the total protein content of RJ (Schimitzova´ et al., 1998). The complexity of the protein complement composition of the RJ and the absence of fundamental sequential data about the most of genes involved with the expression of these proteins, make their biochemical and physiological characterizations difficult, which in turn makes the understanding of their functions not fully known. In addition to this, there has been few substantial biochemical analysis of the proteins synthesized by the hypopharyngeal gland. Four major proteins (50, 56, 57 and 64 kDa) were demonstrated to be selectively synthesized by the hypopharyngeal gland and secreted as RJ proteins (Hanes and Simu´th, 1992; Kubo et al., 1996). These results constitute strong evidences that the some proteins found in RJ are in fact products of the hypopharyngeal gland secretion. Recently, the basic composition of the RJ was analyzed by a proteomic approach, which identified the presence of several different forms of MRJP-1 to MRJP-5 and glucose oxidase, presenting heterogeneities in terms of molecular weights and isoelectric points (Sato et al., 2004). This study offered a wide view of the composition of the proteome complement of the RJ, opening the possibility to develop a similar investigation with the hypopharyngeal gland, in order to compare both compositions considering a large number of proteins. Thus, the aim of the present study was to obtain a proteome profile from the secretion of hypopharyngeal gland of nurse-bees (A. mellifera) by using the honeybee (HB) genome databank as reference for proteins identification and to compare the proteins identified with those already described in RJ, in order to get a better understanding about the contribution of this gland 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 the apiary of the Institute of Biosciences of Sa˜o Paulo State University, at Rio Claro, southeast Brazil, were used. Newly emerged workers were marked (day0) on their thorax using paint and introduced into a normal queenright colony; marked nurse bees were collected 7 days later when they were feeding brood. In order to extract proteins of the hypopharyngeal gland secretion the bees were anesthetized by using carbonic gas and the hypopharyngeal glands were dissected under a binocular microscope. The glands (500 glands/ml) were washed in buffered saline solution (10 mM Tris-HCl pH 7.4, containing 13 mM NaCl, 5 mM KCl and 1 mM CaCl2), containing a mixture of protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml pepstatin and 100 mg/ml leupeptin as described elsewhere (Kubo et al., 1996). The washing extracts were centrifuged at 2000
g during 15 min at 4 1C. The supernatant was collected and the debris discarded; the supernatant was then filtered through a Millex membrane (0.45 mm pore diameter) at 4 1C, lyophilized and dissolved in 1 ml urea/thiourea buffer (2 M thiourea, 7 M urea, 4 w/v DTT, 2% v/v carrier ampholytes pH 3–10). Protein content was determined by using the modified method of Bradford (Sedmak and Groosber, 1977).

2.2. D-polyacrylamide gel electrophoresis (PAGE) 2-D PAGE was performed by using 13 cm gels and IPG strips with immobilized pH gradients (IPG_Dalt); for the first dimension (IPG-IEF) IPG gels were cast with pH gradients of 3–10 (Go¨rg et al., 1999, 2000). Samples (25 ml) were applied by cup-loading close to the anode and the focusings were performed by using the following voltage gradient: 500 V/1 h, 1000 V/ 1 h and 8000 V/2 h permitting the accumulation of 16 000 V h. The runnings were performed at 20 1C, under a current of 0.05 mA with a maximal power of 5.0 W. After equilibration of the IPG strips with SDS buffer were applied to vertical SDS-PAGE gels [12.5% (w/v) polyacrylamide and 0.8%(w/v) Bis(N,N0 -methylenebisacrylamide)]. The second dimension runnings were performed according to the following program: 5 W/gel during 30 min and 17 W/ gel during 5 h, at 10 1C. After the electrophoretic separation the gels were stained with Coomassie Brilliant 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 mode scanner, connected to a PC system, at 24-bit red-greenblue colors and 300 dpi resolution for documentation. Images were analyzed using Image Master 2D Database software version 4.02 (Amersham Biosciences, Uppsala, Sweden). 2.4. Tryptic digestion The CBB stained spots were excised and destained for 30 min using 100 ml acetonitrile (50%) and 25 mM (NH4)HCO3 pH 8 (50%); dried for 20 min with acetonitrile and incubated with 30 ml of 5 mM DTT [in 25 mM (NH4)HCO3] for 30 min at 60 1C. The gel pieces were incubated for 30 min with 30 ml of 55 mM iodoacetamide [in 25 mM (NH4)HCO3] for 30 min at room temperature, washed two times with 100 ml of 25 mM (NH4)HCO3 during 20 min, followed by dehydration with acetonitrile. Finally the spots were dried at 401C for 30 min using a Speed-Vac system. Trypsin solution was prepared by dissolving 20 mg of the enzyme in 100 ml 1 mM HCl. Before use, 150 ml of 5 mM (NH4)HCO3 were added to the trypsin solution (final concentration 12.5 ng/ml); 10 ml of this solution was pipetted on each dried protein spot and incubated for 45 min at 0 1C, the supernatant was discarded to minimize auto digestion of trypsin. Then 20 ml of 5 mM (NH4)HCO3 were added and the sample was incubated for 18 h at 37 1C under soft shaking. To extract the peptide fragments from the tryptic digests 50 ml of 50% (v/v) acetonitrile [containing 0.5% (v/v) TFA] were added and incubated for 30 min at room temperature. Thereafter, 50 ml of 50% (v/v) formic acid were added and incubated for 20 min at room temperature. After each step the supernatants were pooled together and dried using a Speed-Vac system. 2.5. Sample preparation The digested sample was mixed with a matrix solution [10 mg/ml a-cyano-4-hydroxycinnamic acid in 50% (v/v) acetonitrile+0.1% (v/v) TFA]; 0.4 ml of this solution were pipetted on the MALDI slide sampler and airdried. 2.6. Mass spectrometry The masses of the tryptic peptides were determined by using MALDI-TOF mass spectrometry. Ettan z2 MALDI-TOF mass spectrometer (Amersham Biosciences, Uppsala, Sweden) equipped with UV nitrogen laser (334 nm) and harmonic reflectron, was used in the positive ion mode at 20 kV, adjusted to perform delayed extraction and low mass rejection. Calibration was

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performed with peptide samples (angiotensin II, ACTH 1-39). Spectra were acquired by accumulation of 600 single shots. A switch to fragmentation analysis in MALDI instrument allowed the sequencing of some tryptic peptides. Tryptic peptides were derivatized with the CAF-kit (Amersham Biosciences) according to the manufacturer instructions. Peaks were selected from complex spectra using the timed ion gate; each pulse of the laser provided a Post Source Decay (PSD) spectrum over the entire m/z range, without the need for data stitching. The spectra were acquired using an accelerating potential of 20 kV, and adjusting the pulsed laser to 1 shot per second; 300 shots per spectrum were accumulated 2.7. Database queries The sequences of some tryptic peptides from each digested protein were used to interrogate the protein engine search ‘‘Blast of Honeybee Genome’’ at the National Center for Biotechnology Information (NCBI) home page in Internet (URL:http://www.ncbi.nlm.nih.gov/genome/seq/AmeBlast.html). The search parameters were carried out by using the ‘‘Build proteins’’ databases and the ‘‘blastp’’ program. A protein was regarded as identified when the E-value calculated by the search algorithm was lower than 1
103.

3. Results and discussion The preliminary protein sequencing by MALDITOF-PSD/MS and the access to the honeybee genome database through the protein engine search used in the present study revealed suitable for the identification of several proteins from the secretion of the hypopharyngeal gland of nurse-bees. Fig. 1 shows the 2-D electrophoresis pattern of proteins CBB stained from the hypopharyngeal gland. A total of 61 proteins were detected after background subtraction, from which 34 were identified and represented in the Table 1. Among the identified proteins, 27 belong to the MRJPs family, 5 proteins were related to the metabolism of carbohydrates and energy production, 1 protein was related to iron accumulation and 1 protein was identified as a regulator of MRJP-1 oligomerization (Table 1). About 27 protein spots were detected but not identified since their analysis resulted in poor spectral quality due to a noise background and/or low protein amount to get reliable sequencing data . Workers perform the most of tasks in the honeybee hive, except egg laying. The tasks performed by individual workers are susceptible to age-dependent changes, known as age polyethism (Moritz and Southwick, 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.

brood by producing the RJ, while the forager bees (generally older than 10 days post-eclosion) forage for nectar processing it into honey (Ohashi et al., 1997). Apparently, the hypopharyngeal glands seems to exist in two distinct differentiation states, characterized by different patterns of protein expression as function of the age polyethism (Ohashi et al., 1997). Thus, there is a period of time during which there is a transition between the roles of nursing and foraging for the workers, causing an overlapping of functions for the organ. As consequence, in this period of time the hypopharyngeal gland may present an overlapped pattern of proteins expression. It was previously demonstrated that the nurse-bee hypopharyngeal gland is involved with the production of three major groups of RJ proteins, while the hypopharyngeal gland of the forager bees seem to be involved with the production of an a-glucosidase (70 kDa) (Kubo et al., 1996) and selectively it expresses mRNAs for amylase (57 kDa) and glucose oxidase (85 kDa) (Ohashi et al., 1997; Ohashi et al., 1999). Among the identified proteins in the present investigation some aspects are specially important and must be emphasized: Six different forms of MRJP-1 were identified with MW values changing from 48,810 to 59,995 Da and with pI values from 4.23 to 5.50 (spots 21, 23, 24, 36, 38 and 41; Fig. 1), as also reported in a previous publication which demonstrated that MRJP-1 may presents different variant forms with MW around 55,000 Da in the pI range from 4.50 to 5.20 (Hanes and Simu´th, 1992).

However, the NCBI protein databank (http:// www.ncbi.nlm.nih.gov/) reveals three hypothetical glycosylation sites for this protein, which may be used to explain the observed MW heterogeneity. In the present investigation were identified six different forms of MRJP-1 in the proteome complement of the hypopharyngeal gland of nurse-bees, while Sato et al. (2004) identified only two different forms of this protein in the RJ of Africanized honeybees and one form in European honeybees. The experimental approach used in the present investigation did not permit to 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) with MW from 50,673 to 59,995 Da in the pI range from 4.92 to 7.02 in the proteome complement of the secretion from the hypopharyngeal gland of nurse-bees, while 15 and 12 different forms of this protein were observed in the RJ from Africanized and European honeybees, respectively (Sato et al., 2004). Bilikova´ et al, (1999) reported eight isoforms of this protein with MW values around 49 kDa in pI range from 7.5 to 8.5 in RJ from European honeybees. Exploiting the NCBI protein databank (http://www.ncbi.nlm.nih.gov/) is possible to identify two potential glycosylation sites, suggesting the possible existence of different forms of MRJP-2, presenting different degrees of glycosylation. It must be emphasized that all the forms of this protein observed in the secretion of hypopharyngeal gland were also present in the RJ as described by Sato et al, (2004). Thus, the higher number of forms observed in RJ may be due to structural changes suffered by this protein, such as proteolysis, glycosylation/deglycosylation occurred during RJ storage. Five different forms of MRJP-3 were identified in the proteome complement of the hypopharyngeal glands of nurse-bees (spots 4,5,6,7 and 8; Fig. 1), in the MW range from 80,590 to 87,000 Da and pI values from 7.05 to 8.04 (Fig. 1 and Table 1), while 10 and 24 different forms of this protein were reported in RJ from Africanized and European honeybees, respectively (Sato et al., 2004). Thus, taking into account that all forms identified in the hypopharyngeal glands were also present in the RJ and considering that the MRJP-3 may suffer partial proteolysis during the RJ storage (Kimura et al., 1995), it may be speculated that this protein is probably produced by the hypopharyngeal gland, secreted into the RJ, in which it may suffer partial degradation during the storage, originating the exceeding 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 protein identification; however the common sequence of a tryptic 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 23 24 36 38

VGDGGPLLQPYPDWSFAK VGDGGPLLQPYPDWSFAK VGDGGPLLQPYPDWSFAK VGDGGPLLQPYPDWSFAK VGDGGPLLQPYPDWSFAK

Major Major Major Major Major

royal royal royal royal royal

jelly jelly jelly jelly jelly

protein protein protein protein protein

1 1 1 1 1

XP_393380.1 XP_393380.1 XP_393380.1 XP_393380.1 XP_393380.1

2
106 2
106 2
106 2
106 2
106

41 25 26 27 28 29 31 32 37

VGDGGPLLQPYPDWSFAK SLNVIHEWKYFDYDFGSEER SLNVIHEWKYFDYDFGSEER SLNVIHEWKYFDYDFGSEER SLNVIHEWKYFDYDFGSEER SLNVIHEWKYFDYDFGSEER SLNVIHEWKYFDYDFGSEER SLNVIHEWKYFDYDFGSEER SLNVIHEWKYFDYDFGSEER

Major Major Major Major Major Major Major Major Major

royal royal royal royal royal royal royal royal royal

jelly jelly jelly jelly jelly jelly jelly jelly jelly

protein protein protein protein protein protein protein protein protein

1 2 2 2 2 2 2 2 2

XP_393380.1 XP_393061.1 XP_393061.1 XP_393061.1 XP_393061.1 XP_393061.1 XP_393061.1 XP_393061.1 XP_393061.1

2
106 2
107 2
107 2
107 2
107 2
107 2
107 2
107 2
107

4 5 6 7

WLLLVVCLGIACQDVTSAAVNHQRK WLLLVVCLGIACQDVTSAAVNHQRK WLLLVVCLGIACQDVTSAAVNHQRK WLLLVVCLGIACQDVTSAAVNHQRK

Major Major Major Major

royal royal royal royal

jelly jelly jelly jelly

protein protein protein protein

3 3 3 3

XP_391893.1 XP_391893.1 XP_391893.1 XP_391893.1

2
109 2
109 2
109 2
109

8 33 39

WLLLVVCLGIACQDVTSAAVNHQRK LSSHTLNHNSDKMSDQQENLTLK LSSHTLNHNSDKMSDQQENLTLK

Major royal jelly protein 3 n.i.a n.i.a

XP_391893.1 — —

2
109 — —

9 10 11

LANSMNVIHEWKYLDYDFGSDER LANSMNVIHEWKYLDYDFGSDER LANSMNVIHEWKYLDYDFGSDER

Major royal jelly protein 5 Major royal jelly protein 5 Major royal jelly protein 5

XP_396121.1 XP_396121.1 XP_396121.1

5
109 5
109 5
109

40

SSKNLEHSMNVIHEWK

Major royal jelly protein 6

XP_393001.1

1
104

35

GDGLIVYQNSDDSFHR

Major royal jelly protein 7

XP_393063.1

2
104

42

LWVLDNGISGETSVCPSQIVVFDLK

Major royal jelly protein 8

XP_393049.1

2
109

20 17 30 50

PLPENLKEDL IVYQVYPRSF GKNLGGTSSHNGMMYTR ANTYNFDYPQVPYTVKNFHPR HFVLAFSRSLARYYNNTGIR

alpha- glucosidase glucose oxidase alpha-amylase alcohol dehydrogenase

XP_392790.1 XP_392386.1 XP_391983.1 XP_392596.1

1
106 4
105 2
108 2
106

16 53 60

RKNLKPLGQLVSLEMG AINDQINFELHASYIYLSMAYYFDR NISKIQIEPSFLKAIPNSTKIKMSK

aldehyde dehydrogenase ferritin-like apisimin

XP_394614.1 XP_392201.1 XP_393206.1

2
103 2
109 3
108

a In 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.

observed in the C-terminal region of the protein RJP572 (in NCBI protein database; accession number Q17061), which is equivalent to MRJP-4. In the RJ of Africanized and European honeybees were observed 5 and 2 different forms of this protein, respectively (Sato et al., 2004). Three different forms of MRJP-5 were identified in the protein complement of the secretion from the hypopharyngeal gland of nurse-bees, presenting MW from 79,075 to 79,471 Da, and pI values from 6.34 to 6.80 (spots 9, 10 and 11; Fig. 1 and Table 1). In the RJ from Africanized and European honeybees were identified respectively, 7 and 4 different forms of this protein (Sato et al., 2004). All the spots corresponding to MRJP-5 were also detected in the RJ of Africanized

honeybees with similar MW and pI values. In spite to these multiple forms the honeybee genome has a single copy of the gene of MRJP-5, which suggest that this protein may be susceptible to post-translational modifications. 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 a single form. According to Sato et al, (2004) the MRJP-6, -7 and -8 were not observed in the proteome complement of the RJ; however, there are a series of protein spots still not identified in RJ proteome, which could correspond 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 identified in this group are: Glucose-oxidase, alpha-glucosidase and alpha-amylase 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 enzyme in the RJ from Africanized honeybees, with MW and pI values higher than those observed in the present investigation. 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 the energetic metabolism, promoting the formation of reduced 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 may be starting to produce proteins related to the processing of nectar into honey and also proteins related to the catabolism of glucose to produce energy—a behavior typical of workers bees, reflecting the first molecular signs of the age-dependent role changes. However, still must be considered that these enzymes may have originated from the disruption of some cells of hypoharyngeal glands during the manipulation of this material, representing intracellular proteins (not exocrine products) which contaminated the samples, being detected and identified together the secretion of the gland. A protein ferritin-like was identified in the spot 53 (Fig. 1), presenting pI 4.21 and MW: 24,767 Da (Table 1). Ferritin is an intracellular molecule that stores iron in a 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 MW value higher than the expected value from the HB genome suggesting that it may be produced in a precursor form. This peptide seems to be involved with the oligomerization of MRJP-1 (Bilikova´ et al., 2002). Apparently, this polypeptide may be produced by the hypopharyngeal gland of nurse-bees and secreted into the RJ, in spite it was not detected in the RJ by Sato et al, (2004); however, these authors also observed a wide heterogeneity of composition from one sample to another 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 by Sato et al, (2004); these proteins correspond to the spots 1,2,3, 12,14, 15,18,19 and 22. The proteins of all these spots generated a series spectral data which apparently did not permit their identification, because the amount of material was very reduced, resulting poor MALDIPSD/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 old nurse-bees contains at least 61 different proteins, from which 34 were identified through the use of peptide sequencing by MALDI-PSD/MS combined with Blast tool to search for the protein identification in the honeybee genome. When the identified proteins were compared to those previously identified in RJ, it becomes clear that the MRJPs are produced in the hypoharyngeal gland and secreted into the RJ, where some of these proteins may suffer glycosylation and/or partial proteolysis, generating new isoforms of these proteins in stored RJ. It was also demonstrated that some proteins characteristic of the hypopharyngeal gland of worker bees are starting to be synthesized at the seventh day of age, probably as consequence of agecontrolled biochemical and physiological changes to prepare the transition from nurse to worker role of the honeybees. Although the most likeliest candidates in the database to match the spots were found, these identifications need to be physiologically confirmed in order to clearly understand the functional role of these proteins in queen honeybee development.

Acknowledgments This work was supported by the grants from FAPESP and 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 the Brazilian Council for Scientific and Technological Development. References Bilikova´, K., Klaudiny, J., Simu´th, J., 1999. Characterization of the basic major royal jelly protein MRJP2 of honeybee (Apis mellifera L.) and its preparation by heterologous expession in. E. coli. Biologia (Bratislava) 54, 733–739. Bilikova´, K., Hanes, J., Nordhoff, E., Saenger, W., Klaudiny, J., Simuth, J., 2002. Apisimin, a new serine-valine-rich peptide from honeybee (Apis mellifera L.) Royal Jelly: purification and molecular characterization. FEBS Lett 528, 125–129. Go¨rg, A., Obermeier, C., Boguth, G., Weiss, W., 1999. Recent developments in two-dimensional gel electrophoresis with immobilized pH gradients: wide pH gradients up to pH 12, longer separation distances and simplified procedures. Electrophoresis 20, 712–717. Go¨rg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., Weiss, W., 2000. The current state of twodimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053. Hanes, J., Simu´th, J., 1992. Identification and partial characterization of the major royal jelly protein of the honey bee (Apis mellifera L.). J. Apic. Res. 31, 22–26. Kimura, M., Washino, N., Yonekura, M., 1995. N-linked sugar chains of 350 kDa royal jelly glycoprotein. Biosci. Biotechnol. Biochem. 59, 507–509.

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