Proteomic Analysis of the Royal Jelly and Characterization of the

Article pubs.acs.org/jpr

Proteomic Analysis of the Royal Jelly and Characterization of the Functions of its Derivation Glands in the Honeybee Toshiyuki Fujita,† Hiroko Kozuka-Hata,‡ Hiroko Ao-Kondo,‡ Takekazu Kunieda,† Masaaki Oyama,‡ and Takeo Kubo*,† †

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan

‡

S Supporting Information *

ABSTRACT: To identify candidate royal jelly (RJ) proteins that might affect the physiologic status of honeybee colony members, we used shotgun proteomics to comprehensively identify the RJ proteome as well as proteomes of the hypopharyngeal gland (HpG), postcerebral gland (PcG), and thoracic gland (TG), from which RJ proteins are assumed to be derived. We identified a total of 38 nonredundant RJ proteins, including 22 putative secretory proteins and Insulinlike growth factor-binding protein complex acid labile subunit. Among them, 9 proteins were newly identified from RJ. Comparison of the RJ proteome with the HpG, PcG, and TG proteomes revealed that 17 of the 22 putative secretory RJ proteins were derived from some of these glands, suggesting that the RJ proteome is a cocktail of proteins from these three glands. Furthermore, pathway analysis suggested that the HpG proteome represents the molecular basis of the extremely high protein-synthesizing ability, whereas the PcG proteome suggests that the PcG functions as a reservoir for the volatile compounds and a primer pheromone. Finally, to further characterize the possible total RJ proteome, we identified putative secretory proteins in the proteomes of these three glands. This will be useful for predicting novel RJ protein components in future studies. KEYWORDS: royal jelly, hypopharyngeal gland, postcerebral gland, thoracic gland, honeybee, polyphenism, age polyethism, division of labor, insulin-signaling

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INTRODUCTION

exocrine glands, whereas old workers (foragers) are engaged in foraging for nectar and pollen outside the colony.3 RJ is a substance rich in both proteins secreted from the hypopharyngeal glands (HpGs) and lipids secreted from the mandibular glands of nurse bees.3 RJ mainly comprises major royal jelly proteins (MRJPs1−9).5,6 MRJPs have been identified and cloned,7−10 and biochemical studies indicate that at least MRJP1−3 are synthesized in the nurse bee HpGs and secreted into the RJ.11 The biologic functions of MRJPs, however, have remained poorly understood until very recently. In addition, the specific content(s) in the RJ responsible for queen differentiation had not been determined until Kamakura demonstrated in 2011 that MRJP1, which he called “royalactin”, is necessary to induce honeybee larvae to develop into queens and to induce at least the morphologic traits of the queens in an artificial rearing condition.12 It remains unclear, however, whether MRJP1 alone is sufficient for honeybee larvae to develop as functional queens or whether other components in the RJ also contribute to queen differentiation.

Animals exist in a world of stimulation that affects their physiologic and behavioral status. In some animal species, environmental stimulation leads to different phenotypes within a species despite having the same genome (polyphenism).1 Nutrients ingested by broods early in development also lead to different developmental trajectories.1,2 Among them, the most well-characterized and drastic example is the caste differentiation of the Hymenopteran insects.3,4 In the honeybee (Apis mellifera L.), sex is genetically determined: males develop from haploid eggs whereas females develop from diploid eggs.3 Females differentiate into two castes, which are not genetically determined: reproductive queens and sterile workers, depending on the nutrients ingested during the early larval stages. Larvae fed rich royal jelly (RJ) in the queen cell grow up as queens, whereas those fed smaller amounts of RJ in the honeycombs grow to be workers.3,4 The workers are engaged in all tasks needed to support the colony except reproduction, and their tasks change according to the age after eclosion (division of labor of workers).3 Young workers (nurse bees) nurse their broods and queens by synthesizing and secreting RJ from © 2012 American Chemical Society

Received: July 27, 2012 Published: November 16, 2012 404

dx.doi.org/10.1021/pr300700e | J. Proteome Res. 2013, 12, 404−411

Journal of Proteome Research

Article

To identify candidate RJ proteins that could affect the physiologic conditions of the other nestmates through trophallaxis (mouth-to-mouth feeding), we previously performed small-scale proteomics of RJ.13 At the same time, we also analyzed the proteomes of the HpGs, postcerebral glands (PcG), and thoracic glands (TG), the latter two of which comprise the salivary glands that open in the worker mouth,14,15 to determine the derivation glands of the RJ proteins.13 We detected an insect growth factor, imaginal disc growth factor 4 (IDGF4) in the RJ, and immunoblotting analysis using anti-IDGF4 antiserum revealed that it is strongly detectable in PcG extract, whereas only moderately detectable in TG extract and very weakly detectable in HpG extracts. On the basis of these findings, we proposed that IDGF4 is secreted from exocrine glands into the RJ, which might affect the physiologic status of the other nestmates.13 Because IDGF4 was newly identified in the RJ as well as in exocrine glands by our proteome analysis, 13,16−21 we hypothesized the existence of other candidate proteins besides royalactin that could affect the physiologic status of the nestmates. We also speculated that comparison of the proteomes of the RJ and the exocrine glands might allow us to predict the whole RJ proteome more precisely. Therefore, in the present study, we performed a comprehensive proteomic analysis of the RJ and its putative derivation glands: HpG, PcG, and TG, using shotgun proteomics. We also applied proteomics analysis to predict the function of these three exocrine glands to gain more insight into the molecular bases underlying honeybee chemical communication.

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Figure 1. Experimental workflow. Proteins extracted from the RJ and the three exocrine glands: HpG, PcG, and TG, were subjected to a shotgun LC−MS/MS.22 (A) Larva dipped in the RJ in a queen cell. White bar indicates 1 mm. Specimens of (B) PcG, (C) TG and (D) HpG stained with hematoxylin-eosin. Bars indicate 300 μm. Positions of these three exocrine glands are indicated in a cartoon depicting honeybee head and thorax (modified from Goodman, L., 2003).14

the preceding studies, we first focused on putative secretory proteins, assuming that most of the RJ proteins are secretory proteins. We thus performed a protein subcellular localization analysis for the previously reported RJ proteomes as well as the 38 RJ proteins identified in the present study using the protein localization prediction program PSORT.16−21,25 The prediction accuracy of the program, WoLF PSORT, for extracellular proteins is estimated to be around 70%.26 We regarded the “extracellular” proteins predicted by PSORT as putative secretory proteins. A total of 20 proteins (approximately 23%) were predicted to be putative secretory RJ proteins among a total of 88 proteins that had been listed in RJ proteomes in the preceding studies.16−21,25 Similarly, 22 proteins (approximately 58%) were predicted to be putative secretory RJ proteins among the 38 proteins identified in the RJ proteome in the present study (Figure 2A, Table 1). Our list of putative secretory RJ proteins, however, lacked 7 proteins, including MRJPs 5, of 20 previously identified putative secretory RJ proteins, and 13 proteins, including MRJPs 1, 2, 4, 6, 7 and 9, were shared by our and the previously reported putative secretory RJ proteomes (Figure 2B, Table 1). Thus, the present study added 9 proteins to the putative secretory RJ proteome besides the previously identified MRJPs (Figure 2B). On the other hand, we identified MRJP3 and MRJP5, which were not predicted as “extracellular” by PSORT, from the total RJ proteome. Therefore, we identified all MRJPs, except MRJP 8 (Supporting Information Table S1), which is consistent with previous findings16−21,25 that MRJP8 was not detected from the RJ, although all of the MRJP members exhibit high sequence similarity.20 The newly added putative secretory RJ proteins were hypothetical protein LOC726323, hypothetical protein LOC100577210, Toll-like receptor 13-like (isoform 1 and/or isoform 2, not unambiguously identified), Chitinase-like protein idgf4-like (IDGF4), venom acid phosphatase Acph-1-like, protein takeout-like, antithrombin-III, serine proteinase stubble (and/or isoform 1, not unambiguously identified), and multiple inositol polyphosphate phosphatase 1-like (Table 2). When the RJ proteins identified in the present study were listed based on the number of spectral counts, which semiquantitatively

RESULTS AND DISCUSSION

Identification of Proteins from RJ and Three Exocrine Glands

To identify the total proteome of the RJ and their derivation glands, and to analyze the possible functions of these glands, we performed shotgun proteomics analyses using a direct nanoflow liquid chromatography−tandem mass spectrometry system (LC−MS/MS)22 for proteins extracted from the RJ, HpG, PcG, and TG (Figure 1). We identified 38, 632, 711, and 833 proteins from the RJ, HpG, PcG, and TG, respectively (Supporting Information Tables S1−S5). The number of proteins identified from the RJ (38) was much smaller than that from the HpG (632), PcG (711), and TG (833), possibly due to the fact that some MRJPs are extremely abundant in the RJ, representing more than 80% of the total RJ proteins.9,23 We then performed two types of analysis for the 38 identified RJ proteins. First, to search for RJ proteins that could affect the physiologic status of the other nestmates and to determine their derivation glands, we performed localization and derivation analyses for the RJ proteins. Next, to characterize the functions of these three exocrine glands, we performed pathway analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.24 Analysis of the RJ Proteome

The RJ proteome has been analyzed in previous studies.8,9,16−21,23,25 The previously reported RJ proteomes, however, sometimes contained proteins that seemed to be contaminants that were introduced during preparation of the RJ proteins (i.e., human proteins)20 or proteins apparently incorrectly registered (i.e., Apis cerana proteins, although the Apis mellifera proteome was examined in the study).20,25 Therefore, for comparison of our RJ proteome with those of 405



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interleukin-1 receptor domain27 (Supporting Information Figure S1A), and are related to both development and immunity (for review, see refs 28 and 29). In the honeybee, five Tolls have been predicted in the genome: Toll, Toll-10, Toll-6, Toll-8, and 18-w.30,31 In mammals, TLR13 is present only in mice.32−34 Thus, we performed a sequence analysis of honeybee TLR13-like using a BLAST search and found that honeybee TLR13-like does not possess the Toll-interleukin-1 receptor domain, indicating that honeybee TLR13-like cannot be characterized as a TLR (Supporting Information Figure S1A). Rather, TLR13-like is thought of as only a secretory protein containing a leucine-rich repeat domain that exhibits high sequence homology with the ant (Acromyrmex echinatior, Camponotus f loridanus, Harpegnathos saltator) proteins (77, 76, and 78% identities, respectively), whose functions are unknown (Supporting Information Figure S1B). We identified Takeout-like from the RJ. Takeout was originally identified as a differentially expressed gene in a circadian clock mutant fly35 and its expression is modulated by feeding/starvation.36 Takeout is specifically expressed in the male head fat body and is involved in male courtship behaviors.37 Takeout belongs to the Takeout/juvenile hormone (JH) binding protein (To/JHBP) super family and, in Drosophila, 14 of the 23 family members exhibit sexually biased expression.38 In the honeybee, eight Takeout/JHBP family members have been identified and the expression of one family member, GB19811, is modulated by age and JH, but not by the circadian clock.39 The expression pattern and function of Takeout-like in the RJ was unknown. If Takeout is ingested by larvae or nestmates, it may affect their physiologic status, including caste differentiation and the division of labor of workers, by modulating JH function, because JH is involved in both caste differentiation and the division of labor of workers. We identified antithrombin-III/serpin-5 from the RJ. Serpins are serine protease inhibitors that have important roles in the

Figure 2. Percentage proportion of the predicted subcellular localization of the identified RJ proteins and overlap of the predicted proteins in the previous and present studies. (A) Pie diagram indicating the functional categorization of the predicted subcellular localization of the identified RJ proteins. Numbers of proteins in each partition are indicated. PSORT localization site categories are as follows: extr, extracellular; cyto, cytosol; nucl, nuclear; ER, endoplasmic reticulum; plas, plasma membrane; mito, mitochondria; pero, peroxisome; cyto_nucl, cytosol and nuclear. (B) The Venn diagram shows the overlap of the proteins identified in the previous and present studies. Left green circle indicates proteins identified in the previous studies and right orange circle indicates proteins identified in the present study, respectively. Yellow circle indicates MRJPs identified from the RJ. Numbers of proteins in each partition are indicated.

represent the relative amount of protein in the corresponding sample, as expected, the amounts of previously identified RJ proteins were much higher than those of newly identified RJ proteins (Table 2). As for IDGF4, we previously reported that IDGF4 is detectable in the RJ by immunoblotting,13 again supporting our notion that IDGF4, a cell growth factor, in the RJ might affect the physiologic status of the other nestmates through trophallaxis. We also identified TLR 13-like from the RJ. Tolls and TLRs are receptors that are characterized by an amino-terminal leucine-rich repeat domain and a carboxyl-terminal Toll-

Table 1. Summary of Proteins Identified by Proteomics from the RJ in the Previous and Present Reportsa accession no.

protein name

Schoenleben et al., 2007

Furusawa et al., 2008

Han et al., 2011

this study

gi|58585164 gi|254910938 gi|110748686 gi|110764266 gi|166795901 gi|48094573 gi|58585098 gi|58585108 gi|58585170 gi|58585188 gi|62198227 gi|67010041 gi|60115688 gi|94158822 gi|66547819 gi|82527239 gi|110762641 gi|66524161 gi|110772962 gi|58585144

alpha-glucosidase precursor defensin-1 preproprotein p. hypothetical protein LOC726446 p. hypothetical protein apolipophorin-III-like protein precursor p. hypothetical protein LOC408608 major royal jelly protein 1 major royal jelly protein 2 precursor major royal jelly protein 4 precursor major royal jelly protein 6 precursor major royal jelly protein 7 precursor major royal jelly protein 9 precursor icarapin-like precursor odorant binding protein 14 precursor p. s.t. major royal jelly protein MRJP5 yellow-h p. s. t. Ferritin 1 heavy chain homologue CG2216-PE, isoform E p. ferritin heavy chain p. glucose dehydrogenase [acceptor]-like, partial alpha-amylase precursor

● ● ● ● ● ● ● ● ● ● ● ● ●

●

● ●

● ●

● ● ● ● ●

● ● ● ● ● ● ●

● ● ● ● ● ● ● ● ● ●

● ● ● ● ● ● ●

●

● ● ●

●

a

Accession no. represents gene numbers in the honeybee genome database. Protein name represents predicted protein name of the gene product. Dots indicate that the proteins were identified in the corresponding reports. Proteins identified in the present study are in bold. Data are cited from Schoenleben et al. (2007),18 Furusawa et al. (2008),20 and Han et al. (2011),25 respectively. p., putative; s.t., similar to. 406



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such as odorant binding protein 14 and icarapin-like precursor, were previously reported.18,20,25 Besides the putative secretory RJ proteins, we rechecked the remaining 16 proteins whose localization was not predicted to be secretory (Figure 2A, Supporting Information Table S1), because the prediction accuracy for extracellular proteins of the program, WoLF PSORT, is around 70%.26 Among them, we focused on two proteins whose functions are relatively wellcharacterized. One is insulin-like growth factor-binding protein complex acid labile subunit-like (IGFBP-ALS). It is possible that WoLF PSORT missed this protein because IGFBP-ALS is a binding partner of a well-characterized secretory protein, IGFBP.42 The insulin signaling pathway plays a key role in metabolism, growth, reproduction, and aging in insects.43 Especially, in the honeybee, insulin signaling has an important role in queen-specific longevity,44 worker division of labor,45 and caste determination.46,47 The honeybee genome encodes two insulin-like peptides (AmILP-1 and AmILP-2) and they have different expression patterns during the larval stages between queen-destined and worker-destined larvae,46,47 and their patterns show nutritional sensitivity.46 In addition, RJ exhibits insulin bioactivity and insulin immunoreactivity.48−50 In mammals, IGFBP-ALS regulates insulin-like growth factor (IGF) signaling by binding to IGFBPs.42 Therefore, IGFBPALS is one of the most promising candidate RJ proteins that might affect caste determination if ingested by the larvae. Finally, we also focused on mushroom body large-type Kenyon cell-specific protein 1 (Mblk-1), which is a transcription factor that was identified in the search for genes expressed preferentially in the mushroom bodies, a higher cognitive center, in the honeybee brain.51−53 In Drosophila, the mblk-1 homologue, E93 was originally identified as an ecdysone responsive gene,54 and then characterized as a key determinant for both pupal cell death and adult structure patternings during metamorphosis.55,56 Transcription factors such as HIV Tat transactivator and Drosophila melanogaster Antennapedia can translocate across the cell membrane (reviewed in ref 57). Antennapedia has the helix-turn-helix (HTH) type DNA binding domain. Mblk-1 also belongs to HTH type. Thus, it might be that Mblk-1 secreted into the RJ translocates between larval organs through ingestion and affects larval development.

Table 2. List of Putative Secretory RJ Proteins and Their Derivation Glandsa spectral count accession no. gi|58585098 gi|58585108 gi|62198227 gi|58585188 gi|58585170 gi|110772962 gi|58585164 gi|67010041 gi|48094573 gi|166795901 gi|60115688 gi|328784821 gi|110763647 gi|254910938 gi|66514614 gi|328782084 gi|94158822 gi|110755367 (gi| 328787378) gi|328790726 gi|110766389 gi|328776927 (gi| 328776929) gi|328777682

protein name

RJ

HpG

PcG

TG

major royal jelly protein 1 major royal jelly protein 2 precursor major royal jelly protein 7 major royal jelly protein 6 major royal jelly protein 4 precursor p. glucose dehydrogenase [acceptor]-like, partial alpha-glucosidase precursor major royal jelly protein 9 p. hypothetical protein LOC408608 apolipophorin-III-like protein icarapin-like precursor p. hypothetical protein LOC100577210 p. hypothetical protein LOC726323 defensin-1 preproprotein p. Chitinase-like protein Idgf4-like p. antithrombin-III odorant binding protein 14 p. toll-like receptor 13like isoform 1a (p. toll-like receptor 13like isoform 2)b p. venom acid phosphatase Acph-1like p. protein takeout-like p. serine proteinase stubblec (p. serine proteinase stubble isoform 1)d p. multiple inositol polyphosphate phosphatase 1-like

1297 766

298 134

23 16

− −

247 105 91

59 16 31

− − 1

− − −

50

50

−

−

17

−

−

−

15 14

10 −

− 2

− 45

10

−

−

−

8 5

2 2

2 −

2 −

4

1

−

−

4 2

3 −

− 11

− 3

2 1

− −

1 2

− 1

1

−

5

−

(1)

(−)

(5)

(−)

1

−

−

−

1 1

− −

− −

− 1

1

(−)

(−)

(1)

1

−

−

−

a

Analysis of Derivation Glands of Secretory RJ Proteins

control of innate immunity and development (reviewed in ref 40). We also identified serine proteinase stubble/serine proteinase stubble isoform 1/serine protease homologue (SPH) 42 from the RJ. SPHs have roles in defense responses and SPH42 has also been identified in the honeybee.41 Therefore, in addition to the possibility that they affect development or immunity when ingested by the larvae, they might also contribute to the antiseptic effects of RJ. Although we also identified five novel proteins from the RJ, the functions of these proteins in the RJ remain unknown. Some proteins,

Next, to search for derivation glands of the RJ proteins as well as to analyze the function of the glands, we identified the proteomes of HpG, PcG, and TG. We then examined whether the 38 putative secretory RJ proteins are included in some of these three exocrine gland proteomes. Among the 22 putative secretory RJ proteins, 11, 9, and 5 were present in the HpG, PcG, and TG, respectively (Table 2). All eight MRJPs were present in the HpG proteome, consistent with previous reports.11 MRJPs 1, 2, and 4, however, were also present in the PcG proteome, suggesting that they are also synthesized in the PcG. In contrast, no MRJP was detected in the TG proteome, suggesting that the TG does not synthesize MRJPs. Although glucose dehydrogenase-like was present in the HpG proteome, which is consistent with our previous report,58 alphaglucosidase precursor was not detected in the HpG, although we previously purified this protein from the HpG homogenate.11 This indicates that an alpha-glucosidase precursor was accidentally missed in the HpG proteome. IDGF4 was present in both PcG and TG proteomes, which is also consistent with our previous report.13

Accession no. represents gene numbers in the honeybee genome database. Protein name represents predicted protein name of the gene product. Proteins newly identified from the RJ proteome in the present study are in bold. Spectral counts indicate that the peptide numbers of the proteins were identified in the RJ and corresponding gland proteomes, and “−” indicates that the proteins were not identified. Amino acid sequences of proteins labeled with a and b, and proteins labeled with c and d, are very similar to each other, and thus they were not unambiguously identified in this proteomic analysis. p., predicted; s.t., similar to.

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Figure 3. Functional analysis of the three glands by identified proteins from the HpG, PcG, and TG. (A) Venn diagram showing that 137, 138, and 213 proteins were specifically identified from the HpG (orange circle), PcG (Blue circle), and TG (red circle), respectively. Numbers of proteins in each partition are indicated. (B) Comparison of the KEGG pathway categories of proteins specifically identified from the HpG, PcG, and TG, respectively. (C) Comparison of the KEGG pathway subcategories of proteins specifically identified from the HpG, PcG, and TG, and categorized as “metabolism” in panels (B).

Functional Analysis of the Three Exocrine Glands

In addition, hypothetical protein LOC726323, hypothetical protein LOC100577210, and defensine-1 preproprotein were detected only in the HpG proteome, whereas TLR13-like and antithrombin-III were detected only in the PcG proteome, and serine protease stubble/Serine proteinase stubble isoform 1 was detected only in the TG proteome. In contrast, icarapin-like precursor was detected in all of the HpG, PcG, and TG proteomes, and hypothetical protein LOC408608, IDGF4, and odorant binging protein 14 were detected in both the PcG and TG proteomes. These results strongly suggest that RJ is a cocktail of secretory proteins synthesized and secreted from these three exocrine glands and that these three glands have distinct functions. In contrast, major larval or adult hemolymph proteins, such as hexamerins in larval hemolymph or vitellogenin in adult hemolymph,59 were not detected in the present RJ proteomes, strongly arguing against the possibility that the RJ proteome represents honeybee hemolymph proteins, that might have contaminated the RJ during collection of the RJ. The results of semiquantitative analyses of proteins based on spectral counting also supported the above notion: while the amounts of most of the RJ proteins, including MRJPs, were higher in the HpG than in the PcG and TG, the amounts of the hypothetical protein LOC 408608 and IDGF4 were much higher in the TG and PcG, respectively, than in the HpG (Table 2). These results strongly suggested that, while the HpGs mainly synthesize and secrete most of the RJ proteins, hypothetical protein LOC 408608 and IDGF4 in the RJ mainly derived from the TG and PcG, respectively. The possibility that some of the RJ proteins derive from unknown or unexamined organs (i.e., gut or mandibular glands), however, cannot be excluded.

HpG is well characterized as an exocrine gland that produces MRJPs:11,58,60−64 the HpGs are well-developed to produce MRJPs in nurse bees, while they are shrunken and produce carbohydrate-metabolizing enzymes such as alpha-glucosidase and glucose oxidase in worker bees. In contrast, the functions of PcG and TG are poorly understood. PcG contains some volatile compounds that honeybees use for colony memberrecognition,65 and a primer pheromone, ethyl oleate (EO) for adult behavioral maturation,66 suggesting that the PcG functions as a reservoir for volatile compounds and a primer pheromone. Our previous proteomic analysis of the PcG and TG revealed that both aldolase, an enzyme involved in glycolysis, and acetyl-CoA acyltransferase 2, ACAA2, an enzyme involved in lipid metabolism, are expressed most strongly in the PcG, among the HpG, PcG, and TG.13 In addition, both enzymes are more strongly expressed in the forager PcG than the nurse bee PcG, suggesting that EO synthesis is enhanced in the forager PcG.13 Because glycolysis and lipid metabolism are closely related to oleic acid synthesis, we previously suggested that EO might be synthesized in the PcG.13 In the present study, to more precisely examine the functions of PcG and TG, we analyzed their proteomes in detail. First, we analyzed the overlap of proteins identified from the three exocrine glands. We detected 137, 138, and 213 proteins from only the HpG, PcG, and TG proteomes, respectively, which implies that they might be involved in molecular processes specific to each gland (Figure 3A). Therefore, we performed a pathway analysis using the KEGG database24 for these specifically detected proteins. Using the KEGG Mapper (http://www.genome.jp/keg/tool/map_pathway2.html) with NCBI-gi as queries, we mapped 75 among 137 queries from the HpG proteome, 45 among 138 from the PcG proteome, 408



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and 90 among 213 from the TG proteome (Supporting Information Figure S2A, Table S6). The largest part of the proteins detected only in the HpG was categorized as “genetic information processing” (46%) (Figure 3B, Supporting Information Figure S2A, Table S6A), consistent with the fact that the HpG synthesizes a large quantity of MRJPs and carbohydrate-metabolizing enzymes. In contrast, the largest parts of proteins detected only in the PcG and TG were characterized as “metabolism” (60% and 61% for the PcG and TG, respectively) (Figure 3B, Supporting Information Figure S2A). Proteins categorized as “Environmental information processing” were only prominent for the TG (Figure 3B, Supporting Information Figure S2A Table S6C). They are related to ABC transporters (multidrug resistance protein homologue 49-like) and signal transductions such as Wnt signaling (sgg, Camkii, calcyclin-binding protein-like, and dally), mTOR signaling (Rheb and SNF1A), and Hedgehog signaling (sgg), suggesting that TG must communicate with the other organs to exert its functions. Next, to gain more insight into the functions of these three exocrine glands, we further mapped the proteins that were detected only in each gland and further categorized “metabolism” into more specialized categories. The largest part was categorized as combined category of “carbohydrate, lipid and amino acids metabolism” (68%) for the PcG, which was larger than those (58 and 49%) for HpG and TG, respectively (Figure 3C, Supporting Information Figure S2B, Table S6B), consistent with our notion that metabolism of the carbohydrate pheromone, EO, is enhanced in the PcG. In contrast, the category “energy metabolism” (29%) was most prominent in the TG among the three exocrine glands (Figure 3C, Supporting Information S2B, Table S6C), suggesting that honeybees require high TG activity throughout their lives. It is thought that TG has important function for honeybees, but the specific functions of the TG remain unknown. The category “nucleotide metabolism” (17%) was most prominent for the HpG (Figure 3C, Supporting Information Figure S2B, Table S6A), again consistent with the fact that the HpG is specialized for the production of MRJPs and carbohydrate-metabolizing enzymes. The results of semiquantitative analyses of glandspecific proteins based on spectral counting, in which the proteins were categorized based on the multiplication of the number of protein species detected and the number of spectral counts of the protein, also supported the functional characterizations of the three exocrine glands (Supporting Information Figure S3). Finally, to further characterize the possible RJ total proteome, we also performed a PSORT analysis for the proteomes of these three exocrine glands. Similar to identified proteins from the RJ, we regarded the “extr” proteins expected by PSORT as putative secretory proteins. We found 59, 72, and 63 putative secretory proteins in the proteomes of these three exocrine glands (Figure 4). By subtracting overlapping proteins from the three exocrine glands, 119 nonredundant proteins were predicted as putative secretory proteins from these three exocrine glands (Supporting Information Table S7). It is plausible that some of these proteins are also secreted into the RJ as the components of the RJ proteome. Future research will be important to examine whether the putative secretory RJ or exocrine gland proteins identified in the present study are actually actively secreted from the corresponding glands, and whether the putative secretory RJ proteins are ingested from

Figure 4. Overlap of the putative secretory proteins identified from the HpG, PcG, and TG. Venn diagram showing that 59, 72, and 63 proteins were identified specifically from the HpG (orange circle), PcG (blue circle), and TG (red circle), respectively. Numbers of proteins in each partition are indicated. Numbers in parentheses in each partition indicate the number of putative secretory RJ proteins identified in the present study.

the gut into the larval and/or nestmates’ bodies to affect their physiologic status.

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MATERIALS AND METHODS

Animals and Sample Preparation

European honeybees (A. mellifera L.) were purchased from a local dealer (Kumagai Youhou, Saitama, Japan) and maintained at the University of Tokyo (Hongo, Bunkyo-ku, Tokyo). Nurse bees and foragers were collected according to their behaviors and hypopharyngeal gland developmental statuses, as described previously.13 PcGs and TGs dissected from 5−10 workers (mixture of nurse bees and foragers) and HpGs dissected from 5 nurse bees were homogenized in a lysis solution comprising 8 M urea, 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1propanesulfonate, 0.28% dithiothreitol, and 0.5% IPG buffer (pH 3−10, GE Healthcare Bioscience), followed by centrifugation. The supernatants were then subjected to precipitation with 10% trichloroacetic acid, the precipitants were dissolved in rehydration solution comprising 8 M urea and 2 M Tris-HCl, pH 8.2. The protein amounts were then determined using the BCA method (BCA Protein Assay Kit, Pierce Chemical). Raw RJ was diluted with distilled water, subjected to trichloroacetic acid precipitation, dissolved in rehydration solution, and the BCA protein was quantified as described above. Mass Spectrometric Analysis

All four of the above protein extraction solutions were subjected to shotgun proteomics using a direct nanoflow LC−MS/MS as described previously.22 Briefly, the solutions were digested with trypsin, desalted using of a C18 device (ZipTip, Millipore), concentrated, and injected into a direct nanoflow liquid chromatography system (DiNa, KYA Technologies) coupled to a quadruple time-of-flight tandem mass spectrometer (QSTAR Elite, AB SCIEX) as described previously.22 After applying the peptide mixture to a C18 column (800 μm inner diameter × 3 mm long), reversedphase separation of the captured peptides was performed on a column (150 μm inner diameter × 75 mm long) filled with C18 (3 μm particles, 120 Å pore, HiQ sil, KYA Technologies) using DiNa. The peptides were eluted with a linear 5% to 65% gradient of acetonitrile containing 0.1% formic acid over 120 min at a flow rate of 200 nL/min and sprayed into the QSTAR Elite. Protein Identification

The acquired MS/MS signals were searched against the RefSeq downloaded at Jun 2, 2011 through the Mascot algorithm 409



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(version 2.3.02, Matrix Science). The parameters used were as follows: fixed modifications: Carbamidomethylation (cysteine); variable modifications: oxidation (methionine), acetylation (protein N-term) and pyro-glutamination (N-terminal glutamine); maximum missed cleavages: 2; peptide mass tolerance: 200 ppm; MS/MS tolerance: 0.5 Da; as described by Oyama et al.22 The criterion for protein identification was based on at least one MS/MS determination with Mascot scores that exceeded the statistical threshold (p < 0.01). A randomized decoy database created by a Mascot Perl program estimated a false discovery rate (FDR) of 0.66% for all of the identified peptides. Data Analysis

The identified protein lists were attached the whole length of peptide sequences from RefSeq (Jun 2, 2011) based on the accession numbers. The peptide sequences were then applied to the protein subcellular localization analysis tool, WoLF PSORT (http://wolfpsort.org/).26 A pathway analysis was performed using the KEGG Mapper tool (http://www. genome.jp/kegg/tool/map_pathway2.html).24

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-3-5841-4448. Fax: +81-3-5841-4447. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.F. is the recipient of a Grant-in-Aid from Japan Society for the Promotion of Science for young scientists. REFERENCES

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Proteomic Analysis of the Royal Jelly and Characterization of the

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