Origin and function of the major royal jelly proteins of the honeybee (Apis mellifera) as members of the yellow gene family

In the honeybee, Apis mellifera, the queen larvae are fed with a diet exclusively composed of royal jelly (RJ), a secretion of the hypopharyngeal gland of young worker bees that nurse the brood. Up to 15% of RJ is composed of proteins, the nine most abundant of which have been termed major royal jelly proteins (MRJPs). Although it is widely accepted that RJ somehow determines the fate of a female larva and in spite of considerable research efforts, there are surprisingly few studies that address the biochemical characterisation and functions of these MRJPs. Here we review the research on MRJPs not only in honeybees but in hymenopteran insects in general and provide metadata analyses on genome organisation of mrjp genes, corroborating previous reports that MRJPs have important functions for insect development and not just a nutritional value for developing honeybee larvae.     

Antiapoptotic role of major royal jelly protein 8 of honeybee (Apis mellifera) venom

The dominant protein components of honeybee royal jelly (RJ) are major royal jelly proteins (MRJPs), which exhibit various biological properties. However, the biological basis of why bee venom contains MRJPs and what role MRJPs play in bee venom remains to be elucidated. This study reports the antiapoptotic role of MRJP 8 of Apis mellifera venom (AmMRJP 8) in melittin-treated mammalian cells. Recombinant AmMRJP 8 reduced caspase-3 activity and melittin-induced cell apoptosis. Additionally, recombinant AmMRJP 8 decreased the production levels of H₂O₂ and proinflammatory molecules. These results indicate that MRJP in bee venom plays a role in cell protection in bee venom-induced inflammatory responses.     

The Family of Major Royal Jelly Proteins and Its Evolution

A cDNA encoding a new member of the gene family of major royal jelly proteins (MRJPs) from the honeybee, Apis mellifera, was isolated and sequenced. Royal jelly (RJ) is a secretion of the cephalic glands of nurse bees. The origin and biological function of the protein component (12.5%, w/w) of RJ is unknown. We show that the MRJP gene family encodes a group of closely related proteins that share a common evolutionary origin with the yellow protein of Drosophila melanogaster. Yellow protein functions in cuticle pigmentation in D. melanogaster. The MRJPs appear to have evolved a novel nutritional function in the honeybee. Received: 26 September 1998 / Accepted: 28 February 1999     

Proteomics analysis reveals protein expression differences for hypopharyngeal gland activity in the honeybee,Apis mellifera carnica Pollmann

Background

Most of the proteins contained in royal jelly (RJ) are secreted from the hypopharyngeal glands (HG) of young bees. Although generic protein composition of RJ has been investigated, little is known about how age-dependent changes on HG secretion affect RJ composition and their biological consequences. In this study, we identified differentially expressed proteins (DEPs) during HG development by using the isobaric tag for relative and absolute quantification (iTRAQ) labeling technique. This proteomic method increases the potential for new protein discovery by improving the identification of low quantity proteins.

Results

A total of 1282 proteins were identified from five age groups of worker bees, 284 of which were differentially expressed. 43 (15.1%) of the DEPs were identified for the first time. Comparison of samples at day 6, 9, 12, and 16 of development relative to day 3 led to the unambiguous identification of 112, 117, 127, and 127 DEPs, respectively. The majority of these DEPs were up-regulated in the older worker groups, indicating a substantial change in the pattern of proteins expressed after 3 days. DEPs were identified among all the age groups, suggesting that changes in protein expression during HG ontogeny are concomitant with different states of worker development. A total of 649 proteins were mapped to canonical signaling pathways found in the Kyoto Encyclopedia of Genes and Genomes (KEGG), which were preferentially associated with metabolism and biosynthesis of secondary metabolites. More than 10 key high-abundance proteins were involved in signaling pathways related to ribosome function and protein processing in the endoplasmic reticulum. The results were validated by qPCR.

Conclusion

Our approach demonstrates that HG experienced important changes in protein expression during its ontogenic development, which supports the secretion of proteins involved in diverse functions in adult workers beyond its traditional role in royal jelly production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-665) contains supplementary material, which is available to authorized users.     

Identification of major royal jelly proteins in the brain of the honeybee Apis mellifera

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.     

Antibacterial activity of major royal jelly proteins of the honeybee (Apis mellifera) royal jelly

Major royal jelly proteins (MRJPs) are the protein components in royal jelly (RJ). MRJPs 1–7 are detected in the honeybee Apis mellifera RJ. Although A. mellifera MRJP (AmMRJP) 2 exhibited antibacterial activity, the other MRJPs with antimicrobial activities in A. mellifera RJ remains largely unknown. Here, we compared the antibacterial activity of recombinant AmMRJPs 1–7 expressed in baculovirus-infected insect cells. Antibacterial assays of recombinant AmMRJPs 1–7 against the gram-negative bacterium Escherichia coli revealed that AmMRJPs 2–5 and 7 exhibited antibacterial activity, whereas AmMRJPs 1 and 6 displayed almost no antibacterial activity. Consistent with the antibacterial activity of AmMRJPs, AmMRJPs 2–5 and 7 are bound to bacterial cell walls. These results indicated that AmMRJPs 2–5 and 7 contribute directly to the antibacterial property of RJ, suggesting that MRJPs play a role in the antimicrobial property of RJ.     

Towards posttranslational modification proteome of royal jelly

Royal jelly (RJ) is a secretory protein from the hypopharyngeal glands of nurse honeybee workers, which contains a variety of proteins of which major royal jelly proteins (MRJPs) are some of the most important. It plays important roles both for honeybee and human. Each family of MRJP 1-5 displays a string of modified protein spots in the RJ proteome profile, which may be caused by posttranslational modifications (PTMs) of MRJPs. However, information on the RJ PTMs is still limited. Therefore, the PTM status of RJ was identified by using complementary proteome strategies of two-dimensional gel electrophoresis (2-DE), shotgun analysis in combination with high performance liquid chromatography-chip/electrospray ionization quadrupole time-of-flight/tandem mass spectrometry and bioinformatics. Phosphorylation was characterized in MRJP 1, MRJP 2 and apolipophorin-III-like protein for the first time and a new site was localized in venom protein 2 precursor. Methylation and deamidation were also identified in most of the MRJPs. The results indicate that methylation is the most important PTM of MRJPs that triggers the polymorphism of MRJP 1-5 in the RJ proteome. Our data provide a comprehensive catalog of several important PTMs in RJ and add valuable information towards assessing both the biological roles of these PTMs and deciphering the mechanisms underlying the beneficial effects of RJ for human health.     

Honeybee (Apis cerana) major royal jelly protein 4 exhibits antimicrobial activity

Major royal jelly proteins (MRJPs) are important protein components of bee royal jelly (RJ) and exhibit various biological and pharmacological activities. The antimicrobial activities of the royalisin and the jelleines contained within MRJP 1 and MRJP 2 in RJ have been elucidated. However, the antimicrobial effects of other MRJPs remain largely unknown. In this study, we demonstrated the antimicrobial activity of the Asiatic honeybee (Apis cerana) MRJP 4 (AcMRJP4). Recombinant AcMRJP4 was expressed as a 63-kDa protein in baculovirus-infected insect cells. We examined the antimicrobial activity of recombinant AcMRJP4 against bacteria, fungi, and yeast. The mechanisms underlying the antimicrobial activity of AcMRJP4 were assessed using western blot analysis, immunofluorescence staining, and scanning electron microscopy. Recombinant AcMRJP4 bound to the cell walls of bacteria, fungi, and yeast and induced structural damage in the microbial cell walls. AcMRJP4 has an antimicrobial role and exhibits a broad spectrum of antimicrobial activities against bacteria, fungi, and yeast. We demonstrated that AcMRJP4 functions as an antimicrobial agent with activity against bacteria, fungi, and yeast. Together, our data identified a novel function of MRJP 4 as an antimicrobial agent.     

The MRJP/YELLOW protein family of Apis mellifera: identification of new members in the EST library

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.     

蜂王浆主蛋白研究进展

作为社会性昆虫,蜜蜂是研究社会行为和学习记忆的理想模式生物。王浆主蛋白（Major royal jelly protein, MRJP）是蜂王浆中蛋白质的主要成分,该家族一共有9个成员,MRJP1~MRJP9。所有mrjps均以串联排列的形式位于蜜蜂11号染色体上一个大约60 kb的DNA片段上。mrjp的同源体也存在于其他的膜翅目昆虫,均是通过yellow进化而来的。随着不断地进化,MRJPs家族进化出许多重要功能,其中最主要的就是营养功能。本文从MRJPs家族的基因及蛋白质结构、mRNA表达情况、进化和功能等方面进行综述,为今后开展相关研究提供理论支持。     

Antioxidant capacity of major royal jelly proteins of honeybee (Apis mellifera) royal jelly

Major royal jelly proteins (MRJPs) of honeybee royal jelly (RJ) exhibit antimicrobial and antioxidant activities. Although MRJPs of Apis mellifera RJ (AmMRJPs) responsible for antibacterial activity have been identified, AmMRJPs with antioxidant effects remain to be elucidated. Here we identified and compared the antioxidant activities of purified recombinant AmMRJPs 1–7, which are expressed in baculovirus-infected insect cells. Antioxidant assays of recombinant AmMRJPs 1–7 against H₂O₂ revealed that AmMRJPs reduce caspase-3 activity and oxidative stress-induced cell apoptosis and lead to increased cell viability. Consistent with these results, AmMRJPs 1–7 exhibit 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and protect against oxidative DNA damage. These results indicate that AmMRJPs play a role as antioxidants in A. mellifera RJ.     

Major royal jelly protein 2 acts as an antimicrobial agent and antioxidant in royal jelly

Royal jelly (RJ) is a well-known functional and medicinal food for human health promotion. Major royal jelly proteins (MRJPs), which are the major protein components in RJ, exhibit antimicrobial activities. However, the identities of the MRJPs of RJ responsible for its antioxidant effects have remained unclear. Here, we report that honeybee (Apis cerana) MRJP 2 (AcMRJP2) acts as an antimicrobial and antioxidant agent in RJ. Using recombinant AcMRJP2, which was produced in baculovirus-infected insect cells, we established the antimicrobial and antioxidant roles of MRJP 2. AcMRJP2 bound to the surfaces of bacteria, fungi, and yeast, which then induced structural damage in the microbial cell walls and led to a broad spectrum of antimicrobial activities. AcMRJP2 protected mammalian and insect cells via the direct shielding of the cell against oxidative stress, which led to reduced levels of caspase-3 activity and oxidative stress-induced cell apoptosis, followed by increased cell viability. Moreover, AcMRJP2 exhibited DNA protection activity against reactive oxygen species (ROS). Our data indicate that AcMRJP2 could play a crucial role as an antimicrobial agent and antioxidant in RJ, suggesting that MRJP 2 is a component responsible for the antimicrobial and antioxidant activities of RJ.     

Antimicrobial activity of the C-terminal of the major royal jelly protein 4 in a honeybee (Apis cerana)

The protein component of honeybee royal jelly (RJ) is constituted by major royal jelly proteins (MRJPs). The Asiatic honeybee (Apis cerana) MRJP-4 (AcMRJP4) exhibits antimicrobial activities. In this study, we identified the antimicrobial activity of AcMRJP4-15, which is a hydrophilic peptide with 88 amino acid residues in the C-terminal of AcMRJP4 that contains a high content of Asn and positively charged amino acids. Recombinant AcMRJP4-15, which is expressed as a 15-kDa peptide in baculovirus-infected insect cells, induced structural damage to the cell walls of bacteria, fungi, and yeast. Interestingly, the antimicrobial activity of AcMRJP4-15 was greater than that of AcMRJP4, demonstrating that the antimicrobial activity of AcMRJP4 was due in large part to the C-terminal. Our data suggest that AcMRJP4-15 can function as an effective antimicrobial agent.     

王浆主蛋白MRJP7基因在意大利蜜蜂体内的表达

王浆蛋白是蜂王浆生物功能的物质基础,是由王浆蛋白基因家族(mrjps)编码合成的。但部分家族成员如MRJP7在王浆中的含量极少甚至检测不到。基因功能与其在生物体内的时空表达特性相关,为探究mrjp7的生物学功能,本研究利用荧光定量PCR技术对mrjp7在不同发育时期的工蜂和成年工蜂、雄蜂和蜂王的不同组织部位的表达进行定量检测。结果显示mrjp7在成年雄蜂体内的表达水平最低,成年蜂王次之,且在它们的各不同组织部位之间的表达量差异较小。该基因在工蜂幼虫和蛹期的表达同样较低,但在羽化后9日龄前后的哺育蜂王浆腺和头部特异性高表达,这与哺育蜂分泌蜂王浆哺育幼虫和蜂王的功能是相适应的,该结果在转录水平上证实了mrjp7的营养功能,为进一步的研究和应用打下了理论基础。     

More than royal food - <Emphasis Type="Italic">Major royal jelly protein</Emphasis> genes in sexuals and workers of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background

In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee’s genome.

Results

We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals.

Conclusions

The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

     

西方蜜蜂不同级型王浆主蛋白MRJP8基因的表达差异

王浆主蛋白在蜜蜂的级型分化中具有重要的功能。为探究mrjp8在西方蜜蜂Apis mellifera不同级型的表达模式及功能差异。【方法】 利用荧光定量PCR技术对西方蜜蜂工蜂、 雄蜂和蜂王不同发育时期和不同组织的mrjp8表达水平进行检测。【结果】 工蜂体内mrjp8在9日龄前后的毒腺组织内特异性高表达, 为参照基因表达量的上万倍, 在其他发育时期和组织的表达量则明显较低, 其表达具有明显的时空特异性; 在雄蜂体内其表达量与对照相当; 在蜂王体内表达量可达参照的近1 000倍, 没有组织特异性。【结论】 mrjp8的这种表达模式提示其在工蜂防御及维系蜂王长寿命方面有积极作用, 这为进一步研究该基因乃至整个王浆蛋白基因家族的进化和功能分化提供了依据。     

Proteomics analysis of major royal jelly protein changes under different storage conditions

Protein changes in fresh royal jelly (RJ) were compared when stored at -20, 4 degrees C, and room temperature (RT) for 12 months. Protein was partially identified using combinations of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS), gel filtration chromatography, nanoLC MS/MS, and a protein engine identification tool applied to the honeybee genome. Significantly more protein spots were found in fresh (85 spots) and -20 degrees C (81 spots) stored RJ than in samples stored at 4 degrees C (73 spots) and at RT (70 spots) for 1 year. Most identified spots, 56, 57, 51, 46, corresponding to RJ sample of the fresh, -20 degrees C, 4 degrees C, and RT, were assigned to major royal jelly proteins (MRJPs). Marked differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. The quantity of MRJP1 decreased significantly following the temperature trend in all images, but MRJP 2 and -3 did not increase or decrease following the temperature trend, thus, suggesting that MRJP 1-3 are sensitive to temperature. However, MRJP4, 5, glucose oxidase (GOD), peroxiredoxin (PRDX), and glutathione S-transferase (GST) S1 were clearly absent in all images in samples held at RT for 1 year. This indicates that they are the proteins most sensitive to storage temperature and protein markers for freshness of RJ. Combining chromatography and nanoLC MS/MS results, we tentatively conclude that MRJP5 is a reliable freshness marker and that the best way to maintain quality of RJ is under freezing conditions.     

Molecular and biochemical characterization of the major royal jelly protein in bumblebees suggest a non-nutritive function

Honeybee queens are generated on purpose by extensive feeding with a glandular secretion termed royal jelly. Major royal jelly proteins (MRJPs) are the dominant proteinaceous component of royal jelly. One of them, MRJP1, was found to play a central role in honeybee queen development. Genes encoding MRJPs were reported to originate from a single originator, and several of them have evolved nutritive function. Phylogenetic analysis provides evidence that the same originator has multiplied independently in Nasonia and ant lineages. Here we show that bumblebees represent a transition species preserving a single-copy pre-multiplication stage of MRJP evolution. By exploring the single-copy BtRJPL gene, we found striking similarities with MRJPs of the honeybee such as gene structure and expression regulation. At the same time it turned out that BtRJPL does not fulfill criteria for functioning as a nutritive protein. Instead we found evidence that BtRJPL is involved in food digestion or modification, which appears to be the original MRJP function, at least in this lineage. Thus, the evolutionary pattern of MRJPs in hymenopterans constitutes an excellent example of a functional diversification combined with the origin of new properties followed by intensive gene duplication events.