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.     

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.     

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.     

Antimicrobial activity of major royal jelly protein 8 and 9 of honeybee (Apis mellifera) venom

Honeybee venom is a complex mixture of toxic components, including major royal jelly protein (MRJP) 8 and 9. MRJP 8 and MRJP 9 are allergens, and MRJP 8 reduces melittin-induced cell apoptosis. However, their functional roles are poorly understood, and their antimicrobial activities have not been determined. In this study, the antimicrobial role of MRJP 8 and MRJP 9 of honeybee (Apis mellifera) venom (AmMRJP 8 and AmMRJP 9) was demonstrated. The presence of AmMRJP 8 and AmMRJP 9 in the secreted venom was observed using antibodies against recombinant AmMRJP 8 and AmMRJP 9 produced in baculovirus-infected insect cells. Recombinant AmMRJP 8 and AmMRJP 9 exhibited an inhibitory activity against microbial serine proteases. Consistent with their inhibitory activity, they induced structural damage by binding to microbial surfaces, resulting in a broad-spectrum antimicrobial activity against bacteria and fungi. They had little effect on hemolysis. Therefore, AmMRJP 8 and AmMRJP 9 could function as antimicrobial agents in honeybee venom.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

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。     

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.     

Major royal jelly protein 3 modulates immune responses in vitro and in vivo

We have recently shown that royal jelly has potent antiallergic properties in a mouse model of immediate hypersensitivity. However, it is still unclear which components of royal jelly exhibit antiallergic activity. In this study, we have screened for antiallergic factors in royal jelly based on inhibition of IL-4 production by anti-CD3 stimulated spleen cells derived from OVA/alum-immunized mice. Using a series of column chromatographies, we purified a 70 kDa glycoprotein, major royal jelly protein 3 (MRJP3), that suppresses IL-4 production. In in vitro experiments, MRJP3 suppressed the production of not only IL-4 but also that of IL-2 and IFN-gamma by T cells concomitant with inhibition of proliferation. The MRJP3-mediated suppression of IL-4 production was also evident when lymph node cells from OVA/alum-immunized mice were stimulated with OVA plus antigen presenting cells. We next examined the purified suppressive factor on OVA/alum-induced allergic responses in mice. Interestingly, in spite of the antigenicity of MRJP3 itself as an extraneous foreign protein, intraperitoneal administration of MRJP3 inhibited serum anti-OVA IgE and IgG1 levels in immunized mice. In addition, heat-treated soluble MRJP3 treatment reduced its antigenicity while maintaining its inhibitory effects on antibody responses to OVA. These results indicate that MRJP3 can exhibit potent immunoregulatory effects in vitro and in vivo. Furthermore, considering the intriguing immunomodulatory effects of MRJP3, it may be of clinical significance to design MRJP3-derived antiallergic peptides by identifying the associated polypeptide regions.     

Proteome comparison of hypopharyngeal gland development between Italian and royal jelly producing worker honeybees (Apis mellifera L.)

The hypopharyngeal gland (HG) of the honeybee (Apis mellifera L.) produces royal jelly (RJ) that is essential to feed and raise broods and queens. A strain of bees (high royal jelly producing bee, RJb) has been selected for its high RJ production, but the mechanisms of its higher yield are not understood. In this study, we compared HG acini size, RJ production, and protein differential expressions between the RJb and nonselected honeybee (Italian bee, ITb) using proteomics in combination with an electron microscopy, Western blot, and quantitative real-time PCR (qRT-PCR). Generally, the HG of both bees showed age-dependent changes in acini sizes and protein expression as worker behaviors changed from brood nursing to nectar ripening, foraging, and storage activities. The electron microscopic analysis revealed that the HG acini diameter of the RJb strain was large and produced 5 times more RJ than the ITb, demonstrating a positive correlation between the yield and HG acini size. In addition, the proteomic analysis showed that RJb significantly upregulated a large group of proteins involved in carbohydrate metabolism and energy production, those involved in protein biosynthesis, development, amino acid metabolism, nucleotide and fatty acid, transporter, protein folding, cytoskeleton, and antioxidation, which coincides with the fact that the HGs of the RJb strain produce more RJ than the ITb strain that is owing to selection pressure. We also observed age-dependent major royal jelly proteins (MRJPs) changing both in form and expressional intensity concurrent with task-switching. In addition to MRJPs, the RJb overexpressed proteins such as enolase and transitional endoplasmic reticulum ATPase, protein biosynthesis, and development proteins compared to the ITb strain to support its large HG growth and RJ secretion. Because of selection pressure, RJb pursued a different strategy of increased RJ production by involving additional proteins compared to its original counterpart ITb. To our knowledge, this morphological and proteomic comparison study on the HG of the two strains of worker honeybees associated with their age-dependent division of labor is the first of its kind. The study provided not only the quantity and quality differences in the HG from the RJb and the ITb, but also addressed the cellular and behavioral biology development question of how the RJb strain can produce RJ more efficiently than its wild type strain (ITb).     

Quantification of Major Royal Jelly Protein 1 in Fresh Royal Jelly by Indirect Enzyme-Linked Immunosorbent Assay

Rabbit polyclonal antibody produced by a major royal jelly protein 1 (MRJP1) specific peptide reacted only with a MRJP1. Indirect ELISA with the antibody revealed a MRJP1 level of 4.12–4.67 g/100 g in different company's royal jelly, which almost agreed with that of a hexametric form of MRJP1 (apisin) measured by high performance liquid chromatography. These results suggest that MRJP1 exists mainly as apisin in royal jelly.     

Heat Resistant Characteristics of Major Royal Jelly Protein 1 (MRJP1) Oligomer

Soluble royal jelly protein is a candidate factor responsible for mammiferous cell proliferation. Major royal jelly protein 1 (MRJP1), which consists of oligomeric and monomeric forms, is an abundant proliferative protein in royal jelly. We previously reported that MRJP1 oligomer has biochemical heat resistance. Therefore, in the present study, we investigated the effects of several heat treatments (56, 65 and 96°C) on the proliferative activity of MRJP1 oligomer. Heat resistance studies showed that the oligomer molecular forms were slightly maintained until 56℃, but the molecular forms were converted to macromolecular heat-aggregated MRJP1 oligomer at 65℃ and 96℃. But, the growth activity of MRJP1 oligomer treated with 96°C was slightly attenuated when compared to unheated MRJP1 oligomer. On the other hand, the cell proliferation activity was preserved until 96℃ by the cell culture analysis of Jurkat cells. In contrast, those of IEC-6 cells were not preserved even at 56°C. The present observations suggest that the bioactive heat-resistance properties were different by the origin of the cells. The cell proliferation analysis showed that MRJP1 oligomer, but not MRJP2 and MRJP3, significantly increased cell numbers, suggesting that MRJP1 oligomer is the predominant proliferation factor for mammiferous cells.