Functional and Proteomic Investigations Reveal Major Royal Jelly Protein 1 Associated with Anti-hypertension Activity in Mouse Vascular Smooth Muscle Cells.
ABSTRACT: Vascular smooth muscle cells (VSMCs) are a major cell type of the arterial wall and their functionality is associated with blood pressure regulation. Although royal jelly (RJ) has reported effects on anti-hypertension, the mechanism of blood pressure regulation by major royal jelly protein 1 (MRJP1), the most abundant RJ protein, is still unknown. The mrjp1 gene was inserted into mouse VSMCs to investigate how MRJP1 influences VSMC functionality by functional and proteomic analysis. The expression of MRJP1 in VSMCs significantly reduced cell contraction, migration, and proliferation, suggesting a potential role in decreasing hypertension via action on VSMCs. These anti-hypertension activities were further observed in the changes of the proteome setting of mouse VSMCs. Among 675 different proteins after MRJP1 expression, 646 were down-regulated and significantly enriched in pathways implicated in VSMC contraction and migration, which suggest MRJP1 lowers VSMC contraction and migration by inhibiting muscle filament movement. The down-regulated proteins also enriched pathways in proliferation, indicating that MRJP1 hinders VSMC proliferation by reducing the supply of energy and genetic material. This is the first report integrating MRJP1 into VSMC, revealing the function and mechanism correlated with anti-hypertensive activity. This offers a therapeutic potential to control hypertension by gene-therapy using bee-products.
Project description:Vascular smooth muscle cells (VSMCs) are a major cell type of the arterial wall and their functionality is associated with blood pressure regulation. Although royal jelly (RJ) has reported effects on anti-hypertension, the mechanism of blood pressure regulation by major royal jelly protein 1 (MRJP1), the most abundant RJ protein, is still unknown. Therefore, mrjp1 gene was delivered into mouse VSMCs to investigate how MRJP1 influences the VSMC functionality by functional and proteomic analysis.The data here are the proteomic analysis of triplicated control and MRJP1 expressing VSMCs.
Project description:Major royal jelly protein 1 (MRJP1), designated apalbumin 1, has been regarded as a freshness marker of royal jelly (RJ). A MRJP1-specific peptide (IKEALPHVPIFD) identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody (anti-SP-MRJP1 antibody). Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1 (anti-R-MRJP1 antibody) reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay (ELISA) using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ (0 d). Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage (P<0.0001). Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ.
Project description:Honeybee caste development is nutritionally regulated by royal jelly (RJ). Major royal jelly protein 1 (MRJP1), the most abundant glycoprotein among soluble royal jelly proteins, plays pivotal roles in honeybee nutrition and larvae development, and exhibits broad pharmacological activities in humans. However, its structure has long remained unknown. Herein, we identify and report a 16-molecule architecture of native MRJP1 oligomer containing four MRJP1, four apisimin, and eight unanticipated 24-methylenecholesterol molecules at 2.65?Å resolution. MRJP1 has a unique six-bladed ?-propeller fold with three disulfide bonds, and it interacts with apisimin mainly by hydrophobic interaction. Every four 24-methylenecholesterol molecules are packaged by two MRJP1 and two apisimin molecules. This assembly dimerizes to form an H-shaped MRJP14-apisimin4-24-methylenecholesterol8 complex via apisimin in a conserved and pH-dependent fashion. Our findings offer a structural basis for understanding the pharmacological effects of MRJPs and 24-methylenecholesterol, and provide insights into their unique physiological roles in bees.
Project description:Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue ?-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP14 apisimin4) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel ?-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12 apisimin1) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.
Project description: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.
Project description: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.
Project description:Honey adulteration is becoming increasingly alarming incidents in food safety. Monitoring and detecting adulteration face greater challenges. Honey contains the major royal jelly proteins (MRJP) secreted by bee workers. To detect honey adulteration fast and accurately, a rapid gold sandwich immunochromatographic strip (GSIS) was developed based on two specific polyclonal antibodies (PoAbs) against the MRJP1, the most abundant protein of all MRJPs. We determined the best of pH value (pH 8.6) and PoAb SP-1 amount (5 ?g/mL) in conjunction with colloidal. The cut-off value (sensitivity) of GSIS in detecting MRJP1 is 2.0 ?g/mL in solution. Validation analysis with RJ, milk vetch honey, acacia honey and honey adulteration containing rice syrup and corn syrup with different ratios demonstrated that the GSIS could show consistent Test line (T line) when the test samples contain more than 30% pure honey or MRJP1 0.4 mg/g. The validation results by isotope ratio mass spectrometry on the same pure and all adulteration milk vetch honey samples showed the same information of GSIS test. The qualitative assay GSIS provided a valuable new way for honey authenticity and laid the foundation for the future application of GSIS with monoclonal antibodies in honey authentication.
Project description:Honeybees produce royal jelly (RJ) from their cephalic glands. Royal jelly is a source of nutrition for the queen honey bee throughout its lifespan and is also involved in fertility and longevity. Royal jelly has long been considered beneficial to human health. We recently observed that RJ delayed impairment of motor function during aging, affecting muscle fiber size. However, how RJ affects skeletal muscle metabolism and the functional component of RJ is as of yet unidentified. We demonstrate that feeding mice with RJ daily prevents a decrease in myofiber size following denervation without affecting total muscle weight. RJ did not affect atrophy-related genes but stimulated the expression of myogenesis-related genes, including IGF-1 and IGF receptor. Trans-10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), two major fatty acids contained in RJ. After ingestion, 10H2DA and 10HDAA are metabolized into 2-decenedioic acid (2DA) and sebacic acid (SA) respectively. We found that 10H2DA, 10HDAA, 2DA, and SA all regulated myogenesis of C2C12 cells, murine myoblast cells. These novel findings may be useful for potential preventative and therapeutic applications for muscle atrophy disease included in Sarcopenia, an age-related decline in skeletal muscle mass and strength.
Project description:Migration of vascular smooth muscle cells (VSMCs) is essential for vascular reconstruction in hypertension and several vascular diseases. Our recent study showed that extracellular vesicles derived from vascular adventitial fibroblasts of normal rats inhibited VSMC proliferation by delivering miR155-5p to VSMCs. It is unknown whether miR155-5p inhibits cell migration and oxidative stress in VSMCs of spontaneously hypertensive rats (SHR) and in angiotensin II (Ang II)-treated VSMCs. The purpose of this study was to determine the role of miR155-5p in VSMC migration and its underlying mechanisms. Primary VSMCs were isolated from the aortic media of Wistar-Kyoto rats (WKY) and SHR. Wound healing assay and Boyden chamber assay were used to evaluate VSMC migration. A miR155-5p mimic inhibited, and a miR155-5p inhibitor promoted the migration of VSMC of SHR but had no significant effect on the migration of VSMC of WKY. The miR155-5p mimic inhibited angiotensin-converting enzyme (ACE) mRNA and protein expression in VSMCs. It also reduced superoxide anion production, NAD(P)H oxidase (NOX) activity, as well as NOX2, interleukin-1? (IL-1?), and tumor necrosis factor ? (TNF-?) expression levels in VSMCs of SHR but not in VSMCs of WKY rats. Overexpression of miR155-5p inhibited VSMC migration and superoxide anion and IL-1? production in VSMCs of SHR but had no impact on exogenous Ang II-induced VSMC migration and on superoxide anion and IL-1? production in WKY rats and SHR. These results indicate that miR155-5p inhibits VSMC migration in SHR by suppressing ACE expression and its downstream production of Ang II, superoxide anion, and inflammatory factors. However, miR155-5p had no effects on exogenous Ang II-induced VSMC migration.
Project description:The increased proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in pathophysiological remodeling of arteries during hypertension in pregnancy. However, the mechanisms involved in this process remain unclear. We hypothesized that Neuropeptide Y (NPY), which is a potent mitogenic peptide, participates in modulating proliferation and migration of VSMCs during hypertension in pregnancy. Using pregnant hypertensive rats, induced by intraperitoneal injection of L-nitro-arginine methylester (L-NAME), the plasma concentration of NPY was detected. Open angle, which reflects the non-uniform remodeling with high sensitivity, was used to detect the pathophysiological vascular remodeling in vivo. The results revealed that NPY concentration and artery open angle were both significantly increased in rats with hypertension in pregnant. The underlying mechanism of elevated NPY on vascular remodeling were further analyzed by using cultured VSMCs in vitro. In cultured VSMCs, NPY most effectively stimulated the migration and proliferation of VSMCs at 10-6 mol/L, similar to the plasma concentration in L-NAME hypertension in pregnant rats. NPY up-regulated the expressions of both Y1 and Y5 receptors, increased the phosphorylations of STAT3 on Tyr705 and Ser727 residues, and induced the expression of c-Fos. The NPY-induced VSMCs proliferation was reduced by Y5 receptor antagonist, and fully blocked by combinations with other antagonist, such as Y2+Y5, Y1+Y5, and Y1+Y2+Y5. In contrast, the NPY-induced VSMC migration was blocked by either Y receptor antagonist or any combination of Y receptor antagonists. These results suggest that the elevated plasma concentration of NPY during hypertension in pregnancy may induce VSMC proliferation mainly via Y5 receptor, which subsequently modulate STAT3 and c-Fos signaling pathways to result in the vascular remodeling. These results also suggest that NPY mainly acts on VSMCs in vitro via Y1, Y5 receptors and in vascular tissues in vivo via Y5 receptor.