Royal jelly (RJ) intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1), MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine β-sitosterol in rats.

Chlorophyll b is one of the major light-harvesting pigments produced by land plants, green algae and several cyanobacterial species. It is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which in higher plants consists of three domains, namely, A, B, and C. We previously demonstrated that the C domain exhibits a catalytic function, whereas the A domain destabilizes the CAO protein in the presence of chlorophyll b, thus regulating the cellular level of CAO. In a previous study, we also presented genetic evidence demonstrating the involvement of Clp protease in the destabilization of CAO. In this study, in order to gain further insight into the regulatory mechanism of CAO, we screened for mutants defective in the control of CAO accumulation. Seeds from an Arabidopsis transgenic plant overexpressing a chimeric protein consisting of the A and B domains of CAO and green fluorescent protein (GFP) were mutagenized by ethyl methane sulfonate. We screened the progenies of the transgenic plants by laser-scanning confocal microscopy, and isolated a total of 66 mutants exhibiting significant GFP fluorescence. By immunoblotting analysis, we confirmed that these mutants accumulated the fusion protein of the N-terminal domains of CAO and GFP at a high level. We further divided these mutants into seven groups by distribution patterns of the fusion protein, and characterized them by pigment and immunoblotting analyses. Based on these analyses, we proposed a model to describe the regulatory mechanism of CAO.

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