Identification of a novel hypocholesterolemic protein, major royal jelly protein 1, derived from royal jelly.

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

Elution profile of RJ protein using cholic acid-conjugated EAH Sepharose 4B column chromatography and 10% SDS-PAGE patterns of bile acid-binding proteins derived from RJ.

<p>(A) Elution profile of RJ protein using cholic acid-conjugated EAH Sepharose 4B column chromatography. Twenty-five milliliters of RJ protein (10-kDa cut-off RJ) solution (118 mg protein) in 0.02% NaN₃ containing 10 mM Tris-HCl (pH 8.0) were applied to the column and washed with (a) 0.5 M NaCl containing 10 mM Tris-HCl buffer (pH 8.0), (b) 0.5% sodium deoxycholate containing 10 mM Tris-HCl buffer (pH 8.0), and (c) 8 M urea containing 10 mM Tris-HCl buffer (pH 8.0). (B) 10% SDS-PAGE patterns of bile acid-binding proteins derived from RJ by cholic acid-conjugated column chromatography. Lane 1, protein standard; lane 2, bile acid-binding proteins eluted with 0.5% sodium deoxycholate from the column. The amount of applied protein in lane 2 was 4.2 µg. The bile acid-binding proteins consist of MRJP1, MRJP2, and MRJP3.</p

Effects of dietary casein or MRJP1 on body and relative liver weights, food intake, serum and liver lipids, fecal steroid excretion in rats¹.

1<p>Values are means ± SEM, n = 10. Asterisks indicate different from casein (*P<0.05, **P<0.01, ***P<0.001).</p>2<p>Values were calculated as follows: LDL + VLDL cholesterol  =  Total cholesterol - HDL cholesterol.</p>3<p>Atherogenic index calculated by Serum Total cholesterol/Serum HDL cholesterol.</p>4<p>Total steroids  =  neutral steroids + acidic steroids.</p

Effects of MRJP1 or Casein on rat liver CYP7A1 protein level by Western blot analysis.

<p>Total protein extracts from rat liver by MRJP1 or casein treatment for 7 days and used for Western blot analysis. Values are means, with their standard errors represented by vertical bars (<i>n</i> 4 per group).</p

Effects of dietary casein, MRJP¹ or β-sitosterol on body and relative liver weights, food intake, serum choleseterol levels in rats.

1<p>Values are means ± SEM, n = 8. Within a row, means with different superscript letters are significantly different (P<0.05) by Tukey's test.</p

Typical elution profiles of RJ proteins by HPLC and 15% SDS-PAGE patterns of the isolated RJ proteins.

<p>(A) Typical elution profile of RJ protein by HPLC. Elution profile of RJ protein (10-kDa cut-off RJ) by size-exclusion chromatography using a HiLoad 26/60 Superdex 200 p.g. column. Thirteen milliliters of 10-kDa cut-off RJ solution (182 mg protein) in 150 mM NaCl containing 20 mM phosphate (Na₂HPO₄/NaH₂PO₄) buffer (pH 7.5) were applied to the column. The molecular weights of the eluted proteins were calibrated using standard proteins, as follows. Peak A, 515 kDa; peak B, 290 kDa; peak C, 157 kDa; peak D, 79 kDa; peak E, 55 kDa; peak F, 5 kDa. (B) 15% SDS-PAGE patterns of peak B and peak E. Lane 1, molecular weight standards; lane 2, protein containing peak B from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(A)</a>; lane 3, protein containing peak E from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(A)</a>. The amount of applied protein in lanes 2 and 3 was 5 µg each. The protein contained in peak B from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(A)</a> was detected as a 55-kDa protein. The protein contained in peak E of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(A)</a> was detected as 2 major protein bands (55 kDa and 49 kDa)respectively. (C) Elution profile of peak E by anion exchange chromatography using a HiPrep QFF 16/10 column. Five millilters of peak E protein solution (125 mg protein) in 20 mM Tris-HCl (pH 8.0) was applied to the column. (D) 15% SDS-PAGE pattern of proteins derived from anion exchange chromatography. Lane 1, molecular weight standards; lane 2, protein containing the peak E1 and E2 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(C)</a>. The amount of applied protein in lane 2 was 8 µg.</p

Effects of MRJP1 or casein on cholesterol absorption in Caco-2 cells.

<p>Values are means, with their standard errors represented by vertical bars (<i>n</i> 6 per group). Asterisks indicate different from Casein (*P<0.05) by Student's <i>t</i>-test.</p

Effects of MRJP1, casein or their tryptic hydrolysates on hepatic mRNA levels related to cholesterol metabolism in rats or HepG2 cells.

<p>(A) Effects of oral administration of MRJP1 or casein on hepatic mRNA levels related to cholesterol metabolism in rats. Values are means, with their standard errors represented by vertical bars (<i>n</i> 10 per group). Asterisks indicate different from CTH (*P<0.05) by Student's <i>t</i>-test. (B) Effects of MTH or CTH on the mRNA levels mRNA levels related to cholesterol metabolism in HepG2 cells. HepG2 cells were treated with MTH (1 mg/ml) or CTH (1 mg/ml) for 24 h. Total RNA was prepared from the treated cells and used for quantitative real-time PCR analyses. Values are means, with their standard errors represented by vertical bars (<i>n</i> 3 per group). Asterisks indicate different from CTH (*P<0.05, **P<0.01) by Student's <i>t</i>-test.</p

Chemical compositions of casein, FDRJ and MRJP1.

1<p>Casein (Meiji Dairy Corporation).</p>2<p>10-HDA; 10-hydroxy-2-decenoic acid.</p><p>This is included in lipids.</p>3<p>69.3 g lipids/kg contains 0.53 g polyphenols/kg.</p>4<p>0.2 g lipids/kg contains 0.06 mgpolyphenols/kg.</p

Effects of MTH or CTH on CYP7A1 protein level in HepG2 cells by Western blot analysis.

<p>HepG2 cells were treated with MTH (1 mg/ml) or CTH (1 mg/ml) for 48 h. Protein was prepared from the treated cells and used for Western blot analyses. Values are means, with their standard errors represented by vertical bars (<i>n</i> 4 per group). Asterisks indicate different from CTH (*P<0.05) by Student's <i>t</i>-test.</p