Royal Jelly Proteins Inhibit Macrophage Proliferation: Interactions with Native- and Oxidized-Low Density Lipoprotein.

Macrophage proliferation is known to correlate with macrophage accumulation in atherosclerotic plaque, and therefore its inhibition and secondary reduction of plaque inflammation may have therapeutic beneficial effects on atherosclerosis. Recently, we reported that a peptide corresponding to positions 41-51 of royalisin (which consists of 51 amino acid residues), a potent antibacterial protein contained in royal jelly (RJ), can specifically bind to oxidized LDL (Ox-LDL), a major components of atherosclerotic lesions. Here, we investigated the interaction of RJ proteins including royalisin with LDL and Ox-LDL. Measurement of LDL oxidation by the production of thiobarbituric acid reactive substances and conjugated dienes, and by electrophoretic mobility on polyacrylamide gel electrophoresis showed that RJ proteins including royalisin and the degradation products of major RJ protein (MRJP) 1 and MRJP3 can induce oxidation of LDL and Ox-LDL. Surface plasmon resonance experiments showed that these RJ proteins can exhibit much higher binding affinity to LDL than Ox-LDL (the equilibrium dissociation constant, KD = 8.35 and 49.65 µg proteins/mL for LDL and Ox-LDL, respectively). Experiments using cultured mouse J774A.1 macrophage cells proved that these RJ proteins can inhibit macrophage proliferation markedly and concentration-dependently, regardless of the absence or presence of LDL and Ox-LDL, but hardly affect lipid accumulation in macrophages. These results suggest that RJ proteins including royalisin and degradation products of MRJP1/MRJP3 may have therapeutic beneficial effects on atherosclerosis owing to the reduction of plaque inflammation. Further studies of these RJ proteins may lead to the discovery of novel anti-atherosclerotic drugs.

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee-royalisin-for specific detection of oxidized low-density lipoprotein.

The probes for detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox-LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox-LDL, we investigated the interaction (with ox-LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N-terminus to FITC in the presence of 6-amino-n-caproic acid (AC) linker, (FITC-AC)-royalisin P11, which contains both sequences, Phe-Lys-Asp and Asp-Lys-Tyr, similar to Tyr-Lys-Asp in (FITC)KP6. The (FITC-AC)-royalisin P11 bound with high specificity to ox-LDL in a dose-dependent manner, through the binding to major lipid components in ox-LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC-AC)-shuffled royalisin P11 peptide, in which sequences Phe-Lys-Asp and Asp-Lys-Tyr were modified to Lys-Phe-Asp and Asp-Tyr-Lys, respectively, hardly bound to LDL and ox-LDL. These findings strongly suggest that (FITC-AC)-royalisin P11 may be an effective fluorescent probe for specific detection of ox-LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox-LDL.