Phosphoproteome Map of Royal Jelly

In-depth Phosphoproteomic Analysis of Royal Jelly Derived from Western and Eastern Honeybee Species

J Proteome Res, 2014 Sep 29

The protein components of royal jelly (RJ) play a pivotal role in the nutrition, immune defense and cast determination of honeybee larvae. RJ also has a wide range of pharmacological and health-promoting functions for humans. Although the importance of post translational modifications (PTMs) in protein function is known, investigation of protein phosphorylation of RJ proteins is still very limited. To this end, two complementary phosphopeptide enrichment materials (Ti4+-IMAC and TiO2) and high sensitivity mass spectrometer were applied to establish a detailed phosphoproteome map and qualitatively and quantitatively compare the phosphoproteomes of RJ produced by Apis mellifera ligustica (Aml) and Apis cerana cerana (Acc). In total, 16 phosphoproteins carrying 67 phosphorylation sites were identified in RJ derived from western bees, and nine proteins phosphorylated on 71 sites were found in RJ produced by eastern honeybees. Of which, eight phosphorylated proteins were shared in both RJ samples and the same motif ([S-x-E]) was extracted, suggesting the function of major RJ proteins as nutrients and immune agents are evolutionary preserved in both honeybee species.

All eight overlapping phosphoproteins showed significantly higher abundance in Acc-RJ than in Aml-RJ, and the phosphorylated Jelleine-II (an antimicrobial peptide: TPFKLSLHL) at site on T1 in Acc-RJ had stronger antimicrobial properties than the one at site on S6 in Aml-RJ even the overall antimicrobial activity of Jelleine-II was found to decrease after phosphorylation. The differences in phosphosites, peptide abundance, and antimicrobial activity of the phosphorylated RJ proteins indicate that the two major honeybee species employ distinct phosphorylation strategies that align with their different biological characteristics shaped by evolution. The phosphorylation of RJ proteins are supposed to be driven by enzymatic activity of FAM20C-like protein kinase through recognizing [S-x-E] motif, which is supported by the evidence that mRNA and protein expression of FAM20C-like protein kinase are both found in the highest level in hypopharyngeal glands of nurse bees.

Our data represent a first comprehensive RJ phosphorylation atlas, recording patterns of phosphorylated RJ protein abundance and antibacterial activity of some RJ proteins in two major managed honeybee species. These data constitute a firm basis for future research to better understand the biological roles of each RJ protein for honeybee biology and human health care