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

Abstract

The proteins in royal jelly (RJ) play a pivotal role in the nutrition, immune defense, and cast determination of honeybee larvae and have a wide range of pharmacological and health-promoting functions for humans as well. 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 (Ti(4+)-IMAC and TiO2) and high-sensitivity mass spectrometry were applied to establish a detailed phosphoproteome map and to 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 common to both RJ samples, and the same motif ([S-x-E]) was extracted, suggesting that the function of major RJ proteins as nutrients and immune agents is evolutionary preserved in both of these honeybee species. All eight overlapping phosphoproteins showed significantly higher abundance in Acc-RJ than in Aml-RJ, and the phosphorylation of Jelleine-II (an antimicrobial peptide, TPFKLSLHL) at S(6) in Acc-RJ had stronger antimicrobial properties than that at T(1) in Aml-RJ even though 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 potentially driven by the activity of extracellular serine/threonine protein kinase FAM20C-like protein (FAM20C-like) through the [S-x-E] motif, which is supported by evidence that mRNA and protein expression of FAM20C-like protein kinase are both found in the highest level in the hypopharyngeal gland of nurse bees. Our data represent the 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.

Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools

“…The sequence stretches covered by trypsin mapping resemble those detected after pepsin digestion. The limited sequence coverage seen with both proteases is consistent with the fact that MRJP1 experiences various post-translational modifications 9,20 in addition to glycosylation. 24 Any such covalent modifications will interfere with peptide matching, which relies on comparisons with the cDNA-derived MRJP1 sequence.…”

“…During maturation, MRJP1 undergoes additional post-translational modifications. 20 Isoelectric focusing reveals the presence of nine MRJP1 isoforms that share a similar MW but have slightly different pIs between 4.7 and 5.2. 21,22 Differences in the nature and extent of post-translational modifications are thought to be chiefly responsible for this heterogeneity.…”

Phosphoproteome Analysis Reveals Phosphorylation Underpinnings in the Brains of Nurse and Forager Honeybees (Apis mellifera)

“…To enrich the phosphopeptides in the brains of nurse and forager worker bees, a high efficiency Ti 4+ -IMAC material was applied as previously described 28 . Specifically, the immobilized Ti 4+ polymer beads (Ti 4+ -IMAC) were prepared by overnight incubation of 10 mg of polymer beads in 100 mM Ti(SO 4 ) 2 solution at room temperature (RT) under gentle stirring.…”

New Insights into the Biological and Pharmaceutical Properties of Royal Jelly

“…RJ also contains a calcium-binding protein, known as regucalcin, and a lipid-binding protein, such as apolipophorin-III [38]. Phosphorylated icarapin (venom protein-II) and apolipophorin-III-like proteins are identified in RJ may promote the strength of immunity [39]. There are 53 N-glycosylation sites residing on 25 N-glycosylated proteins in RJ.…”

“…They can be identified by proteomics, such as jelleines-I, jelleines-II, jelleines-III, jelleines-IV, and jelleines, are identical to the C-terminal of the MRJP-1 [43]. Moreover, RJ also contains peptides including apidaecin, defensin, hymenoptaecin, jelleine-II, jelleine-II (pT), and jelleine-II (pS) [39,44]. Phosphorylated jelleine-I (pS), jelleine-II (pS), and jelleine-IV (pS) are found in Apis cerana RJ while jelleine-II (pT) and jelleine-IV (pT) in Apis mellifera RJ [39].…”