Novel Aspects Of Understanding Post-translational Modifications And Proteins In Royal Jelly

Royal jelly (RJ) is a proteinaceous secretion from the hypopharyngeal and mandibular glands ofnurse honeybee workers. It not only provides the essential nutrition to egg-laying queens and larvae lessthan72h old, but also plays important health promotion roles for human. Although most of proteins inroyal jelly have been identified, analyzing the potential novel proteins hidden in RJ is of vital importantboth for understanding its roles for honeybee biology and health promotion effects for human. To fullyaccomplish the biofunction of proteins, post-translational modifications (PTMs) such as phosphorylation,methylation, deamidation and N-glycosylation are a must process. While identification the types andmapping the sites of PTMs are an essential first step towards better understanding the biochemicalnatures and biofunction of proteins. However, the investigation of PTMs in RJ proteins is still lacking sofar. Therefore, this study is aim to investigate the potential unknown proteins and the type and positionof phosphorylation, methylation, deamidation and N-glycosylation in RJ proteins using gel-base orgel-free proteomics strategies using liquid chromatography tandem mass spectrometry (LC-MSn) andbioinformatic approaches.A total of four phosphorylated proteins were found in RJ. The phosphorylated MRJP1-2are thoughtto increase the nutrition efficiency in supporting the egg-laying honeybee queens and the fast developingyoung larvae. The rapid dephosphorylation of the phosphorylated venom protein2precursor on serineresidues may function as a regulatory mechanism to protect the young larvae from deseases infectionwhile their immune system is immature. In addition, the phosphorylated apolipophorin-III protein in RJsuggests a role in strengthening the immunity of the larvae and queens.Methylation occurred on most of RJ proteins is likely reason that causes the visible drift of pI of RJprotein profile on two-dimensional electrophoresis. This helps the RJ retaining high viscosity and formprotein micelles, this thus further helps young larvae to develop normally through adhering to thevertical face-down queen and horizontal opened worker cells in the hive. The high rate of deamidation inRJ proteins explains the main reason why RJ is sensitive to temperature at the molecular level. To ensurethe fast developing larvae and egg-laying queen receive the freshest and most efficient food at34-350Cin the hive, the honeybee has an evolutionary strategy that the nurse bees feed the young larvae andqueens with RJ shoot period of time (0.5-3min) to prevent the RJ proteins from degradation.A total of27N-glycosylated proteins carried57N-glycosylation sites were confidently identified inRJ proteins using18O-water labling and complementary enriched method and high sensitive andresolution LC-MSn system. Among them,46N-linked glycosylation sites in RJ proteins were mappedfor the first time. In addition,9potential N-glycosyltional sites predicted by bioinformatics in Uniprotdatabase are verified in this study. This suggests that our complementary strategies in this study isefficient to profile the N-glycosylation in RJ. Glycosylation occurred in7major royal jelly proteins(MRJPs) indicates its roles in promoting MRJPs to be well dissolved in RJ, this thus efficiently tobenefit for the better utilization by the honeybee and human. Most of proteins carried multiple N-linkedglycosylation sites were related to metabolism acitivity. This suggests that glycosylated modification of this protein family plays the main roles in regulating the metabolism process in honeybees and satisfyingthe energy needs of the fast developing larvae and egg-laying queen. Meanwhile, most RJ proteins withhealth promotion acitivity were glycosylated, suggesting this may help RJ proteins gain the antibacterialand immune acitivity both for honeybees and human. RJ contains about two third the N-X-T motif ofN-glycosylated peptides. This well explains the biochemistry essence of N-link glycosylated RJ proteins.Simultaneously, a total of16proteins, almost associated with metabolism and health promotionacitivity, were first reported in RJ. This significant extends the proteome coverage of RJ.The present study comprehensive identifies a number of novel proteins and PTMs in RJ. Thisprovides vital knowledge not only towards deep understanding the protein components in RJ but alsosignificantly advancing our level of understanding how PTMs benefit honeybee biology and humanhealth at the systemic level.