Comprehensive identification of novel proteins and N-glycosylation sites in royal jelly

Abstract

BackgroundRoyal jelly (RJ) is a proteinaceous secretion produced from the hypopharyngeal and mandibular glands of nurse bees. It plays vital roles in honeybee biology and in the improvement of human health. However, some proteins remain unknown in RJ, and mapping N-glycosylation modification sites on RJ proteins demands further investigation. We used two different liquid chromatography-tandem mass spectrometry techniques, complementary N-glycopeptide enrichment strategies, and bioinformatic approaches to gain a better understanding of novel and glycosylated proteins in RJ.ResultsA total of 25 N-glycosylated proteins, carrying 53 N-glycosylation sites, were identified in RJ proteins, of which 42 N-linked glycosylation sites were mapped as novel on RJ proteins. Most of the glycosylated proteins were related to metabolic activities and health improvement. The 13 newly identified proteins were also mainly associated with metabolic processes and health improvement activities.ConclusionOur in-depth, large-scale mapping of novel glycosylation sites represents a crucial step toward systematically revealing the functionality of N-glycosylated RJ proteins, and is potentially useful for producing a protein with desirable pharmacokinetic and biological activity using a genetic engineering approach. The newly-identified proteins significantly extend the proteome coverage of RJ. These findings contribute vital and new knowledge to our understanding of the innate biochemical nature of RJ at both the proteome and glycoproteome levels.

Oral transfer of chemical cues, growth proteins and hormones in social insects

“…Most of these appear to have functions related to digestion, except for apolipophorin, which is involved in lipid/nutrient transport (Kutty et al, 1996). For ortholog groups found in the TF of the three ant species, most were also digestion-related with the notable exception of CREG1, a secreted glycoprotein that has been implicated in cell growth control (Di Bacco et al, 2003) and insect JH response (Li Y et al, 2007; Barchuk et al, 2007; Zhang et al, 2014). Genus- and species-specific proteins were frequently associated with growth or developmental roles.…”

“…Across the TF of four species of social insect, we have found diverse molecules intimately involved in insect growth regulation: JH, JHE, JH-binding protein, vitellogenin, hexamerin, apolipophorin, and MRJPs. Several of the proteins and microRNAs identified in ant and bee TF are also found in royal and worker jelly (Guo et al, 2013; Zhang et al, 2014). Notably, there are also some similarities between proteins in social insect TF and mammalian milk (Zhang et al, 2015), such as the cell growth regulator CREG1.…”

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

“…Recently, our group comprehensively identified N-glycosylation modification in RJ. 22 However, mapping the phosphorylation status in RJ proteins still remains in its infancy and has been conducted only in Aml so far. 14,15,23 Among all PTMs, reversible phosphorylation of proteins at serine, threonine, and tyrosine residues is one of the most important and pleiotropic modifications.…”

Comparative analyses of the major royal jelly protein gene cluster in three Apis species with long amplicon sequencing

“…The gene cluster is located on chromosome 11 between two yellow genes ( y-e3 and y-h ) 4 , 5 consisting of nine protein coding genes mrjp1 to 9 and one pseudogene – mrjp- Ψ, recently renamed into mrjp 2 -like . 4–7 All major royal jelly proteins (MRJP) show a remarkable pairwise identity ranging from 47% to 74% 8 and have mostly been identified in royal jelly (RJ), 9 , 10 a hypopharyngeal gland secretion produced by nurse bees feeding the brood 11 . In A. mellifera , the RJ-feeding regime determines the prospective caste (queen or worker) of developing female larvae and although it has been under discussion that a specific MRJP in RJ might interfere with queen determination there is according to newer investigations no indication that MRJPs transcend the long proposed nutritional value in RJ 6 , 12 …”

“…Whereas the nine mrjps found in the genome have been confirmed on mRNA and protein level in A. mellifera , 10 , 13 similar information about the mrjps in other honeybee species is missing and only available for some mrjp homologues 14–17 . Based on draft genome sequences, first insights in the gene cluster organization of the most basal species A. florea suggested a highly conserved gene order except for the position of the pseudogene ( mrjp-Ψ/2-like ) 8 .…”