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

Lan Zhang1,2†, Bin Han1†, Rongli Li1, Xiaoshan Lu1,3, Aiying Nie4, Lihai Guo5, Yu Fang1, Mao Feng1 and Jianke Li1*

Abstract

Background: Royal 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. Results: A 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.

Conclusion: Our 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.

Keywords: Royal jelly, N-glycosylation, Hydrazide chemistry, Lectin affinity, Tandem mass spectrometry

Background

Royal jelly (RJ) is a proteinaceous secretion derived from the hypopharyngeal and mandibular glands of young worker bees [1,2]. It is the sole food fed to the queen throughout her lifetime, and is also fed to all young larvae for the first three days after hatching [2]. RJ possesses various biological attributes beneficial for human health, such as antioxidant activities [3], antibacterial effects [4], enhancement of immune activity [5], and antitumor effects [6]. Protein accounts for >50% of RJ by dry weight [2]. It has been reported that nine members of major royal jelly proteins (MRJPs, MRJP1-9) [7,8] account for 80-90% of the total protein in RJ [9]. Other proteins, such as alpha-glucosidase, glucose oxidase, and alphaamylase have also been detected in RJ [1,10-12]. Although several studies have indicated that the proteins in RJ have undergone glycosylation modification [12-16], we do not yet know the types or site assignments of this glycoprotein. With the development of new technologies in protein separation and identification, dozens of novel proteins have been recently identified in RJ by our group and by others [1,11,16,17]. Advances in resolution and sensitivity (double high) of liquid chromatography-tandem mass spectrometry (LC-MS/MS) have made it a powerful platform. These advances have made it possible to profile the proteome of RJ more deeply, while allowing for systemlevel mapping of glycosylation sites of RJ proteins.

Asparagine-linked (N-linked) protein glycosylation is the most abundant of all posttranslational modifications in eukaryotes, with nearly 70% of all eukaryotic proteins predicted to be N-glycoproteins [18]. N-linked glycosylation is an enzymatically catalyzed process that occurs in the endoplasmic reticulum (ER). It involves the assembly of glycans on a lipid carrier in the ER membrane, followed by a transfer to specific asparagine residues of target polypeptides [19]. The attachment of N-glycans to a peptide backbone has been reported to assist in protein folding, stability, solubility, oligomerization, quality control, sorting, and transport [20,21]. Glycoproteins mediate many important biological processes by their involvement in cell adhesion, cell differentiation, cell growth, and immunity [22,23].

To identify N-glycosylated peptides from the more abundant non-glycosylated peptides in complex biological samples, specific enrichment methods, such as lectin affinity [24] or hydrazide chemistry [25], are required before they are subjected to double high LC-MS/MS analysis. Since a consensus sequence motif of N-X-S/T exists in Nglycosylation [20,21] (N = asparagine, X = any amino acid except proline, S/T = serine or threonine), the digested asparagine residue in N-X-S/T resulting from deglycosylation of the enzyme (Peptide N Glycosidase, PNGase F, commonly used) usually increases the mass by 0.98 Da. This basic scientific evidence is used to locate the Nglycosylation sites on a protein [26]. For more exact mapping of N-glycosylation sites, deglycosylation is usually done by introduction of 18O-water (H2 ¹⁸O), which increases a mass shift in the MS spectra of 2.99 Da, thus

adding confidence to the site assignment [27].

It is well-known that mapping residue-specific glycosylation sites is the first step towards a detailed and functional understanding of proteins [20]. However, information on N-glycosylation site assignment in RJ proteins is still very limited, thus demanding a powerful glycoproteomics approach to large-scale comprehensive mapping N-glycosylated sites in RJ proteins. Until now, RJ proteins have been documented to contain a series of glycoproteins [12,14,15], and are potentially glycosylated by a gel stain [28]. Only MRJP 2 is reported to carry two N-glycosylated sites attached a high-mannose structure and complex type antennary structures [16].

In an effort to identify hidden proteins and to map the N-linked glycosylation sites in RJ, two different double high LC-MS/MS systems, Q-Exactive coupled to EasynLC 1000 (orbitrap-based MS) and Triple TOF 5600 coupled with an Eksigent nLC (triple TOF-based MS), as well as complementary glycopeptide enrichment protocols (hydrazide and lectin), were employed. Overall, 25 N-glycosylated

proteins carrying 53 N-glycosylation sites were confidently identified, of which novel 42 N-linked glycosylation sites were mapped in RJ proteins, and 13 novel proteins were identified in RJ.