Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Site-Specific Analysis of N‑Linked Glycosylation Heterogeneity from Royal Jelly Glycoproteins Na Lin, Junmin Li, Rouming Shao, and Hong Zhang* School of Food Science and Biological Engineering, Zhejiang Gongshang University, Hangzhou, Zhejiang Province 310018, P. R. China
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ABSTRACT: Royal jelly (RJ) is secreted by young worker bees, and it plays key roles in the development and physiological function in honeybees and can improve human health. Although there have been analyses on the glycosylation modification of RJ proteins, none of these methods have been conducted on a site-specific analysis of glycosylation from these glycoproteins. Here, a combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity of RJ glycoproteins. First, global characterization of the N-glycome of RJ was performed using a direct infusion ion trap−sequential mass spectrometry (IT-MSn) method. Second, tryptic glycopeptides were enriched and separated by hydrophilic interaction liquid chromatography−ion trap−sequential mass spectrometry (HILIC-IT-MSn). A total of 50 Nglycopeptides and 30 N-glycans have been site-specific glycosylation profiled in major royal jelly protein 1 (MRJP1) and MRJP2 of RJ for the first time. Eighteen of the identified N-glycans have been structurally characterized by IT-MSn, including oligosaccharide composition, sequence, branching, and linkage. Two N-glycosylation sites (N177 and N394), 3 sites (N145, N178, and N92), and 1 site of N183 were identified in MRJP1, MRJP2, and MRJP3, respectively. There were 18, 17, and 2 N-glycans attached to MRJP1, MRJP2, and MRJP3, respectively. The diversity of N-glycans attached to each single glycosylation site of these glycoproteins confirmed that MRJP1 and MRJP2 heterogeneity was mostly associated with their glycoform populations. Understanding the properties of the site-specific glycosylation heterogeneity of the RJ glycoproteins can be potentially useful for producing a glycoprotein with desirable pharmacokinetic and biological activity. KEYWORDS: royal jelly, N-glycans, N-glycosylation sites, glycopeptides, hydrophilic interaction liquid chromatography−sequential mass spectrometry (HILIC-MSn)
1. INTRODUCTION Royal jelly (RJ) is produced by worker bees, and it is the principal food of honeybee larvae within the first 3 days and also the exclusive food for the honeybee queen during her lifetime.1 RJ has many biological activities, such as antioxidant activity, antibacterial activity, enhanced immunomodulation, and antitumor activity.2−4 Protein content in RJ (by its dry weight) was >50%, and among these proteins, major royal jelly proteins (MRJPs, MRJP1−MRJP9) account for 80%−90% of the total proteins in RJ.5,6 MRJPs are glycoproteins that are covalently bound to oligosaccharides at the N-terminal residue,7 and they are considered to be the most specific physiological functions in RJ, especially in the development and growth of bee queens. Glycosylation is the most obvious and complex modification pathway in protein post-translational modification. It is estimated that >50% of proteins in nature are glycosylated; among this, >70% of glycoproteins are N-glycosylated.8 Nlinked glycans are covalently attached to the asparagine (N) side chain with an Asn-X-Ser/Thr (sometimes also Cys, X ≠ Pro) motif and can be divided into three classes according to the monosaccharide composition and their branching.9 Protein glycosylation plays a very important role in regulating many biological processes, including cell proliferation, cell growth, and immune activity.10 The normal physiological function of glycoprotein is directly related to the nature of the glycan chain monomer on its protein. The abnormal regulation of © XXXX American Chemical Society
glycosylation type on glycoprotein plays a key role in the pathogenesis of the body and the development of different diseases. It has been reported that the increment of fucosylation and branching to form new antennae is a characteristic feature of the carbohydrate chains associated with disease occurrence.11 Despite being reported as glycoproteins, only 12 different N-glycans have been identified in RJ with the high mannose as the main type, and a low level of complex/hybrid type was also observed.12,13 Hayashi et al. reported that the carbohydrates in the IgE-binding proteins (MRJP1 in honey) are a major epitope for patient IgE.14 ́ Biliková et al.15 demonstrated that the glycosylation of a minority homologue of MRJP2 (apalbumin2a) could inhibit the growth of bacteria P. larvae. They also found that, in addition to high-mannose glycan, apalbumin2a carried complex-type antennary carbohydrate structure.15 Although the sites of glycosylation on MRJPs and other proteins from RJ have been analyzed, there is less information regarding the structures of the glycans on the specific glycoproteins.15−17 The analysis of protein glycosylation using mass spectrometry (MS) is typically by glycoproteomics or glycomics, that is, the glycoproteins and sites of attachment are identified from Received: May 16, 2019 Revised: July 20, 2019 Accepted: July 24, 2019
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DOI: 10.1021/acs.jafc.9b03080 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry deglycosylated peptides17,18 or the released glycans are characterized without reference to their former attachment site.19−21 However, neither of the two methods provide sitespecific information about the analyzed glycoprotein. Sitespecific profiling of a protein glycosylation is usually achieved by various LC-MS/MS fragmentation techniques (such as CID,22,23 ETD,24,25 ECD,26 and HCD26) with or without a specific or nonspecific protease (or glycosidase) treatment.27,28 The improvements in the global analysis of intact glycopeptides have enabled unambiguous identification of peptide− glycan compositions, but complete structural characterization of some proteins has not been achieved.29,30 In view of the lack of systematic studies on the site-specific glycan−peptide structure assignment of glycoproteins from RJ, this article aims to perform a detailed structural characterization of RJ glycoproteins, including their glycan structure, glycosylation sites, and glycopeptides sequence. Mastering the structural features of these glycosylated proteins allows us to better understand how RJ glycoproteins benefit honeybee physiology and human health.
the freeze-dried glycopeptides were resuspended in an equilibration buffer and loaded 3 times repeatedly on the column. Third, glycopeptides bound to the column were eluted with the elution buffer (elution buffer was the same as the equilibration buffer, but it also included 0.1−0.3 M methyl α-D-mannopyranoside). The glycopeptides eluent was collected and then desalted by a Sep-Pak C18 cartridge (purification steps were the same as described earlier). Finally, the obtained glycopeptide enrichment was lyophilized for further analysis. 2.4. N-Glycan Release and Permethylation. The lyophilized glycopeptides (samples without lectin enrichment) were resuspended in 1 mL of 50 mM NH4HCO3 (pH 8.4) buffer and followed by addition of 500 U of PNGase F. Incubation takes place for 48 h at 37 °C. Released N-glycans were purified using a Sep-Pak C18 cartridge. Condition and equilibration steps were the same as described earlier. Then the glycans were loaded into the cartridge and eluted with 9 mL of 5% acetic acid solution. The 5% acetic acid fraction was lyophilized, and this fraction contained the N-glycans. Three milliliters of sodium hydroxide slurry in dimethyl sulfoxide (DMSO) was added to the lyophilized N-glycans, and then 1 mL of CH3I was added to this mixture. The reaction mixture was mixed vigorously and vortexed at room temperature for 15 min. Subsequently, the reaction was stopped by adding cold water dropwise and constantly shaking between the additions. Two mL of chloroform and 2 mL of deionized water were added to the sample, mixed thoroughly, and left to settle until two layers were formed. The upper aqueous layer was removed, and the chloroform was washed 4 times with cold water until the removed water was clear. The chloroform layer was dried off, and the sample was dissolved in 1:1 MeOH/H2O and then desalted using a Sep-Pak C18 cartridge. The cartridge was conditioned with MeOH, water, and ACN. Then the permethylated glycans were loaded into the cartridge and washed with 7 mL of water and 1 mL of 10% ACN/H2O. Finally, the derivatized glycans were eluted sequentially with 2 mL of 35%, 50%, 75%, and 80% of ACN/H2O. The eluting fractions were collected and dried in a vacuum centrifuge. These purified permethylated N-glycans were ready for MS analysis.31,32 2.5. IT-MSn Analysis of Released N-Glycans. Direct infusion MS experiments were performed using an LCQ (Thermo) instrument. Permethylated N-glycans were dissolved in 50 μL of MeOH, and 2 μL/min were loaded into the electrospray ionization (ESI) spray capillary. The parameters used in the ESI (+) source included a sheath gas flow rate of 7 arb, an auxiliary gas flow rate of 0, a spray voltage of 4.5 kV, a capillary temperature of 300 °C, and a tube lens offset voltage of 5 V. The experiments used in the mass analyzer were full scan (m/z 350−2000), MS/MS, and MSn analysis with a collisioninduced dissociation (CID) fragmentation mode. The collision energy (CE) was varied between 50 and 70 V. 2.6. HILIC-MSn Analysis of Intact Glycopeptides from RJ. The enriched and purified glycopeptides were redissolved in 300 μL of ACN/H2O (50:50, v/v) for LC-MSn analysis. High-performance liquid chromatography with a diode-array detector (HPLC-DAD) separation of glycopeptides was performed on a HILIC column (XBridge Amide column, 2.1 × 150 mm, 3.5 μm) and a 2695 Waters system that was equipped with a 2996 DAD detector whose detection wavelength was 280 nm. The column temperature was set as 35 °C, the injection volume was 10 μL, and the flow rate was 0.3 mL/min. Mobile phase A was 10 mM ammonium formate (pH 4.5), and mobile phase B was ACN mixed with 100 mM ammonium formate (pH 4.5) in a ratio of 9:1 (v/v). The gradient was from 90% to 50% B in 50 min and then from 50% to 90% B in 5 min. The column was equilibrated with 90% B for 15 min before the next sample injection. LC-IT-MSn analysis was performed on an LCQ (Thermo) instrument. The ESI source parameters were optimized to maximize the signal/noise in the positive mode. The optimized parameters used in the ion source included a spray voltage of 5 kV, a capillary voltage of 17 V, a capillary temperature of 300 °C, a sheath gas flow rate of 80 arb, an auxiliary gas flow rate of 10 arb, and a tube lens offset voltage of 5 V. The experiments used in the mass analyzer were full scan (m/z 300−2000), auto data-dependent MS/MS, and manual MS3 analysis
2. MATERIALS AND METHODS 2.1. Materials. Iodomethane (CH3I), dithiothreitol (DTT), iodoacetamide (IAA), and ammonium bicarbonate (NH4HCO3) were analytical-grade and obtained from Sigma-Aldrich. Methyl α-Dmannopyranoside (≥99% GC grade) and ConA-Sepharose 4B were also purchased from Sigma-Aldrich. Formic acid (FA), ammonium formate, methanol (MeOH), and acetonitrile (ACN) were of HPLCgrade and purchased from Tedia (Fairfield, U.S.A.) and Merck (Darmstadt, Germany). Sequencing-grade trypsin (specific activity, >8000 units/mg protein) was purchased from Sigma-Aldrich. PNGase F (500 U/μL) was obtained from NEB (Beijing, LTD). Ultrapure water was obtained from a Milli-Q purification system (Millipore, MA, U.S.A.). C18 Sep-Pak SPE column (3 cc/200 mg) was purchased from Waters Company. Fresh RJ samples (source Apis mellifera) were kindly provided from Quzhou Entry−Exit Inspection and Quarantine Bureau (Zhejiang, China). 2.2. Extraction of RJ Glycoproteins. Appropriate RJ was diluted in deionized water by a ratio of 1:1 (w/w), stirred at room temperature for 20 min, and centrifuged at 12 000g at 4 °C for 50 min.6 The supernatant was collected and dialyzed against water at 4 °C for 24 h. Then the obtained glycoproteins were freeze-dried and stored at −20 °C until further analysis. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified RJ glycoproteins showed that these proteins are mainly composed of MRJP1, MRJP2, and MRJP3.6 2.3. Peptide Preparation and Glycopeptide Enrichment. Four milligrams of RJ glycoproteins was suspended in 500 μL of 50 mM NH4HCO3 (pH 8.4) and then reduced with 500 mM DTT at 37 °C for 1 h, followed by alkylation with 500 mM IAA at room temperature for 1 h in the dark. Subsequently, 80 μg of trypsin (1:50, w/w, enzyme-to-substrate ratio) was added to the solution and incubated overnight in 37 °C water bath. Glycopeptides were then purified using a Sep-Pak C18 cartridge after trypsin digestion. First, the C18 column was conditioned with methanol, 5% acetic acid, and 100% n-propanol, and then glycopeptide samples (pH of the samples was adjusted to
