pH-dependent stability of honey bee (Apis mellifera) major royal jelly proteins

Honey bee larval food jelly is a secretion of the hypopharyngeal and mandibular glands of young worker bees that take care of the growing brood in the hive. Food jelly is fed to all larvae (workers, drones and queens) and as royal jelly to the queen bee for her entire life. Up to 18% of the food jelly account for proteins the majority of which belongs to the major royal jelly protein (MRJP) family. These proteins are produced in the hypopharyngeal glands at a pH value of 7.0. Before being fed to the larvae, they are mixed with the fatty acids secreted by the mandibular glands of the worker bees resulting at a pH of 4.0 in the food jelly. Thus, MRJPs are exposed to a broad pH range from their site of synthesis to the actual secreted larval food. We therefore determined the pH-dependent stability of MRJP1, MRJP2 and MRJP3 purified from royal jelly using differential scanning fluorimetry. All MRJPs were much more stable at acidic pH values compared to neutral ones with all proteins showing highest stability at pH 4.0 or 4.5, the native pH of royal jelly.

The stability of proteins can be assessed by experimentally denaturing their native structure 36 . Transitions between the native and the disordered state are typically induced by denaturing agents, e.g. guanidinium chloride or heat. We determined the melting temperature (Tm), where the fraction of folded and unfolded protein is equal 36,37 , of purified oligoMRJP1/apisimin, monoMRJP1, MRJP2 and MRJP3 in terms of their pH-dependent stability using differential scanning fluorimetry (ThermoFluor) [37][38][39] . All tested MRJPs exhibited within their natural pH range (4.0 to 7.0) Tm values (≥43 °C) above the maximum temperature of 35 °C normally occurring in the hive. In addition, all tested MRJPs were much more stable at pH 4.0, the native pH of the food jelly, compared to pH 7.0 (ΔTm = 14.3-18.6 °C) ensuring that MRJPs in the food jelly are not denatured by heat even at elevated ambient temperatures.

Results
pH dependent stability of oligoMRJP1/apisimin, monoMRJP1, MRJP2 and the MRJP3 isoforms was determined covering a pH range from 2.5 to 11. At the beginning of the experiment, at low temperature, a low fluorescence is expected as an indicator of a well folded protein. Within the physiological pH range for MRJPs (pH 4.0-7.0), monoMRJP1, MRJP2 and the MRJP3 isoforms possess at 20 °C a rather low fluorescence around 3,000-3,500 relative fluorescence units (RFU) (Fig. 1B-D). Contrary to that, the complex of oligoMRJP1/apisimin exhibited at the same pH values already at 20 °C a fluorescence between 15,000-20,000 RFU (Fig. 1A) indicating exposed hydrophobic regions. For pH 4.0 to 6.0, this fluorescence declines continuously until approximately 60 °C, followed by a further fluorescence increase peaking between 67 to 74 °C. For pH 6.5 and 7.0, the decline in fluorescence was constant. In addition, MRJP2 has a higher fluorescence intensity increase than monoMRJP1, and MRJP3 within the physiological pH range (Δfluorescence: monoMRJP1, ~3,000; MRJP2, ~10,000; MRJP3, ~4,000) implying a tighter packed hydrophobic core for MRJP2.
At pH 2.5 and 3.0, the curves recorded for monoMRJP1, MRJP2 and MRJP3 did either not show any increase in fluorescence at all or just a very slight one of approximately 500 RFU (Fig. 1B-D). Either the proteins do not unfold at these acidic pH values or the increased solvent polarity at decreased pH values leads to lower fluorescence intensity or shifting of the maximum fluorescence emission to other wavelengths as shown for other fluorescent dyes than SYPRO Orange 40 . Again, the complex of oligoMRJP1/apisimin exhibited also at these pH values an increased starting fluorescence (~10,000-12,000 RFU) which decreased at elevated temperatures (Fig. 1A). 9  www.nature.com/scientificreports www.nature.com/scientificreports/ At pH values above 9.0, starting fluorescence gradually increased with higher pH also for monoMRJP1, MRJP2 and MRJP3 indicating exposed hydrophobic residues and protein unfolding even at lower temperatures (monoMRJP1 pH ≥10.0, MRJP2 pH ≥10.5, MRJP3 ≥9.0).

Discussion
Transition midpoints could be determined for all proteins except for the complex of oligoMRJP1 and apisimin, which showed elevated starting fluorescence already at low temperatures indicating exposed hydrophobic regions. As monoMRJP1 does not show any peculiarity regarding hydrophobicity, the high hydrophobicity of oligoMRJP1/apisimin might be attributed to several reasons: (1) Either to the bound apisimin itself, which consists of ~40% hydrophobic amino acids, or (2) to hydrophobic residues of MRJP1 which are exposed only after apisimin binding. Hydrogen/deuterium exchange experiments indeed revealed that the N-terminal part of MRJP1 within oligoMRJP1/apisimin is highly disordered 11 but a comparison to the structure of monoMRJP1 is missing. (3) Only very recently it has been revealed that the complex of oligoMRJP1 and apisimin binds in addition eight molecules of 24-methylenecholesterol 41 which explains most likely the high hydrophobicity. However, also a combination of the reasons mentioned might be possible. Still, the melting curves of oligoMRJP1/apisimin have marked transitions points at pH values between 4.0 and 6.0 (Tm ~ 66-70 °C) which might be caused by the unfolding of the structured part of the complex. OligoMRJP1/apisimin (pH 4.0-6.0; Tm ~ 66-70 °C) appears to have an increased stability compared to monoMRJP1 (pH 4.0-6.0; Tm = 59.3-48.5 °C), irrespective of the fact that some hydrophobic residues are already exposed at low temperatures. At neutral pH, fluorescence of oligoM-RJP1/apisimin constantly decreased and no evaluation of the curves was possible in this study. However, it was shown that incubation of oligoMRJP1/apisimin in phosphate buffered saline (pH 7.5) at 56 °C for 30 min did not lead to a dissociation of the complex 42 and that the heat treatment did not impair proliferation activity of the complex on human lymphoid cells 43 . Circular dichroism spectroscopy in 2 mM HEPES, pH 7.0 showed even a slight structural gain from 20 °C to 95 °C 44 . This suggests that oligoMRJP1/apisimin is still folded at elevated temperatures at near neutral pH and illustrates that differential scanning fluorimetry is not an appropriate method to analyze the stability of the complex of oligoMRJP1 and apisimin. In addition, no strong difference was observed between fluorescence curves recorded above and below pH 5.0 although oligoMRJP1/apisimin starts to assemble into fibrillary structures below pH 5.0 16,17 .
MonoMRJP1, MRJP2 and the MRJP3 isoforms were all much more stable at pH 4.0 than at neutral pH (ΔTm: 14.3-18.6 °C). At elevated alkaline pH (≥9.0), hydrophobic residues were already exposed at 20 °C, residues which were buried at lower pH values. Indeed MRJPs have been shown to be more stable against limited proteolysis at acidic rather than alkaline conditions 45 . The different MRJPs showed at pH 4.0 a remarkable stability with melting temperatures ranging between 59.3 to 64.2 °C. This is in accord with accelerated protein degradation in fresh RJ above 65 °C 46 as the proteins unfold at these temperatures. www.nature.com/scientificreports www.nature.com/scientificreports/ Minimum solubility often coincides with the isoelectric point (pI) of proteins 47 where the electrostatic forces are at minimum and the proteins might precipitate out of solution. MRJPs exhibit theoretical pIs between 5.0 and 6.7 (monoMRJP1 and oligoMRJP1/apisimin-5.0, MRJP2-6.7, MRJP3-6.5) and should thus show lowest solubility at a pH of 5.0 (monoMRJP1 and oligoMRJP1/apisimin) and ~6.5 (MRJP2 and MRJP3). In the worker bee, all MRJPs reach their highest concentration in the hypopharyngeal gland secretion, before being mixed with the mandibular gland secretion to produce the final food jelly. Interestingly, the hypopharyngeal gland secretion has a pH of 5.1 ± 0.1 21 , which is exactly at the pI of monoMRJP1 and oligoMRJP1/apisimin. Thus, the increased protein stability at the acidic pH values is essential to ensure that MRJPs, and especially oligoMRJP1/apisimin, do not precipitate in the hypopharyngeal gland secretion as this would lead to blocking of the glands and prevent further food jelly secretion.
Due to a repetitive region with length polymorphisms 51 , MRJP3 occurs in different isoforms showing as multiple bands on SDS PA gels between 60 and 70 kDa 15,45 (Fig. S1, [37][38][39] were conducted. This method is based on monitoring the fluorescence of specific dyes, e.g. Sypro Orange, which is quenched in aqueous solutions but is highly fluorescent in presence of hydrophobic sites of unfolded proteins 37,39 . Thus, upon proceeding protein unfolding, fluorescence intensity increases and can be plotted as function of temperature. Experiments were performed in Hard-Shell 96-well microplates sealed with Microseal 'B' seals in a CFF Connect Real-Time System (all Bio-Rad, Hercules, CA, USA). Measurements were performed (3 biological replicates (proteins purified from three different royal jellies), 4 technical replicates per protein) in a total volume of 20 µl with 2 µM oligoMRJP1/apisimin, monoMRJP1, MRJP2 and MRJP3 and SYPRO Orange (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:1000 in 50 mM Na 2 HPO 4 /citric acid pH 2.5-8.0 or 50 mM Na 2 CO 3 /NaHCO 3 pH 9.0-11.0. Samples were heated from 20 °C to 95 °C at 1 °C per min and fluorescence intensity of SYPRO Orange was measured every degree using the FRET channel of the Real-Time System (excitation: 450-490 nm, detection: 560-580 nm). To determine the melting temperature (Tm -temperature at which the concentrations of folded and unfolded protein are equal), fluorescence intensities (FI) were plotted as a function of temperature (T) and fitted with Origin 5.0 (Microcal Software Inc., Northampton, MA, USA) according to the Boltzmann equation.