Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent

Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent

  • Christian Seutter von Loetzen, 
  • Thessa Jacob, 
  • Olivia Hartl-Spiegelhauer, 
  • Lothar Vogel, 
  • Dirk Schiller, 
  • Cornelia Spörlein-Güttler, 
  • Rainer Schobert, 
  • Stefan Vieths, 
  • Maximilian Johannes Hartl, 
  • Paul Rösch
PLOS
x

Abstract

Each spring millions of patients suffer from allergies when birch pollen is released into the air. In most cases, the major pollen allergen Bet v 1 is the elicitor of the allergy symptoms. Bet v 1 comes in a variety of isoforms that share virtually identical conformations, but their relative concentrations are plant-specific. Glycosylated flavonoids, such as quercetin-3-O-sophoroside, are the physiological ligands of Bet v 1, and here we found that three isoforms differing in their allergenic potential also show an individual, highly specific binding behaviour for the different ligands. This specificity is driven by the sugar moieties of the ligands rather than the flavonols. While the influence of the ligands on the allergenicity of the Bet v 1 isoforms may be limited, the isoform and ligand mixtures add up to a complex and thus individual fingerprint of the pollen. We suggest that this mixture is not only acting as an effective chemical sunscreen for pollen DNA, but may also play an important role in recognition processes during pollination.

Introduction

Allergies are a major health problem worldwide. In particular, type I or immediate type allergies [1] that involve proteins as causative agents are very widespread and potentially severe. The major birch pollen allergen Bet v 1 from the European white birch (Betula verrucosa) alone [2] affects an estimated 100 million people [3]. Although birch pollen contain a variety of allergens from different protein families, more than 60% of all birch pollen-allergic patients react exclusively to Bet v 1 [4]. Up to 90% of the Bet v 1-sensitized patients also exhibit IgE-mediated allergic cross-reactions (oral allergy syndrome) to Bet v 1-homologous food allergens, with fruits, vegetables, and nuts as the most important elicitors of the allergy [5,6].

On the basis of sequence similarities and the protein three-dimensional structures, Bet v 1 and related pollen and food allergens belong to the family of class 10 pathogenesis-related proteins (PR-10) within the Bet v 1 superfamily. It was suggested that proteins in this family are involved in plant defense mechanisms, since expression of the respective genes is induced upon attacks of pathogens and by environmental stress [7]. However, the physiological roles of PR-10 proteins seem to extend beyond stress and pathogen response. Thus, the PR-10 strawberry allergen Fra a 1 is involved in controlling flavonoid biosynthesis and this protein is capable of binding different metabolic intermediates [8]. In general, PR-10 proteins often co-occur with flavonoids in vivo [915] and interact with flavonoids in vitro [8,16], as clearly evidenced, for example, for Bet v 1 [17,18]. Why many, if not all, PR-10 proteins appear as mixtures of isoforms, however, remains elusive [1921].

The first Bet v 1 isoform described on the DNA level was Bet v 1a [22] followed by the identification of numerous other isoform sequences [2325]. At least 18 Bet v 1 variants found in pollen on the mRNA or protein level [23,26,27] are officially listed as isoallergens (http://www.allergen.org). Studies on the proteomic profile of birch pollen extracts of different origin or species revealed significant differences of isoform composition and quantity [26,27]. For example, Bet v 1 constitutes up to 30% of the total protein content in Swedish pollen and 12% in Austrian pollen. In all cases so far, the most abundant isoform is Bet v 1a (50% to 70%), followed by Bet v 1d (20%), Bet v 1b (3% to 20%), Bet v 1f (2% to 8%), and Bet v 1j (~1%) [26].

Bet v 1a is well characterized by biochemical [2,18,28] and structural [2931] studies. The large hydrophobic pocket formed by the secondary structure elements of Bet v 1 suggested that this allergen acts as storage or carrier protein [29,32,33]. Previous research work focused on trial-and-error approaches or docking simulations to test various ligands for binding to recombinant Bet v 1 [18,30,34]. We recently purified Bet v 1 in complex with its natural ligand quercetin-3-O-sophoroside (Q3OS) directly from mature birch pollen and confirmed binding by reconstitution of the Bet v 1a:Q3OS complex from its recombinant protein and synthetic ligand component [17]. We hypothesized that this complex may be involved in UV-protection of the pollen DNA and that Q3OS may stimulate pollen tube formation upon rehydration of the pollen. We then asked why different isoforms exist and whether there are physiological ligands other than Q3OS. Although it is tempting to believe on the basis of the high sequence identities of 87.4%–99.4% to Bet v 1a that all isoforms specifically interact with Q3OS, Bet v 1 isoforms are strikingly different in their immunological and allergenic properties [35] and, although allergenicity is mainly correlated with binding epitopes at the surface of allergens [36] it has always been speculated that Bet v 1 proteins as such are only part of the story, and that IgE binding needs to be tested in complex with their natural binding partners to arrive at meaningful results [30].

In order to characterize serological IgE binding as a measure for allergenicity as well as the physiological function of Bet v 1, we thoroughly studied ligand- and antibody-binding behaviour of the Bet v 1 isoforms a (hyperallergen), m (intermediate), and d (hypoallergen). Surprisingly, while none of the ligands significantly alters the allergenicity of Bet v 1, ligand binding to the different isoforms is diverse and highly dependent on the composition of the ligands’ sugar moieties.

Results and Discussion

Bet v 1:Q3OS interaction is isoform-dependent

We were asking whether isoforms a, d, and m form identical complexes with the Bet v 1a natural ligand Q3OS [17]. In an initial experiment we noticed that Q3OS exhibits slightly different shades of yellow when incubated with these Bet v 1 isoforms. After incubation we removed excess Q3OS with a G25 column and recorded UV/VIS absorption spectra of the protein fractions (Fig 1A) and of unbound Q3OS (Fig 1B). In the presence of Bet v 1a, the UV/VIS spectrum of Q3OS shows a clear shoulder around 360 nm, while this is not the case for Bet v 1 isoforms d or m. These absorbance differences suggest that the putative Bet v 1d:Q3OS and Bet v 1m:Q3OS complexes are different from the Bet v 1a:Q3OS complex.

thumbnail
Fig 1. UV/VIS spectroscopy of flavonoids and Bet v 1 isoforms.

All spectra were recorded at 298 K with 50 mM sodium phosphate, 50 mM NaCl at pH 7.0 and 10% DMSO as sample buffer. A Binding of Q3OS to Bet v 1 isoforms. UV/VIS spectra of 20 μM Bet v 1a (-) and Q3OS incubated with Bet v 1a (-), Bet v 1d (-) and Bet v 1m (-) concentrated and subsequently eluted from a G25 column. B UV/VIS spectra of 20 μM Q3OS (-), quercetin (-), Q3OGlc (-) and Q3OGal (-) reveal differences in absorption maxima and intensities. C Sequence alignment of the Bet v 1 isoforms a, d and m as performed with ClustalW [91]. Amino acids are marked with asterisks (identical), colons (conserved) and dots (semi-conserved). Residues that vary compared to Bet v 1a are highlighted in red for Bet v 1d (95.6% sequence identity to Bet v 1a) and in blue for Bet v 1m (89.3% sequence identity to Bet v 1a).

https://doi.org/10.1371/journal.pone.0128677.g001

Binding of unglycosylated flavonoids to Bet v 1 isoforms

Since the determination of the three-dimensional structure of Bet v 1a in 1996 [29] it has been suggested that the protein functions as a carrier or storage protein. The existence of various highly similar, structurally almost identical isoforms could be evidence for a complex network of different acceptors, targeted to bind chemically similar ligands. Hitherto, there is only limited comparable information available about differences in ligand binding behaviour between Bet v 1 isoforms of different allergenic potential. Recent approaches used indirect methods (ANS replacement assay, [18]) or analysed ligand binding in protein crystals [30,37]. We now used UV/VIS and NMR spectroscopy to systematically analyse and compare binding of physiologically relevant ligands to three different Bet v 1 isoforms (Fig 1C) in solution, with a focus on flavonoids.

A set of five different flavonoids was used to analyse the influence of number and position of hydroxyl groups of the flavonoid moiety during binding to Bet v 1 isoforms (Table 1 and S1A to S1E Fig). UV/VIS and chemical shift perturbation (CSP) measurements with 1H-15N HSQC NMR spectroscopy were performed to study affinities and binding sites of various flavonoids. The UV/VIS spectra from the titration experiment of naringenin and Bet v 1a show isosbestic points indicating a two-state binding process with a Kd of roughly 60 μM (Fig 2A and 2B). In the 1H-15N HSQC spectra of 15N-Bet v 1a with increasing concentration of naringenin, the G140 resonance was in the intermediate exchange regime, but gradual CSPs were observed for the majority of affected resonances (Fig 2C, S1 Table), from which a Kd value of approximately 30 μM could be estimated (Fig 2D and 2E). The CSP mapping on the Bet v 1a:naringenin structure (pdb code 4A87, [30]) agreed well with the results from X-ray crystallography (Fig 2F). We confirmed F22, Y83, I102, and E141 as interacting residues (S1 Table) with CSPs > 0.12 ppm and the reported change in side chain conformation of K137 [30] could also be observed as large CSP with a Δδnorm value of 0.27 ppm.

thumbnail
Fig 2. Binding of naringenin to Bet v 1a.

A, UV/VIS spectra of the equilibrium titration of 20 μM naringenin with Bet v 1a. All spectra were recorded at 298 K with 50 mM sodium phosphate, 50 mM NaCl at pH 7.0 and 10% DMSO as sample buffer. B, Absorbance changes at 325 nm plotted against the Bet v 1a concentration as shown for the data in A. The curve represents the best fit to Eq (1) resulting in a Kd value of 60.6 ± 3.2 μM. C, Overlay of six 1H-15N HSQC spectra of 100 μM Bet v 1a in the presence of increasing naringenin concentrations from light to dark red. The experiments were performed with a Bruker Avance 700 MHz spectrometer in 50 M sodium phosphate, 50 mM NaCl, pH 7.0 and 10% 2H2O at 298 K. Naringenin was added from a stock prepared in deuterated DMSO to a final excess of 1:4.5 over Bet v 1a and a final DMSO concentration of 10%. D, Chemical shift changes (Δδnorm) calculated with Eq (2) for residues A15 (○) and G89 (●) plotted against the ration of naringenin:Bet v 1a during titration. The curves represent the best fit to a quadric binding equation from the analysis software of NMRViewJ [89] (S1 Table). E, Calculated Δδnorm values upon naringenin addition plotted against the Bet v 1a amino acid sequence and F, mapped on a cartoon representation of the complex structure of Bet v 1a:naringenin (pdb code: 4A87) with 0.04 ppm ≤ Δδ ≤ 0.08 ppm shown as yellow; 0.08 ppm ≤ Δδ ≤ 0.12 ppm shown as orange; and 0.12 ppm < Δδ shown as red. Bet v 1a in grey, naringenin in green sticks, oxygen in red.

https://doi.org/10.1371/journal.pone.0128677.g002

thumbnail
Table 1. Dissociation constants for Bet v 1 isoform interaction with flavonoids and sugars.

https://doi.org/10.1371/journal.pone.0128677.t001

The Kd values of all tested flavonoids were in the medium to low micromolar range (Table 1, S1 to S3 Tables). We observed shifts of the UV/VIS absorption maxima and isosbestic points in the spectra upon Bet v 1 addition for all isoforms and flavonoids (S4 Table). During 1H-15N HSQC titration, the majority of affected Bet v 1 resonances were in the fast exchange regime with the highest Kd generally for the non-hydroxylated flavone. Thereby, the significant CSPs obtained during titration were generally spread over the sequence of each isoform, making it difficult to predict a precise binding site for flavone. Due to hydrophobic interactions, flavone seems to bind more flexibly and somewhat more weakly inside the hydrophobic pocket.

In general, we obtained the best results (lowest standard error) for our titration experiments by fitting the data to an equation corresponding to a simple bimolecular reaction (Eq 1 and a similar equation provided by the NMRviewJ software). Prior experiments on flavonoid binding to other allergens of the PR-10 class performed so far also suggested a single site binding scheme to be valid [8,30]. Therefore, it seems as there is only one binding site for flavonoids inside the Bet v 1 hydrophobic pocket.

While the position of hydroxyl groups is insignificant, the addition of such leads to a significant decrease of Kd for flavonoids interacting with Bet v 1 isoforms a and d. Myricetin contains six hydroxyl groups and shows a 15-fold higher affinity to Bet v 1a (4.2 μM) and an even 60-fold higher affinity to Bet v 1d (1.2 μM) than flavone (Table 1). Those affinities are characteristic for a change of resonance positions and shapes in the form in the fast exchange regime to the intermediate exchange regime on the NMR time scale ([38], S1 Table). Accordingly, in the presence of myricetin, almost half of the affected resonances (11 of 28 residues) of Bet v 1a are in the intermediate exchange regime (S1 Table). Bet v 1m generally shows lower affinities towards the tested flavonoids compared to Bet v 1 isoforms a and d. Furthermore, the Kd values seem to be independent of the number of flavonoid hydroxyl groups. However, the presence of a hydroxyl group at C5’ in the B-ring of fisetin and myricetin decreases the affinity towards Bet v 1m compared to naringenin and quercetin (Table 1).

The interaction surfaces of all flavonoids are located inside the hydrophobic pocket of Bet v 1 but vary between Bet v 1a (T7 to S11, I23 to N28, F64, G89 to I91, I102, K115 to N118, K137 to E141, and R145) and Bet v 1m (T57, G89 to G92, I102, K137 to L143). Most likely, flavonoids enter the hydrophobic pocket via one of the two gaps formed by the mostly nonpolar residues F62, P63, F64, P90, Q132, A135, S136, and M139 (entrance 1) or by residues I23, L24, D25, D27, T52, K54, Y81, and I102. The third gap, Y5, T7, V133, and K137 with a diameter of ~6 Å, is probably too small for flavonoids to enter the cavity [30].

Despite the observed differences between the three isoforms with respect to binding of unglycosylated flavonoids, the hydrophobic cavity of Bet v 1 isoforms seem to be promiscuous acceptors of small hydrophobic and amphiphilic molecules in vitro. However, the vast majority of naturally occurring flavonoids are modified with additional functional groups such as methyl ether groups, glycosylations, or combinations of these [39]. In addition, the low water solubility of unglycosylated flavonoids [40] and their low potential physiological concentration in pollen [41] is not necessarily indicative of a major physiological importance of these complexes.

Binding of glycosylated flavonoids is governed by the sugar moiety

As no isoform-specific binding pattern for unglycosylated flavonoids could be derived, we focused on the sugar moiety of the quercetin glycosides quercetin-3-O-sophoroside (Q3OS), quercetin-3-O-glucoside (Q3OGlc), and quercetin-3-O-galactoside (Q3OGal) as binding partners of Bet v 1 isoforms (Table 1 and S1F to S1H Fig). UV/VIS absorption spectra show maxima of different intensities at physiological pH for Q3OGlc at 364 nm, and for Q3OGal and Q3OS at 358 nm (Fig 1B), but the spectral changes on Bet v 1 binding were too small to be analysed with confidence. Thus we resorted to 1H-15N HSQC spectroscopy for further studies.

Titration of Bet v 1a with Q3OS resulted in a change of resonance positions on the intermediate to slow exchange limit on the NMR time-scale for 16 residues (F22, L29, I38, K55, R70, E73, V74, N82, S84, V85, K115, Y120, K137, E138, G140, and L144), with a resulting Kd of 566 ± 85 nM ([17], Fig 3A and 3B, S1 Table). Although Q3OGlc is simply shortened by a single glucose moiety compared to Q3OS, the Bet v 1a:Q3OGlc Kd of 288 μM is three orders of magnitude higher than that of Bet v 1a:Q3OS (Fig 3C and S2A Fig). In contrast, Bet v 1a shows high affinity to Q3OGal with resonances of 16 residues in intermediate exchange (F22, I23, G26, K54, F64, R70, E73, D93, K115, S136 to E141, and L144) and 11 residues in the fast exchange regime (T10, I53, T66, G92, L95, V128, Q132, V133, A135, T142, and V147) showing CSPs > 0.04 ppm (Fig 3D and S2B Fig, S1 Table). According to docking simulations, Q3OGal binds in the hydrophobic pocket of Bet v 1a, with the sugar moiety either completely inside or at the opening of the pocket (entry ε1, [30]) at the flexible loop connecting β7 with α3. Since we observed the majority of affected resonances in the intermediate exchange regime, we concluded that the affinity of Bet v 1a to Q3OGal is higher than for its aglycon quercetin (9.6 μM) and estimated the Kd-value < 5 μM. Affinity scores of the models resulted in Kd values from 3.2 μM to 14.8 μM (Table 1). Obviously, stereochemical changes in the sugar moiety of flavonol glycosides can strongly influence the affinity to Bet v 1a.

thumbnail
Fig 3. Binding of quercetin glycosides to Bet v 1 isoforms.

All experiments were performed with 50 μM (Q3OS) or 100 μM (Q3OGlc, Q3OGal) 15N-uniformly labelled Bet v 1 isoforms at 298 K in 50 mM sodium phosphate buffer, 50 mM NaCl at pH 7.0, and 10% 2H2O with Bruker Avance 700 MHz and Avance 800 MHz spectrometers. Chemical shift changes were mapped on Bet v 1a (pdb code: 1BV1, grey) or models of Bet v 1d and Bet v 1m as in Fig 2F. Models of Bet v 1d and Bet v 1m were created using the Phyre server [92]. Docked ligands [93] are illustrated in green sticks, oxygen in red. A Overlay of two 1H-15N HSQC spectra of Bet v 1a in the absence (black) and presence of a 15-fold excess of Q3OS (red). B Disappearing resonances after addition of Q3OS mapped on Bet v 1a in red. Q3OS is docked inside the hydrophobic pocket [17]. C Mapping of chemical shift changes of (weak) Q3OClc or D (strong) Q3OGal interaction on Bet v 1a. E Overlay of two 1H-15N HSQC spectra of Bet v 1d in the absence (black) and presence of a 15-fold excess of Q3OS (red) and F occurring chemical shift changes mapped on a model of Bet v 1d. Weak affinity is observed for interaction of Bet v 1d with G Q3OGlc of H Q3OGal. I Overlay of two 1H-15N HSQC spectra of Bet v 1m in the absence (black) and presence of a 15-fold excess of Q3OS (red) and J occurring chemical shift changes mapped on a model of Bet v 1m. High affinity is observed for the interaction of Bet v 1m with K Q3OGlc and L Q3OGal. Regions of the 1H-15N HSQC spectra during titration of Bet v 1d or Bet v 1m with Q3OGlc and Q3OGal are provided in the S1 Fig

https://doi.org/10.1371/journal.pone.0128677.g003

Although Bet v 1d binds flavonoids with affinities comparable to those of Bet v 1a (Table 1), it shows only very weak affinity for the glycosylated flavonoids that we have analysed here. Remarkably, even a 15-fold excess of Q3OS, Q3OGlc or Q3OGal (Fig 3E to 3H, S2C and S2D Fig) did not produce significant CSPs for Bet v 1d.

Furthermore, titration of Bet v 1m with Q3OS also did not lead to significant CSPs (Fig 3I and 3J), suggesting that Bet v 1a:Q3OS formation is highly specific. However, in contrast to Bet v 1a and d, Bet v 1m strongly binds to Q3OGlc (estimated Kd < 5 μM), with resonances of 21 residues in intermediate exchange (E6, I23, G26, I38, T57, F64, Y66, G89 to G92, I98, and I136 to L144) and four residues (A34, E87, E96, and V147) with CSPs > 0.04 ppm (Fig 3K and S2E Fig, S2 Table). The docking simulation suggested Q3OGlc to bind in the hydrophobic pocket of Bet v 1m with Kd values of 0.4 μM to 10.4 μM. Bet v 1m also shows high affinity for Q3OGal with 21 intermediate exchanging residues (I38, S39, T57, F64, Y66, M85 to E87, G89 to G92, E96, K134, I136 to E141, L143, and L144) and 14 residues (D25, A34, A37, V41, N47, I56, E87, G88, T94, L95, K115, T122, K123, and A135) with CSPs > 0.04 ppm (Fig 3L and S2F Fig, S2 Table) and Kd values obtained from docking simulations between 0.2 μM and 6.1 μM (Table 1).

Although glycosylation drastically changed the binding behaviour of quercetin to the various Bet v 1 isoforms, glucose and galactose alone showed no detectable affinity to any isoform (Table 1).

Bet v 1d varies in seven amino acids (T7I, F30V, S57N, I91V, S112C, I113V, and D125N; Fig 1C) compared to Bet v 1a. Thus, strong specific binding and virtual lack of such is achieved by variation of just seven or even fewer amino acids. None of those seven variable residues, however, is directly involved in Q3OS or Q3OGal binding in Bet v 1a or is part of the amino acids which form the potential entrances. T7 is part of the third opening in Bet v 1a, which is presumably too small for glycosylated flavonoids entrance. The loss of affinity might be explained by a slightly different structural arrangement of Bet v 1d, which could result in variations in the openings to the hydrophobic pocket. In contrast to Bet v 1d, Bet v 1m shows four variations in entrance 1 (F62S, P90A, Q132H, and S136I compared to Bet v 1a; Fig 1C) which are likely to directly block the access route for Q3OS, but not for Q3OGlc and Q3OGal, into the hydrophobic pocket. Substitutions of amino acids in the C-terminal helix (S136I, M139K, and T142A) could contribute to an increased affinity to Q3OGlc as compared to Bet v 1a as the C-terminal helix determines size and character of the hydrophobic cavity in PR-10 proteins [33].

In addition to structural aspects, a phenomenon known as enthalpy-entropy compensation [42] can explain the binding behaviour of the isoforms to glycosylated flavonoids and the sugars alone. Upon Bet v 1 isoform–ligand complexation, water molecules that form the hydration shell of the sugar moiety and the binding cavity will tend to escape to the bulk with a concomitant decrease or increase in entropic energy contribution, depending on the pre-existing molecular interactions. This event is accompanied by the increase or decrease of degrees of freedom for the ligand and the residues forming the binding site. The setup of solvent clusters on the surface of the protein-ligand complex also contributes to the overall binding affinity with enthalpy/entropy gains (Bet v 1a:Q3OS or Q3OGal; Bet v 1m:Q3OGlc or Q3OGal), penalties (Bet v 1a:Q3OGlc), or even complete abolishment of observable binding (Bet v 1d:Q3OS, Q3OGlc or Q3OGal; Bet v 1m:Q3OS) compared to the aglycon quercetin. Similar effects have been reported and seem to be generally characteristic for each ligand/receptor involved [4345]. In addition, glucose and galactose alone showed no detectable affinity to any isoform (Table 1). The potential enthalpy gains upon carbohydrate interaction with proteins are often counteracted by the above described change of entropy [42], resulting in the abolishment of binding. We observed this effect already for the binding of sophorose to Bet v 1a [17].

In summary, our results firmly suggest that Bet v 1:ligand binding is isoform-specific and that the binding specificity is entropically driven by the sugar moiety. Glycosylation of quercetin can thereby significantly increase the affinity compared to the aglycon (Table 1). The hydrophobic pockets formed by Bet v 1 isoforms are obviously designed for specific discrimination between the sugar moieties of glycosylated flavonoids.

Allergenicity of Bet v 1 isoforms is unaffected by ligands

Bet v 1 isoforms can be grouped into three classes with molecules showing high (isoforms a, e, and j), intermediate (isoforms b, c, and f), and low/no IgE-binding activities (d, g, and l) [35]. A study on the modulation of IgE reactivity by site-directed mutagenesis revealed a limited number of crucial amino acid positions (residues F30, S57, S112, I113, and D125 in the Bet v 1a sequence) that strongly influence IgE binding [36]. Although Bet v 1 isoforms d, g, and l are highly similar in sequence to Bet v 1a (95.6%, 95.0%, 94.3% identity, respectively), those hypoallergenic isoforms show variations in each of these positions. A small subset of critical amino acids can drastically modulate the binding of IgE to an epitope and consequently change the allergenicity of Bet v 1 isoforms as exemplified by Bet v 1 isoforms a and d [35,46]. In the absence of ligands, we observed comparable IgE interactions (Fig 4A) and mediator release activities (Fig 4B) for isoforms a and m as measured by indirect ELISA and β-hexosaminidase release from humanized rat basophil leukaemia (RBL) cells. Sequence and allergenicity of Bet v 1m and the intermediate IgE-binding isoform Bet v 1b are nearly identical (Bet v 1.0201, 98.1% identity; [23]). The IgE-binding capacity of Bet v 1d is only marginal in the ELISA, and consequently an approximately 10-fold shift to a higher Bet v 1d concentration is needed for half-maximum release of β-hexosaminidase in comparison to the other isoforms (Fig 4A and 4B). Comparable results concerning the allergenicity of these Bet v 1 isoforms were also obtained in previous experiment [35,36,46].

thumbnail
Fig 4. Interaction of Bet v 1 isoforms with serum IgE in the absence and presence of Q3OS.

A, Binding of serial dilutions of pool serum IgE to equimolar amounts of surface-coated Bet v 1a, Bet v 1d, and Bet v 1m. Allergen-specific human IgE was detected with a horseradish peroxidase-conjugated mouse anti-human IgE antibody. As substrate 3,3′,5,5′-tetramethylbenzidine was used and the absorbance at 450 nm was measured after stopping the reaction with 25% H2SO4. B, Mediator release induced by recombinant Bet v 1 isoforms. Humanized rat basophil leukemia cells were sensitized with a pool of human birch-specific sera. Cross-linking of membrane-bound human IgE by IgE-Bet v 1 isoform interaction and subsequent release of β-hexosaminidase was determined with serial dilutions of Bet v 1 a, d and m. The β-hexosaminidase activity in the culture supernatants was quantified by photometric measurements. The percentage of β-hexosaminidase activity relative to cells lysed with Triton X-100 was calculated and corrected for spontaneous release. C, Binding of serial dilutions of pool serum IgE to equimolar amounts of surface-coated Bet v 1a, Bet v 1d, and Bet v 1m (as described in A) and D, mediator release (as described in B) in the presence of a 5-molar excess of Q3OS.

https://doi.org/10.1371/journal.pone.0128677.g004

X-ray crystallography revealed that Bet v 1:ligand interaction could lead to an increase in volume of the hydrophobic pocket, thus altering the protein surface [30,37], an effect that was hypothesized to influence IgE epitopes. Our results, however, do not indicate any significant influence of high-affinity ligands on the IgE binding properties of Bet v 1. Presence of a 5-fold molar excess of Q3OS does not significantly influence the interaction of IgE with any of the three isoforms (Fig 4C and 4D), and rutin, quercetin, Q3OGlc, Q3OGal, and sophorose did not modify IgE-binding of the Bet v 1 isoforms either (S3 Fig). Our results are in agreement with a recent study on the influence of deoxycholate on the allergenic properties of Bet v 1a [47].

Although recognition of an allergen by IgE is the key step in the allergic response, numerous other factors such as functional activity, presence of infective agents or chemical substances can induce non-specific inflammatory responses or will augment the immunological shift towards an allergic reaction [48]. We suggest the lack of a direct ligand effect on IgE recognition of Bet v 1, but leave open the possibility of indirect influences or sensitization [49]. Indeed, flavonoids influence the inflammatory pathway in human cells [50], and their uptake by the human body may be facilitated by Bet v 1 [51,52].

Bet v 1:flavonol-glycosides—adaptable sunscreens for birch pollen DNA?

The Bet v 1:Q3OS complex was suggested to protect pollen DNA from UV-damage, and the mixture of different isoforms was suggested to provide an individual fingerprint to prevent self-pollination [17]. Indeed, glycosylated flavonoids are common in plant pollen. Flavonol-3-O-glycosides, e. g., were found in pollen from alder, ragweed, buttercup, date palm, narrowleaf cattail, hazelnut, petunia, maize, and ophrys [11,5360], and quercetin-3-O-glycosylgalactoside was identified in pollen from Betula verrucosa [12] along with the Bet v 1a ligand Q3OS. Interactions of glycosylated flavonoids with different Bet v 1 isoforms in combination with variations in the production and composition of isoforms during maturation of pollen are probably dependent on a set of parameters like climate, location, and solar radiation, as the Bet v 1 levels in pollen are not constant over time [61], show variable IgE reactivity [27], and vary geographically [26,62]. Upon UV-B radiation flavonoids (mostly quercetin derivatives) are produced to protect the DNA from radiation damage [63] and glycosylation increases the UV tolerance of a flavonoid compared to the corresponding aglycon [64,65]. As we observed a shift of the absorption maximum of quercetin depending on the sugar moiety (Fig 1B) and the absorption maxima of different unglycosylated flavonoids shift towards higher (myricetin, quercetin, fisetin) or lower (naringenin) wavelengths during UV/VIS titration with Bet v 1 isoforms (S4 Table), Bet v 1 complex formation combined with variation of isoform composition in pollen may be a means to expand or to optimize the absorption spectrum for sunlight-emitted UV-A radiation.

After maturation and before dispersing into the environment, the pollen dehydrate [66] to reduce their water content to 20% [67], thus forming highly viscous intracellular glass-like structures [68]. In this milieu of highly concentrated biomolecules, glycosylated flavonoids may be protected from degradation or chemical modulation by complex formation with Bet v 1.

Although flavonoids are considered most effective UV-B screening compounds because of their strong absorbance in the UV region [69], continuous UV-irradiation leads to their degradation [64]. Existence of functional complexes of glycosylated flavonoids and Bet v 1 in high concentration may serve as an important signal for unharmed pollen DNA as UV-damage of the flavonoid moiety may modify the complex and prevent pollination. The pollen–pistil interaction before fertilization comprises a series of complex cellular interactions involving a continuous exchange of signals between pollen and the pistil of the stigma [70,71]. Upon contact, birch pollen get rehydrated, and the Bet v 1-ligand complexes are released onto the stigma surface [10,66] with the specific mixture of the isoforms and ligands possibly serving as molecular fingerprints to prevent self-pollination.

This means that isoforms of PR-10-allergen do not simply just exist by chance, but have been selected through evolution with each isoform fulfilling a particular function. Isoforms from other Bet v 1 homologs like Ara h 8 [72,73], Dau c 1 [74,75], Api g 1 [76,77], Pru av 1 [78,79] or Fra a 1 [80,81] seem to have less diverse functions in vivo without the necessity to provide such a complex individual fingerprint. In those cases, the amount of (so far identified) genetically available and actually expressed isoforms seems to be significantly lower than observed for example for Mal d 1 in apple [8287] or Bet v 1 in birch pollen [23,26,27].

Materials and Methods

Flavonoids

All nonglycosylated and monoglycosylated flavonoids as well as glucose and galactose were purchased in analytical grade from Sigma-Aldrich. Q3OS was obtained from ALNuMed (Germany) or AApin Chemicals Limited (UK).

Protein preparation

The genes coding for Bet v 1d (Bet v 1.0102; UniProt P43177) and Bet v 1m (Bet v 1.0204; UniProt P43186) were purchased from GeneScript and cloned into the bacterial expression vector pET11a (Novagen) using the restriction enzymes NdeI and BamHI-HF (New England Biolabs). The expression for all isoforms was performed as previously described for Bet v 1a (Bet v 1.0101, UniProt P15494, [17]) with minor modifications. For purification, Bet v 1 isoforms d and m were regained from protein pellets after cell lysis with 50 mM sodium phosphate, pH 7.4, 200 mM NaCl, and 8 M urea and refolded by subsequently lowering the urea concentration during dialysis in 20 mM Hepes buffer, pH 8.0 and 500 mM NaCl at 4°C (Bet v 1d) or 20 mM Hepes buffer, pH 8.0 at RT (Bet v 1m).

Refolded Bet v 1d was further purified via hydrophobic interaction chromatography on a 4 ml octyl sepharose column (HiTrap, Octyl Fast flow, GE Healthcare) equilibrated with loading buffer (20 mM Hepes, pH 8.0, 1 M ammonium sulphate) and eluted stepwise with elution buffer (20 mM Hepes, pH 8.0). Refolded Bet v 1m was loaded on a 25 ml Q sepharose column (Q sepharose Fast flow, GE Healthcare) equilibrated with loading buffer (20 mM Hepes, pH 8.0) followed by elution with 20 mM Hepes, pH 8.0, 300 mM NaCl. Bet v 1a was purified as previously described [17]. Fractions containing the respective Bet v 1 isoform were pooled and dialyzed at 4°C against 50 mM sodium phosphate, pH 7.0, 50 mM NaCl, concentrated and stored at -80°C. Protein concentrations were determined by the DC protein assay (BioRad) and UV/VIS spectroscopy using the molar extinction coefficient ε280 = 10430 M-1 cm-1. Standard methods were used to analyse purity (SDS/PAGE), oligomeric state (size exclusion chromatography), and signal dispersion (1H-15N HSQC spectroscopy) of all isoforms (S4 Fig).

UV/VIS spectroscopy

All flavonoids and Bet v 1 isoforms were dissolved in 50 mM sodium phosphate, 50 mM NaCl, 10% (v/v) DMSO, pH 7.0, to a final concentration of 10 to 20 μM in 500 μl. Absorption spectra from 200–800 nm were recorded at 25°C in a 1 cm quartz cuvette (Hellma GmbH) using a 8453 UV-visible spectrophotometer (Agilent).

To observe binding of Q3OS to Bet v 1 isoforms a, d, and m, 20 μM Q3OS were initially incubated with 20 μM of the respective isoform in buffer without DMSO for 30 min at room temperature (RT) in a total volume of 550 μl. Samples were concentrated to a final volume of 100 μl using a Vivaspin concentrator (Sartorius, molecular mass cut off 10 kDa). The concentrated samples where loaded on a G25 spin trap column (GE Healthcare) and eluted as described in the manual. Absorption spectra of the eluted fractions were normalized at 280 nm and set to zero at 700 nm.

To further characterize flavonoid binding to Bet v 1, titration experiments were performed by adding small amounts of concentrated Bet v 1 isoform a, d or m to different flavonoids. Changes of flavonoid absorption occurring at specific wavelengths were plotted against the protein concentration. Prior to curve-fitting, absorbance data were corrected for dilution. If possible, the equilibrium dissociation constant (Kd) was determined by non-linear regression analysis of the data with GraFit-5 (Version 5.0, Erithacus Software, UK) using the following Eq (1): (1) ΔAmax, maximum change in absorbance at specific wavelengths; B, Bet v 1a concentration; Q, total flavonoid concentration.

NMR spectroscopy

All NMR experiments were performed at 298 K in 50 mM sodium phosphate buffer, 50 mM NaCl, pH 7.0, 10% deuterium oxide (2H2O) with 15N-uniformly labelled Bet v 1 isoforms using Bruker Avance 700 MHz and Avance 800 MHz spectrometers with cryogenically cooled triple-resonance probes equipped with pulsed field-gradient capabilities. NMR data were processed using NMRPipe [88] and visualized with NMRViewJ [89]. Three-dimensional 15N-edited NOESY (nuclear Overhauser enhancement spectroscopy, mixing times 120 ms) experiments to assign chemical shifts were obtained with 500 μM 15N-labeled samples of Bet v 1 isoform d or m and yielded 91% of assigned residues for Bet v 1d and 89% for Bet v 1m. The sequence-specific assignments of the amide resonances of Bet v 1a are reported elsewhere [90].

Interaction of Q3OS with the Bet v 1 isoforms was measured by incubating 700 μM Q3OS with 50 μM of each 15N-labeled Bet v 1 isoform in 50 mM sodium phosphate, 50 mM NaCl buffer, pH 7.0.

For titration experiments all other flavonoids were dissolved in deuterated DMSO, while glucose and galactose were dissolved in 50 mM sodium phosphate buffer, 50 mM NaCl, pH 7.0, 10% deuterium oxide and titrated stepwise to a final excess of up to 17-fold to protein samples (ca. 100 μM). Final DMSO concentrations did not exceed 10% (v/v). Chemical shift perturbations caused by increasing DMSO concentrations during measurements were identified by titrating DMSO in comparable steps. CSPs for ligand binding were calculated based on Eq (2): (2) ΔδHN and ΔδN, chemical shift differences of amide proton and nitrogen resonances, respectively, in ppm.

Kd values for flavonoid binding were determined with NMRViewJ [89]. All analysable amino acid residues that were unaffected by DMSO addition and showing CSPs > 0.08 ppm were fitted to a quadratic binding curve with default settings, and an average Kd app was calculated (Table 1 and S1S3 Tables). The CSPs of all residues showing CSPs > 0.04 ppm were mapped either on models of Bet v 1d and Bet v 1m or on the Bet v 1a structure (pdb code 1BV1, [29]).

Sequence alignments, modelling and docking simulation

Sequence alignments of the Bet v 1 isoforms a, d and m were performed with ClustalW [91]. Models of Bet v 1d and Bet v 1m were created using the Phyre2 server [92]. The calculated models are based on the structural fold of PR-10 proteins with a confidence of 99% and a coverage of 92% (Bet v 1d) and 87% (Bet v 1m) compared to the template sequence. We used AutoDockVina [93] to dock ligands into the hydrophobic pocket of Bet v 1a and the model of Bet v 1m. The PDB files for Q3OGlc and Q3OGal were created with ProDrg [94]. Furthermore, input files for Bet v 1a (pdb code 1BV1), the model of Bet v 1m, Q3OGlc, and Q3OGal were generated with AutoDockTools [95]. The grid box (2.0 nm×2.4 nm×2.8 nm, or 13.44 nm3) was centred over the hydrophobic pocket of the isoforms and AutoDockVina was run with default settings. Affinity scores were given by AutoDockVina as binding energies (ΔG), which were subsequently used to calculate an equilibrium dissociation constant by Eq (3) with R = 0.001968 kcal·mol−1·K−1 and T = 298.15 K: (3)

Ligand docking was performed only if more than five amino acids with Δδ ≥ 0.12 ppm or intermediate exchange rates were observed during NMR titrations. The output of the docking simulation lists up to nine energetically most favourable orientations of the respective ligand in the Bet v 1 pocket. The models in best agreement with our experimental NMR data were chosen to illustrate ligand binding to the Bet v 1 isoforms a or m.

Sera used in the study

Fifteen sera of birch pollen-allergic subjects were collected, tested, and pooled according to the guideline of the European Medicines Agency (EMEA/CHMP/BWP/304831/2007). The serum pool is routinely used for batch-release testing of birch pollen-derived allergenic products at the Paul-Ehrlich-Institut. The same serum pool was used for both, ELISA and mediator release assays.

Indirect ELISA for IgE binding to Bet v 1 isoforms

For IgE-ELISA experiments, Maxisorp plates (Nunc via Fisher Scientific) were coated overnight at room temperature with 50 ng/100 μl recombinant Bet v 1 isoforms a, d, or m with a 5-fold molar excess of quercetin-3-O-sophorose, rutin, quercetin, quercetin-3-O-glucoside, quercetin-3-O-galactoside, or sophorose, respectively, in phosphate-buffered saline (PBS). After blocking with PBS containing 2% bovine serum albumin (BSA) these plates and an uncoated control were incubated with a dilution series of a serum pool of birch-pollen allergic subjects for 1 h at room temperature with PBS containing 0.05% Tween 20 and 0.1% BSA. Allergen-specific human IgE was detected with a horseradish peroxidase-conjugated mouse anti-human IgE antibody (Clone B3102E8, Southern biotech via Biozol, Eching, Germany) diluted 1:1000. 3,3′,5,5′-tetramethylbenzidine (Roth, Karlsruhe) was used as substrate for horseradish peroxidase, and the absorbance at 450 nm was measured after stopping the reaction with 25% H2SO4.

β-Hexosaminidase release from humanized rat basophil leukemia (RBL) cells

The mediator release assay was performed according to an established protocol [96]. Briefly, RBL cells expressing the α-chain of the high-affinity receptor for human IgE were sensitized overnight at 37°C (5%CO2) with a serum pool of birch pollen-allergic subjects (diluted 1:40 in culture medium). After washing, cells were stimulated with serial dilutions of Bet v 1 isoforms a, d, or m in Tyrode's buffer containing 50% 2H2O. For complex formation, the Bet v 1 isoforms were incubated overnight with a 5-fold molar excess of Q3OS, rutin, quercetin, Q3OGlc, Q3OGal, or sophorose, respectively, before stimulating the cells. Degranulation was quantified by photometric measurement of β-hexosaminidase activity in the culture supernatants. The percentage of β-hexosaminidase activity relative to cells lysed with Triton X-100 (Sigma-Aldrich, Steinheim, Germany) was calculated and corrected for spontaneous release (sensitized cells without allergen).

Supporting Information

S1 Fig. Chemical structures of flavonoids used in this study.

A flavone, B naringenin, C fisetin, D quercetin, E myricetin, F quercetin-3-O-glucoside, G quercetin-3-O-galactoside, H quercetin-3-O-sophoroside.

https://doi.org/10.1371/journal.pone.0128677.s001

(TIF)

S2 Fig. NMR titration experiments of Q3OGlc and Q3OGal with the Bet v 1 isoforms.

The experiments were performed with 100 μM 15N-uniformly labelled Bet v 1 isoforms at 298 K in 50 mM sodium phosphate buffer, 50 mM NaCl at pH 7.0, and 10% 2H2O with Bruker Avance 700 MHz and Avance 800 MHz spectrometers. Q3OGlc and Q3OGal were dissolved in deuterated DMSO and titrated stepwise to a final excess of up to 1:17 to protein samples. Final DMSO concentrations did not exceed 10% (v/v). Spectra are illustrated in a divergent colour scheme from red (absence of ligand) to blue (final excess of ligand). Intermediate exchanging residues are labelled. Titration experiments of Bet v 1a with A Q3OGlc and B Q3OGal, Bet v 1d with C Q3OGlc, D Q3OGal and Bet v1m with E Q3OGlc and F Q3OGal.

https://doi.org/10.1371/journal.pone.0128677.s002

(TIF)

S3 Fig. Interaction of Bet v 1 isoforms with IgE in the presence of different flavonoids and sophorose.

The left panel shows binding of serial dilutions of serum IgE to equimolar amounts of surface-coated Bet v 1a (■), Bet v 1d (●), and Bet v 1m (▲) with 5-molar excess of A quercetin, C Q3OGlc, E Q3OGal, G sophorose, and I rutin respectively. Mediator release induced by recombinant Bet v 1 isoforms is illustrated in the right panel. Humanized RBL cells were sensitized with a pool of human birch-specific sera. Cross-linking of membrane-bound human IgE by IgE-Bet v 1 isoform interaction and subsequent release of β-hexosaminidase was determined with serial dilutions of Bet v 1a (■), Bet v 1d (●), and Bet v 1m (▲) with 5-molar excess of B quercetin, D Q3OGlc, F Q3OGal, H sophorose, and J rutin respectively.

https://doi.org/10.1371/journal.pone.0128677.s003

(TIF)

S4 Fig. Protein analytics.

A SDS/PAGE on 19% gels of ca. 1 μg Bet v 1 isoforms (MW 17.4 kDa) after purification. M, molecular-mass standard (Low Range, Bio-Rad Laboratories). B SEC of the isoforms performed with a Superdex S75 GL 10/300 column (total bed volume: 24 ml; GE Healthcare) in 50 mM sodium phosphate, 50 mM NaCl, pH 7.0 at RT. Column calibration was performed with conalbumin (75.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (29.0 kDa) and ribonuclease (13.7 kDa). The elution profile of 0.5 mg Bet v 1a is shown in black, 0.25 mg of Bet v 1d in red and 2.4 mg of Bet v 1m in blue. The peaks correspond to monomeric proteins with molecular masses of 19.66 kDa (Bet v 1a), 17.63 kDa (Bet v 1d) and 19.74 kDa (Bet v 1m). C 1H-15N HSQC spectra of 100 μM Bet v 1a (black), Bet v 1d (red) and Bet v 1m (blue) in 50 mM sodium phosphate, 50 mM NaCl, pH 7.0 and 10% 2H2O at 298 K.

https://doi.org/10.1371/journal.pone.0128677.s004

(TIF)

S1 Table. Bet v 1a residues affected from addition of flavonoids with CSPs showing Δδnorm > 0.08 ppm.

https://doi.org/10.1371/journal.pone.0128677.s005

(DOCX)

S2 Table. Bet v 1m residues affected from addition of flavonoids with CSPs showing Δδnorm > 0.08 ppm.

https://doi.org/10.1371/journal.pone.0128677.s006

(DOCX)

S3 Table. Bet v 1d residues affected from addition of flavonoids with CSPs showing Δδnorm > 0.08 ppm.

https://doi.org/10.1371/journal.pone.0128677.s007

(DOCX)

S4 Table. Absorption maxima of unglycosylated flavonoids and their Bet v 1-complexes.

https://doi.org/10.1371/journal.pone.0128677.s008

(DOCX)

Acknowledgments

We thank Detlef Bartel and Frank Führer for providing the serum pool and Stefanie Randow, Ulrike Persau, and Ramona Heissmann for excellent technical assistance.

Author Contributions

Conceived and designed the experiments: CSvL OHS DS RS SV MJH PR. Performed the experiments: CSvL TJ OHS LV CSG. Analyzed the data: CSvL TJ LV DS. Contributed reagents/materials/analysis tools: DS CSG RS SV PR. Wrote the paper: CSvL DS MJH PR.

References

  1. 1. Gell PGH, Coombs RRA. Clinical aspects of immunology. Oxford: Blackwell; 1963.
  2. 2. Ipsen H, Lowenstein H. Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa). J Allergy Clin Immunol. 1983;72: 150–159. pmid:6886253
  3. 3. Vrtala S, Hirtenlehner K, Susani M, Akdis M, Kussebi F, Akdis CA, et al. Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen Bet v 1. FASEB J. 2001;15: 2045–2047. pmid:11511511
  4. 4. Jarolim E, Rumpold H, Endler AT, Ebner H, Breitenbach M, Scheiner O, et al. IgE and IgG antibodies of patients with allergy to birch pollen as tools to define the allergen profile of Betula verrucosa. Allergy. 1989;44: 385–395. pmid:2802112
  5. 5. Dreborg S. Food allergy in pollen-sensitive patients. Ann Allergy. 1988;61: 41–46. pmid:3061321
  6. 6. Geroldinger-Simic M, Zelniker T, Aberer W, Ebner C, Egger C, Greiderer A, et al. Birch pollen–related food allergy: Clinical aspects and the role of allergen-specific IgE and IgG4 antibodies. J Allergy Clin Immunol. 2011;127: 616–622.e1. pmid:21251701
  7. 7. Liu J, Ekramoddoullah AKM. The family 10 of plant pathogenesis-related proteins: Their structure, regulation, and function in response to biotic and abiotic stresses. Physiol Mol Plant Pathol. 2006;68: 3–13.
  8. 8. Casañal A, Zander U, Muñoz C, Dupeux F, Luque I, Botella MA, et al. The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding to Metabolic Intermediates. Journal of Biological Chemistry. 2013;288: 35322–35332. pmid:24133217
  9. 9. Bollen MA, Garcia A, Cordewener JH, Wichers HJ, Helsper JP, Savelkoul HF, et al. Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery. Mol Nutr Food Res. 2007;51: 1527–1536. pmid:17979095
  10. 10. Grote M, Vrtala S, Valenta R. Monitoring of two allergens, Bet v I and profilin, in dry and rehydrated birch pollen by immunogold electron microscopy and immunoblotting. J Histochem Cytochem. 1993;41: 745–750. pmid:8468456
  11. 11. Strack D, Meurer B, Wray V, Grotjahn L, Austenfeld FA, Wiermann R. Quercetin 3-glucosylgalactoside from pollen of Corylus avellana. Phytochemistry. 1984;23: 2970–2971.
  12. 12. Meurer B, Wiermann R, Strack D. Phenylpropanoid patterns in fagales pollen and their phylogenetic relevance. Phytochemistry. 1988;27: 823–828.
  13. 13. Breiteneder H, Ferreira F, Hoffmann-Sommergruber K, Ebner C, Breitenbach M, Rumpold H, et al. Four recombinant isoforms of Cor a I, the major allergen of hazel pollen, show different IgE-binding properties. 1993;212: 355–362. pmid:7916686
  14. 14. Leja M, Kamińska I, Kramer M, Maksylewicz-Kaul A, Kammerer D, Carle R, et al. The Content of Phenolic Compounds and Radical Scavenging Activity Varies with Carrot Origin and Root Color. 2013;68: 163–170. pmid:23613033
  15. 15. Hostetler GL, Riedl KM, Schwartz SJ. Effects of food formulation and thermal processing on flavones in celery and chamomile. Food Chem. 2013;141: 1406–1411. pmid:23790931
  16. 16. Koistinen KM, Soininen P, Venalainen TA, Hayrinen J, Laatikainen R, Perakyla M, et al. Birch PR-10c interacts with several biologically important ligands. Phytochemistry. 2005;66: 2524–2533. pmid:16246382
  17. 17. Seutter von Loetzen C, Hoffmann T, Hartl MJ, Schweimer K, Schwab W, Rösch P, et al. Secret of the major birch pollen allergen Bet v 1: identification of the physiological ligand. Biochemical Journal. 2014;457: 379–390. pmid:24171862
  18. 18. Mogensen JE, Wimmer R, Larsen JN, Spangfort MD, Otzen DE. The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands. J Biol Chem. 2002;277: 23684–23692. pmid:11953433
  19. 19. Hoffmann-Sommergruber K. Plant allergens and pathogenesis-related proteins. What do they have in common? Int Arch Allergy Immunol. 2000;122: 155–166. pmid:10899758
  20. 20. Agarwal P, Agarwal P. Pathogenesis related-10 proteins are small, structurally similar but with diverse role in stress signaling. Mol Biol Rep. 2014;41: 599–611. pmid:24343423
  21. 21. Lebel S, Schellenbaum P, Walter B, Maillot P. Characterisation of the Vitis vinifera PR10 multigene family. 2010;10: 184. pmid:20727162
  22. 22. Breiteneder H, Pettenburger K, Bito A, Valenta R, Kraft D, Rumpold H, et al. The gene coding for the major birch pollen allergen BetvI, is highly homologous to a pea disease resistance response gene. EMBO J. 1989;8: 1935–1938. pmid:2571499
  23. 23. Swoboda I, Jilek A, Ferreira F, Engel E, Hoffmann-Sommergruber K, Scheiner O, et al. Isoforms of Bet v 1, the major birch pollen allergen, analyzed by liquid chromatography, mass spectrometry, and cDNA cloning. J Biol Chem. 1995;270: 2607–2613. pmid:7852325
  24. 24. Schenk M, Gilissen L, Esselink G, Smulders M. Seven different genes encode a diverse mixture of isoforms of Bet v 1, the major birch pollen allergen. BMC Genomics. 2006;7: 168. pmid:16820045
  25. 25. Swoboda I, Scheiner O, Heberle-Bors E, Vicente O. cDNA cloning and characterization of three genes in the Bet v 1 gene family that encode pathogenesis-related proteins. 1995;18: 865–874.
  26. 26. Erler A, Hawranek T, Krückemeier L, Asam C, Egger M, Ferreira F, et al. Proteomic profiling of birch (Betula verrucosa) pollen extracts from different origins. Proteomics. 2011;11: 1486–1498. pmid:21360672
  27. 27. Schenk MF, Cordewener JH, America AH, Van't Westende WP, Smulders MJ, Gilissen LJ. Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen. BMC Plant Biol. 2009;9: 24. pmid:19257882
  28. 28. Ferreira FD, Hoffmann-Sommergruber K, Breiteneder H, Pettenburger K, Ebner C, Sommergruber W, et al. Purification and characterization of recombinant Bet v I, the major birch pollen allergen. Immunological equivalence to natural Bet v I. J Biol Chem. 1993;268: 19574–19580. pmid:8366100
  29. 29. Gajhede M, Osmark P, Poulsen FM, Ipsen H, Larsen JN, Joost van Neerven RJ, et al. X-ray and NMR structure of Bet v 1, the origin of birch pollen allergy. Nat Struct Biol. 1996;3: 1040–1045. pmid:8946858
  30. 30. Kofler S, Asam C, Eckhard U, Wallner M, Ferreira F, Brandstetter H. Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1. J Mol Biol. 2012;422: 109–123. pmid:22634284
  31. 31. Faber C, Lindemann A, Sticht H, Ejchart A, Kungl A, Susani M, et al. Secondary structure and tertiary fold of the birch pollen allergen Bet v 1 in solution. J Biol Chem. 1996;271: 19243–19250. pmid:8702605
  32. 32. Radauer C, Lackner P, Breiteneder H. The Bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands. BMC Evol Biol. 2008;8: 286. pmid:18922149
  33. 33. Fernandes H, Michalska K, Sikorski M, Jaskolski M. Structural and functional aspects of PR-10 proteins. 2013;280: 1169–1199. pmid:23289796
  34. 34. Roth-Walter F, Gomez-Casado C, Pacios LF, Mothes-Luksch N, Roth GA, Singer J, et al. Bet v 1 from lipocalin-like protein acting as allergen only when devoid of iron by promoting Th2 lymphocytes. Journal of Biological Chemistry. 2014.
  35. 35. Ferreira F, Hirtenlehner K, Jilek A, Godnik-Cvar J, Breiteneder H, Grimm R, et al. Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy. J Exp Med. 1996;183: 599–609. pmid:8627171
  36. 36. Ferreira F, Ebner C, Kramer B, Casari G, Briza P, Kungl AJ, et al. Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J. 1998;12: 231–242. pmid:9472988
  37. 37. Markovic-Housley Z, Degano M, Lamba D, von Roepenack-Lahaye E, Clemens S, Susani M, et al. Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier. J Mol Biol. 2003;325: 123–133. pmid:12473456
  38. 38. Göbl C, Madl T, Simon B, Sattler M. NMR approaches for structural analysis of multidomain proteins and complexes in solution. Prog Nucl Magn Reson Spectrosc. 2014;80: 26–63. pmid:24924266
  39. 39. Bohm BA, editor. Introduction to flavonoids. Vol. 2 ed. Amsterdam: Harwood Academic Publishers; 1998.
  40. 40. Srinivas K, King JW, Howard LR, Monrad JK. Solubility and solution thermodynamic properties of quercetin and quercetin dihydrate in subcritical water. J Food Eng. 2010;100: 208–218.
  41. 41. Tao W, Yang N, Duan JA, Wu D, Guo J, Tang Y, et al. Simultaneous determination of eleven major flavonoids in the pollen of Typha angustifolia by HPLC-PDA-MS. Phytochem Anal. 2011;22: 455–461. pmid:22033915
  42. 42. Holgersson J, Gustafsson A, Breimer ME. Characteristics of protein-carbohydrate interactions as a basis for developing novel carbohydrate-based antirejection therapies. Immunol Cell Biol. 2005;83: 694–708. pmid:16266322
  43. 43. Toone EJ. Structure and energetics of protein-carbohydrate complexes. Curr Opin Struct Biol. 1994;4: 719–728.
  44. 44. Lemieux RU. How Water Provides the Impetus for Molecular Recognition in Aqueous Solution. Acc Chem Res. 1999;32: 631–631.
  45. 45. Swaminathan CP, Surolia N, Surolia A. Role of Water in the Specific Binding of Mannose and Mannooligosaccharides to Concanavalin A. J Am Chem Soc. 1998;120: 5153–5159.
  46. 46. Arquint O, Helbling A, Crameri R, Ferreira F, Breitenbach M, Pichler WJ. Reduced in vivo allergenicity of Bet v 1d isoform, a natural component of birch pollen. J Allergy Clin Immunol. 1999;104: 1239–1243. pmid:10589007
  47. 47. Asam C, Batista AL, Moraes AH, de Paula VS, Almeida FC, Aglas L, et al. Bet v 1—a Trojan horse for small ligands boosting allergic sensitization? Clin Exp Allergy. 2014.
  48. 48. Bufe A. The biological function of allergens: relevant for the induction of allergic diseases? Int Arch Allergy Immunol. 1998;117: 215–219. pmid:9876222
  49. 49. Bublin M, Eiwegger T, Breiteneder H. Do lipids influence the allergic sensitization process? J Allergy Clin Immunol. 2014.
  50. 50. Chirumbolo S. The role of quercetin, flavonols and flavones in modulating inflammatory cell function. Inflammation & Allergy-Drug Targets. 2010;9: 263–285.
  51. 51. Mogensen JE, Ferreras M, Wimmer R, Petersen SV, Enghild JJ, Otzen DE. The Major Allergen from Birch Tree Pollen, Bet v 1, Binds and Permeabilizes Membranes. Biochemistry. 2007;46: 3356–3365. pmid:17311414
  52. 52. Golebski K, Roschmann KI, Toppila-Salmi S, Hammad H, Lambrecht BN, Renkonen R, et al. The multi-faceted role of allergen exposure to the local airway mucosa. Allergy. 2013;68: 152–160. pmid:23240614
  53. 53. Karioti A, Kitsaki CK, Zygouraki S, Ziobora M, Djeddi S, Skaltsa H, et al. Occurrence of flavonoids in Ophrys (Orchidaceae) flower parts. 2008;203: 602–609.
  54. 54. Mo Y, Nagel C, Taylor LP. Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen. Proc Natl Acad Sci U S A. 1992;89: 7213–7217. pmid:11607312
  55. 55. Ceska O, Styles ED. Flavonoids from Zea mays pollen. Phytochemistry. 1984;23: 1822–1823.
  56. 56. STEVENS FA, MOORE D, BAER H. The isolation of isoquercitrin from giant ragweed pollen; the electrophoretic pattern and biologic activity of the pigment. J Allergy. 1951;22: 165–169. pmid:14823829
  57. 57. Sosa F, Percheron F. Isolation and identification of a quercetol-3-sophoroside in Alnus cordata Desf. pollen. C R Acad Sci Hebd Seances Acad Sci D. 1965;261: 4544–4546. pmid:4954804
  58. 58. EL RIDI MS, STRAIT LA, ABOUL WAFA MH. Isolation of rutin from the pollen grain of the date palm (Dactylifera Palma L.). Arch Biochem Biophys. 1952;39: 317–321. pmid:12997166
  59. 59. Jia SS, Liu YL, Ma CM, Yang SL, Zhou HM, Zhao DC, et al. Studies on the constituents of the flavonoids from the pollen of Typha angustfolia L. (puhuang). Yao Xue Xue Bao. 1986;21: 441–446. pmid:3811929
  60. 60. Arraez-Roman D, Zurek G, Bässmann C, Almaraz-Abarca N, Quirantes R, Segura-Carretero A, et al. Identification of phenolic compounds from pollen extracts using capillary electrophoresis-electrospray time-of-flight mass spectrometry. Anal Bioanal Chem. 2007;389: 1909–1917. pmid:17899027
  61. 61. Buters JTM, Weichenmeier I, Ochs S, Pusch G, Kreyling W, Boere AJF, et al. The allergen Bet v 1 in fractions of ambient air deviates from birch pollen counts. Allergy. 2010;65: 850–858. pmid:20132158
  62. 62. Buters JTM, Kasche A, Weichenmeier I, Schober W, Klaus S, Traidl-Hoffmann C, et al. Year-to-Year Variation in Release of Bet v 1 Allergen from Birch Pollen: Evidence for Geographical Differences between West and South Germany. Int Arch Allergy Immunol. 2008;145: 122–130. pmid:17848805
  63. 63. Lavola A, Nybakken L, Rousi M, Pusenius J, Petrelius M, Kellomäki S, et al. Combination treatment of elevated UVB radiation, CO2 and temperature has little effect on silver birch (Betula pendula) growth and phytochemistry. Physiol Plantarum. 2013;149: 499–514.
  64. 64. Cvetković D, Markovic D, Cvetković D, Radovanovic B. Effects of continuous UV-irradiation on the antioxidant activities of quercetin and rutin in solution in the presence of lecithin as the protective target. J Serb Chem Soc. 2011;76: 973–985.
  65. 65. Smith GJ, Thomsen SJ, Markham KR, Andary C, Cardon D. The photostabilities of naturally occurring 5-hydroxyflavones, flavonols, their glycosides and their aluminium complexes. J Photochem Photobiol A. 2000;136: 87–91.
  66. 66. Firon N, Nepi M, Pacini E. Water status and associated processes mark critical stages in pollen development and functioning. Annals of Botany. 2012;109: 1201–1214. pmid:22523424
  67. 67. Pacini E. Cell biology of anther and pollen development. In: Williams E, Clarke A, Knox RB, editors.: Springer Netherlands; 1994. pp. 289–308.
  68. 68. Buitink J, Claessens MMAE, Hemminga MA, Hoekstra FA. Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen. Plant Physiol. 1998;118: 531–541. pmid:9765538
  69. 69. Morales LO, Tegelberg R, Brosché M, Lindfors A, Siipola S, Aphalo PJ. Temporal variation in epidermal flavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula. Physiol Plantarum. 2011;143: 261–270. pmid:21883252
  70. 70. Heslop-Harrison J. Incompatibility and the Pollen-Stigma Interaction. Annu Rev Plant Physiol. 1975;26: 403–425.
  71. 71. Hiscock SJ, Allen AM. Diverse cell signalling pathways regulate pollen-stigma interactions: the search for consensus. New Phytol. 2008;179: 286–317. pmid:19086285
  72. 72. Mittag D, Akkerdaas J, Ballmer-Weber B, Vogel L, Wensing M, Becker W, et al. Ara h 8, a Bet v 1–homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy. J Allergy Clin Immunol. 2004;114: 1410–1417. pmid:15577846
  73. 73. Riecken S, Lindner B, Petersen A, Jappe U, Becker WM. Purification and characterization of natural Ara h 8, the Bet v 1 homologous allergen from peanut, provides a novel isoform. Biol Chem. 2008;389: 415–423. pmid:18208358
  74. 74. Yamamoto M, Torikai S, Oeda K. A major root protein of carrots with high homology to intracellular pathogenesis-related (PR) proteins and pollen allergens. Plant Cell Physiol. 1997;38: 1080–1086. pmid:9360325
  75. 75. Ballmer-Weber BK, Wangorsch A, Bohle B, Kaul S, Kündig T, Fötisch K, et al. Component-resolved in vitro diagnosis in carrot allergy: Does the use of recombinant carrot allergens improve the reliability of the diagnostic procedure?. 2005;35: 970–978. pmid:16008686
  76. 76. Breiteneder H, Hoffmann-Sommergruber K, O'Riordain G, Susani M, Ahorn H, Ebner C, et al. Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and structural relationships to a group of 17-kDa tree pollen allergens. Eur J Biochem. 1995;233: 484–489. pmid:7588792
  77. 77. Hoffmann-Sommergruber K, Ferris R, Pec M, Radauer C, O’Riordain G, Laimer da CM, et al. Characterization of Api g 1.0201, a New Member of the Api g 1 Family of Celery Allergens. Int Arch Allergy Immunol. 2000;122: 115–123. pmid:10878490
  78. 78. Scheurer S, Metzner K, Haustein D, Vieths S. Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen. Mol Immunol. 1997;34: 619–629. pmid:9393965
  79. 79. Reuter A, Fortunato D, Garoffo LP, Napolitano L, Scheurer S, Giuffrida MG, et al. Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics. Proteomics. 2005;5: 282–289. pmid:15593144
  80. 80. Karlsson AL, Alm R, Ekstrand B, Fjelkner-Modig S, Schiott A, Bengtsson U, et al. Bet v 1 homologues in strawberry identified as IgE-binding proteins and presumptive allergens. Allergy. 2004;59: 1277–1284. pmid:15507096
  81. 81. Muñoz C, Hoffmann T, Escobar NM, Ludemann F, Botella MA, Valpuesta V, et al. The strawberry fruit Fra a allergen functions in flavonoid biosynthesis. Mol Plant. 2010;3: 113–124. pmid:19969523
  82. 82. Vanekkrebitz M, Hoffmannsommergruber K, Machado MLD, Susani M, Ebner C, Kraft D, et al. Cloning and Sequencing of Mal d 1, the Major Allergen from Apple (Malus domestica), and Its Immunological Relationship to Bet v 1, the Major Birch Pollen Allergen. Biochem Biophys Res Commun. 1995;214: 538–551. pmid:7677763
  83. 83. Ziadi S, Poupard P, Brisset M, Paulin J, Simoneau P. Characterization in apple leaves of two subclasses of PR-10 transcripts inducible by acibenzolar-S-methyl, a functional analogue of salicylic acid. Physiol Mol Plant Pathol. 2001;59: 33–43.
  84. 84. Atkinson RG, Perry J, Matsui T, Ross GS, Macrae EA. A stress-, pathogenesis-, and allergen-related cDNA in apple fruit is also ripening-related. N Z J Crop Hortic Sci. 1996;24: 103–107.
  85. 85. Pühringer H, Moll D, Hoffmann-Sommergruber K, Watillon B, Katinger H, da Câmara Machado, Margit Laimer. The promoter of an apple Ypr10 gene, encoding the major allergen Mal d 1, is stress- and pathogen-inducible. 2000;152: 35–50.
  86. 86. Holm J, Baerentzen G, Gajhede M, Ipsen H, Larsen JN, Lowenstein H, et al. Molecular basis of allergic cross-reactivity between group 1 major allergens from birch and apple. J Chromatogr B Biomed Sci Appl. 2001;756: 307–313. pmid:11419722
  87. 87. Hoffmann-Sommergruber K, Vanek-Krebitz M, Radauer C, Wen J, Ferreira F, Scheiner O, et al. Genomic characterization of members of the Bet v 1 family: genes coding for allergens and pathogenesis-related proteins share intron positions. Gene. 1997;197: 91–100. pmid:9332353
  88. 88. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995;6: 277–293. pmid:8520220
  89. 89. Johnson BA, Blevins RA. NMRview: A computer program for the visualization and analysis of NMR data. J Biomol NMR. 1994;4: 603–614. pmid:22911360
  90. 90. Schweimer K, Sticht H, Nerkamp J, Boehm M, Breitenbach M, Vieths S, et al. NMR Spectroscopy Reveals Common Structural Features of the Birch Pollen Allergen Bet v 1 and the Cherry Allergen Pru a 1. Appl Magn Reson. 1999;17: 449–456.
  91. 91. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23: 2947–2948. pmid:17846036
  92. 92. Kelley LA, Sternberg MJ. Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc. 2009;4: 363–371. pmid:19247286
  93. 93. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31: 455–461. pmid:19499576
  94. 94. Schüttelkopf AW, van Aalten DM. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr. 2004;60: 1355–1363. pmid:15272157
  95. 95. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. 2009;30: 2785–2791. pmid:19399780
  96. 96. Vogel L, Lüttkopf D, Hatahet L, Haustein D, Vieths S. Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system. Allergy. 2005;60: 1021–1028. pmid:15969682