A total of 84 participants with fruit allergies (including allergies except peaches) were enrolled in the Fruits Allergy Component Study Group (http://www.fruit-allergy.jp/) from June 2014 to December 2015. After enrolment, we applied to the ethical review boards of each facility and examined only those who were approved after obtaining written informed consent. Exclusion criteria included participants who had no symptoms when they ate peach or those whose symptoms were unclear or not reproducible. After obtaining informed consent, the participants answered the questionnaire and then underwent a skin-prick test with peach. This study was conducted according to the World Medical Association's Declaration of Helsinki. Ethical approval was obtained from the Fujita Health University Ethics Committee in July 2014 (reference number: 14-075) for all sites taking part in this study.

We evaluated the following types of symptoms induced by the ingestion of peach according to the participant's questionnaire as follows: Oral reaction (OR): Itching or tingling sensations in the oral mucosa, palate, or throat that developed within 5–10min after peach ingestion and localized symptoms with no SR.27 SR: Symptoms presented in ≥2 organs after peach ingestion. Systemic symptoms included anaphylaxis and food-dependent exercise-induced anaphylaxis.

Peaches (P. persica, cultivar Asama–Hakutou strain) at the commercial ripening stage were obtained from a local store. Entire peach fruits (peel and pulp) were homogenized with an extraction solution (2mM disodium ethylenediamine tetraacetate, 10mM sodium N,N-diethyldithiocarbamate, 3mM sodium azide, and 2% solid polyvinyl polypyrrolidone) at a 1:1 [w:v] ratio. After filtration through gauze, the homogenate was centrifuged at 10,000×g for 15min at 4°C. A cation exchange resin (Toyopearl CM-650M; TOSO, Tokyo, Japan) was added to the supernatant and mixed overnight at 4°C. The resin was collected by centrifugation, packed, and washed with 20mM phosphate buffer at pH 5.0 in a column. The proteins absorbed to the resin were eluted as the crude extract with 0.5M sodium chloride in the same buffer.

The production of hybridomas producing mAbs specific to peach Pru p 7 or Pru p 3 and the purification of natural antigens were essentially as described previously.28 The hybridomas that produced IgG reactive to the crude extract were cloned twice with the limiting dilution method and inoculated into pristine-primed mice. Then, mAbs were prepared from the ascitic fluids and purified on Protein G-Sepharose columns (GE Healthcare, Fairfield, Connecticut, USA). The mAbs specific to peach Pru p 7 or Pru p 3 were selected using an immunoblotting analysis with the crude extract under non-reducing conditions. The animal experiments were performed under the guidelines of the Animal Experiment Committee of Kyoto Women's University following a bulletin (No. 71, 2006) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan.

Pru p 7 or Pru p 3 was purified from the crude extract using a HiTrap NHS-activated HP column coupled with each mAb according to the manufacturer's instructions (GE Healthcare).

Total RNA was isolated from peach from 200 to 400mg net weight using Sepasol (Nacalai Tesque Inc., Kyoto, Japan) according to the manufacturer's protocol. Complementary DNA (cDNA) was obtained using 0.5μg total RNA, 100U reverse transcriptase II (Invitrogen, Carlsbad, CA, USA), a random primer, 10mM dithiothreitol (DTT), and 20U RNase inhibitor (Nacalai Tesque Inc.) in the presence of a reaction buffer provided from the manufacturer. The full-length Pru p 7 (locus name ppa014086 m.g, Phytozome site, https://phytozome.jgi.doe.g.,ov/pz/portal.html) was obtained by polymerase chain reaction (PCR) amplification. The primers used were 5′-GGTTCATCTTTCTGTGACTCCAAGT-3′ and 5′-TTAAGGGCATTTGGGGTTGCCCTTG-3′. Restriction endonuclease sites were extended at the 5′ end of both primers. The PCR reaction was performed through 25 cycles at 94°C for denaturing, 55°C for annealing, and 68°C for extension. The amplified product was visualized using agarose electrophoresis and ethidium bromide staining. First, the product was cloned into pZERO2 (Invitrogen), and the correct construction of the plasmids was confirmed by sequencing. The Pru p 7 cDNA was digested using BamHI and XhoI and cloned into the BamHI and XhoI sites of pGEX-6p-2 (GE Healthcare) to make pGEX-Pru p 7 for the expression in E. coli and cloned into the SnaBI site of pPIC9K (Invitrogen) to make pPIC9K-Pru p 7 for the expression and secretion into the medium of P. pastoris strain GS115 (Invitrogen).

To induce the expression of GST Pru p 7, 0.2mM IPTG was added in the Luria–Bertani media/50μg/mL ampicillin and incubated at 30°C for 4h. E. coli was pelleted by centrifugation at 10,000×g for 8min and was suspended in PBS at pH 7.4 containing 0.1mM DTT and 0.2mM phenylmethylsulfonyl fluoride. After sonication four times at a power of 12W×30s each time, the clear lysate was obtained by centrifugation at 10,000×g for 30min. After the addition of glutathione beads to the lysate and overnight incubation, erPru p 7 was released from the beads by treatment with PreScission Protease according to the manufacturer's instructions (GE Healthcare).

For the expression of prPru p 7, pPIC9K-Pru p 7 was transformed into P. pastoris. Land transformants that did not require histidine were selected and expanded in buffered minimal glycerol medium (100mM potassium phosphate, pH 6.0, 1.34% Yeast Nitrogen Base, 4×10−5% biotin, and 1% glycerol). The induction of prPru p 7 was achieved using buffered minimal methanol medium (100mM potassium phosphate, pH 6.0, 1.34% Yeast Nitrogen Base, 4×10−5% biotin, 0.5% methanol) at 30°C, and the expression of prPru p 7 was examined in the culture supernatant.

ErPru p 7 and prPru p 7 were purified using HiTrap NHS-activated HP columns coupled with anti-Pru p 7 mAb similar to the purification of nPru p 7.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at a constant current of 80mA under reducing or non-reducing conditions. The gel was dyed using Coomassie Brilliant Blue R-250.

The proteins on the gel were transferred to a PVDF membrane (BIO-RAD, Hercules, California, USA), and the membrane was blocked with PBS containing 5% skim milk for 1h at room temperature. After incubation of the membrane with anti-Pru p 7 or Pru p 3 mAb (hybridoma culture supernatant) for 1h at room temperature, alkaline phosphatase-labeled anti-mouse IgG (American Qualex, San Clemente, CA, USA) was added and incubated for 1h at room temperature. The color reaction was performed using an alkaline phosphatase coloring kit (Nacalai Tesque Inc.).

Protein concentration was determined using a DC Protein Assay kit (Bio-Rad, Bradford, UK). The N-terminal amino acid sequences of Pru p 7 and Pru p 3 were determined using a 476A gas-phase protein sequencer (Applied Biosystems, CA, USA). The molecular mass and purity of Pru p 7 and Pru p 3 were also checked using an Autoflex III matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, MA, USA). Amino acid sequences were analyzed using the Phytozome and BLAST programs to identify the proteins in the databases.

A skin-prick test using a bifurcated needle with fresh peach was performed with the prick-to-prick technique. A wheal diameter of ≥3mm was defined as positive.29 The entire peach, including the skin, was used for pricking. Peaches used for the prick test were prepared at each facility.

The specific IgE to peach, Pru p 3, Bet v 1, and Bet v 2, were measured by 3gAllergy™ (Siemens Healthcare Diagnostic Inc., Tarrytown, NY, USA). In this study, to simplify calculations, 0.08IUA/mL was substituted for all values <0.1IUA/mL.

IgE in patients’ sera specific to Pru p 7 or Pru p 3 was determined using ELISA as described previously with some modifications.6 The wells of a microtiter plate (Nunc A/S, Roskilde, Denmark) were coated with Pru p 7 or Pru p 3 (2μg/mL in PBS) at 50μL/well. After blocking, the individual sera (1:10 dilution with the solution containing 0.1% bovine serum albumin and 0.1% Tween 20 in 10mM Tris–HCl buffer, pH 7.4) were added at 50μL/well and incubated for 1h at 37°C. The IgE Abs bound to the antigen were detected using alkaline phosphatase-labeled anti-human IgE (American Qualex, San Clemente, CA, USA) and p-nitrophenylphosphate (1mg/mL) as the substrate. Absorbance was read at 405nm using a microplate reader (BioRad). Mean+10 standard deviations of the negative control values were defined as positive.

Correlations between the clinical symptoms of peach-allergic patients and sensitization to peach, Bet v 1, Bet v 2, nPru p 3, and Pru p 7 were analyzed using the Mann–Whitney U-test. Data sets of nPru p 7, erPru p 7, and prPru p 7 were analyzed for normal distribution using the Kolmogorov–Smirnov normality test. Because most of the data sets were not normally distributed, the non-parametric Friedman test with Dunn's multiple comparison post-test was used. All data were analyzed using GraphPad Prism version 6.0. (GraphPad Software Inc., San Diego, CA, USA).

We examined 27 participants who met the criteria (9 men and 18 women; age range, 7–54 years; median age, 13 years). The participants were classified into the SR (n=17, 6 men and 11 women; age range, 7–21 years; median age, 12.5 years) and OR groups (n=10, 3 men and 7 women; age range, 9–54 years; median age, 13 years) (Table 1).

All the participants were positive for the skin-prick test against peach. The level of the specific IgE to Bet v 1 in the OR group was significantly higher than that in the SR group (P<0.0001). The Mann–Whitney U-test showed no significant difference in the level of the specific IgE to peach, Bet v 2, and nPru p 3 between the SR and OR groups (Fig. 1). It was noted that only patient no. 18 in the SR group showed a positive response against nPru p 3, consistent with the results of the 3gAllergy™ test. As reported previously,6 the level of the specific IgE to nPru p 7 in the SR group was significantly higher than that in the OR group (P=0.0011).

Figure 1.

Specific IgE to peach, Bet v 1, Bet v 2, erPru p 3, n Pru p 3, nPru p 7.

Specific IgE levels for Peach, Bet v 1, Bet v 2, erPru p 3 in patients’ sera were determined using 3gAllergy™ and those for n Pru p 3, n Pru 7were determined using ELISA.

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The crude extract solution was prepared using the cation-exchange resin from fully ripened peaches (Fig. 2, lane 1), and a one-step purification of nPru p 7 was performed using an anti-Pru p 7 mAb column (Fig. 2, lane 3). Pru p 3 was passed through the antibody column (Fig. 2, lane 2). On the basis of the results of western blotting, it was evident that nPru p 7 was completely purified and free from Pru p 3. nPru p 3 was also purified using the anti-Pru p 3 mAb column (data not shown). MALDI-TOF MS analysis also indicated the purity and molecular weight as 6898Da and 9136Da for nPru p 7 and nPru p 3, respectively (Fig. 3).

Figure 2.

Purification of nPru p 7 by using the anti-Pru p 7 monoclonal antibody column.

Peach crude extract was applied to the anti-Pru p 7 column. The flow through and the eluate fractions were examined on SDS-PAGE under reducing conditions.

(A) Stained with Coomassie Brilliant Blue, (B) Western analysis by using anti-Pru p 7 monoclonal antibody, (C) Western analysis by using anti-Pru p 3 monoclonal antibody.

Lane

apply (peach crude extract), Lane

antibody column flow through, Lane

antibody column eluate. MW indicates molecular weight marker (in kilodaltons).

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Figure 3.

MALDI-TOF-MS analysis of nPru p 7 and nPru p 3.

Purity and molecular mass of nPru p 7 (left) and nPru p 3 (right) were analyzed by MALDI-TOF-MS. Small peaks with half and dimeric sizes are artifacts.

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We extracted RNA from peaches (P. persica, cultivar Asama–Hakutou strain) before they fully ripened and cloned the cDNA of Pru p 7 using PCR. Isolation and sequencing of the cDNA revealed a 189-bp open reading frame coding for a 63-amino acid polypeptide. The nucleotide sequence of the open reading frame was entirely consistent with that obtained from a database. Moreover, the expected molecular weight was consistent with the value mentioned above obtained by MALDI-TOF MS.

The cDNA clone was first expressed in E. coli as a fusion protein with glutathione S-transferase, with a molecular weight of approximately 34kDa (Fig. 4A-a, lanes 2 and 3). The lysate of the recombinant E. coli was applied to glutathione-Sepharose beads, and erPru p 7 was released from the beads by protease treatment. erPru p 7 was electrophoresed at almost the same position as nPru p 7 in SDS-PAGE (Fig. 4A-a, lanes 4 and 6) and stained with monoclonal anti-Pru p 7 antibody on western blot analysis (Fig. 4A-b). The N-terminal amino acid sequence analysis of erPru p 7 revealed the sequence (GPL)GSSFCDSKCGVRCSKAG, where the original sequence of nPru p 7 (GSSFCD) was correctly expressed following the first tripeptide (GPL) derived from the vector and added by recombination.

Figure 4.

Expression of rPru p 7 by E. Coli and P. pastoris.

(A) Detection of erPru p 7 expressed in E. coli.

(a) SDS-PAGE under reducing conditions stained with Coomassie Brilliant Blue.

(b) Western analysis by using anti-Pru p 7 monoclonal antibody.

before induction of expression,

after induction of expression,

glutathione beads before cleavage,

eluate after cleavage,

beads after cleavage,

nPru p 7.

(B) Detection of prPru p 7 secreted in the medium of P. pastoris on western analysis by using anti-Pru p 7 monoclonal antibody.

culture supernatant after 24h,

after 48h,

after 72h,

after 96h,

after 120h,

nPru p 7 100ng,

nPru p 7 50ng.

MW indicates molecular weight marker (in kilodaltons).

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Pru p 7 cDNA was also expressed in P. pastoris, and prPru p 7 was secreted into the medium by the signal sequence of the vector. Secreted prPru p 7 in the medium was too faint to be detected by Coomassie staining but was visible on western blot analysis (Fig. 4B). Secreted prPru p 7 migrated slightly slower than nPru p 7 probably because of the addition of the extra pentapeptide (EAEAY) at recombination. The N-terminal amino acid sequence analysis of prPru p 7 revealed the sequence (EAEAY) GSSFCD, where the original sequence of nPru p 7 was correctly expressed following the first pentapeptide.

To compare the reactivities of erPru p 7 and prPry p 7 with serum IgE in peach-allergic patients, erPru p 7 and prPry p 7 were purified with mAb columns, similar to nPru p 7. As the reference, Pru p 3 was cloned, and erPru p 3 was expressed in E. coli and purified using an anti-Pru p 3 mAb column.

Finally, in the SR group, we compared the reactivity of patients’ IgE to nPru p 7 and rPru p 7 using ELISA (Fig. 1). Because the data did not exhibit normal distribution by the Kolmogorov–Smirnov test, we decided to perform non-parametric analysis. First, a significant difference was observed between each antigen by the Friedman test (P<0.05). Then, Dunn's multiple comparison post-test revealed that a significant difference was not observed between nPru p 7 and prPru p 7 (P>0.05) but was observed among nPru p 7 and erPru p 7 (P<0.05), erPru p 7 and prPru p 7 (P<0.001) (Fig. 5).

Figure 5.

Reactivity of patients’ IgE to natural and recombinant Pru p 7 in ELISA.

IgE in the sera of the systemic group (No. 11–27 in Table 1) was determined by ELISA with nPru p 7, erPru p 7, prPru p 7.

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