Major Peach Allergen Research Articles

R-mandelonitrile-lyase, homolog to Pru d 10, is a major peach allergen in peach allergic Spanish population.

Peach allergy is a prevalent cause of food allergy. Despite the repertoire of allergens available for molecular diagnosis, there are still patients with undetectable IgE levels to peach allergens but presenting symptoms after its ingestion. The objective of this study was to investigate the allergenic profile in a patient population with symptoms produced by peach. An exploratory retrospective study was performed with patients presenting symptoms after the ingestion of peach. Forty-two patients were included in the study. The allergenic profile of individual patients was investigated by immunoblot. A serum pool was prepared with the sera that recognized a 70 kDa band. This pool was used to detect this protein in peach peel and pulp and to identify the 70 kDa protein in 2D immunoblot. Spots recognized in the 2D immunoblot were sequenced by LC-MS/MS. Inhibition studies were performed between peach peel and almond. Twenty-two patients (52.4%) recognized the 70 kDa protein in immunoblot. This protein was recognized in peel and pulp. Two different spots were observed in 2D-PAGE, both were identified as (R)-mandelonitrile lyases (RML) with high amino acid similarity with Pru du 10. Peach RML were partially inhibited with an almond extract. No association was found between any reported symptom and sensitization to RML. RML-sensitized patients were older and reported pollen associated respiratory symptoms more frequently than negative patients. A new peach allergen, a RML, homologous of Pru du 10, recognized by 52% of the population has been identified.

A synthetic glycodendropeptide induces methylation changes on regulatory T cells linked to tolerant responses in anaphylactic-mice.

Lipid transfer proteins (LTPs) are allergens found in a wide range of plant-foods. Specifically, Pru p 3, the major allergen of peach, is commonly responsible for severe allergic reactions. The need for new alternatives to conventional food allergy treatments, like restrictive diets, suggests allergen immunotherapy as a promising option. It has been demonstrated that sublingual immunotherapy (SLIT) with synthetic glycodendropeptides, such as D1ManPrup3, containing mannose and Pru p 3 peptides induced tolerance in mice and that the persistence of this effect depends on treatment dose (2nM or 5nM). Moreover, it produces changes associated with differential gene expression and methylation profile of dendritic cells, as well as phenotypical changes in regulatory T cells (Treg). However, there are no works addressing the study of epigenetic changes in terms of methylation in the cell subsets that sustain tolerant responses, Treg. Therefore, in this work, DNA methylation changes in splenic-Treg from Pru p 3 anaphylactic mice were evaluated. It was performed by whole genome bisulphite sequencing comparing SLIT-D1ManPrup3 treated mice: tolerant (2nM D1ManPrup3), desensitized (5nM D1ManPrup3), and sensitized but not treated (antigen-only), with anaphylactic mice. Most of the methylation changes were found in the gene promoters from both SLIT-treated groups, desensitized (1,580) and tolerant (1,576), followed by the antigen-only (1,151) group. Although tolerant and desensitized mice showed a similar number of methylation changes, only 445 genes were shared in both. Remarkably, interesting methylation changes were observed on the promoter regions of critical transcription factors for Treg function like Stat4, Stat5a, Stat5b, Foxp3, and Gata3. In fact, Foxp3 was observed exclusively as hypomethylated in tolerant group, whereas Gata3 was only hypomethylated in the desensitized mice. In conclusion, diverse D1ManPrup3 doses induce different responses (tolerance or desensitization) in mice, which are reflected by differential methylation changes in Tregs.

Impact of Different Lipid Ligands on the Stability and IgE-Binding Capacity of the Lentil Allergen Len c 3.

Previously, we isolated the lentil allergen Len c 3, belonging to the class of lipid transfer proteins, cross-reacting with the major peach allergen Pru p 3 and binding lipid ligands. In this work, the allergenic capacity of Len c 3 and effects of different lipid ligands on the protein stability and IgE-binding capacity were investigated. Impacts of pH and heat treating on ligand binding with Len c 3 were also studied. It was shown that the recombinant Len c 3 (rLen c 3) IgE-binding capacity is sensitive to heating and simulating of gastroduodenal digestion. While being heated or digested, the protein showed a considerably lower capacity to bind specific IgE in sera of allergic patients. The presence of lipid ligands increased the thermostability and resistance of rLen c 3 to digestion, but the level of these effects was dependent upon the ligand’s nature. The anionic lysolipid LPPG showed the most pronounced protective effect which correlated well with experimental data on ligand binding. Thus, the Len c 3 stability and allergenic capacity can be retained in the conditions of food heat cooking and gastroduodenal digestion due to the presence of certain lipid ligands.

Transcriptional Profiling of Dendritic Cells in a Mouse Model of Food-Antigen-Induced Anaphylaxis Reveals the Upregulation of Multiple Immune-Related Pathways.

Much of the knowledge about gene expression during anaphylaxis comes from candidate gene studies. Despite their potential role, expression changes in dendritic cells (DCs) have not been studied in this context using high throughput methods. The molecular mechanisms underlying food-antigen-induced anaphylaxis are investigated using DCs from an animal model. RNA sequencing is used to study gene expression in lymph-node-derived DCs from anaphylactic mice sensitized intranasally with the major peach allergen Pru p 3 during the acute reaction phase, induced intraperitoneally. In total, 237 genes changed significantly, 181 showing at least twofold changes. Almost three-quarters of these increase during anaphylaxis. A subset is confirmed using RT-PCR in a second set of samples obtained from a new batch of mice. Enrichment analysis shows an overrepresentation of genes involved in key immune system and inflammatory processes, including TGF-β signaling. Comparison with a study using anaphylactic human subjects show significant overlap. The findings provide a comprehensive overview of the transcriptional changes occurring in DCs during anaphylaxis and help elucidate the mechanisms involved. They add further weight to the putative role of these cells in anaphylaxis and highlight genes that may represent potential therapeutic targets.

Immunotherapy with Native Molecule rather than Hypoallergenic Variant of Pru p 3, the Major Peach Allergen, Shows Beneficial Effects in Mice.

Background The use of hypoallergenic derivatives is considered beneficial to promote the safety and efficacy of allergen-specific immunotherapy. We aimed to assess the efficacy of reduced and alkylated (R/A) Pru p 3, a hypoallergenic folding variant of the major peach allergen, in subcutaneous immunotherapy (SCIT) using a murine model of peach allergy. Methods and Results After sensitization with Pru p 3, BALB/c mice received SCIT with Pru p 3 or R/A Pru p 3 and were challenged with Pru p 3. SCIT with Pru p 3, but not with R/A Pru p 3, suppressed anaphylaxis upon the challenge significantly. SCIT with Pru p 3 did not suppress Pru p 3-specific IgE and IgG1 production, but enhanced IgG2a production. In contrast, SCIT with R/A Pru p 3 suppressed IgE and IgG1 production, but enhanced IgG2a production only moderately. The therapeutic efficacy of SCIT with Pru p 3 was associated with induction of IL-10 and IFN-γ. Conclusion Hypoallergenic folding variant of Pru p 3 is not likely an efficacious therapeutic component in SCIT of peach allergy. The lower efficacy of R/A Pru p 3 might be attributed to poor antigenicity and/or weak stability due to its unfolded conformation.

LPS promotes Th2 dependent sensitisation leading to anaphylaxis in a Pru p 3 mouse model

Pru p 3 is the major peach allergen in the Mediterranean area. It frequently elicits severe reactions, limiting its study in humans, raising the need for animal models to investigate the immunological mechanisms involved. However, no anaphylaxis model exists for Pru p 3. We aimed to develop a model of peach anaphylaxis by sensitising mice with Pru p 3 in combination with lipopolysaccharide (LPS) as an adjuvant. Four groups of mice were sensitised intranasally: untreated; treated with Pru p 3; treated with LPS; treated with Pru p 3 + LPS. After sensitisation mice were intraperitoneally challenged with Pru p 3 and in vivo and in vitro parameters were evaluated. Only mice in the Pru p 3 + LPS group showed anaphylaxis symptoms, including a decrease in temperature. Determination of in vitro parameters showed a Th2 response with an increase of Pru p 3-specific IgE and IgG1. Moreover, at the cellular level, we found increased levels of IgE and IgG1 secreting Pru p 3-specific cells and a proliferative CD4+ T-cell response. These results demonstrate that Pru p 3-specific anaphylaxis can be generated after nasal sensitisation to Pru p 3 in combination with LPS. This is a promising model for evaluating food allergy immunotherapies.

Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein

The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity.

Hypoallergenic allergen derivatives of Pru p 3 for immunotherapy of IgE-mediated peach allergy

The major peach allergen Pru p 3 is responsible for allergies occurring mainly in the Mediterranean area. Hypoallergenic derivatives of Pru p 3 are disclosed which can be used as a vaccine in the treatment of allergic patients.

Component-resolved diagnosis of peach allergy and its relationship with prevalent allergenic pollens in China

Component-resolved diagnosis of peach allergy and its relationship with prevalent allergenic pollens in China

Peel LTP (Pru p 3) – the major allergen of peach – is methylated. A proteomic study

Lipid transfer protein (LTP, Pru p 3) is the major allergen of peach (Prunus persica), and is in a greater abundance in the peel than in the pulp of the fruit. Peel LTP is more allergenic than pulp LTP, but it is not clear whether this is due to its specific allergenic properties or to its higher concentration.In this study, we have used a new one-step, rapid procedure for the purification of LTP from peel and pulp of four peach varieties [Gladys (white flesh), California (nectarine yellow flesh), Plusplus (yellow flesh), Red Fair (nectarine yellow flesh)] harvested in a field grown in Southern Italy.Purification was based on miniature reversed-phase chromatography, a procedure suitable for proteomic study. Proteomic analysis of purified LTPs revealed that the amino acid sequence of LTP was identical in all peach genotypes but, for the first time, peel LTP was found to be methylated.

복숭아, 앵두, 고추의 교차반응성 및 소화효소안정성

복숭아는 오래 전부터 알레르기를 일으키는 식품으로 알려져 있으나 특정 식품과의 교차반응에 대한 연구는 부족한 실정이다. 본 연구에서는 한국에서 재배되고 있는 복숭아, 앵두, 고추의 단백질을 추출하여 복숭아 알레르기를 가진 환자가 앵두와 고추에 대해 교차반응성을 일으키는지 확인하고 이 단백질들의 소화효소처리에 따른 안정성을 조사하였다. 복숭아, 앵두 및 고추 단백질 추출물은 Tricine-SDS-PAGE상에서 3 kDa부터 26 kDa 이상까지 넓은 범위의 단백질 분포를 보였으나 각기 다른 패턴을 가지는 것을 확인하였다. 복숭아 단백질의 항원성을 확인하기 위해 복숭아 민감성 환자의 혈청과의 IgE반응성을 확인한 결과, 환자 혈청은 모두 복숭아의 주요 항원성 단백질 lipid transfer protein로 예상되는 9 kDa 부근에서 강한 IgE결합력을 보였으며 또한 9 kDa 단백질이 anti-LTP1 polyclonal antibody에 의해 검출되는 것을 확인하였다. 앵두와 고추 단백질 추출물은 23 kDa 부근에서의 IgE결합력이 9 kDa과의 반응성보다 크게 나타났다. 복숭아, 앵두 및 고추의 소화효소에 대한 안정성을 SDS-PAGE 상에서 확인한 결과, 복숭아, 앵두 및 고추의 항원성 단백질은 인공 위액 및 장액에 의해 완전히 분해되지 않아 알레르기를 유발할 잠재성이 있는 것을 확인할 수 있었다. 본 연구 결과를 바탕으로, 복숭아, 앵두 및 고추 단백질 추출물이 소화효소처리에 안정성을 가지는 특성을 가지며 복숭아 알레르기 증상을 가지는 사람에게 앵두 및 고추는 교차반응성을 일으킬 가능성이 크다는 것을 확인할 수 있었다. Peach (Prunus persica) has been recognized as a food allergen for over 20 years. However, there is little information about cross-reactivity with other foods. The aim of this study was to research cross-reactivity of Korean cherry and hot pepper on patients allergic to peach and its stability by digestive enzyme treatment. Peach, Korean cherry, and hot pepper proteins were extracted and separated by Tricine-SDS-PAGE analysis. The protein extracts had a wide range of molecular weight, from 3 kDa to more than 26 kDa, and displayed different patterns of protein bands on Tricine-SDS-PAGE. Peach allergic patients' sera were used to detect the allergenic protein in three samples. Three peach allergic patients' sera reacted strongly with 9 kDa protein of peach, which was the expected lipid transfer protein (LTP) as the major allergen of peach and was detected with anti-LTP1 polyclonal antibody. However, the reactivity of the 23 kDa protein in Korean cherry and hot pepper protein was stronger than that of the 9 kDa protein. The stability of protein extracts on digestive enzyme treatment was examined using simulated gastric fluids (SGF) and simulated intestinal fluids (SIF), in which digestive enzyme stability is one of the characteristics of allergen potentially causing food allergy. Findings confirmed that allergenic proteins in peach, Korean cherry, and hot pepper were not completely digested by SGF and SIF treatments from results of SDS-PAGE analysis. These results confirmed that Korean cherry and hot pepper might cause cross-reactivity in peach allergic patients, and its allergenic proteins have stability against digestive enzymes.

Effect of Pru p 3 on dendritic cell maturation and T-lymphocyte proliferation in peach allergic patients

BackgroundPru p 3 is the major peach allergen and the most frequent cause of food allergy in adults in the Mediterranean area. Although its allergenicity is well characterized, its ability to generate a T-cell response is not completely known. ObjectiveTo investigate the influence of Pru p 3 allergen on dendritic cell (DC) maturation and specific T-cell response (TH1/TH2) in peach allergic patients. MethodsPeach allergic patients (n = 11) and tolerant controls (n = 14) were included in the study. Monocyte-derived DC maturation after incubation with Pru p 3 was evaluated by the increase of maturational markers (CD80, CD86, and CD83) by flow cytometry. Lymphocyte proliferation was evaluated by coculturing monocyte-derived DCs and 5,6-carboxyfluorescein diacetate N-succinimidyl ester–stained lymphocytes with different concentrations of Pru p 3 (25, 10, and 1 μg/mL) by flow cytometry and cytokine production. ResultsPru p 3 induced a significant increase in the CD80, CD86, and CD83 expression on stimulated DCs from patients compared with controls. The lymphocyte proliferative response after Pru p 3 stimulation was also significantly higher along with an increase in interleukin 8 in patients compared with tolerant controls. ConclusionPru p 3 allergen induces changes in DC maturational status mainly in peach allergic patients. An increase in lymphocyte proliferative response accompanied with a different cytokine pattern was also observed compared with healthy controls.

Extraction and mass spectrometry identification of a major peach allergen Pru p 1

Peach allergy can be caused by the allergen Pru p 1. This occurs by cross-reactivity with the homologous birch pollen allergen Bet v 1. However, the direct identification of Pru p 1 as an immunoglobulin E (IgE)-binding protein extracted from peach fruit has never been reported. Phosphate-buffered saline (PBS) and phenol extractions were applied to solubilise the proteins from peach peel and pulp, and IgE immunoblotting with sera of individual peach-allergic patients was used to detect the potential allergens. Most of the patients showed binding to an 18 kDa band in IgE immunoblotting performed with the phenolic extracts of peach peel and pulp, but not when the PBS extracts were used. Mass spectrometry of the 18 kDa spot excised from a two-dimensional electrophoretic gel showed this protein to correspond to the peach allergen Pru p 1. Phenol extraction was necessary to detect by IgE immunoblotting a major peach allergen, which showed very low extractability with PBS, indicating the appropriateness of adopting different extraction procedures to identify plant allergens. The 18 kDa peach protein was definitively identified as the Bet v 1-homologous peach allergen Pru p 1.

Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen

Allergen-specific immunotherapy for food allergies, including peach allergy, has not been established. Use of allergens with reduced allergenic potential and preserved immunogenicity could improve the safety and efficacy of allergen-specific immunotherapy. We sought to create a hypoallergenic derivative of the major peach allergen Pru p 3 and to characterize its biochemical and immunologic properties. A Pru p 3 folding variant generated by means of reduction and alkylation was investigated for structural integrity and stability to gastrointestinal enzymes. IgE reactivity and allergenic potency were determined by means of immunoblotting, ELISA, and in vitro mediator release assay with sera from patients with peach allergy. T-cell immunogenicity was investigated by using human allergen-specific T cells and CBA/J mice immunized with either native Pru p 3 (nPru p 3) or reduced and alkylated (R/A) Pru p 3. Pru p 3 processing by endolysosomal fractions of dendritic cells and antigenicity was examined in mice. Unfolding of Pru p 3 reduced its high resistance to gastrointestinal proteolysis and almost completely abrogated its IgE reactivity and allergenic potency. However, R/A Pru p 3 was capable of stimulating human and murine T cells. Endolysosomal degradation of R/A Pru p 3 was accelerated in comparison with nPru p 3, but similar peptides were generated. IgG and IgE antibodies raised against nPru p 3 showed almost no cross-reactivity with R/A Pru p 3. Moreover, the antigenicity of R/A Pru p 3 was strongly reduced. Unfolded Pru p 3 showed reduced allergenicity and antigenicity and preserved T-cell immunogenicity. The hypoallergenic variant of Pru p 3 could be a promising vaccine candidate for specific immunotherapy of peach allergy.

In vitro gastrointestinal digestion of the major peach allergen Pru p 3, a lipid transfer protein: Molecular characterization of the products and assessment of their IgE binding abilities

A simulated gastrointestinal digestion has been carried out on purified peach lipid transfer protein, one of the main allergens among the population of the Mediterranean area and the major allergen of peach allergic patients. The percentage of intact protein, after extensive digestion, measured by comparison with a non-digestible peptide analogue used as internal standard, was found to be about one-third of the original protein content. The peptides formed in digested fraction were characterized by means of LC/MS. The products of the digestion essentially derived from trypsin action, whereas the protein appeared to be resistant to pepsin and chymotrypsin. The identified peptides could be classified as low molecular weight and high molecular weight peptides. The latter consisted of the full protein, with the disulfide bridges still intact, deprived of the smaller peptides. The different digestion products, including the high and low molecular weight peptides, were purified by LC and assessed, together with the intact protein, by dot-blot analysis with sera of allergic patients, allowing to estimate their potential allergenicity. The intact protein and the high molecular weight peptides were found to be recognized by patients' sera, whereas the small peptides were found to be not reactive.

Characterization of the T-Cell Epitopes of the Major Peach Allergen Pru p 3

Background: Pru p 3 is the major peach allergen recognized by more than 90% of peach-allergic individuals of the Mediterranean area. Identification of the dominant Pru p 3 T-cell epitopes can improve our understanding of the immune responses against this protein and could be helpful in the development of hypoallergenic immunotherapy. For this purpose, we examined the phenotypes, specificities and cytokine secretion profiles of proliferating T cells in response to Pru p 3 in peach-allergic individuals. Methods: Peripheral blood mononuclear cells from 15 peach-allergic patients were incubated with Pru p 3. The proliferation of antigen-specific T-cell lines (TCLs) was assessed by tritiated methylthymidine incorporation. T-cell epitopes were identified by analyzing the reactivity of TCLs against 8 overlapping peptides spanning the entire length of Pru p 3. We characterized the phenotype of Pru-p-3-specific TCLs by flow cytometry and analyzed their production of interleukin (IL) 4 and γ-interferon (IFN-γ) by ELISA. Results: Ninety-two Pru-p-3-specific TCLs were isolated (stimulation index ≧5). These TCLs proliferated mainly in response to Pru p 3_12–27 and Pru p 3_57–72. Pru-p-3-specific TCLs were mainly CD4+ (81%) and expressed cell surface CD30. In addition, TCLs produced high levels of IL-4 and low levels of IFN-γ, indicating a Th2 phenotype. Conclusions: Two immunodominant T-cell-reactive regions of Pru p 3 were identified: Pru p 3_12–27 and Pru p 3_57–72. These peptides showed a differential ability to elicit a Th2 response. Taken together, our results provide a better understanding of the immunological T-cell reactivity against Pru p 3.

T-cell epitopes of the major peach allergen, Pru p 3: Identification and differential T-cell response of peach-allergic and non-allergic subjects

Lipid transfer proteins (LTPs), particularly peach Pru p 3, are the most relevant plant food allergens in the South of Europe, and, therefore, their allergic properties have been extensively studied. However, neither T-cell epitopes nor their effect on the patients’ T-cell response has been investigated in any member of the LTP panallergen family. The objective of the present study was to map the major T-cell epitopes of Pru p 3, as well as to evaluate their induced T-cell response in peach-allergic versus control subjects. Thus, peripheral blood mononuclear cells (PBMCs) from 18 peach-allergic patients and Pru p 3-specific T-cell lines (TCLs) from 9 of them were cultured with Pru p 3 and with a panel of 17 derived peptides (10-mer overlapping in 5 amino acids representing the full sequence of Pru p 3). Proliferation in 5-day assays was carried out via tritiated-thymidine incorporation, while IL4 and IFNγ production was assessed via sandwich enzyme-linked immunosorbent tests (ELISA) of TCL culture supernatants. The results were compared to those obtained from 10 non-peach allergic control volunteers. Two consecutive peptides showed the highest activation capacity. About 74% of PBMCs and TCLs recognized them, forming a single T-epitope: Pru p 365–80. Additionally, other specific T-cell epitopes were observed. Pru p 325–35 was detected by more than 60% of TCLs from peach-allergic patients, and Pru p 345–55 only activated PBMCs from control subjects. Interestingly, TCLs from patients were associated with a Th2-type, whereas control TCLs presented a Th1-type cytokine response. The major immunogenic T-cell epitope identified in Pru p 3, Pru p 365–80, is a good candidate to develop new vaccines for hypersensitivity reactions associated with LTP allergens from Rosaceae fruits.

Mimotope mapping as a complementary strategy to define allergen IgE-epitopes: Peach Pru p 3 allergen as a model

Lipid transfer proteins (LTPs) are the major allergens of Rosaceae fruits in the Mediterranean area. Pru p 3, the LTP and major allergen of peach, is a suitable model for studying food allergy and amino acid sequences related with its IgE-binding capacity. In this work, we sought to map IgE mimotopes on the structure of Pru p 3, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms (OAS-pool or SYS-pool), and used for screening of a random dodecapeptide phage display library. Positive clones were further confirmed by ELISA assays testing individual sera from each pool. Three-dimensional modelling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surface. Twenty-one phage clones were selected using Pru p 3-specific IgE, 9 of which were chosen using OAS-specific IgE while the other 12 were selected with systemic-specific IgE. Peptide alignments revealed consensus sequences for each pool: L37 R39 T40 P42 D43 R44 A46 P70 S76 P78 Y79 for OAS-IgE, and N35 N36 L37 R39 T40 D43 A46 S76 I77 P78 for systemic-IgE. These 2 consensus sequences were mapped on the same surface of Pru p 3, corresponding to the helix 2-loop-helix 3 region and part of the non-structured C-terminal coil. Thus, 2 relevant conformational IgE-binding regions of Pru p 3 were identified using a random peptide phage display library. Mimotopes can be used to study the interaction between allergens and IgE, and to accelerate the process to design new vaccines and new immunotherapy strategies.

Assessing allergen levels in peach and nectarine cultivars

The lipid transfer protein Pru p 3 has been identified as a major peach fruit allergen. However, the putative peach member of the Bet v 1 family, Pru p 1, has been neither identified nor characterized. To determine the distribution and solubility properties of the main peach allergens and to quantify Pru p 3 and Pru p 1 levels in peach and nectarine cultivars. Peach peel and pulp were extracted using different buffers, and extracts were analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection using polyclonal antibodies against lipid transfer proteins, profilins, and Bet v 1 homologues. Pru p 3 was quantified in peach and nectarine cultivars using a sandwich enzyme-linked immunosorbent assay method. A similar method was developed to quantify Pru p 1. A differential distribution between peel and pulp and different solubility properties were found for Pru p 3, Pru p 1, and peach profilin. Mean Pru p 3 levels were 132.86, 0.61, and 16.92 microg/g of fresh weight of peels, pulps, and whole fruits, respectively. The corresponding mean Pru p 1 levels were 0.62, 0.26, and 0.09 microg/g of fresh weight. Most US cultivars showed higher levels of both allergens than Spanish cultivars. The different distribution and solubility properties of the main peach allergens can determine the quality of fruit extracts used as diagnostic tools. These differences, together with the natural variation of Pru p 3 and Pru p 1 levels among peach and nectarine cultivars, can be exploited to reduce peach allergenicity by means of industrial processing and plant breeding.

Lipid Transfer Proteins and Allergy to Oranges

Background: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy. Methods: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3. Results: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens. Conclusions: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30–50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.