Search

Ara h 2 is a relevant peanut allergen linked to severe allergic reactions. The interaction of Ara h 2 with components of the sensitization phase of food allergy (e.g., dendritic cells) has not been investigated, and could be key to understanding the allergenic potential of this allergen. In this study, we aimed to analyze such interactions and the possible mechanism involved. Ara h 2 was purified from two forms of peanut, raw and roasted, and labeled with a fluorescent dye. Human monocyte-derived dendritic cells (MDDCs) were obtained, and experiments of Ara h 2 internalization by MDDCs were carried out. The role of the mannose receptor in the internalization of Ara h 2 from raw and roasted peanuts was also investigated. Results showed that Ara h 2 internalization by MDDCs was both time and dose dependent. Mannose receptors in MDDCs had a greater implication in the internalization of Ara h 2 from roasted peanuts. However, this receptor was also important in the internalization of Ara h 2 from raw peanuts, as opposed to other allergens such as raw Ara h 3.

Oral allergy syndrome (OAS) describes an allergic reaction where an individual sensitized by pollen allergens develops symptoms after eating certain foods. OAS is caused by cross-reactivity among a class of proteins ubiquitous in plants called pathogenesis related class 10 (PR-10) proteins. The best characterized PR-10 protein is Bet v 1 from birch pollen and its putative function is binding hydrophobic ligands. We cloned a subset of seven recombinant PR-10 proteins from pollens, peanuts, and hazelnuts and developed a standard purification method for them. Immunoglobulin E (IgE) binding of purified PR-10 proteins was analyzed by ImmunoCAP ISAC microarray and enzyme-linked immunosorbent assays (ELISAs) with sera from allergic patients. We investigated the binding activities of PR10s by testing 16 different ligands with each protein and compared their secondary structures using circular dichroism (CD). The PR-10s in this study had very similar CD spectra, but bound IgE with very different affinities. All seven proteins showed a similar pattern of binding to the polyphenol ligands (resveratrol, flavonoids, and isoflavones) and variable binding to other potential ligands (fatty acids, sterols, and plant hormones). We suggest our protocol has the potential to be a near-universal method for PR-10 purification that will facilitate further research into this important class of panallergens., Competing Interests: The authors declare no conflict of interest.

Roasting is known to change the allergenic properties of peanuts. To study these observations at a molecular level, the relationship of IgE binding to the structure of Ara h 3 from raw and roasted peanuts was assessed. Ara h 3 (A3) was purified from raw (R), light roast (LR) and dark roast (DR) peanuts, the purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the secondary structures were compared with circular dichroism (CD) spectroscopy. In order to understand the contribution of structure to IgE binding, the R A3 was partially denatured (PD) by heat treatment (65 °C for 2 h), subjected to CD spectroscopy and IgE spot blot analysis with sera from peanut- allergic individuals. While we observed that the secondary structure of purified A3 from R and LR peanut in solution was affected by the reduction of disulfide bonds and heat treatment when purified from the peanut following the roasting process, only small alterations were seen in the secondary structure. The purified LR A3 bound higher levels of IgE than the RA3. CD spectroscopy of PD A3 revealed a reduction in the percentage of alpha helices, and serum IgE binding. Therefore, while A3 purified from roasted peanuts did not show significant changes in secondary structure, it showed higher IgE binding than R A3. Therefore, the higher IgE binding to LR A3 was more likely to be due to chemical modifications than structural changes. However, a decrease in the IgE binding was seen if R A3 was deliberately unfolded, indicating that the structure played an important role in IgE binding to A3.

Allergic reactions to food are on the rise worldwide and there is a corresponding increase in interest to understand the molecular mechanisms responsible. Peanut allergies are the most problematic because the reaction often persists into adulthood and can be as severe as anaphylaxis and death . The purpose of the work presented here was to develop a reproducible method to produce large quantities of pure recombinant Ara h 1(rAra h 1) that will enable standardization of immunological tests for patients and allow structural and immunological studies on the wild type and mutagenized forms of the protein. Ara h 1 is initially a pre-pro-protein which, following two endoproteolytic cleavages, becomes the mature form found in peanut. The mature form however has flexible regions that make it refractory to some structural studies including crystallography. Therefore, independent purification of the mature and core regions was desirable. Expression constructs were synthesized cDNA clones for each in a pET plasmid vector without tags. Codons were optimized for expression in E. coli . High-level expression was achieved in BL21 strains. Purification to near homogeneity was achieved by a combination of ammonium sulfate precipitation and ion exchange chromatography. The purified rAra h 1 was then compared with natural Ara h 1 for IgE binding. All patients recognized both the folded natural and rAra h 1, but the IgE binding to the rArah1 was significantly reduced in comparison to the natural allergen, which could potentially make it useful for immunotherapeutic purposes.

It has been suggested that the boiling or frying of peanuts leads to less allergenic products than roasting. Here, we have compared the digestibility of the major peanut allergens in the context of peanuts subjected to boiling, frying or roasting and in purified form. The soluble peanut extracts and the purified allergens were digested with either trypsin or pepsin and analyzed by gel electrophoresis and western blot. T-cell proliferation was measured for the purified allergens. In most cases, boiled and raw peanut proteins were similarly digestible, but the Ara h 1 protein in the boiled extracts was more resistant to digestion. Most proteins from fried and roasted peanuts were more resistant to digestion than in raw and boiled samples, and more IgE binding fragments survived digestion. High-molecular-weight fragments of Ara h1 were resistant to digestion in fried and roasted samples. Ara h 1 and Ara h 2 purified from roasted peanuts were the most resistant to digestion, but differed in their ability to stimulate T-cells. The differences in digestibility and IgE binding properties of the major allergens in roasted, fried and boiled peanuts may not explain the difference between the prevalence of peanut allergy in different countries that consume peanut following these varied processing methods.