The cashew allergens : a molecular and serological characterization

Cashew nut allergy can be a severe food allergy of which the prevalence appears to be increasing. The aim of this thesis was a comprehensive molecular and serological characterisation of the cashew nut allergens Ana o 1, 2 and 3 for improved diagnosis and characterisation of patient populations. <strong>Chapter 1 </strong>in this thesis provides background information on cashew nuts, allergy, the allergens Ana o 1, 2 and 3, the effect of heat treatments on cashew nut proteins, the digestibility of cashew nut proteins, cross-reactivity between cashew nut proteins and other tree nuts, and the detection of cashew nut in food products. Subsequently, in <strong>Chapter 2</strong>, a review is presented on the topic of epithelial protein and allergen transport. This review describes multiple pathways of intestinal protein transport, sums up existing experimental data concerning protein and peptide transport, and presents different methods to study this. Interestingly, the pathway of (allergenic) protein transport can differ between sensitized and non-sensitized persons. In sensitized persons, protein transport occurs transcellularly via enterocytes, and paracellularly with the involvement of mast cells, while in non-sensitized persons microfold cells and enterocytes are considered most important. In the next three chapters, cashew nut allergens were studied. Cashew nut allergy and cashew nut allergens were chosen because of a high number of undiagnosed cashew nut allergic children reported at the children’s hospital “Kinderhaven”, in Rotterdam, an outpatient clinic that is involved in this study. <strong>Chapter 3 </strong>describes a protocol for the purification of Ana o 1, 2 and 3 from cashew nuts. Ana o 1 and 3 were purified by protein extraction, salt precipitation and filtering over a 30kDa molecular weight membrane. Ana o 2 was purified by protein extraction followed by gel filtration chromatography. These purified proteins were characterised by SDS-PAGE, western blot, glycoprotein stain, and protein identification. In this chapter also more in-depth analysis was performed on the N- and C-termini of the large and small subunits of Ana o 3. These N- and C-termini of Ana o 3, as well as the SDS-PAGE protein profiles were compared between cashew nuts of different origins in <strong>Chapter 4</strong>. In this chapter also the effects of different heat treatments on the electrophoretic behaviour of cashew nut allergens from various origins were studied, using both 1D and 2D electrophoresis. In these data no significant differences were detected between the electrophoresis patterns of Ana o 1, 2 or 3 in the various origins of cashew nuts. Some small but significant differences in Ana o 1, 2 and 3 content, however, were detected between the differently heated cashew nuts. No major differences in N- and C-terminal micro-heterogeneity were detected between cashew nuts of different origins. Next, in <strong>Chapter 5</strong>, the cashew nut allergens Ana o 1, 2 and 3 were produced as recombinant proteins using a yeast (<em>P. pastoris</em>) production system. This procedure was used as recombinant allergens often produce higher yields of higher purity compared to native purified allergens. The recombinant proteins were compared to the native cashew nut proteins for their glycosylation pattern, IgE binding capacity, and 2D electrophoresis profile. In <strong>Chapter 6</strong>, the major findings of this thesis are discussed. An overview of the protein characteristics (e.g. 1D and 2D electrophoresis profile, glycosylation, IgE binding, pepsin-digestibility) was provided, as well as a discussion on the clinical benefits that can be derived from the results obtained in this thesis. Also some additional results are presented, studying the serologic cross-reactivity between cashew nuts and other tree nuts and Anacardiaceae nuts and fruits. This thesis provides an in-depth study regarding the protein characteristics of the cashew nut allergens Ana o 1, 2 and 3. Using the allergens that were purified in this thesis project, the serum IgE levels of Ana o 1, 2 and 3 could be measured in cashew nut-allergic children. The allergens were also recombinantly produced to obtain higher quantity of allergens for regular use in diagnostics of cashew nut allergy. The results from this thesis can potentially expand clinical patient characterisation with measurements of IgE levels to purified and recombinantly produced major cashew nut allergens. These results might have applications for other food allergens or patient populations.