Distinct Lipid Transfer Proteins display different IgE‐binding activities that are affected by fatty acid binding

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Additional supporting information may be found online in the Supporting Information section at the end of the article. The different natural and recombinant nsLTPs were purified and characterized ( Figure S1). rMal d 3, rCor a 8, and nHel a 3 bound the ANS probe to varying extent ( Figure 1A). All proteins showed the lowest preference for STE ( Figure 1B pronounced reduction of ANS fluorescence, equal to 59%, 23%, and 46% at 50 μmol/L for rMal d 3, rCor a 8, and nHel a 3, respectively.
Eighteen patients' sera containing specific IgE to Pru p 3 and/or Cor a 8 (Table S1) were tested by direct IgE-ELISA applying rMal d 3, rCor a 8, and nHel a 3 ( Figure S2). We used selected sera (n = 6 for rMal d 3 and rCor a 8 each) to test if the IgE binding to the proteins is influenced by the interaction with OLE and STE, chosen as representatives of unsaturated and saturated fatty acid, respectively.
Due to its very low IgE reactivity, nHel a 3 was excluded from this analysis. Preincubation of rMal d 3 with OLE significantly increased IgE-binding (P < 0.05), whereas STE did not affect rMal d 3 IgE-binding properties ( Figure 1E). Regarding rCor a 8, both fatty acids induced an increase in IgE-binding, but it was statistically significant only for OLE (P < 0.01; Figure 1F).
We performed computational calculations, detecting some differ- important residues are highlighted. Note: The Mal d 3 model used for computations has one additional amino acid, Ala, at the N-terminus and, hence, the indexes of protein residues discussed for the 3D model are by 1 larger with respect to the sequence reported in Figure S1 Recently, the direct immunomodulatory effect of food-derived lipids has gained increasing interest. Tordesillas et al showed the adjuvant activity of the natural ligand of Pru p 3 in the allergen sensitization. 9 Summarizing the recent findings, including our data, it still remains to be fully elucidated whether lipids alone or in complexes can activate key players of both, the innate and adaptive immune system within an allergic response.
In conclusion, this multidisciplinary study analyzed for the first time the ligand-binding capacities of Mal d 3, Cor a 8, and Hel a 3 and confirmed that these individual homologous allergens display different, higher and lower, IgE-binding activities, due to differences in their epitope structure.  items, the FAQLQ-TF contains 23 items, and the FAQLQ-CF contains 24 items. Each item is scored on a 7-point scale and ranges from 1 (minimal impairment in HRQL) to 7 (maximal impairment in HRQL). The FAQLQs have been shown to be valid, reliable and responsive instruments. [1][2][3][4][5][6][7] In order to ascertain whether HRQL items of the FAQLQ-CF, FAQLQ-TF and FAQLQ-AF were suitable to peanut-allergic patients, some retrospective analysis was performed using databases generated during the development and validation of the FAQLQs.
Firstly, all patients with a physician-diagnosed peanut allergy were selected from the development database to identify FAQLQ items that were reported most frequently and had the highest impact scores (ie, overall importance) for peanut-allergic patients (ranging from 1.0 "not" to 5.0 "extremely"). 1-3 Additionally, the overall importance of each FAQLQ item was compared and correlated (Pearson correlation coefficient) between peanut-allergic patients and non-peanut-allergic patients and tested for significance (P < 0.05).
Secondly, the cross-sectional validity 1-3 of the FAQLQs was determined in a peanut-allergic subgroup. Spearman correlation coefficients (ρ) were calculated between the FAQLQ mean scores and the Food Allergy Independent Measure (FAIM) mean score. The FAIM is an instrument that measures the patients perceived disease severity and has previously been used successfully in validating the FAQLQs. 6 Moderate correlation was to be expected (0.40-0.60). [1][2][3][4] Thirdly, the longitudinal validity 4 of the FAQLQs was determined in a peanut-allergic subgroup. Correlations were calculated between the change in total FAQLQ and the change in total FAIM score of patients who underwent a double-blind placebo-controlled food challenge (DBPCFC) with peanut (longitudinal validity). HRQL was measured 1 month before (baseline) and 6 months after (follow-up) a DBPCFC.
Data were analysed with SPSS software for Windows. Table 1 shows the participant characteristics.
Overall importance analysis showed that all items exceeded the previously used cut-off level of >1.37 in the peanut-allergic subgroup. 1-3 Figure 1 shows that peanut-allergic patients reported a higher overall impact than non-peanut-allergic patients (P < 0.05). Thus, all items may be considered to be important. The Pearson correlation coefficient between the overall importance of peanut-allergic patients and nonpeanut-allergic patients was 0.70, P < 0.001 (FAQLQ-AF), 0.71, P < 0.001 (FAQLQ-TF) and 0.80, P < 0.001 (FAQLQ-CF).
Cross-sectional validity analysis showed that the total FAQLQ score correlated significantly with the total FAIM score in the LETTERS TO THE EDITOR