SummaryBackground: Little is known on the clinical relevance of peanut 2S albumin Ara h 7.
Summary Background Screening for specific IgE against 2S albumin proteins Ara h 2 and 6 has good positive predictive value in diagnosing peanut allergy. From the third 2S member Ara h 7, 3 isoforms have been identified. Their allergenicity has not been elucidated. Objective This study investigated the allergenicity of Ara h 7 isoforms compared to Ara h 2 and 6. Methods Sensitization of 15 DBPCFC‐confirmed peanut‐allergic patients to recombinant Ara h 2.0201, Ara h 6.01 and isoforms of recombinant Ara h 7 was determined by IgE immunoblotting strips. A basophil activation test (BAT) was performed in 9 patients to determine IgE‐cross‐linking capacities of the allergens. Sensitivity to the allergens was tested in 5 patients who were sensitized to at least 1 Ara h 7 isoform, by a concentration range in the BAT. 3D prediction models and sequence alignments were used to visualize differences between isoforms and to predict allergenic epitope regions. Results Sensitization to Ara h 7.0201 was most frequent (80%) and showed to be equally potent as Ara h 2.0201 and 6.01 in inducing basophil degranulation. Sensitization to Ara h 7.0201 together with Ara h 2.0201 and/or 6.01 was observed, indicating the presence of unique epitopes compared to the other 2 isoforms. Differences between the 3 Ara h 7 isoforms were observed in C‐terminal cysteine residues, pepsin and trypsin cleavage sites and 3 single amino acid substitutions. Conclusion & clinical relevance The majority of peanut‐allergic patients are sensitized to isoform Ara h 7.0201, which is functionally as active as Ara h 2.0201 and 6.01. Unique epitopes are most likely located in the C‐terminus or an allergenic loop region which is a known allergenic epitope region for Ara h 2.0201 and 6.01. Due to its unique epitopes and allergenicity, it is an interesting candidate to improve the diagnostic accuracy for peanut allergy.
Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus were cultivated in media containing "MOO$-. The distribution of "Mo in cells grown under conditions of repression and derepression of nitrogenase synthesis, was investigated by anion-exchange (DEAE-Sephacel) chromatography, Cells of K. pneumoniae took up MOO:-only under conditions of derepression of nitrogenase thus serving the formation of the FeMo cofactor of the MoFe protein (Kpl) as the predominant Mo-containing species. In the case of A. vinelandii, under diazotrophic growth conditions, molybdenum was preferably incorporated into the nitrogenase MoFe protein (Avl). However, if excess amounts of molybdate were present in the medium, molybdenum was also bound to the Mo-storage protein. In the presence of 20 mM NH,', conditions which completely repress nitrogenase formation, molybdenum accumulated in the Mo-storage protein exclusively. This protein proved to be unstable towards DEAE-Sephacel, apparently releasing all the molybdenum in form of MOO:-during the fractionation procedure. R. capsulatus contained, in addition to the MoFe protein (Rcl), significant amounts of other not-yet-identified Mo species, which partially are formed under conditions of both, repression and derepression of nitrogenase.The Mo centers of all these compounds were characterized by measuring the nuclear quadrupole interaction of the process "Mo(P-)"Tc using time differential perturbed angular correlation spectroscopy. Keywords: nitrogenase ; molybdenum-iron protein ; molybdenum-storage protein; time differential perturbed angular correlation (TDPAC) spectroscopy.The biological nitrogen fixation is one of the most important processes in nature, because it is essential for the N-cycle and thus for the life of all organisms (for reviews see [l-31). The conventional nitrogenase enzyme system responsible for the reduction of molecular N, to NHf consists of two separable proteins, the MoFe protein (component 1) and the Fe protein (com- whose primary role is the mediation of electrons in nitrogenase, and (b) the iron-molybdenum cofactor (FeMo cofactor, FeMoco), which is located in the a-subunits and has been postulated to be the active site for substrate (e.g. N,) reduction. Based on X-ray structure analyses of MoFe proteins from Azotobacter vinelandii and Clostridium pasteurianum, a structural cofactor model has recently been published [6, 71. According to this model the FeMo cofactor consists of two cubane fragments (Fe,S, and MoFe,S,) bridged by three inorganic sulfide ligands. The Mo center is ligated by two 0 atoms from homocitrate and one N atom from histidine of the protein side chain. In this work we determined the nuclear quadrupole interacprotein and in other Mo-containing proteins present in different (TDPAC) spectroscopy Of y-rays. This method detects the hyperfine interaction between the nuclear quadruPole moment of the probe nucleus (in our case 9 9 M~) and an electric field gradient (EFG). Since the EFG depends on the distribution tion (NQI) parameters of...
Background The peanut allergens Ara h 2, h 6, and h 7 are potent allergens and can trigger severe reactions. Ara h 7 consists of three isoforms differing in their ability to induce basophil degranulation, whereas the ability of Ara h 7.0201 is comparable to Ara h 2 and 6 as shown in previous literature. Objective To identify linear epitopes of Ara h 7.0101, Ara h 7.0201 and Ara h 7.0301 recognized by IgE and IgG4 from patients sensitized to Ara h 7 and to investigate their potential to elucidate divergent abilities of the Ara h 7 isoforms in inducing basophil activation. Methods Linear epitopes recognized by IgE and IgG4 were mapped by peptide microarray analysis containing 15‐mer peptides of Ara h 2.0201, 6, 7.0101, 7.0201 and 7.0301 and 39 peanut allergic patients sensitized to Ara h 7 (discovery). For validation, 20‐mer peptides containing the minimal epitope and surrounding amino acids were incubated with 25 sensitized patients and 10 controls (validation). Results Three out of 14 linear epitopes were unique for each isoform (Ara h 7.0101: aa 97‐109; Ara h 7.0201: aa 122‐133; Ara h 7.0301: aa 65‐74) but scarcely recognized by IgE. The main linear IgE epitope (aa 51‐57) located in the long flexible loop of all Ara h 7 isoforms was bound by antibodies from 31% of the patients (discovery and validation cohort). Regarding IgG4, 55% of the patients recognized an epitope present on all isoforms (aa 55‐65), whereas epitope aa 129‐137, only present on Ara h 7.0101/0.0301, was recognized by 38% of the patients. Recognition was highly individual, although 20% of the patients recognized any linear epitope neither by IgE nor by IgG4 despite a low mean z‐score of ≥ 1.7. Remarkably, only 50% of the patients recognized one or more epitopes by IgE. Conclusion & Clinical Relevance Ara h 7 isoforms share many linear epitopes being easily accessible for antibody binding. Unique epitopes, essential to elucidate divergent potencies, were scarcely recognized, suggesting a crucial involvement of conformational epitopes.
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Background In daily practice, one-third of sesame allergic patients, confirmed by clinical history or food challenge, do not show any detectable specific IgE using current diagnostics. Currently used sesame extracts are water-based and therefore lacking hydrophobic proteins like oleosins. Oleosins, the stabilizer of lipid droplets in plants, are described as allergens in sesame, peanut and hazelnut. In this study, we examine the role of oleosins in sesame allergy and their potential cross-reactivity between sesame and (pea)nuts. Methods Specific IgE and IgG sensitisation to native and heterologously expressed sesame components and oleosins from other nuts, free of seed storage proteins, was assessed by line blot and sera from 17 sesame allergic patients without detectable specific IgE sensitisation to sesame, and compared to 18 sesame allergic and 13 tolerant patients with specific IgE sensitisation to sesame. Results Sesame allergic patients without sensitisation showed no specific IgE to the tested sesame oleosins or components. Low levels of specific IgE to sesame oleosins were detected in 17% of sesame allergic and 15% of tolerant patients with sIgE sensitisation. Oleosins were recognised by serum IgG from multiple patients confirming immune reactivity and excluding technical issues leading to lack of specific IgE-binding to oleosins. Conclusion Sesame oleosins are minor allergens and appear to have no additonal value in diagnosing sesame allergy in adults based on sIgE and sIgG detection. There is a high need for additional diagnostic tools in those patients to minimize the number of required food challenges. Electronic supplementary material The online version of this article (10.1186/s13601-019-0271-x) contains supplementary material, which is available to authorized users.
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