Characterization of Low Molecular Weight Allergens from English Walnut (Juglans regia)

Although English walnut is a commonly allergenic tree nut, walnut allergens have been poorly characterized to date. The objective of this work was to characterize the natural, low molecular weight (LMW) allergens from walnut. A protocol was developed to purify LMW allergens (specifically 2S albumins) from English walnuts. In addition to 2S albumins, a series of peptides from the N-terminal region of the 7S seed storage globulin proprotein were also identified and characterized. These peptides comprised a four-cysteine motif (C-X-X-X-C-X_10–12-C-X-X-X-C) repeated throughout the 7S N-terminal region. Upon IgE immunoblotting, 3/11 and 5/11 sera from walnut-allergic subjects showed IgE reactivity to the 7S N-terminal fragments and 2S albumin, respectively. The mature 7S protein and the newly described 7S N-terminal peptides represent two distinct types of allergens. Because the proteolytic processing of 7S globulins has not been elucidated in many edible plant species, similar protein fragments may be present in other nuts and seeds

Clinical data of patients included in this study.

<p>Clinical data of patients included in this study.</p

Average weight of allergens included in the co-sensitization graph.

<p>Average weight of allergens included in the co-sensitization graph.</p

Recognition frequencies of food and pollen LTPs by geographical area.

<p>The recognition frequencies, shown as percentage of positive response (%), were obtained incubating the LTP microarray with single sera from allergic patients. Only LTPs with a positive response of more than 20% (taking all patients into account) are represented. Percentage positive responses and significant differences (p<0.05) are indicated.</p

Purified proteins included in the LTP microarray.

<p>r recombinant protein;</p>*<p>non-LTPs or control proteins included in the array are marked with an asterisk.</p

Lipid transfer proteins (LTP) included in the homemade array were separated by SDS-PAGE and stained with Coomassie Blue.

<p>Replicas were electrotransferred and incubated with polyclonal antibodies produced against peach LTP (dilution 1∶500). The name of the proteins corresponds to Table I.</p

Frequency of LTP sensitization.

<p><b>A</b>. Recognition frequencies of food and pollen LTPs comparing fruit allergic patients with and without pollen sensitization (Pollen Fruit Allergy and Fruit Allergy, respectively). The recognition frequencies, shown as percentage of positive response (%), were obtained incubating the LTP microarray with single sera from allergic patients. <b>B</b>. Analysis of LTP crossreactivities by inhibition assays using the LTP microarray as solid phase and Pru p 3, Art v 3 and Cas s 8 (5, 1, 0.1, 0.01 µg/mL) as inhibitors. The inhibition percentage of the IgE binding capacity is indicated. Means (n = 3) and SDs (bars) are represented. All tests were performed in triplicate.</p

Pollen counts (grains/m³ of air) of the regions included in the study.

*<p>Average pollen counts (grains/m³). The period is indicated in brackets.</p><p>Data were obtained as the average of the previous years (period in parentheses), from the Comité de Aerobiología-SEAIC (<a href="http://www.polenes.com/concentraciones.html" target="_blank">http://www.polenes.com/concentraciones.html</a>) and the PIA-Punto de información de Aerobiología-UAB (<a href="http://lap.uab.cat/aerobiologia/" target="_blank">http://lap.uab.cat/aerobiologia/</a>).</p

Co-sensitization graph of LTP allergens.

<p>Each node represents one allergen (LTP, white ovals non-LTP allergens, blue squares) and the links represent co-sensitization of one or more sera for the linked allergens. The weight of each link, between 0 and 1, measures the degree of co-sensitization. For the sake of clarity, only the 35 links of weights greater than 0.55 of the total 190 existing links are plotted.</p

Co-sensitization graph of TLP allergens.

<p>Each node represents one allergen (TLP as white ellipses, non-TLP allergens as blue square nodes, and LTP-allergen Pru p 3 as a green diamond) and the links represent co-sensitization of one or more sera for the linked allergens. The weight of each link, ranging between 0 and 1, measures the degree of co-sensitization. For the sake of clarity, only the 25 links of weight greater than 0.50 out of the total 253 existing links were plotted.</p