Kidney Bean: A Major Sensitizer among Legumes in Asthma and Rhinitis Patients from India

BACKGROUND: The prevalence of IgE mediated food allergies has increased over the last two decades. Food allergy has been reported to be fatal in highly sensitive individuals. Legumes are important food allergens but their prevalence may vary among different populations. The present study identifies sensitization to common legumes among Indian population, characterizes allergens of kidney bean and establishes its cross reactivity with other legumes. METHODOLOGY: Patients (n = 355) with history of legume allergy were skin prick tested (SPT) with 10 legumes. Specific IgE (sIgE) and total IgE were estimated in sera by enzyme-linked immunosorbent assay. Characterization of kidney bean allergens and their cross reactivity was investigated by immunobiochemical methods. Identification of major allergens of kidney bean was carried out by mass spectrometry. PRINCIPAL FINDINGS: Kidney bean exhibited sensitization in 78 (22.0%) patients followed by chickpea 65 (18.0%) and peanut 53 (15%). SPT positive patients depicted significantly elevated sIgE levels against different legumes (r = 0.85, p<0.0001). Sera from 30 kidney bean sensitive individuals exhibited basophil histamine release (16-54%) which significantly correlated with their SPT (r = 0.83, p<0.0001) and sIgE (r = 0.99, p<0.0001). Kidney bean showed eight major allergens of 58, 50, 45, 42, 40, 37, 34 and 18 kDa on immunoblot and required 67.3±2.51 ng of homologous protein for 50% IgE inhibition. Inhibition assays revealed extensive cross reactivity among kidney bean, peanut, black gram and pigeon pea. nLC-MS/MS analysis identified four allergens of kidney bean showing significant matches with known proteins namely lectin (phytohemagglutinin), phaseolin, alpha-amylase inhibitor precursor and group 3 late embryogenesis abundant protein. CONCLUSION/SIGNIFICANCE: Among legumes, kidney bean followed by chick pea and peanut are the major allergic triggers in asthma and rhinitis patients in India. Kidney bean showed eight major allergens and cross reacted with other legumes. A combination of SPT, sIgE and histamine release assay is helpful in allergy diagnosis

Purification and immunobiochemical characterization of a 31 kDa cross-reactive allergen from Phaseolus vulgaris (kidney bean).

BACKGROUND: Legumes are a rich source of proteins but are also potential elicitors of IgE-mediated food allergy. This study aimed to isolate and characterize a major allergen of Phaseolus vulgaris (kidney bean) and determine its allergenicity. METHODOLOGY: Kidney bean allergen was purified using Q Sepharose column (anion exchanger) and eluates with high intensity were pooled to purify protein using Superdex 75 (gel filtration) and C18 column (RP-HPLC). Patients with history of kidney bean allergy were skin prick tested (SPT) with crude kidney bean extract and the purified protein. Specific IgE was estimated in sera by enzyme-linked immunosorbent assay (ELISA). Characterization of purified protein and its cross-reactivity was investigated by immunobiochemical methods. Identification of purified protein was carried out by tandem mass spectrometry. PRINCIPAL FINDINGS: Purified protein appeared as a single band at 31 kDa on SDS-PAGE and showed IgE binding to 88% patients' sera by ELISA and immunoblotting. SPT with purified protein identified 78% hypersensitive patients of kidney bean. Significant release of histamine from sensitized basophils was observed after challenge with purified protein. PAS staining suggested it to be a glycoprotein, but no change in IgE binding was observed after periodate oxidation. The 31 kDa protein remained stable for 60 min on incubation with pepsin. The purified protein had high allergenic potential since it required only 102 ng of self protein for 50% IgE inhibition. Mass spectrometric analysis identified it as Phytohemagglutinin. It also showed hemagglutination with human RBCs. Cross-reactivity was observed with peanut and black gram with IC50 of 185 and 228 ng respectively. CONCLUSION/SIGNIFICANCE: A 31 kDa major allergen of kidney bean was purified and identified as phytohemagglutinin with cross-reactivity to peanut and black gram

Peptide mass fingerprint database search of selected spots from 2-DE of purified 31 kDa kidney bean proteins.

<p>Peptide mass fingerprint database search of selected spots from 2-DE of purified 31 kDa kidney bean proteins.</p

Histamine released from stripped basophils re-sensitized with individual patients’ sera (n = 15) on challenge with kidney bean extract and 31 kDa purified protein, separately.

<p>Lane C1−C5: controls (A). Scatter plot of the correlation analysis between % histamine released by patients (n = 15) and controls on challenge with crude kidney bean extract and purified protein (B). 2-DE of purified 31 kDa protein showing two close spots (1 & 2) at the same molecular weight. Both the spots were identified as PHA-E after mass spectrometric analysis (C).</p

Glycoprotein detection by Periodic Acid Schiff’s (PAS) staining.

<p>Lane 1: kidney bean extract, lane 2: purified 31 kDa protein (A). Immunoblot of purified protein after periodate treatment. The 31 kDa protein was electro-transfered onto nitrocellulose membrane and then periodate oxidation was done. The strip was washed, blocked with 3% defatted milk and immunoblotted with pooled patients’ sera. Lane 1: 31 kDa protein (untreated), lane 2: 31 kDa protein after periodate treatment (B). Hemagglutination assay of 31 kDa protein, PHA (positive control) and PBS (negative control). Both 31 kDa and PHA formed uniform reddish color across the well with a minimum concentration of 15.62 µg/ml (C). Agglutination of human erythrocytes using purified protein and PHA (Sigma) (D).</p

IgE binding of purified 31 kDa protein by immunoblotting.

<p>Lane 1−25: with individual patients’ sera and lane C: purified protein probed with normal human sera (control).</p

SPT and specific IgE among the patients (1−25) and controls (C1−C5) against raw kidney bean extract and 31 kDa protein.

a<p>specific IgE cut off 0.189 OD.</p>b<p>specific IgE cut off 0.126 OD.</p>*<p>used for making patient’s pooled sera.</p><p>n.d. = SPT not done.</p

Protein profile of eluted fractions containing 31 kDa as a major eluted kidney bean protein.

<p>Kidney bean extract was subjected to anion exchange chromatography (A). Lane 1−5: fractions (10 to 14). Protein profile of gel filtration eluted fractions containing 31 kDa as a major eluted protein (B). Lane 1−3: fractions (17 to 19). Elution profile of purified 31 kDa protein after HPLC (C). Lane 1: purified protein after silver staining on SDS-PAGE, lane 2: immunoblot of purified protein using kidney bean hypersensitive pooled patients’ sera.</p

SDS-PAGE profile and immunoblot of 31 kDa kidney bean protein.

<p>Lane 1 : untreated, Lane 2−5 : boiled at 100°C for 15, 30, 45 and 60 min, Lane M: molecular weight markers. Protein fractions were stained with Coomassie brilliant blue (A) and immunoblotted (B). SGF digestion of 31 kDa protein Lane M:molecular weight markers, Lane 1: 31 kDa protein (undigested), lanes 2−8: 31 kDa protein incubated in SGF for 0.5, 1, 5, 10, 15, 30 and 60 min, lane 9: pepsin. The digested protein was electrophoresed on SDS-PAGE and visualized by CBB staining (C).</p

Sensitization to legume allergens detected by sIgE estimation in ELISA.

<p>*Cut off value for sIgE positivity = 0.138 OD (≥3 times of control).</p