Decreased Immunoglobulin E (IgE) Binding to Cashew Allergens

Jun 13, 2014 - (14, 16) Immunoglobulin E (IgE) binding to these cashew allergens is ..... To determine if sodium sulfite treatment alters IgE binding ...
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Decreased Immunoglobulin E (IgE) Binding to Cashew Allergens following Sodium Sulfite Treatment and Heating Christopher P. Mattison,*,† Wendy A. Desormeaux,† Richard L. Wasserman,§ Megumi Yoshioka-Tarver,# Brian Condon,† and Casey C. Grimm† †

Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, Louisiana 70124, United States § Allergy Immunology Research Center of North Texas, Department of Pediatrics, University of Texas Southwestern Medical School, Dallas, Texas 75390, United States # Southern University and A&M College, Baton Rouge, Louisiana 70813, United States ABSTRACT: Cashew nut and other nut allergies can result in serious and sometimes life-threatening reactions. Linear and conformational epitopes within food allergens are important for immunoglobulin E (IgE) binding. Methods that disrupt allergen structure can lower IgE binding and lessen the likelihood of food allergy reactions. Previous structural and biochemical data have indicated that 2S albumins from tree nuts and peanuts are potent allergens, and that their structures are sensitive to strong reducing agents such as dithiothreitol. This study demonstrates that the generally regarded as safe (GRAS) compound sodium sulfite effectively disrupted the structure of the cashew 2S albumin, Ana o 3, in a temperature-dependent manner. This study also showed that sulfite is effective at disrupting the disulfide bond within the cashew legumin, Ana o 2. Immunoblotting and ELISA demonstrated that the binding of cashew proteins by rabbit IgG or IgE from cashew-allergic patients was markedly lowered following treatment with sodium sulfite and heating. The results indicate that incorporation of sodium sulfite, or other food grade reagents with similar redox potential, may be useful processing methods to lower or eliminate IgE binding to food allergens. KEYWORDS: allergen, allergy, cashew, protein structure, sulfite, immunoglobulin E, epitope, antibodies

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INTRODUCTION Food allergies affect up to 8% of children in the United States,1 and the self-reported frequency of peanut and tree nut allergy has grown over the past decade.2 Currently, the only accepted management practice for food allergy patients is strict avoidance of the allergy-causing food. Food allergies place significant burdens on patients and families,3−5 and medically related costs in the United States associated with food allergies are estimated to be $25 billion annually.6 Congress has enacted the Food Allergen Labeling and Consumer Protection Act (2004) and the School Access to Emergency Epinephrine Act (2013) to help food allergy sufferers. Reactions to nuts, including peanuts, pecans, walnuts, pistachios, and cashews, can be severe and are rarely outgrown.7 In particular, cashew allergens often cause very serious reactions.8,9 Three seed storage proteins, including vicilins, legumins, and 2S albumins, are known nut allergens.10 Vicilins are trimeric proteins, whereas legumins are hexameric, composed of legumin monomers with acidic and basic subunits linked by a disulfide bond. The 2S albumins are composed of two subunits joined by a conserved structural framework of cysteine disulfide bonds.11 The cashew Ana o 1 protein is a vicilin,12 the Ana o 2 protein a legumin,13 and the Ana o 3 protein a 2S albumin.14 Under reducing conditions Ana o 2 dissociates into basic ∼20−25 kDa and acidic ∼30−35 kDa subunits.15 Ana o 3 subunits also dissociate under reducing conditions, although only the large subunit (∼6−10 kDa) is easily resolved on SDS-PAGE.14,16 Immunoglobulin E (IgE) binding to these cashew allergens is retained after mechanical © 2014 American Chemical Society

and physical processing methods such as blanching, microwaving, roasting, autoclaving, and irradiation.17,18 The 2S albumin seed storage proteins, members of the prolamin superfamily, have been shown to be some of the most potent allergens in peanuts. For example, the peanut 2S albumins Ara h 2 and Ara h 6 have been demonstrated to account for most of the basophil degranulation capacity within peanut extracts.19 2S albumins are resistant to enzymatic digestion and processing methods, owing, at least in part, to the tertiary structure of the protein imparted by the disulfide bond framework. The importance of the disulfide bonds to the structural integrity of the 2S albumins presents an obvious target for processing methods designed to lower IgE binding and subsequent allergic responses. Enzymatic and chemical methods targeting the reduction of disulfide bonds in allergens to lower their ability to cause an allergic response have been described. For example, thioredoxins have been used to lower the allergenic potential of wheat, milk, and castor bean.20−22 Similarly, the reducing agent dithiothreitol has been shown to destabilize several 2S albumins and decrease their ability to cause an allergic response.16,23−25 Generally recognized as safe (GRAS) compounds, when used at the minimum amount required for the intended purpose within human foods and cosmetics, are considered safe by the Received: Revised: Accepted: Published: 6746

March 12, 2014 May 20, 2014 June 13, 2014 June 13, 2014 dx.doi.org/10.1021/jf501117p | J. Agric. Food Chem. 2014, 62, 6746−6755

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Cashew Extract Preparation. Raw and roasted cashews were ground with a coffee grinder and defatted with petroleum ether in a Soxhlet apparatus overnight. Defatted cashew flour (0.4 g) was placed in 10 mL of 0.2 M Tris, 0.2 M NaCl, pH 8.0, and vortexed for 30 s and then sonicated twice on ice for 15 s using a Sonic Dismembrator (Fisher Scientific Co., Orlando, FL, USA). Extract solutions were centrifuged for 30 min at 12000 rpm at 4 °C, and clarified cashew extracts were collected. Protein concentrations were determined using a NanoDrop (ThermoFisher, Pittsburgh, PA, USA), and samples were stored at −20 °C. SDS-PAGE. Proteins were resolved by SDS-PAGE with either a Mini Protean system II (Bio-Rad) or a Novex Mini Cell gel rig (Life Technologies) and prestained Precision Plus molecular weight markers (Bio-Rad). Prior to electrophoresis, 4× NuPAGE LDS sample buffer (Life Technologies) was added to the protein samples using a 1:4 (v/ v) ratio. Samples were heated at 65 °C for 15 min, and after electrophoresis, protein bands were visualized with Safe Stain (Invitrogen, Grand Island, NY, USA). Gel images were captured, and normalization of protein load in each lane was confirmed by quantifying the 680 nm channel signal on an Odyssey CLX infrared imaging system (LI-COR). Cashew Extract Treatment. Cashew extract (125 μg) was incubated with various concentrations of sodium sulfite (0−100 mM) and at different temperatures (0−100 °C) for 15 min. Control samples were heated with or without added 5 mM DTT. Sample aliquots were removed and combined with SDS-PAGE sample buffer lacking reducing agent prior to analysis with SDS-PAGE or immunoblotting. Rabbit Antibodies. Rabbit antibody production was performed by Pierce Biotechnology Inc. (Rockford, IL, USA). Briefly, prescreened rabbits were immunized with 1 mg of cashew extract made from “ready to eat” cashew nuts and then boosted at days 14, 28, and 42 with 0.5 mg of cashew extract at each injection. Test rabbits were phlebotomized, and sera were screened for antibodies against total cashew protein. Large-volume phlebotomies were collected from hightiter rabbits, and serum was stored at −80 °C for later use. Human Sera. Clinically relevant sera from cashew-allergic patients (from >7 to 55 years of age) with a documented history of cashew allergy supported by in vivo (skin test) or in vitro (serum immunoassay) cashew-specific IgE were collected at Dallas-Allergy Immunology (Dallas, TX, USA). The study protocol was approved by the North Texas Institutional Review Board. Immunoblotting. Cashew extract samples (25 μg) treated with DTT, water, or sodium sulfite were subjected to SDS-PAGE (with loading buffer lacking reducing agent) and transferred to a PVDF membrane for Western blot or spotted (5 μg) on to PVDF membrane for dot-blot. Blotted PVDF (Millipore, Billerica, MA, USA) was blocked for 1 h at room temperature in PBST with 2% (w/v) nonfat dry milk. Rabbit anti-cashew antibody was diluted 1:1000 in PBST and incubated overnight at 4 °C. The membranes were then washed three times for 5 min in PBST and incubated for 30 min with anti-rabbit IRdye-680 (1:10000 in PBST) at room temperature. The membranes were washed as above and visualized using an Odyssey CLx (LI-COR) infrared imaging system. Immunoblotting for IgE (Western blot and dot blot) was performed overnight using similar methods with sera pooled from six cashew allergic patients. Membranes were washed as above and incubated for 1 h at room temperature with biotinylated anti-IgE (1:1000 in PBST). Membranes were then washed and incubated for 30 min at room temperature with IRdye-680-labeled streptavidin (1:5000 in PBST) and visualized with an Odyssey CLx instrument. Circular Dichroism (CD). Ana o 3 was purified as described in Mattison et al.16 Purified native and chemically treated Ana o 3 samples were analyzed by far-UV CD (185−250 nm) on a Jasco-815 spectropolarimeter (JASCO, Oklahoma City, OK, USA) as described previously16 with minor changes. Briefly, treated Ana o 3 samples were buffer exchanged into 5 mM sodium phosphate (pH 6.8) with a 3 kDa spin column and then diluted to a protein concentration of 0.1 mg/mL for analysis. CD spectra were acquired using a quartz cuvette with a 1 cm path length at 25 °C with a 2 nm bandwidth and a response time of 1 s. Three acquisitions were obtained for each sample, and background

U.S. Food and Drug Administration (FDA). Sulfite-containing compounds including sodium sulfite (GRAS 182.3798), sodium bisulfite (GRAS 182.3739), and sodium metabisulfite (GRAS 182.3766) are on the FDA’s GRAS list. Sulfites are multipurpose compounds commonly used as calcium, potassium, or sodium salts in the food and pharmaceutical industries as preservatives or antioxidants. They are also used to slow food oxidation and can sometimes affect the smell and taste of foods.26 Sulfite-containing compounds occur naturally in foods, such as garlic, onions, cheeses, and eggs, and drinks, such as wine, beer, and some teas. Although they are generally considered safe, sulfites can cause contact dermatitis and have been highlighted as a cause of cosmetic allergy.27−29 However, allergies to preservatives such as sodium sulfite and its derivatives are considered rare.30,31 The FDA requires labeling when sulfites are used as preservatives in a final product; however, when sulfites are used in food-processing steps, labeling is required only when the sulfite residual is ≥10 ppm.32 The use of sulfite-containing compounds on fresh fruits and vegetables was banned in 1986 because of potential allergic reactions, and those suffering from asthma are at elevated risk of sulfite sensitivity.33 Nevertheless, sodium metabisulfite is currently included as a preservative in therapeutic epinephrine,34 and according to the prescription information for EpiPen, sodium metabisulfite is included as a preservative in the epinephrine shot used to counteract a severe food allergy reaction. Each 0.3 mL dose from an EpiPen autoinjector contains 0.5 mg of sodium metabisulfite. Individuals suffering from sulfide oxidase deficiency are also sulfite sensitive because of their inability to convert sulfites to sulfates.35 The development of new, cost-effective processing methods that can lower or eliminate the ability of tree nuts and peanuts to cause allergic reactions is important for the prevention of severe reactions. The reaction between sulfite and cysteine disulfides within proteins is reversible at alkaline pH and has been shown to disrupt disulfide bonds of several proteins.36 Standard protocols using sodium sulfite to break disulfide bonds, referred to as sulfitolysis, can be used to introduce a negative charge on modified cysteines.37 Here, using cashew extracts and purified Ana o 3, we evaluated the ability of sodium sulfite to target cashew allergens having structure and stability that are dependent upon cysteine disulfide bonds. We assessed the effect of sulfite addition on the structure of cashew allergens and their innate capacity to bind IgE from cashew allergic individuals.

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EXPERIMENTAL PROCEDURES

Materials. “Ready to eat” cashews were purchased from Nuts Online (http://www.nuts.com, Cranford, NJ, USA). Roasted cashews were prepared by heating “ready to eat” cashews in an oven at 177 °C for 20 min. Sodium sulfite and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA), spin columns from Millipore (Carrigtwohill, CO, USA), SDS-PAGE Any kD MiniProtean TGX precast Tris-glycine gels from Bio-Rad (Hercules, CA, USA), and Novex 4−20% Tris−glycine gels from Life Technologies (Carlsbad, CA, USA). Sequencing-grade modified trypsin from Promega (Madison, WI, USA) was used for sample digestion prior to mass spectrometry. Biotinylated anti-human IgE antibody was purchased from Southern Biotech (Birmingham, AL, USA). IRdye680-labeled streptavidin, IRdye-680, and IRdye-800 secondary antibodies were purchased from LI-COR (Lincoln, NE, USA). Nonsterile Maxisorp 96-well Nunc microtiter plates were purchased from Thermo Scientific (Rochester, NY, USA). 6747

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Figure 1. Sodium sulfite alters cashew protein migration and lowers antibody binding. Cashew nut extract (125 μg) subjected to treatment with various amounts of sodium sulfite and temperature was analyzed by nonreducing SDS-PAGE and rabbit anti-cashew antibodies. Cashew extract incubated for 15 min at 100 °C with sodium sulfite or 5 mM DTT was analyzed by SDS-PAGE (A) and immunoblot with rabbit anticashew antibodies (C). Lanes: (1, 1′) 5 mM DTT; (2, 2′, 10, 10′) buffer control, 100 °C; (3, 3′) 1 mM sodium sulfite; (4, 4′) 2.5 mM sodium sulfite; (5, 5′) 5 mM sodium sulfite; (6, 6′) 10 mM sodium sulfite; (7, 7′) 25 mM sodium sulfite; (8, 8′) 50 mM sodium sulfite; (9, 9′) 100 mM sodium sulfite. Sodium sulfite treatment of cashew extracts is temperature dependent. Cashew extract (125 μg) was subjected to treatment with 50 mM sodium sulfite at various temperatures and analyzed by SDS-PAGE (B) and immunoblot with rabbit anti-cashew antibodies (D). Lanes: (11, 11′) 5 mM DTT at 100 °C; (12, 12′) buffer control, 100 °C; (13, 13′) 50 mM sodium sulfite, 37 °C; (14, 14′) 50 mM sodium sulfite, 50 °C; (15, 15′) 50 mM sodium sulfite, 65 °C; (16, 16′) 50 mM sodium sulfite, 100 °C. Rabbit IgG binding was visualized with IRdye-680-labeled anti-rabbit IgG secondary antibody. Quantification of IgG antibody binding in each respective lane (C and D) is shown in panels E and F, and molecular weight markers are indicated on the left side of each gel or immunoblot.

measurements were subtracted. Mean residue ellipticity was calculated using a mean residue weight of 114.5 g/mol, and the K2D3 web server

(http://www.ogic.ca/projects/k2d3/) was used to estimate secondary structure.33 6748

dx.doi.org/10.1021/jf501117p | J. Agric. Food Chem. 2014, 62, 6746−6755

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LC-MS/MS Mass Spectrometry. Following treatment with sodium sulfite, ammonium bicarbonate (100 mM) and calcium chloride (1 mM) were added to defatted cashew extracts made from “ready to eat” cashew nuts. Sequencing-grade modified trypsin (0.2 μg) was added, and samples were allowed to digest for 1 h at 37 °C with gentle mixing. Following acidification with formic acid, samples were analyzed via LC-MS/MS with an Agilent 1200 LC system, an Agilent Chip Cube interface, and an Agilent 6520 Q-TOF tandem mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) as described previously.16 Raw data files were extracted, sequenced, and searched against a custom food allergen database containing the Ana o 1, Ana o 2, and Ana o 3 sequences. Peptides were searched for sulfite (SO3) cysteine modifications resulting in an 80 Da mass shift after incorporation of the corresponding mass change into the Spectrum Mill search algorithm. Competetive ELISA. Microtiter plates were coated with 1 μg of raw cashew extract per well in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and incubated overnight at 4 °C. Cashew extract was discarded, and the plate was blocked with PBST containing 1% BSA (w/v) at room temperature for 1 h. Plates were washed three times with PBST following blocking and all experimental incubation steps to remove unbound proteins. Treated or untreated cashew extract was centrifuged with 3 kDa spin columns to remove unreacted sodium sulfite or DTT. Samples were then buffer exchanged in 100 mM Tris, pH 8.3, by 10-fold dilution and centrifugation four times using the spin columns to further dilute any remaining sodium sulfite or DTT. Samples were resuspended in 200 μL of buffer and serially diluted 10-fold with 100 mM Tris, pH 8.3. Competition samples, to evaluate IgE binding, were made using 12.5 μL of treated cashew sample, 12.5 μL of pooled sera, and 25 μL of 100 mM Tris, pH 8.3. Samples were added to the microtiter plates and incubated at 37 °C for 1 h. Plates were washed, and 50 μL of biotinylated anti-IgE (1:1000 in PBST) was added to each well and then incubated at 37 °C for 30 min. Plates were washed with PBST, and 50 μL of streptavidin-labeled IRdye-680 (1:5000 in PBST) was added to each well. Plates were incubated at 37 °C for 30 min, washed, and then imaged with the Odyssey CLx instrument. The data in the plot are presented as percent of IgE inhibition using the following formula: IR680 value of uninhibited control − IR680 value of inhibited sample/IR680 value of uninhibited control × 100.

3 allergens in cashew extracts treated with sodium sulfite were dependent upon temperature. Disruption of a small portion of Ana o 3 in the extract was observed at 37 °C in the presence of 50 mM sodium sulfite. More of the dissociated large subunit of Ana o 3 can be observed just below the 10 kDa marker as the temperature was increased to 50, 65, or 100 °C (Figure 1B). Extensive disruption of Ana o 2 and alteration in its migration pattern were not observed until the incubation temperature reached 100 °C (Figure 1B). These results indicated that Ana o 3 was more sensitive to sodium sulfite-induced disruption compared to Ana o 2 in the extracts. We did not observe a noticeable shift in the cashew extract protein migration pattern when samples were treated at temperatures below 37 °C in the presence of sodium sulfite. To evaluate the immunological consequences of sodium sulfite treatment on cashew extracts, we raised antibodies against cashew proteins in rabbits. Western blots using these rabbit polyclonal antibodies indicated that there was a reduction in IgG binding to cashew proteins in sulfite-treated extracts (Figure 1C). We quantified the antibody binding signal and found that overall IgG binding was lowered by 60−70% at sodium sulfite concentrations of ≥25 mM and incubation at 100 °C (Figure 1E). In particular, we noticed a reduction in antibody binding to the slower migrating, higher molecular weight forms >75 kDa (Figure 1C,E). These forms have been associated with heating-induced cross-linking of peanut and other food allergens during processing and can alter IgE binding.38−44 We also observed that binding to Ana o 3 was lowered at sodium sulfite concentrations as low as 1 mM and was undetectable at higher concentrations (Figure 1C,E). Similarly, antibody binding to Ana o 2 and other higher molecular weight proteins was lowered as sodium sulfite concentration increased. At ≥37 °C, we observed a reduction in antibody binding to cashew proteins treated with 50 mM sodium sulfite (Figure 1D,F). However, when extracts were incubated at 100 °C in the presence of 50 mM sodium sulfite, we observed the dissociation of Ana o 2 subunits and the largest reduction in overall binding to cashew proteins (Figure 1D,F). Different food preparation methods have been shown to alter antibody binding, and nuts are commonly roasted before consumption. We next tested the effect of sodium sulfite on extracts made from roasted cashews. Extracts made from previously roasted cashews were treated with sodium sulfite, and we observed similar results. Figure 2A shows the disruption of Ana o 2 and Ana o 3 proteins into subunits from both the raw and roasted cashew samples after the sodium sulfite treatment. Whereas there was greater IgG binding to the roasted extracts, overall IgG binding was lowered in both raw and roasted sodium sulfite-treated extracts (Figure 2B). Although IgG binding to higher molecular weight cross-linked forms was lowered as it was to DTT-treated extracts, the reduction in IgG binding was not as great in the roasted extracts. We observed an approximate 50% reduction in binding to the extracts of roasted nuts after the IgG signal was normalized to the roasted nut control extract (Figure 2C). Notably, in each of our experiments, binding to the large subunit of Ana o 3 was markedly lowered in sodium sulfitetreated samples compared to control treated extracts. Reducing agents have been shown to disrupt the structure of 2S albumins, including Ara h 2, Ber e 1, and Ana o 3, increasing their sensitivity to digestive proteases.16,23,25 We compared the structure of purified Ana o 3 after treatment with water, DTT, or sodium sulfite using far-UV CD (Figure 3). The DTT-

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RESULTS Some nut allergens, including Ana o 2 and Ana o 3, contain disulfide bonds important for their structure; disruption of these bonds can result in lowered antibody binding. To determine if sulfite compounds could disrupt cashew nut IgE binding epitopes, we incubated cashew extracts with sodium sulfite. As shown in Figure 1A, addition of sodium sulfite and heating at 100 °C dissociated Ana o 2 and Ana o 3 and altered their SDS-PAGE migration patterns in cashew extracts. Concentrations as low as 1 mM sodium sulfite disrupted disulfide bonds and altered the migration pattern of at least a portion of the Ana o 2 and Ana o 3 proteins in the extract. Increasing the sodium sulfite concentration resulted in a gradient of dissociation and in the migration pattern of Ana o 2 and Ana o 3 proteins. Elevated sodium sulfite resulted in an increasing appearance of the Ana o 2 acidic (∼33 kDa) and basic (∼20−25 kDa) subunits and the large Ana o 3 subunit migrating just below the 10 kDa marker. At higher concentrations of sodium sulfite (25−100 mM), we observed a migration pattern very similar to that seen using 5 mM DTT (Figure 1A). Similar results were obtained when cashew extracts were incubated with 50 mM sodium bisulfite, sodium metabisulfite, and potassium sulfite, but not sodium thiosulfate (not shown). Next, we altered the incubation temperature to determine if the dissociation and altered migration of the Ana o 2 and Ana o 6749

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Figure 3. Ana o 3 protein structure is disrupted by sodium sulfite. Purified Ana o 3 was analyzed by circular dichroism from 185 to 250 nm in native form or after treatment with either DTT or sodium sulfite. Open circles represent native Ana o 3 after mock treatment at 65 °C, gray squares represent Ana o 3 after treatment with 5 mM DTT at 65 °C, and black triangles represent Ana o 3 after sodium sulfite treatment (50 mM) at 65 °C.

treated sample had an estimated 4% and the sodium sulfitetreated protein, 7%, α-helical content. The β-sheet content of the protein was also lowered slightly to 12% for DTT-treated and to 13% for sodium sulfite-treated extracts. Thus, sodium sulfite disrupts Ana o 3 structure to an extent similar to treatment of the protein with the reducing agent DTT. Sodium sulfite is expected to attack the disulfide bond containing cysteine residues within the cashew allergens Ana o 2 and Ana o 3. To confirm that these bonds were targets of sodium sulfite, we digested treated extracts and analyzed the peptide mixture by mass spectrometry to find evidence of sulfite-modified cysteine-containing peptides. Indeed, tryptic peptide masses consistent with modification of Cys76 or Cys77 and Cys 89 in Ana o 3 were observed (Table 1). Peptides containing the expected modified cysteines were detected with an 80 Da shift, corresponding to the addition of sulfite ion (SO3). These cysteine residues are expected to be part of the disulfide bond network important for Ana o 3 structure and stability. Importantly, the Cys76 and Cys77 residues in Ana o 3 lie within a previously mapped dominant IgE epitope.14 We were unable to obtain confirmation of the precise location of the modified residue(s) within peptides because of the facile loss of the SO3 modification prior to or during peptide fragmentation. A similar difficulty has been observed in attempts to specifically identify modified residues in insulin and BSA modified by sodium sulfite.45 We also observed evidence of a SO3 modification within several Ana o 2 cysteinecontaining peptides (Table 1). The Cys25 and Cys101 SO3 modified residues lie within or near previously mapped IgE epitopes that strongly react with Ana o 2.13 Our mass spectrometry analysis further indicated that sodium sulfite disrupted cysteine disulfide bonds within Ana o 3 in cashew extracts. It is evident from the analysis that sodium sulfite treatment can alter the structure of some cashew allergens, as well as the ability of rabbit IgG antibodies to bind these allergens. To determine if sodium sulfite treatment alters IgE binding to cashew allergens, we performed several IgE binding assays.

Figure 2. Sodium sulfite alters roasted cashew extract migration and lowers antibody binding. Raw or roasted cashew nut extract (125 μg), subjected to treatment with 5 mM DTT, buffer control, or 50 mM sodium sulfite at 100 °C, was analyzed by nonreducing SDS-PAGE (A) and rabbit anti-cashew antibodies (B). Lanes: (1, 1′) raw extract with 5 mM DTT; (2, 2′) raw extract buffer control; (3, 3′) raw extract 50 mM sodium sulfite; (4, 4′) roasted extract with 5 mM DTT; (5, 5′) roasted extract buffer control; (6, 6′) roasted extract 50 mM sodium sulfite. Rabbit IgG binding was visualized with IRdye-680-labeled antirabbit IgG secondary antibody (C). Quantification of rabbit anticashew IgG antibody binding in each respective lane is indicated in panel C, and molecular weight indicators are indicated on the side of each gel or immunoblot. 6750

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Table 1. Potential Sites of Sulfite-Modified Cysteine Residues in Cashew Allergens Ana o 2 and Ana o 3 protein

Cys residue

Ana o 2 Ana o 2 Ana o 2

25 58 101

Ana o 2 Ana o 3 Ana o 3

146 76, 77 89

peptide (R)QEWQQQDECQIDRLDALEPDNRVEYEAGTVEAWDPNHEQFR(C) (R)CAGVALVR(H) (R) HTIQPNGLLLPQYSNAPQLIYVVQGEGMTGISYPGCPETYQAPQQGR(Q) (R)FRRGDIIAIPAGVAHWCYNEGNSPVVTVTLLDVSNSQNQLDRTPR(K) (R)ECCQELQEVDR(R) (R)CQNLEQMVR(Q)

m/z obs (Da)

z

MH+ (Da)

calcd MH+ (Da)

Δ mass (Da)

1014.237 434.7047 1039.708

5 2 5

5067.1557 868.4016 5194.5102

4987.225 788.445 5114.514

79.9307 79.9566 79.9962

1012.709 716.2723 600.7483

5 2 2

5059.5147 1431.5368 1200.4888

4979.534 1351.562 1120.524

79.9807 79.9748 79.9648

pronounced with DTT, sodium sulfite-treated extracts were a weaker competitor for IgE binding compared to untreated control extracts (Figure 6). Again, the reduction in IgE binding was greater when extracts were heated at 100 °C. Even at the highest protein concentration used, the sodium sulfite or DTT 100 °C treated extracts could not inhibit 50% of the IgE binding. These findings are consistent with the reduction of IgE binding we observed in the Western blot analysis (Figure 4).

Figure 4 shows Western blot binding of IgE from a pool of six cashew-allergic patients’ sera to sodium sulfite treated cashew extracts. Overall IgE binding to cashew proteins was lowered in both the DTT- and sodium sulfite-treated samples compared to the mock treated control. Quantification of the IgE signal indicated that binding to sodium sulfite- and DTT-treated extracts was lowered by 80−90% compared to mock treated extracts (Figure 4). In particular, reduction of Ana o 2 and Ana o 3 disulfide bonds with either DTT or sodium sulfite clearly lowered binding to both of these allergens. IgE binding to both of the faster migrating dissociated Ana o 2 subunits and the large subunit of Ana o 3 was markedly lowered (Figure 4). Individual variation in IgE binding to nut allergens by allergy patients is well documented. We continued the IgE binding analysis by comparing IgE binding of individual cashew allergic sera to treated cashew extracts using dot-blots. Aliquots of treated or untreated cashew extract were spotted onto a PVDF membrane, allowed to dry, and extensively washed. We evaluated IgE binding to the bound material using six individual cashew-allergic patients’ sera, and in every case IgE binding to the sodium sulfite-treated extract was lowered compared to the untreated control (Figure 5). When samples were incubated at 65 °C in the presence of sodium sulfite or DTT, the IgE signal was lowered to approximately half of the control sample with most of the sera samples we tested. Five of the six sodium sulfite-treated samples had IgE binding values ranging from 50 to 58% of the untreated control. The remaining sample was lowered to only 67% of the untreated control. IgE binding to the DTT-treated samples was also lowered to an extent equivalent to or greater than that of the sodium sulfite treatment for each of the sera. The DTT-treated samples had IgE binding values ranging from 16 to 53% of the untreated control. When extracts were incubated at 100 °C, the results were more dramatic. In both the sodium sulfite- and DTTtreated extracts, IgE binding was lowered to ≤10% of control treated extracts in five of six serum samples. In the remaining sample, IgE binding was lowered to 10% for sodium sulfite treatment and 16% for DTT. Our analysis of the cashewallergic patients’ serum samples indicated that treatment of cashew extracts with sodium sulfite could effectively lower IgE binding to cashew allergens. To confirm these findings, IgE binding to the control and sodium sulfite-treated extracts was evaluated using competitive ELISA with the pooled cashew-allergic patients’ sera. For these experiments, unreacted DTT or sodium sulfite was removed from extracts treated at 65 or 100 °C with centrifugation and buffer exchange over a course of 3 h using a 3 kDa cutoff spin filter to lower the remaining concentration to