The Seed Biotinylated Protein of Soybean (Glycine max): A Boiling

Apr 25, 2016 - In the United States most legume allergic reactions are caused by exposure to soybeans and peanuts, with peanut allergy affecting ...
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The Seed Biotinylated Protein of Soybean (Glycine max): A BoilingResistant New Allergen (Gly m 7) with the Capacity To Induce IgEMediated Allergic Responses John J. Riascos,*,†,§ Sandra M. Weissinger,† Arthur K. Weissinger,† Michael Kulis,§,# A. Wesley Burks,§,# and Laurent Pons§ †

Department of Crop Science, North Carolina State University, Raleigh, North Carolina, United States Department of Pediatric Allergy and Immunology, Duke University Medical Center, Durham, North Carolina, United States

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ABSTRACT: Soybean is a common allergenic food; thus, a comprehensive characterization of all the proteins that cause allergy is crucial to the development of effective diagnostic and immunotherapeutic strategies. A cDNA library was constructed from seven stages of developing soybean seeds to investigate candidate allergens. We searched the library for cDNAs encoding a seedspecific biotinylated protein (SBP) based on its allergenicity in boiled lentils. A full-length cDNA clone was retrieved and expressed as a 75.6-kDa His-tagged recombinant protein (rSBP) in Escherichia coli. Western immunoblotting of boiled bacterial extracts demonstrated specific IgE binding to rSBP, which was further purified by metal affinity and anion exchange chromatographies. Of the 23 allergic sera screened by ELISA, 12 contained IgEs specific to the purified rSBP. Circular dichroism spectroscopy revealed a predominantly unordered structure consistent with SBP’s heat stability. The natural homologues (nSBP) were the main proteins isolated from soybean and peanut embryos after streptavidin affinity purification, yet they remained lowabundance proteins in the seed as confirmed by LC-MS/MS. Using capture ELISAs, the soybean and peanut nSBPs were bound by IgEs in 78 and 87% of the allergic sera tested. The soybean nSBP was purified to homogeneity and treatments with different denaturing agents before immunoblotting highlighted the diversity of its IgE epitopes. In vitro activation of basophils was assessed by flow cytometry in a cohort of peanut-allergic children sensitized to soybean. Stronger and more frequent (38%) activations were induced by nSBP-soy compared to the major soybean allergen, Gly m 5. SBPs may represent a novel class of biologically active legume allergens with the structural resilience to withstand many food-manufacturing processes. KEYWORDS: Glycine max, Arachis hypogaea, biotin, allergenic orthologues

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INTRODUCTION In the United States most legume allergic reactions are caused by exposure to soybeans and peanuts, with peanut allergy affecting approximately 1% of children and 0.6% of adults and soybean allergy affecting 0.4% of children and 0.3% of adults.1 As a consequence, these legumes are listed among the eight most allergenic foods by the Food and Agricultural Organization.2 Recent studies3 suggest that approximately 50% of children with soy allergy outgrew their allergy by age 7 years and that the majority of patients with soy allergy will eventually develop soybean tolerance. Still, there are recurring reports of children and adults having anaphylactic reactions to soybean in foodstuffs,4−6 which adds to the stress level of many sensitized individuals who have been told to avoid dietary soybean based on the sole presence of soy-specific IgEs in their sera. Furthermore, the wide variety of soy-based products in the typical diet makes it very difficult for soy-allergic patients to avoid exposure. Several soybean allergens have been described in the literature. They belong to a diverse group of protein families and superfamilies, such as the cupin superfamily (11S and 7S seed storage globulins), the prolamin superfamily (composed of nonspecific lipid transfer proteins and the 2S storage albumins), pathogenesis-related proteins (PR proteins), and profilins. Importantly, many of these protein families have been characterized as allergenic in various plant food species.7,8 As © XXXX American Chemical Society

a consequence, understanding the distribution of common allergenic protein families across species is key to the prediction of novel allergens. Despite the number of reported soy allergens, their immunologic characterizations have not been as extensive as those of allergens from other foods, such as peanuts.9 Most of the soybean allergens were characterized by serological studies that described an allergen as “major” if it caused sensitization in >50% of patients. Belonging to this category were the abundant seed storage proteins Gly m 5 (β-conglycinin) and Gly m 6 (glycinin), both respective homologues of the major peanut allergens, Ara h 1 and Ara h 3.9 Although much less abundant, the vacuole-associated cysteine protease, P34, was also identified as a major allergen, Gly m Bd 30 K.10,11 However, the lack of information regarding any type of biological activity (e.g., skin testing, histamine release, or basophil activation) can raise doubts about their legitimacy as “major” allergens. It is therefore important to hold the characterization of soy allergens to standards similar to those defining allergens from the other main allergenic foods. We believe that a better understanding of the soybean proteins that cause allergy would contribute to the Received: December 17, 2015 Revised: April 13, 2016 Accepted: April 24, 2016

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DOI: 10.1021/acs.jafc.5b05873 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

(47A11) using the primers SBP-F 5′-TACTGGAGATGGCGTCTGAACAATTGGC-3′ and SBP-R 5′-ATGAATTCTCAAGCACCGTGCGACCCT-3′, which included the restriction sites PstI and EcoRI, respectively (underlined sequences). The PCR product was subcloned into the PstI/EcoRI sites of the pBAD/His C plasmid. TOP10 E. coli cells (Invitrogen) were transformed with the recombinant construct, and integrity of the insert was verified by DNA sequencing. A negative control was made by transforming E. coli with the empty vector (EV). Cells transformed with each of the expression vectors were stored at −80 °C until use. Bacterial expression of the SBP cDNA was optimized by Western blot screening of the His-tagged protein (SuperSignal West HisProbe kit, Pierce, Rockford, IL, USA). The protein extracts were prepared from exponential cultures wherein different concentrations and induction times with arabinose were tested (pBAD/His kit, Invitrogen). The optimal expression level of rSBP was obtained when bacteria were grown at room temperature for 2 h in the presence of 0.5% arabinose inducer. Crude extracts of bacteria expressing the soy SBP cDNA or the EV were prepared from pellets of centrifuged cultures (300 mL) and homogenized in 5 mL of 20 mM N-tris(hydroxymethyl)methyl-2aminomethanesulfonic acid (TES)−KOH buffer (pH 8.0) containing 500 mM NaCl.16 The extract was boiled for 10 min and then cooled on ice, and the precipitated material was removed by centrifugation at 10000g for 20 min. Protein concentration was determined in the supernatant using the Bradford assay (Bio-Rad, Hercules, CA, USA). Purification of the Recombinant Soybean SBP. Cell pellets collected after centrifugation of bacterial cultures (4 × 2.5 L pool) were solubilized in 150 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 5 mM imidazole, 0.5 mM PMSF, and 8 M urea and then stirred overnight at 4 °C. The cell lysate was clarified by centrifugation at 30000g for 30 min at 4 °C (rotor JA-14, Beckman Coulter, Brea, CA, USA) and filtration through a 0.8-μm hydrophilic poly(ether sulfone) membrane (Supor PES filters, Pall Life Sciences, VWR). The clarified extract was loaded onto a glass column (2.5 cm i.d.) packed with 25 mL of Ni-NTA His bind resin (EMD Millipore, Billerica, MA, USA) equilibrated in the extract buffer. After washing, urea was progressively exchanged to 0.5 M NaCl using a decreasing linear gradient. Bound proteins were eluted using a 5−500 mM imidazole linear gradient at a flow rate of 2 mL/min, while collecting 5.5 mL fractions. The protein concentration in each fraction was measured by using the bicinchoninic acid assay (BCA, Pierce). Fractions corresponding to the main elution peak were pooled and extensively dialyzed in a 3500 MW cutoff tubing (Pierce) for 2 days against 2 × 15 L of 50 mM Tris-HCl buffer (pH 8.5) at room temperature. The dialyzed IMAC pool was then loaded onto a glass column (2.5 cm i.d.) packed with 20 mL of diethylaminoethyl methacrylate support (DEAE Macro-Prep, Bio-Rad) equilibrated in 50 mM Tris-HCl buffer (pH 8.5). After washing with 250 mL of equilibration buffer, bound proteins were eluted using a 0−500 mM NaCl linear gradient at a flow rate of 2 mL/min. Protein concentration in each collected fraction (5 mL) was measured by BCA. Selected fractions were further analyzed by SDS-PAGE (4−12% Bis-Tris NuPAGE, Invitrogen) with colloidal Coomassie blue staining (LabSafe Gel Blue, G-Biosciences, VWR). Fractions containing pure rSBP were carefully pooled and dialyzed at room temperature for 2 days against 2 × 15 L of 10 mM ammonium bicarbonate (pH 8.0). After lyophilization, rSBP was reconstituted to 1 mg/mL with 10 mM phosphate buffer (pH 7.4), filter-sterilized, and kept at 4 °C until use. Circular Dichroism Analysis of the Purified Recombinant Soybean SBP. Far-UV CD spectra (190−250 nm) were recorded at 25 °C on purified rSBP (IMAC + AEC) at 1 mg/mL in 10 mM phosphate buffer (pH 7.4), using a Pi-Star stopped-flow spectrometer and a 0.2 mm path length cylindrical quartz cuvette (Applied Photophysics, Leatherhead, UK). The final spectrum was the average of eight scans recorded at a 0.2 nm resolution and corrected for baseline and buffer. The secondary structure content of rSBP was estimated using the deconvolution program CDSSTR,20 which gave the best fit compared to other algorithms accessible through the DichroWeb server.21

development of new immunotherapies for the treatment of soy allergy. Furthermore, preliminary results in mice indicated that immunotherapy using homologous allergens from soybean could contribute to the treatment of peanut allergy.12 Comparison between homologous proteins could therefore provide relevant information to advance alternative immunotherapies. We selected new soy candidate allergens by identifying proteins that bind IgEs in other food plant species but did not have allergenic orthologues in soybean. Among the proteins of interest was a lentil polypeptide13 with significant sequence identity to the seed-specific biotinylated proteins (SBP) from both peas14,15 and soy.16 We used a comprehensive approach to characterize this protein, encompassing the construction of a soybean library, the production and purification of the recombinant protein, allergenicity testing using a large panel of serum samples, comparison to the purified natural counterparts in two species, and assessment of its biological activity alongside known reference allergens.

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MATERIALS AND METHODS

Isolation of a Full-Length SBP Clone from a Soybean cDNA Library. A cDNA library was constructed using the ZAP cDNA synthesis kit (Stratagene, La Jolla, CA, USA) from a pool of the mRNAs purified from soybean seeds (cv. Williams 82) at various stages of development until maturity, that is, 17, 23, 30, 40, 50, 60, and 70 days after flowering. To increase the chances of recovering lowabundant transcripts, an aliquot of the unamplified, excised library (approximately 2 × 104 pfu) was used to infect Escherichia coli cells (SOLR strain) and select white colonies on LB agar plates. A total of 24960 white colonies containing individual clones were subsequently picked using a Q-bot colony picker robot (Genetix, New Milton, Hampshire, UK) and stored at −80 °C in 384-well plates. Of these, 18048 were randomly chosen and spotted at high density onto Hybond N+ filters according to a 4 × 4 grid pattern using Genetix Qbot plates. The probes used to screen the library were also spotted onto the membranes and used as positive controls. Further details about the construction of this library can be found elsewhere.17 A 775-bp cDNA probe encompassing 40% of the SBP coding sequence (positions 653−1428, accession no. U59626) was generated from mature seed mRNAs using the primer pair F 5′-AAAGATCAGCGTGGGAACAG-3′ and R 5′-CTGAGGGTTTCCTTCACTTG-3′ and an Omniscript reverse transcription kit (Qiagen, Hilden, Germany). This probe was cloned in the vector pCR 2.1, using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) and served as template for PCR labeling with α32P-dCTP and the Taq PCR Master Mix kit (Qiagen). Before hybridization, the probe was purified with QIAquick nucleotide removal kit (Qiagen) and used to screen the library.17 All clones retrieved after hybridization were first sequenced using the M13 and T7 primer binding sites present in the pBluescript SK(−) vector. Those containing the complete SBP coding cDNAs were fully sequenced at least three times on both strands. All of the Sanger DNA sequencing was carried out by the Iowa State DNA Sequencing Facility (Ames, IA, USA). The cDNA sequences were analyzed using the Vector NTI Advance v10.1 software (Invitrogen). All library-derived DNA sequences were compared to available GenBank sequences, using BLAST.18 Pairwise comparisons between sequences were done using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). The molecular mass and isoelectric point (pI) were calculated from the cDNA-deduced amino acid sequence using the ProtParam tool of ExPASy.19 NGlycosylation sites in the amino acid sequence were predicted using NetNGlyc accessible through the ExPASy resource portal (http:// www.expasy.org). Subcloning, Bacterial Expression, and Extraction of the Recombinant Soybean SBP. The SBP coding sequence was PCR amplified from the pBluescript SK(−) phagemid of a single clone B

DOI: 10.1021/acs.jafc.5b05873 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. IgE Levels and Reactivities in the Serological Samples of the Allergic Subjects IgE level in kU/L (Phadia ImmunoCAP) allergic subject

sample source

sample type

peanut

soy

total

IgE reactivity (Immunoblot) soy rSBPa

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23

PlasmaLab PlasmaLab PlasmaLab PlasmaLab PlasmaLab PlasmaLab Duke PAIDb PlasmaLab PlasmaLab Duke PAID Duke PAID Duke PAID Duke PAID Duke PAID PlasmaLab PlasmaLab Duke PAID Duke PAID Duke PAID Duke PAID Duke PAID Duke PAID Duke PAID

plasma plasma plasma plasma plasma plasma serum/plasma plasma plasma serum/plasma serum/plasma serum/plasma serum/plasma serum/plasma plasma plasma serum/plasma serum/plasma serum/plasma serum/plasma serum/plasma serum/plasma serum/plasma

14.8 12.4 13.8 35.4 30.2 62.5 234.0 406.0 68.6 391.0 122.1 268.5 335.5 298.0 23.0 84.5 626.0 766.0 102.0 223.0 295.5 135.0 313.0

7.9 11.3 12.2 25.9 21.6 4.9 44.4 21.2 4.8 6.3 2.3 11.7 14.6 2.9 98.7 34.2 46.9 14.7 43.0 24.2 12.6 16.3 11.8

2992 522 239 1148 1243 359 1442 1510 145 448 165 392 727 966 4138 5000 598 809 722 672 1319 2093 353

+ − +++ ++ ++ + ++ − +++ − +++ +++ − +++ ++ +++ − − +++ ++ − − −

a Examples of the semiquantitative evaluation of IgE-reactive bands in Western immunoblotting are shown in Figure 4C. bDuke PAID, Pediatric Allergy and Immunology Division at Duke University. IgE values for the Duke samples were measured in the sera. PlasmaLab and Duke PAID samples represent adults and children, respectively.

Purification of Natural SBPs from Soybean and Peanut Seeds. Raw mature seeds of soybean (cv. Hutcheson) and peanut (cv. Florunner) were used as starting materials. For soybean only, seeds were soaked overnight at 4 °C in Milli-Q water (EMD Millipore) to ease removal of the embryo from the cotyledons. Embryos (50 g) were coarsely ground with a mortar and pestle, mixed with 250 mL of 100 mM Tris-HCl buffer (pH 8.5) containing 2 mM EDTA, 5 mM DTT, and 5 M urea, and then finely ground using a VirTishear homogenizer equipped with a 25 mm diameter probe (The VirTis Co., Gardiner, NY, USA) for 5 min on ice. After overnight stirring at 4 °C, the extract was clarified by centrifugation at 40000g for 30 min at 4 °C (JA-14 rotor, Beckman Coulter) and by filtration through a 0.8-μm membrane (Supor PES filters, Pall). Streptavidin−agarose beads (30 mL of a 50% slurry washed with PBS, Pierce) were added to the clarified extract and incubated for 3 h at 4 °C under slow rotation. The bead suspension was transferred to a dripping glass column (2.5 cm i.d.) and thoroughly washed with 500 mL of extraction buffer without DTT, but supplemented with 0.2% SDS. Streptavidin-bound proteins were competitively eluted from the washed beads by two successive incubations with 45 mL and then 15 mL of PBS containing 30 mM Dbiotin, 6 M urea, 2 M thiourea, and 2% SDS (pH 12.0 with NaOH) and, each time, boiling the suspension for 15 min with occasional shaking. The pooled eluate was dialyzed in 3500 MW cutoff tubing against 2 × 5.5 L of 10 mM ammonium bicarbonate (pH 8.0), overnight at room temperature, and concentrated by lyophilization. Alternatively, soybean nSBP eluate was dialyzed in 50 mM Tris-HCl buffer (pH 8.5) to be further purified by AEC. Chromatography and fraction analyses, dialysis, lyophilization, and buffer reconstitution were carried out similarly to the recombinant soy SBP purification. Protein Identification by LC-MS/MS. Colloidal Coomassie bluestained gel bands were excised after SDS-PAGE (4−12% Bis-Tris NuPAGE, Invitrogen) and processed by the Duke Proteomics Core Facility. Briefly, proteins underwent in-gel trypsin digestion and were identified using liquid chromatography−tandem mass spectrometry (LC-MS/MS) on a Q-ToF Premier mass spectrometer (Waters, Milford, MA, USA) coupled to a Nano Acquity liquid chromatography

(Waters). All MS/MS spectra were then searched using Mascot v2.2 (Matrix Science, Boston, MA, USA) against the Viridi plante (green plant) taxonomy of a SwissProt v57.2 database supplemented with the peanut SBP peptide SENVAASDAQAQHHNVGKFESGEEFEGRTREVTGSVPERSGEN.22 Results were then imported into Scaffold v2.5 (Proteome Software, Portland, OR, USA), and peptide identifications were accepted if they could be established at >80% probability as specified by the Peptide Prophet algorithm.22 Protein identifications were accepted if they could be established at >95% probability and contained at least two identified peptides. Protein Composition by LC-MS/MS. The relative abundance of the soybean nSBP purified after streptavidin affinity chromatography (SAC) alone or in combination with anion exchange chromatography (AEC) was quantified by LC-MS/MS. Each purified fraction (5 μg) was buffer exchanged into 50 mM ammonium bicarbonate (pH 8.0) using a 7000 MW cutoff gel filtration spin column, and Rapigest SF (Waters) acid-cleavable surfactant was added to a final concentration of 0.1%. Samples were reduced in 5 mM DTT at 70 °C for 20 min and then alkylated in 10 mM iodoacetamide at room temperature in the dark for 45 min. Following proteolytic digestion with trypsin (1:50 enzyme to protein ratio) (Promega, Madison, WI, USA) for 18 h at 37 °C, the samples were acidified to 0.5% TFA and incubated at 60 °C for 1 h to hydrolyze the Rapigest SF surfactant. LC-MS/MS was performed on a nanoAcquity UPLC coupled to a Q-ToF Premier mass spectrometer (Waters). The mass spectrometer was operated in a data-independent acquisition mode (MSE acquisition). All high-energy spectra were searched using the Identity database within the ProteinLynx Global Server 2.4 (Waters) against the Glycine max taxonomy of an NCBI protein database appended with reverse sequences of each forward protein entry. Relative protein abundances were determined by analyzing each raw LC-MS file in Rosetta Elucidator (Rosetta Biosoftware, Cambridge, MA, USA). Following peak annotation, the intensities (area under curve) for all annotated peaks were summed to yield the total signal intensity. The percent contribution of each protein from the total summed peak intensity was C

DOI: 10.1021/acs.jafc.5b05873 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 2. Percentages of CD63 Positive Basophils Following Stimulation with nSBP-Soy and Other Known Allergens peanut extract (μg/mL)

IgE levela (kU/L) subject b

Pba1 Pba2 Pba3 Pba4e Pba5 Pba6 Pba7 Pba8 Pba9e Pba10 Pba11 Pba12 Pba13 Pba14 Pba15 P14e P19

peanut 3% of CD63+ basophils based on the unstimulated condition values (medium alone) and the negative control responses (Pba1). Three samples were identified as “non responders” (Pba4, Pba9, and P14) because they reacted with low percentages to the anti-IgE positive control (≤3%) and to the peanut extract (7-kDa size discrepancy between the natural SBP and its sequence-deduced calculation could accommodate carbohydrate moieties. However, no signal peptide cleavage site was predicted in the soy SBP sequence after computation through the SignalP algorithm of the NetNGlyc server. Mapping of the six potential Nglycosylation sites shows that five of them belong to regions not covered by any of the tryptic peptides identified by LC-MS/MS (Figure 3). Complex carbohydrates can interfere with peptide identification by either hindering the proteolytic digestion or increasing unpredictably the theoretical mass of peptides.38 Because we boiled the seed extracts before purification, glycation by Maillard reactions could have added reducing sugars to the numerous lysine (9.5%) and histidine (2.2%) residues of the soybean nSBP (Figure 3). This process was also shown to enhance IgE binding for several legume allergens.39 The interaction of nSBP-Soy and IgEs appears to be mediated by both conformational and linear epitopes (Figure 6). For some sera (P4 and P16), when the structure of nSBPSoy was partly disrupted by a chaotropic agent (urea) or fully unfolded by the addition of a strong detergent (SDS) and boiling, it increased IgE binding compared to the native structure (PBS). Conversely, the native nSBP-Soy (PBS) bound more IgEs from serum P5 than when fully denatured

superficial. We selected soy candidate allergens by searching the literature for orthologous proteins that were binding IgEs in other plant species. To optimize the acquisition of the different sequences and their possible variations, we constructed a cDNA library from developing soy seeds. Following this strategy, we isolated the sequence encoding a soybean seed biotinylated protein, which was highly similar to a previously reported sequence.16 Through the characterization of the recombinant and natural proteins, we have demonstrated its allergenic activity. Seed biotinylated proteins were first characterized as allergens in boiled lentils, in which a 66-kDa heat-soluble protein with marked IgE reactivity could be identified as SBP through its sequence similarities to the pea SBP65.13 SBP proteins are only found within the seed embryo, and data collected from pea seeds27 show that they account for 50%) of random coils.30−33 Our CD results on rSBP also reflected this predominantly unorganized conformation in aqueous solution, providing further evidence that SBPs belong to the LEA protein family. The structural stability of the LEA proteins would come from their high content of hydrophilic and small amino acid residues, which promotes flexibility and prevents aggregation caused by the exposure of hydrophobic regions during heat treatment.30 This unusual property of SBP to resist heat denaturation suggests that it could persist, or even be enriched, under certain cooking/food processing conditions. So far, there is no information about SBP’s resistance to acidic pH, digestion by proteases, or other destructive conditions encountered in the gastrointestinal environment. However, during affinity purification of nSBPs, we had to use harsh conditions, especially to break the avidin−biotin complex, one of strongest noncovalent interactions in nature (Kd = 10−15 M). Successful recovery of SBPs after streptavidin affinity shows the remarkable resilience of these proteins. Furthermore, the high percentage of sera reacting against SBP suggests that despite its I

DOI: 10.1021/acs.jafc.5b05873 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Funding

(urea + SDS + heat). Despite the crude nature of our approach and small sample size, the diversity of situations in which IgEs bind to nSBP-Soy reflects the structural versatility of these proteins. Unlike many food allergens with more constrained secondary structures that harbor mostly linear IgE epitopes,40 the flexible structure of SBP makes it a resilient target for the immune system, especially during sensitization. The inverse relationship between SBP’s denaturation state and IgE binding level for sera P5 and P16 (Figure 6) may also explain their ELISA results (Figure 5). Under the non-denaturing conditions of the ELISAs, serum P5 was highly positive to nSBP-Soy, whereas P16 was negative. Conversely, P16 reacted much more to the rSBP-Soy than P5, suggesting that the recombinant protein was more unfolded than its natural counterpart. The relevance of the soybean seed biotinylated protein as a biologically active allergen was supported by its capacity to cross-link FcεRI-bound IgEs and up-regulate CD63 expression on the basophil surface (Table 2). No statistical correlations were found between the specific IgE levels and the percentages of CD63+ basophils (data not shown). The nSBP-Soy was always a stronger basophil activator than the major soybean allergen Gly m 5 and, in two specific instances (P19 and Pba11) even stronger than the major peanut allergen, Ara h 1. Also, two samples (Pba3 and Pbs5) that were activated by nSBP-Soy did not react with Gly m 5, and one (Pba5) did not respond to the soybean extract. These results illustrate the immunospecificity of the basophil reaction to nSBP-Soy. On the basis of its broad IgE recognition and clear ability to stimulate basophils, we believe that nSBP-Soy could trigger IgE-mediated allergic reactions. Our results also show that SBP is a new allergen in peanuts, which together with the original finding in lentils13 and the recent IgE reactivity associated with soybean LEA proteins31 may define a novel family of allergens across different legume species. It is difficult to appraise the clinical significance of this new allergen family considering the small concentrations of SBPs in the seed. This particular point could be misleading in assessing the potential threat posed by SBPs as allergens. Indeed, the food industry broadly uses soybean protein concentrates as supplements, which are often obtained through heat treatment and enrichment processes that could very much increase the amount of SBP available for allergic sensitization. Finally, we developed a purification method to generate working amounts of highly purified natural SBP that can be used to further study the role of seed biotinylated proteins for both the plant science and food allergy communities. The seed biotinylated protein from soybean was registered as the allergen Gly m 7 by the IUIS allergen nomenclature subcommittee (http://www.allergen.org).

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This work was supported by the Food Allergy Initiative at Duke University and a grant from the North Carolina Soybean Producers Association to S.M.W. J.J.R. was awarded a COLCIENCIAS-FULBRIGHT-DNP scholarship from the Colombian Administrative Department of Science, Technology and Innovation. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sarah MacKenzie at North Carolina State University for her valuable contribution to the circular dichroism analysis. ABBREVIATIONS USED rSBP, recombinant seed biotinylated protein; nSBP, natural seed biotinylated protein; Pn, peanut; LEA, late embryogenesis abundant; IMAC, immobilized metal ion affinity chromatography; SAC, streptavidin affinity chromatography; AEC, anion exchange chromatography; DEAE, diethylaminoethyl-; TES, Ntris(hydroxymethyl)methyl-2-aminomethanesulfonic acid; LCMS/MS, liquid chromatography-tandem mass spectrometry

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REFERENCES

(1) Sicherer, S. H.; Sampson, H. A. Food allergy. J. Allergy Clin. Immunol. 2010, 125, S116−S125. (2) Technical Consultation on Food Allergies; Food and Agriculture Organization of the United Nations: Rome, Italy, Nov 13−14, 1995; No. 365907, pp 1−56. (3) Savage, J. H.; Kaeding, A. J.; Matsui, E. C.; Wood, R. A. The natural history of soy allergy. J. Allergy Clin. Immunol. 2010, 125, 683− 686. (4) El-Hifnawi, D.; Neudert, M.; Hardt, A.; Hauswald, B.; Zahnert, T. Anaphylactic reaction after soya intake in a patient with birch pollen allergy. HNO 2012, 60, 117−119. (5) Oei, H. D.; Kartanegara, W. L. Anaphylactic reaction to soy drink in two patients with birch pollen allergy. J. Allergy Clin. Immunol. 2010, 125, AB27. (6) Moroz, L. A.; Yang, W. H. Kunitz soybean trypsin inhibitor: a specific allergen in food anaphylaxis. N. Engl. J. Med. 1980, 302, 1126− 1128. (7) Riascos, J. J.; Weissinger, A. K.; Weissinger, S. M.; Burks, A. W. Hypoallergenic legume crops and food allergy: factors affecting feasibility and risk. J. Agric. Food Chem. 2010, 58, 20−27. (8) Verma, A. K.; Kumar, S.; Das, M.; Dwivedi, P. D. A comprehensive review of legume allergy. Clin. Rev. Allergy Immunol. 2013, 45, 30−46. (9) Holzhauser, T.; Wackermann, O.; Ballmer-Weber, B. K.; Bindslev-Jensen, C.; Scibilia, J.; Perono-Garoffo, L.; Utsumi, S.; Poulsen, L. K.; Vieths, S. Soybean (Glycine max) allergy in Europe: Gly m 5 (β-conglycinin) and Gly m 6 (glycinin) are potential diagnostic markers for severe allergic reactions to soy. J. Allergy Clin. Immunol. 2009, 123, 452−458. (10) Helm, R. M.; Cockrell, G.; Connaughton, C.; Sampson, H. A.; Bannon, G. A.; Beilinson, V.; Nielsen, N. C.; Burks, A. W. A soybean G2 glycinin allergen. 2. Epitope mapping and three-dimensional modeling. Int. Arch. Allergy Immunol. 2000, 123, 213−219. (11) Helm, R.; Cockrell, G.; Herman, E.; Burks, A.; Sampson, H.; Bannon, G. Cellular and molecular characterization of a major soybean allergen. Int. Arch. Allergy Immunol. 1998, 117, 29−37. (12) Pons, L.; Ponnappan, U.; Hall, R. A.; Simpson, P.; Cockrell, G.; West, C. M.; Sampson, H. A.; Helm, R. M.; Burks, A. W. Soy immunotherapy for peanut-allergic mice: modulation of the peanutallergic response. J. Allergy Clin. Immunol. 2004, 114, 915−921. (13) Sanchez-Monge, R.; Pascual, C. Y.; Diaz-Perales, A.; FernandezCrespo, J.; Martin-Esteban, M.; Salcedo, G. Isolation and character-

AUTHOR INFORMATION

Corresponding Author

*(J.J.R.) Present address: Laboratory of Biotechnology, Centro de Investigación de la Cañ a de Azúcar de Colombia (Cenicaña), Calle 58 Norte No. 3BN-110, Cali, Colombia. Phone: (57) 2 687 6611, ext. 5142. Fax: (57) 2 260 7853. Email: [email protected]. Present Address #

(M.K.; A.W.B.) Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. J

DOI: 10.1021/acs.jafc.5b05873 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b05873 J. Agric. Food Chem. XXXX, XXX, XXX−XXX