Gly m 2S albumin is a major allergen with a high diagnostic value in soybean-allergic children

Abstract

Several soybean proteins have been suggested to have IgE reactivity but only 6 of them have so far been recognized by the WHO/IUIS Allergen Nomenclature Subcommittee.1Ballmer-Weber B.K. Vieths S. Soy allergy in perspective.Curr Opin Allergy Clin Immunol. 2008; 8: 270-275Crossref PubMed Scopus (64) Google Scholar Gly m 4, a pollen-related allergen, and the storage proteins Gly m 5 and Gly m 6 have been found to be associated with food allergy.2Fukutomi Y. Sjölander S. Nakazawa T. Borres M.P. Ishii T. Nakayama S. et al.Clinical relevance of IgE to recombinant Gly m 4 in the diagnosis of adult soybean allergy.J Allergy Clin Immunol. 2012; 129: 860-863Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar, 3Ito K. Sjölander S. Sato S. Movérare R. Tanaka A. Söderström L. et al.IgE to Gly m 5 and Gly m 6 is associated with severe allergic reactions to soybean in Japanese children.J Allergy Clin Immunol. 2011; 128: 673-675Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar The storage protein 2S albumin has not yet been recognized as an important allergen for soybean (Gly m 2S albumin), and its association with clinical symptoms is not clearly established.4Lin J.P.R. Shewry P.R. Archer D.B. Beyer K. Niggemann B. Haas H. et al.The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.Int Arch Allergy Immunol. 2006; 141: 91-102Crossref PubMed Scopus (18) Google Scholar Biochemically, the 2S albumins are characterized by a conserved 3-dimensional structure, high stability to the gastrointestinal tract environment, and high resistance to thermal processing.5Moreno F.J. Clemente A. 2S Albumin storage proteins: what makes them food allergens?.Open Biochem J. 2008; 2: 16-28Crossref PubMed Scopus (90) Google Scholar The amino acid sequence identity between 2S albumins from different species is low, often less than 40%. Altogether these properties may explain the generally high diagnostic value of IgE measurements.6Ebisawa M. Movérare R. Sato S. Maruyama N. Borres M.P. Komata T. Measurement of Ara h 1-, 2-, and 3-specific IgE antibodies is useful in diagnosis of peanut allergy in Japanese children.Pediatr Allergy Immunol. 2012; 23: 573-581Crossref PubMed Scopus (65) Google Scholar In this study, the diagnostic value of Gly m 2S albumin was investigated and compared with Gly m 5 and Gly m 6, by analysis of IgE in sera from soybean-allergic children. Sera from 55 soybean-sensitized Japanese children were collected (Table I). Nineteen were diagnosed with soybean allergy by positive oral food challenge (OFC) (n = 16) or a definitive history (n = 3) of urticaria within 1 hour after intake. The remaining 36 children were nonsymptomatic and were either diagnosed by negative OFC (n = 17) or a regular consumption in their daily life (n = 19). Ethical approvals were obtained through the institutional review boards at Sagamihara National Hospital and Aichi Children's Health and Medical Center. Among the symptomatic children, 16 had skin symptoms, 5 oral symptoms, 6 respiratory symptoms (coughing and wheeze), 1 gastrointestinal symptom (diarrhea), and 1 neurologic symptom (sleep). Intramuscular adrenaline injection was used in 1 case, which was considered as an anaphylaxis. No allergies to nuts were documented among the children in the study. OFCs were conducted in accordance with Japanese guidelines.7Ito K. Urisu A. Diagnosis of food allergy based on oral food challenge test.Allergol Int. 2009; 58: 467-474Crossref PubMed Scopus (30) Google ScholarTable IDemographic, serologic, and clinical characterization of 55 soybean-sensitized Japanese children with or without symptomsPatient characteristicsSymptomaticNonsymptomaticP value∗Comparison between the study groups regarding IgE level to Gly m 2S albumin, soybean, Gly m 5, and Gly m 6 using the Mann-Whitney U test (P < .05 was considered significant).AUC†Area under the curve (AUC) from receiver operating characteristic (ROC) curve analysis comparing children in the symptomatic and nonsymptomatic groups.(n = 19)(n = 36)Sex,12/728/8 male/femaleAge (y), median (range)2.4 (0.7-9.8)1.9 (0.6-10.3)Specific IgE to (kUA/L), median (range) Soybean23.7 (0.4-92.0)4.1 (0.5-77.3).02580.69 2S albumin6.8 (0.1-120.0)0.5 (0.0-46.6).00240.75 Gly m 520.4 (0.1-58.3)1.7 (0.1-81.9).05210.69 Gly m 615.5 (0.3-70.8)2.5 (0.4-93.5).10160.64Diagnosis of soybean allergy OFC1619 History317Symptoms Skin16— Oral5— Respiratory6— Gastrointestinal1— Anaphylaxis1—∗ Comparison between the study groups regarding IgE level to Gly m 2S albumin, soybean, Gly m 5, and Gly m 6 using the Mann-Whitney U test (P < .05 was considered significant).† Area under the curve (AUC) from receiver operating characteristic (ROC) curve analysis comparing children in the symptomatic and nonsymptomatic groups. Open table in a new tab Gly m 2S albumin was purified from soybeans by anion exchange chromatography followed by gel filtration. Purity and identity of Gly m 2S albumin were determined by SDS-PAGE, analytical gel filtration, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight ion separation (positive linear mode and peptide mass fingerprinting), and N-terminal protein sequencing (10 cycles). Experimental ImmunoCAP with Gly m 2S albumin was developed by Phadia AB (Uppsala, Sweden).8Marknell DeWitt A. Niederberger V. Lehtonen P. Spitzauer S. Sperr W.R. Valent P. et al.Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11.Clin Exp Allergy. 2002; 32: 1329-1340Crossref PubMed Scopus (72) Google Scholar Soybean, Gly m 5, and Gly m 6 are commercially available ImmunoCAP products. The detection limit of the test was 0.1 kUA/L. The Methods are described more fully in this article's Online Repository at www.jacionline.org. The recovery of pure Gly m 2S albumin was 1.1 mg/g of soybeans. Analytical gel filtration showed a homogenous dimer with a molecular size of 28 kDa (see Fig E1 in the Online Repository at www.jacionline.org). Matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight ion separation revealed the mass 14 kDa (m/z) (Fig 1). Peptide mass fingerprinting and N-terminal sequencing showed agreement with UniProt accession no. P19594, and 80 amino acids of 120 (75%) were identified (see Fig E2 in the Online Repository at www.jacionline.org). The N-terminal protein sequencing did not indicate the presence of any impurities. No evidence of the presence of carbohydrate chains was found. The allergenicity of Gly m 2S albumin was investigated by analysis of IgE antibody in sera (Table I). Seventeen out of 19 children with soybean allergy and 31 out of 36 nonsymptomatic children had IgE levels of more than 0.1 kUA/L to Gly m 2S albumin. A significant differentiation between IgE levels in the symptomatic and the nonsymptomatic children was found (P < .01). Of the 2 sera with IgE levels of less than 0.1 kUA/L in the symptomatic children, both had IgE to Gly m 5 and 6, and of the 5 sera with IgE levels of less than 0.1 kUA/L in the nonsymptomatic children, all had IgE to Gly m 6 and 4 to Gly m 5. No significant differences in IgE levels between those who were OFC negative and history negative could be seen. Receiver operating characteristic analysis showed that Gly m 2S albumin had an area under the curve of 0.75, followed by soybean and Gly m 5 (0.69 for both) and Gly m 6 (0.64) (see Fig E3 in the Online Repository at www.jacionline.org). Receiver operating characteristic analysis by combining the 3 components, 2 or all 3, did not improve the area under the curve. Gly m 2S albumin, Gly m 5, and Gly m 6 were found to be major allergens in the study group. Gly m 5 and Gly m 6 have in earlier studies been classified as major allergens in children and have also been correlated with severe symptoms from soybean.3Ito K. Sjölander S. Sato S. Movérare R. Tanaka A. Söderström L. et al.IgE to Gly m 5 and Gly m 6 is associated with severe allergic reactions to soybean in Japanese children.J Allergy Clin Immunol. 2011; 128: 673-675Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 9Holzhauser T. Wacermann O. Ballmer-Weber B.K. Bindslev-Jensen C. Scibilia J. Perono-Garoffo L. et al.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: 452-458Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar In this study, IgE to Gly m 2S albumin was significantly higher in the symptomatic group, while IgE to Gly m 5 and Gly m 6 was not, although a trend toward a significant differentiation was seen. IgE reactivity to Gly m 2S albumin has been investigated earlier.4Lin J.P.R. Shewry P.R. Archer D.B. Beyer K. Niggemann B. Haas H. et al.The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.Int Arch Allergy Immunol. 2006; 141: 91-102Crossref PubMed Scopus (18) Google Scholar Lin et al4Lin J.P.R. Shewry P.R. Archer D.B. Beyer K. Niggemann B. Haas H. et al.The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.Int Arch Allergy Immunol. 2006; 141: 91-102Crossref PubMed Scopus (18) Google Scholar reported no IgE to 2S albumin in the soybean-allergic patients. This discrepancy between the study of Lin et al4Lin J.P.R. Shewry P.R. Archer D.B. Beyer K. Niggemann B. Haas H. et al.The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.Int Arch Allergy Immunol. 2006; 141: 91-102Crossref PubMed Scopus (18) Google Scholar and this study may, for example, depend on the study population, geographical reasons, and different 2S albumin preparations, and is further discussed in the Online Repository at www.jacionline.org. Soybean allergy in adults and adolescents may be pollen-related, caused by IgE cross-reactivity between the PR-10 allergen component Bet v 1 from Betulaceae pollen and its homologue in soybean, Gly m 4.2Fukutomi Y. Sjölander S. Nakazawa T. Borres M.P. Ishii T. Nakayama S. et al.Clinical relevance of IgE to recombinant Gly m 4 in the diagnosis of adult soybean allergy.J Allergy Clin Immunol. 2012; 129: 860-863Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar The Gly m 2S albumin in this study has proved to be pure and homogeneous, and the identity correlates best with the reviewed 2S albumin from soybean with accession no. P19594. Proteins from the lipid transfer protein (LTP) group and the 2S albumin group generally have similar molecular weights, and it is therefore important to eliminate cross-contamination. Extensive efforts have been made to prove that LTP and other contaminating proteins are not present in Gly m 2S albumin. In addition, efforts to purify LTP from soybean have failed (data not shown). Others have shown that soybean LTP is present in the hull of the soybean, and it is therefore not considered a food allergen.1Ballmer-Weber B.K. Vieths S. Soy allergy in perspective.Curr Opin Allergy Clin Immunol. 2008; 8: 270-275Crossref PubMed Scopus (64) Google Scholar Soybean is 1 of 8 foods thought to cause more than 90% of food allergy reactions in children. Nevertheless, knowledge about specific soybean allergens associated with clinical symptoms is in large part uninvestigated. Earlier studies have shown that analysis of IgE to both Gly m 5 and Gly m 6 will facilitate the diagnosis of soybean allergy in children.3Ito K. Sjölander S. Sato S. Movérare R. Tanaka A. Söderström L. et al.IgE to Gly m 5 and Gly m 6 is associated with severe allergic reactions to soybean in Japanese children.J Allergy Clin Immunol. 2011; 128: 673-675Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 9Holzhauser T. Wacermann O. Ballmer-Weber B.K. Bindslev-Jensen C. Scibilia J. Perono-Garoffo L. et al.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: 452-458Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar In this study, we show that 2S albumin from soybean is a major allergen in Japanese children with soybean allergy. The biochemical features of 2S albumin allergens with generally low cross-reactivity between species may be advantageous in the differentiation between clinical soybean allergy and allergies caused by other legumes, for example, peanut. We put forward that analysis of IgE to Gly m 2S albumin will provide a high diagnostic value and will even further facilitate the diagnosis of soybean allergy. We are grateful to Raimo Carlsson for help with the glycosylation analysis and Fredrik Bernhardsson for help with the running of the immunoassays. Oral soy challenges were conducted in accordance with Japanese guidelines by using an increasing amount of tofu (traditional soy paste containing 6.6% of soy protein) or boiled soybeans (16% protein). Increasing amount in 5 to 6 doses was given every 15 to 20 minutes, up to the cumulative dose of more than 3 g soy protein. Challenge results were considered as positive when obvious objective symptoms were observed. Negative challenge was confirmed after follow-up visits to ensure the absence of any symptoms after the ingestion of soy products. Preparation of Gly m 2S albumin was performed from defatted and freeze-dried soybeans, Glycine max (Allergon, Ängelholm, Sweden). Extract of soybeans was applied to anion exchange chromatography (Q Sepharose HP; GE Healthcare, Uppsala, Sweden), and adsorbed material containing Gly m 2S albumin was eluted by a linear sodium chloride gradient. Further purification was performed by gel filtration on Superdex 75 pg (GE Healthcare). Fractions containing purified Gly m 2S albumin were pooled and concentrated by using an Amicon cell with regenerated cellulose (5 kDa) membrane filter (Millipore, Billerica, Mass). The protein concentration was determined by using the bicinochroninic acid (BCA) method (BCA protein assay kit, Thermo Scientific, Rockford, Ill). The prepared Gly m 2S albumin solution was frozen at −20°C until use. SDS-PAGE was performed on NuPAGE 4% to 12% Bis-Tris Gel from Invitrogen (Carlsbad, Calif). Prepared Gly m 2S albumin was treated with NuPAGE lithium dodecyl sulfate sample buffer, and for the reducing condition dithiothreitol was added followed by alkylation with iodoacetamide, and heated at 97°C for 5 minutes. The gel was loaded with 0.5 μg Gly m 2S albumin (reduced and nonreduced). The molecular weight calibration of the gel was achieved by using Mark 12 unstained standard (Invitrogen). Electrophoresis was performed in 2-(N-morpholino) ethanesulfonic acid running buffer following the manufacturer's instruction. The gel was silver stained according to Blum et alE1Blum H. Beier H. Gross H.J. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels.Electrophoresis. 1987; 8: 93-99Crossref Scopus (3734) Google Scholar in a Hoefer Processor Plus (GE Healthcare). The glycosylation status of 2S albumin was investigated by enzymatic deglycosylation of the protein with several glycosidases (PNGase F, Neuraminidase, GPase A, 3 O-glycosidases) followed by SDS-PAGE, where 5 and 10 μg of treated and untreated Gly m 2S albumin and 5 μg fetuin (positive control) were run on the gel under reducing and nonreducing conditions. The gel was stained by using a glycoprotein detection kit (Sigma-Aldrich, St Louis, Mo) based on the periodic acid-Schiff method. Matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight ion separation (MALDI-TOF MS) in the positive linear mode and peptide mass fingerprinting (PMF) were performed by using a Autoflex, II system (Bruker Daltonic, Bremen, Germany) from prepared Gly m 2S albumin in solution. For the PMF analysis, the Gly m 2S albumin solution was reduced with dithiothreitol and alkylated with iodoacetamide before it was digested with trypsin. Salt was removed by reversed phase chromatography on a C18 column (Millipore, Billerica, Mass). When the whole Gly m 2S albumin protein was analyzed in the linear mode, salt was removed directly on a C18 column. The molecular weight of Gly m 2S albumin was estimated by using a calibrated Superdex 75 PC 3.2/30 column (GE Healthcare) on an Ettan LC system (GE Healthcare). Twenty microliters was applied to the column, and the Unicorn analysis module 5.0 (GE Healthcare) was used to estimate molecular size. Five micrograms of Gly m 2S albumin was diluted in 1 mL 6 M guanidine-HCl, 0.1 M Tris, pH 8, and incubated with 5 μL 0.5 M dithiothreitol for 2 hours at 37°C. After cooling the solution to room temperature, 20 μL 0.5 M iodoacetamide was added and the solution was incubated in the dark for 15 minutes. N-terminal protein sequencing (10 cycles) was then performed by using the Edman degradation method in a G1000A sequencer (Hewlett-Packard, Palo Alto, Calif). The obtained sequence was compared with other known 2S albumins in UniProtKBE2The UniProt ConsortiumReorganizing the protein space at the Universal Protein Resource (UniProt).Nucleic Acids Res. 2012; 40 (The UniProtKB database at): D71-D75http://www.uniprot.orgCrossref PubMed Scopus (1096) Google Scholar by using the BLAST function. Analytical gel filtration of Gly m 2S albumin confirms that the preparation is homogeneous, but it also shows that it acts as a dimer with a molecular size of 28 kDa in the native state (Fig E1) in contrast to the analysis with MALDI-TOF MS in the positive linear mode, which reveals the mass determination, m/z, to be 14 kDa. The N-terminal protein sequencing was originally performed to obtain identity of the Gly m 2S albumin. By using the peak area from the internal standard, it was possible to calculate the mole content reflected by the obtained peak areas from the first 10 amino acids from the 2 subunits of Gly m 2S albumin. In this way, it could be shown that the total obtained amount mole corresponds to the amount mole of Gly m 2S albumin used in the analysis given an indirect proof of its purity. The identity of the prepared material was verified by N-terminal protein sequencing and PMF using MALDI-TOF MS. The N-terminal protein sequencing showed that the first 10 amino acids from the 2 subunits corresponded to 100% with 2S albumin (Glycine max) with accession no. P19594,E2The UniProt ConsortiumReorganizing the protein space at the Universal Protein Resource (UniProt).Nucleic Acids Res. 2012; 40 (The UniProtKB database at): D71-D75http://www.uniprot.orgCrossref PubMed Scopus (1096) Google Scholar amino acid 22 to 31 and 82 to 91, respectively. Using PMF, peptides containing amino acids 24 to 50, 97 to 105, and 117 to 158, corresponding to accession no. P19594, could be shown, and together with the N-terminal sequences, a total identification of 80 amino acids of a total of 120 (75%) was obtained (Fig E2). IgE reactivity to Gly m 2S albumin has been investigated earlier.E3Lin J.P.R. Shewry P.R. Archer D.B. Beyer K. Niggemann B. Haas H. et al.The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.Int Arch Allergy Immunol. 2006; 141: 91-102Crossref PubMed Scopus (33) Google Scholar In the study by Lin et al,E3Lin J.P.R. Shewry P.R. Archer D.B. Beyer K. Niggemann B. Haas H. et al.The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.Int Arch Allergy Immunol. 2006; 141: 91-102Crossref PubMed Scopus (33) Google Scholar none of the soybean-allergic patients including both children and adults from Europe was found to have IgE to 2S albumin. This discrepancy may depend on several factors, one being the different study group compositions. In the study by Lin et al,E3Lin J.P.R. Shewry P.R. Archer D.B. Beyer K. Niggemann B. Haas H. et al.The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.Int Arch Allergy Immunol. 2006; 141: 91-102Crossref PubMed Scopus (33) Google Scholar the patients are a mixture of both children and adults, while the patients in this study are composed of children only. Soybean allergy in adults and adolescents has in several studies been shown to be pollen-related, caused by IgE cross-reactivity between the PR-10 allergen component Bet v 1 from Betulaceae pollen (birch) and its homologue in soybean, Gly m 4.E6Fukutomi Y. Sjölander S. Nakazawa T. Borres M.P. Ishii T. Nakayama S. et al.Clinical relevance of IgE to recombinant Gly m 4 in the diagnosis of adult soybean allergy.J Allergy Clin Immunol. 2012; 129: 860-863Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar The disagreement may also depend on geographic reasons, with different dietary habits regarding soybean products, and different prevalences regarding Betulaceae pollen–related allergies. Japan is one of the largest soybean-consuming populations in the world but has a low prevalence of Betulaceae pollen–related allergies, while the soybean consumption in Europe is low but with a high prevalence of Betulaceae pollen–related allergies, especially in central and northern parts. However, the reason may also be that different isoforms of 2S albumin have been used in the 2 studies. In the study by Lin et al,E3Lin J.P.R. Shewry P.R. Archer D.B. Beyer K. Niggemann B. Haas H. et al.The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.Int Arch Allergy Immunol. 2006; 141: 91-102Crossref PubMed Scopus (33) Google Scholar both native and recombinantly produced 2S albumin from soybean have been used for IgE measurements, while the Gly m 2S albumin used in this study was purified from soybeans.Fig E2The short and long chains from the amino acid sequence of the soybean 2S albumin accession no. P19594. The gray boxes indicate identified amino acid residues from N-terminal sequencing and PMF of the prepared Gly m 2S albumin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Comparison of ROC curves for soybean, Gly m 2S albumin (Gly m 2S), Gly m 5, and Gly m 6 in symptomatic versus nonsymptomatic children. AUC, Area under the curve; ROC, receiver operating characteristic.View Large Image Figure ViewerDownload Hi-res image Download (PPT)