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Identification of sesame seed allergens by 2-dimensional proteomics and Edman sequencing: Seed storage proteins as common food allergens
Food and drug reactions and anaphylaxis Identification of sesame seed allergens by 2-dimensional proteomics and Edman sequencing: Seed storage proteins as common food allergens Kirsten Beyer, MD, Ludmilla Bardina, MS, Galina Grishina, MS, and Hugh A. Sampson, MD New York, NY
Background: Sesame seed allergy is becoming increasingly prevalent, probably because of its use in international fast-food and bakery products. Despite this fact, few studies have focused on the identification of its major allergenic proteins. Objective: The aim of this study was to identify allergenic proteins of sesame seeds (Sesamum indicum). Methods: Extracted sesame seed proteins were separated by means of SDS-PAGE and 2-dimensional (2-D) PAGE. Immunolabeling was performed with individual patient sera from 20 patients with sesame seed allergy. Selected proteins were further analyzed by means of Edman sequencing. Results: IgE-binding proteins were identified at 78, 52, 45, 34, 32, 29, 25, 20, 9, and 7 kd. Analyzing internal sequences, the protein at 45 kd, which was recognized by 75% of the patients, was found to be a 7S vicilin-type globulin, a seed storage protein of sesame and named Ses i 3. The protein at 7 kd was found to be a 2S albumin, another seed storage protein of sesame and named Ses i 2. Seed storage proteins are known food allergens in peanut, walnut, Brazil nut, and soybean. Interestingly, one known IgE-binding epitope of the peanut allergen Ara h 1 has 80% homology with the corresponding area of Ses i 3. The different amino acids were previously shown not to be critical for IgE binding in Ara h 1. In addition, the proteins at 78 and 34 kd were found to be homologous to the embryonic abundant protein and the seed maturation protein of soybeans, respectively. Conclusion: The identification of 4 sesame seed allergens is the first step toward generating recombinant allergens for use in future immunotherapeutic approaches. In addition, the detection of conserved IgE binding epitopes in common food allergens might be a useful tool for predicting cross-reactivity to certain foods. (J Allergy Clin Immunol 2002;110:154-9.)
Food and drug reactions and anaphylaxis
From the Division of Pediatric Allergy and Immunology and the Jaffe Institute for Food Allergy, The Mount Sinai School of Medicine, New York. Supported in part by a grant from the Food Allergy Initiative and in part by a grant from the National Center of Research Resources-National Institutes of Health (MO1 RR-00071) awarded to the Mount Sinai School of Medicine. Received for publication October 29, 2001; revised March 28, 2002; accepted for publication April 1, 2002. Reprint requests: Kirsten Beyer, MD, The Mount Sinai School of Medicine, Division of Pediatric Allergy and Immunology, Box-No. 1198, One Gustave L. Levy Place, New York, NY 10029-6574. © 2002 Mosby, Inc. All rights reserved. 0091-6749/2002 $35.00 + 0 1/87/125487 doi:10.1067/mai.2002.125487
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Key words: Sesame seed, allergen, 2-dimensional, proteomics, immunolabeling, IgE, seed storage protein, vicilin, 2S albumin, 7S globulin, Ses i 3, Ses i 2
An increase in sesame seed allergy in children and adults has been reported in recent years, with several reports found throughout the literature.1-9 In a more complex study investigating 4078 Australian children, the sensitization rate to sesame seed was found to be one third that of the peanut sensitization rate and higher than that to any tree nut.8 The majority of the sensitized children were less than 2 years of age.8 Similar results were found in a study from Israel.9 However, sesame seed allergy has also been described as an occupational allergy.10,11 Common symptoms seen in sesame seed allergy are urticaria, angioedema, and respiratory distress.1,8,9 Similar to peanut and tree nut allergy, hypersensitivity reactions tend to be more severe in nature, often resulting in an anaphylactic reaction.1-4,6,7 The increase in sesame seed allergy might be explained by increasing consumption of sesame seed–containing foods, such as international fast-food and bakery products. In North America and Europe sesame seeds generally are used for toppings on breads, such as hamburger buns, bagels, bread sticks, and other baked goods. Restaurants purchase sesame seeds for use in ethnic dishes. In 1995, the United States imported 39,366 metric tons of sesame seeds.12 In the same year the member countries from the European Union (EU) imported 56,529 metric tons of sesame seeds, an increase of 100% by volume since 1988.12 The most significant sesame seed importers in the EU are Germany, the United Kingdom, and the Netherlands. The sesame species of greatest economic importance in the United States and the EU is Sesamum indicum. Despite the importance of sesame as a food allergen, the identification of its clinically relevant allergens is still incomplete. Very recently, one major allergen of sesame seeds, a 2S albumin seed storage protein, has been identified.13 Through the use of SDS-PAGE, 2-dimensional (2-D) proteomics, mass spectrometry, and internal sequencing, the present article reports 4 new sesame seed allergens.
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METHODS Preparation of sesame seed extract Sesame seeds (S indicum) were ground in a coffee grinder (Krups), followed by mortar and pestle, until a smooth paste was achieved. The paste was defatted by washing with at least 20 volumes (wt/vol) of cold acetone and dried overnight at 4°C. Dried acetone powder was stored at –80°C. Protein was extracted from the defatted, dried, powdered seed pulp by agitating with PBS and a protease inhibitor cocktail without EDTA (Roche) overnight at 4°C. After centrifugation at 2500g for 15 minutes at 4°C, supernatant was collected, filtered, and centrifuged at 12,000g for 3 minutes. Protein concentrations were determined by means of spectrophotometry (SmartSpec, Biorad) by using the Coomassie Plus Protein Assay (Pierce). All extracts were stored at –80°C.
SDS-PAGE analysis and 2-D proteomics mapping For SDS analysis, proteins were separated by means of SDSPAGE (Nu-Page MES Bis-Tris, Invitrogen), according to the protocol from the manufacturer. Ten micrograms of protein extract was loaded into each well. For 2-D gel electrophoresis, the crude extract was desalted and concentrated with Perfect Focus (GenoTechology Inc) and resuspended in rehydration buffer (9.8 mol/L urea, 4% chaps, 2 mmol/L tributylphosphine, 0.2% ampholytes, and 0.001% bromophenol blue; Biorad). The 50 µg of protein extract was applied to 7 cm of immobilized pH gradient strips (Biorad) for rehydration and focusing for 17 hours with the linear voltage slope of up to 20,000 volt-hours, followed by 2-D gel electrophoresis with Invitrogen Zoom Gels (Invitrogen). Before running the SDS-PAGE, immobilized pH gradient strips were equilibrated with urea, SDS, and glycerol containing TrisHCl buffer in the presence of 5 mmol/L tributylphosphine for 10 minutes and then 135 mmol/L iodoacetamide for 10 minutes. The proteins resolved by using both techniques were subsequently transferred to Immobilon-P membrane (Millipore) and then stained with 2.2% Coomassie blue for total protein analysis. For molecular weight determination, MultiMark Multi-Colored Standard (Invitrogen) was used.
Probing immunoblots with sera from patients with sesame seed allergy For detection of IgE binding to the separated sesame seed proteins, immunolabelling was performed with individual patient sera from 20 individuals with sesame seed allergy. Clinical history, age of the patients, and sesame seed–specific IgE levels detected with the Pharmacia FEIA-Cap System are shown in Table I. Normal human serum (Pharmacia) was used as a negative control. Patient and control sera diluted from 1:4 to 1:10, depending on the level of serum IgE, in PBS-Tween plus 1% BSA and 10% normal goat serum were incubated with immunoblots by means of gentle agitation at room temperature. After 1 hour, 2-D immunoblots were briefly rinsed with PBS, and phosphatase-labeled goat anti-human IgE (KPL) at a concentration of 1 µg/mL goat IgG per milliliter in PBS-Tween plus 1% BSA and 10% normal goat serum was applied. After agitation for 45 minutes at room temperature and washing with PBS, immunoblots were developed with phosphatase substrate 5-bromo-4-chloroindolyl-phosphatase/nitroblue tetrazolium (KPL). For SDS-PAGE analysis, immunoblots were rinsed after 2 hours with PBS, and
iodine 125–labeled goat anti-human IgE (DiaMed) diluted as per the manufacturer was applied. After agitation for 1 hour at room temperature and washing with PBS, immunoblots were mounted on filter paper and exposed to Kodak X-OMAT Imaging Film for 1 to 5 days. Protein images were analyzed with GelDoc 2000 (Biorad) by using Quantity One Quantitation Software.
MALDI-MS analysis and Edman sequencing Following the procedure described under 2-D proteomics mapping, the proteins were separated with 2-D PAGE, and the gels were stained with Coomassie blue. Proteins in spots of interest were analyzed at the Wistar Institute Protein Microchemistry/Mass Spectrometry Facility. Briefly, the proteins of interest were excised for in-gel trypsin digestion by using a sequencing-grade modified trypsin (Promega) and were alkylated with iodoacetamide (Sigma) as part of the digestion protocol. The resulting peptide mixture was separated by means of reverse-phase HPLC on a Zorbax 2.1 × 150–mm C-18 column (Agilent Technologies). The solvent gradient was delivered and controlled with System Gold HPLC (Beckman). Solvent A was 0.1% trifluoracetic acid in MilliQ water, and solvent B was 0.085% trifluoracetic acid in 95% acetonitrile. Selected HPLC fractions were then analyzed by means of MALDI-MS on a PerSeptive DE Pro, and Edman sequence analysis was completed on the Applied BioSystems Procise sequencer.
RESULTS Proteins were separated by means of SDS-PAGE, as well as 2-D gel electrophoresis, and either stained with Coomassie blue for total protein analysis or transferred to membranes and immunolabeled with sera from 20 individuals with sesame seed allergy to identify allergenic proteins of sesame seeds. Fig 1 shows the SDSPAGE and the results of the immunolabeling for all 20 patients and the control subject. IgE-binding proteins were identified at 78, 52, 45, 34, 32, 29, 25, 20, 9, and 7 kd. Fig 2 shows all sesame seed proteins separated by means of 2-D PAGE and the results of the immunolabeling for 4 representative patients and normal control serum. Four IgE-binding protein fractions at 7, 34, 45, and 78 kd that were recognized by 30%, 60%, 75%, and 50% of the patients, respectively, were chosen for further analysis by means of Edman sequencing (Figure 1 and 2). The internal sequences of these 4 proteins are shown in Table II. However, the protein at 78 kd that was recognized by half of the patients in 2-D gel electrophoresis showed weak binding in most patients and was not visible in the majority of patients in SDS-PAGE analysis. Analyzing internal sequences, the protein at 45 kd was found to be a 7S vicilin-type globulin, and the 7-kd protein was found to be a 2S albumin of S indicum (Table II). The 2 new sesame seed allergens were named Ses i 3* and Ses i 2.* 7S vicilin-type globulins or 2S albumins are known food allergens in several plants, including peanut, walnut, Brazil nut, and soybean. Looking at the entire sequence, the 2S albumin precursor of sesame seed (Ses i 2) has 38% homology to the walnut allergen Jug r *Allergen
data have been submitted to the World Health Organization/International Union of Immunological Societies Allergen Nomenclature SubCommittee for approval of the new name.
Food and drug reactions and anaphylaxis
Abbreviation used EU: European Union
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FIG 1. SDS-PAGE analysis for sesame seed proteins: lane 2, labeling with Coomassie blue stain for total protein analysis; lanes 4 to 23, immunolabeling with sera from 20 patients with sesame seed allergy; lane 24, labeling with control sera. Molecular weight standards (MW) are shown in lanes 1, 3, and 25.
Food and drug reactions and anaphylaxis
FIG 2. Two-dimensional proteomics map for sesame seed proteins. Shown is labeling with Coomassie blue for total protein analysis and immunolabeling of 4 representative patients and the control serum. Molecular weight standards (MW) are shown at left. Red circles indicate proteins recognized at 7, 34, 45, and 78 kd, for which Edman sequencing was performed.
1, 40% homology to the Brazil nut allergen Ber e 1, and 34% homology to the peanut allergen Ara h 2. The 7S vicilin-type globulin of sesame (Ses i 3) has 36% homology to the peanut allergen Ara h 1 and 41% homology to
the walnut allergen Jug r 2. It is of interest that one of the 22 known IgE-binding epitopes of Ara h 1 has 80% homology with the corresponding area of the sesame vicilin (Fig 3). The amino acids that differed were previ-
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FIG 3. Shown is the homology in amino acid sequences for the sesame seed 7S globulin and Ara h 1 in the area of an IgE-binding epitope of Ara h 1. Identical amino acids are shown in bold and underlined, and homologous amino acids are underlined only. Amino acids highlighted in gray are known to lead to a loss of IgE binding when altered.14
FIG 4. Shown are the amino acid sequences for the large subunits of the two 2S albumins of sesame seed. Identical amino acids are shown in bold and underlined, and homologous amino acids are underlined only. The two 2S albumins of sesame seed are, in general, only 35% identical and 47% homologous. Amino acids highlighted in light gray were found to be the sequence of an internal fragment of the 7-kd allergen in the present study, and amino acids are highlighted in dark gray in the sequence of an internal fragment of the allergen in the study by Pastorello et al.13 Adapted from Pastorello et al.13 Copyright 2001 with permission from Elsevier Science.
TABLE I. Shown are clinical reactions, age, and sesame seed–specific IgE levels for the 20 individuals with sesame seed allergy Patient
A B C D E F G I J K L M P Q R S T U V W
Clinical reaction
Angioedema, worsening of eczema Angioedema, urticaria, GI symptoms Angioedema, urticaria, rhinitis Angioedema, GI symptoms Mouth and throat itchiness GI symptoms, worsening of eczema Angioedema, urticaria Angioedema, respiratory symptoms Angioedema, GI symptoms Urticaria Angioedema Mouth and throat itchiness Angioedema, worsening of eczema Angioedema, GI symptoms Urticaria, angioedema, worsening of eczema Urticaria, angioedema, GI symptoms Urticaria GI symptoms GI symptoms, respiratory symptoms, throat tightness Urticaria, rhinoconjunctivitis
Age (y)
IgE (kU/L)
6 7 3 28 6 6 15 2 9 9 3 14 7 12 11 3 4 6 10 2
>100.00 >100.00 >100.00 >100.00 73.90 22.40 5.92 4.85 3.83 29.90 >100.00 64.70 37.50 >100.00 29.1 >100.00 6.65 8.78 13.10 8.23
ously shown not to be critical for IgE binding to Ara h 1 by using single-amino-acid substitution.14 In addition, the proteins at 78 and 34 kd were found to be homologous to the embryonic abundant protein and the seed maturation protein of soybeans, respectively (Table II).
DISCUSSION In the present study we identified 4 allergens for sesame seeds. Two of them, the 7S vicilin-type globulin
(Ses i 3) and the 2S albumin (Ses i 2), belong to the family of seed storage proteins. Sesame seed contains approximately 20% protein. 2S albumins are the major soluble part and constitute approximately 25% of the total sesame proteins.15 2S albumins are typically heterodimeric proteins with small and large subunits linked by disulfide bonds, both encoded as part of a large precursor polypeptide. Recently, one of the homologous genes encoding the 2S albumin precursor has been reported.16 Deduced amino acid sequences indicated that the corresponding 2S
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GI, Gastrointestinal.
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TABLE II. Shown are internal sequences for identified allergens (top) and sequences of the homologous proteins (bottom) Spot
7 kd
34 kd
45 kd
78 kd
Amino acid sequences
Homologous to:
1 108 1 108
QMQQEYGMEQX 10 QMQQEYGMEQE 118 QMQQEYGMEQEMQQMQQMMQYLPR 24 QMQQEYGMEQEMQQMQQMMQYLPR 131
1 127 1 116
IDILVNNAAEQYEASTVEEIDEPR 24 IDILVNNAAEQYECGTVEDIDEPR 150 VVEEVVNNYGR 11 VVDEVVSAYGC 126
1 336 1 565
SFSDEILEAAFNTR 14 SFSDEILEAAFNTR 350 SQQEEFFFK 9 SQQEEFFFK 574
1 435 1 16
EADQLTGQTFNDVGR 15 AADQIAGQTFNDVGR 449 TAADELSDVNR 11 VAAKELEQVNR 26
2S albumin precursor (sesame seed)
Seed maturation protein (glucose and ribitol dehydrogenases; soybean)
7S globulin (sesame seed)
Embryonic abundant protein (soybean)
Underlined amino acids are identical.
Food and drug reactions and anaphylaxis
albumin is a sulfur-rich protein.16 Even more recently, the same group cloned another cDNA encoding a second 2S albumin precursor. 17 Deduced amino acid sequences indicated that this 2S albumin is not sulfur rich.17 Comparison of the amino acid sequences of both 2S albumins of sesame seed showed only 47% homology and 35% identity. Previously, Pastorello et al13 identified a major allergen of sesame seed. The short amino acid sequence of their allergenic protein was reported to be homologous to the 2S albumin of Brazil nut, castor bean, and sunflower13 and was submitted to the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Sub-Committee, which designated it Ses i 1. Comparing the amino acid sequence of this allergen13 with both 2S albumins of sesame seed published by Tai et al,16,17 we were able to show that the sequence has high homology to the second, sulfur-poor form (Fig 3). In contrast to this, the 2S albumin allergen in the present study (Ses i 2) is identical with the first published, sulfur-rich form (Fig 4). The 2S albumin seed storage proteins from Castor bean, walnut, Brazil nut, and mustard seed have previously been identified as allergens. The pairwise homology between these proteins ranges from 16.6% to 49.3% (8.9%-38.8% identity). With our present study and the previous study by Pastorello et al,13 both 2S albumins of sesame could be identified as food allergens. Because the overall homology between these 2S albumins is moderate and they do not appear to be cross-reactive,18 they can be viewed as universal allergens. However, Ses i 2 is only recognized by 30% of the patients in the present study. In addition, only a minority of the 20 patients with systemic reactions in the present study recognized proteins in the molecular weight range of Ses i 1. This stands in striking contrast to the findings of Pastorello et al, in which Ses i 1 was recognized in 10 of 10 patients.13 There are some minor differences between the study groups: age (4-36
years [median, 10 years] in the Pastorello study vs 2-28 years [median, 6.5 years] in the present study) and specific IgE levels (0.8->100 kU/L [median, 10.4 kU/L] vs 3.83->100 kU/L [median, 33.7 kU/L], respectively). Further studies with recombinant Ses i 1 and Ses i 2 will be necessary to determine the importance of both 2S albumins in sesame seed allergy. We also identified the 7S vicilin-type globulin of sesame seed as another major food allergen and named it Ses i 3. In contrast to the 2S albumin, the 7S globulin appears only to be a minor constituent in sesame seeds and comprises a single polypeptide instead of 2 disulfidelinked subunits in its mature protein.17 Recently, a cDNA sequence encoding the 7S globulin was obtained.17 Similar to 2S albumins, 7S vicilin-type globulins have been described as food allergens in plants. Both the major peanut allergen Ara h 1 and the major walnut allergen Jug r 2 are 7S vicilin-type globulins. It is still not clear why some individuals have clinical reactions to only one nut or seed, whereas others reacts to several. The 7S globulin of sesame has only 36% homology with Ara h 1, but more interestingly, it seems to share an IgE-binding site with Ara h 1. It can be speculated that the recognition of this IgE-binding site in one patient might result in clinical reactivity to both peanut and sesame seed or that recognition of this binding site might explain the presence of measurable IgE levels to sesame without clinical reactivity in patients with peanut allergy. However, future studies will be necessary to identify Bcell epitopes on sesame seed allergenic proteins and then screen for similar IgE-binding sites with sera from patients with allergy to peanut, sesame seed, or both. The allergens at 34 and 78 kd were homologous to the seed maturation protein and the embryonic abundant protein of soybean, respectively. Neither protein is described as a food allergen in other plants. Future work will be necessary to obtain the full sequence of these proteins in sesame seed and to verify their role as sesame seed allergens.
In the present study we were able to identify 4 allergens in sesame seeds. Two of them belong to the group of seed storage proteins that are well-known food allergens. Interestingly, we could show sequence homology between an IgE-binding site of Ara h 1 and the 7S globulin of sesame seed that might explain cross-reactivity in certain patients. We thank Kaye Speicher and David Reim from the Wistar Institute Protein Microchemistry/Mass Spectrometry Facility for their great work and helpful discussions, as well as Dana Stalcup for technical assistance.
REFERENCES 1. Kolopp-Sarda MN, Moneret-Vautrin DA, Gobert B, Kanny G, Brodschii M, Bene MC, et al. Specific humoral immune responses in 12 cases of food sensitization to sesame seed. Clin Exp Allergy 1997;27:128-91. 2. Kagi M, Wuthrich B. Falafel-burger anaphylaxis due to sesame seed allergy. Lancet 1991;338:582. 3. Pajno GB, Passalacquag G, Magazzu G, Barberio G, Vita D, Canonica GW. Anaphylaxis to sesame. Allergy 2000;55:199-201. 4. Asero R, Mistrello G, Roncarolo D, Antoniotti PL, Falagiani P. A case of sesame seed-induced anaphylaxis. Allergy 1999;54:526-33. 5. Perkins MS. Peanut and nut allergy: sesame allergy is also a problem. BMJ 1996;313:300. 6. Stern A, Wuthrich. Non-IgE mediated anaphylaxis to sesame. Allergy 1998;53:325-6. 7. Chiu JT, Haydik IB. Sesame seed oil anaphylaxis. J Allergy Clin Immunol 1991;88:414-5.
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8. Sporik R, Hill D. Allergy to peanut, nuts, and sesame seed in Australian children. BMJ 1996;313:1477-8. 9. Levy Y, Danon YL. Allergy to sesame seed in infants. Allergy 2001;56:193-4. 10. Alday E, Curiel G, Lopez-Gil MJ, Carreno D, Moneo I. Occupational hypersensitivity to sesame seeds. Allergy 1996;51:69-70. 11. Keskinen H, Ostman P, Vaheri E, Tarvainen K, Grenquist-Norden B, Arppinen O, et al. A case of occupational ashma, rhinitis and urticaria due to sesame seed. Clin Exp Allergy 1991;21:623-4. 12. World market for sesame seeds. Available at: www.marketag.com. Accessed September 20, 2001. 13. Pastorello E, Varin E, Farioli L, Pravettoni V, Ortolani C, Trambaioli C, et al. The major allergen of sesame seeds (Sesamum indicum) is a 2S albumin. J Chromatogr 2001;756:85-93. 14. Shin DS, Compadre CM, Maleki SJ, Kopper RA, Sampson HA, Huang SK, et al. Biochemical and structural analysis of the IgE binding sites on Ara h 1, an abundant and highly allergenic peanut protein. J Biol Chem 1998;273:13753-9. 15. Rajendran S, Prakash V. Isolation and characterization of β-globulin low molecular weight protein fraction from sesame seeds (Sesamum indicum). J Agric Food Chem 1988;36:269-75. 16. Tai SSK, Wu LSH, Chen ECF, Tzen JTC. Molecular cloning of 11S globulin and 2S albumin, the major seed storage proteins in sesame. J Agric Food Chem 1999;47:4932-8. 17. Tai SSK, Li TTT, Tsai CCY, Yiu TJ, Tzen JTC. Expression pattern and deposition of the three storage proteins, 11S globulin, 2S albumin, and 7S globulin in maturing sesame seeds. Plant Physiol Biochem 2001;39:981-92. 18. Teuber SS, Dandekar AM, Peterson R, Sellers CL. Cloning and sequencing of a gene encoding a 2S albumin seed storage protein precursor from English walnut (Juglans regia), a major food allergen. J Allergy Clin Immunol 1998;101:807-14.
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J ALLERGY CLIN IMMUNOL VOLUME 110, NUMBER 1
Background: Sesame seed allergy is becoming increasingly prevalent, probably because of its use in international fast-food and bakery products. Despite this fact, few studies have focused on the identification of its major allergenic proteins. Objective: The aim of this study was to identify allergenic proteins of sesame seeds (Sesamum indicum). Methods: Extracted sesame seed proteins were separated by means of SDS-PAGE and 2-dimensional (2-D) PAGE. Immunolabeling was performed with individual patient sera from 20 patients with sesame seed allergy. Selected proteins were further analyzed by means of Edman sequencing. Results: IgE-binding proteins were identified at 78, 52, 45, 34, 32, 29, 25, 20, 9, and 7 kd. Analyzing internal sequences, the protein at 45 kd, which was recognized by 75% of the patients, was found to be a 7S vicilin-type globulin, a seed storage protein of sesame and named Ses i 3. The protein at 7 kd was found to be a 2S albumin, another seed storage protein of sesame and named Ses i 2. Seed storage proteins are known food allergens in peanut, walnut, Brazil nut, and soybean. Interestingly, one known IgE-binding epitope of the peanut allergen Ara h 1 has 80% homology with the corresponding area of Ses i 3. The different amino acids were previously shown not to be critical for IgE binding in Ara h 1. In addition, the proteins at 78 and 34 kd were found to be homologous to the embryonic abundant protein and the seed maturation protein of soybeans, respectively. Conclusion: The identification of 4 sesame seed allergens is the first step toward generating recombinant allergens for use in future immunotherapeutic approaches. In addition, the detection of conserved IgE binding epitopes in common food allergens might be a useful tool for predicting cross-reactivity to certain foods. (J Allergy Clin Immunol 2002;110:154-9.)
Food and drug reactions and anaphylaxis
From the Division of Pediatric Allergy and Immunology and the Jaffe Institute for Food Allergy, The Mount Sinai School of Medicine, New York. Supported in part by a grant from the Food Allergy Initiative and in part by a grant from the National Center of Research Resources-National Institutes of Health (MO1 RR-00071) awarded to the Mount Sinai School of Medicine. Received for publication October 29, 2001; revised March 28, 2002; accepted for publication April 1, 2002. Reprint requests: Kirsten Beyer, MD, The Mount Sinai School of Medicine, Division of Pediatric Allergy and Immunology, Box-No. 1198, One Gustave L. Levy Place, New York, NY 10029-6574. © 2002 Mosby, Inc. All rights reserved. 0091-6749/2002 $35.00 + 0 1/87/125487 doi:10.1067/mai.2002.125487
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Key words: Sesame seed, allergen, 2-dimensional, proteomics, immunolabeling, IgE, seed storage protein, vicilin, 2S albumin, 7S globulin, Ses i 3, Ses i 2
An increase in sesame seed allergy in children and adults has been reported in recent years, with several reports found throughout the literature.1-9 In a more complex study investigating 4078 Australian children, the sensitization rate to sesame seed was found to be one third that of the peanut sensitization rate and higher than that to any tree nut.8 The majority of the sensitized children were less than 2 years of age.8 Similar results were found in a study from Israel.9 However, sesame seed allergy has also been described as an occupational allergy.10,11 Common symptoms seen in sesame seed allergy are urticaria, angioedema, and respiratory distress.1,8,9 Similar to peanut and tree nut allergy, hypersensitivity reactions tend to be more severe in nature, often resulting in an anaphylactic reaction.1-4,6,7 The increase in sesame seed allergy might be explained by increasing consumption of sesame seed–containing foods, such as international fast-food and bakery products. In North America and Europe sesame seeds generally are used for toppings on breads, such as hamburger buns, bagels, bread sticks, and other baked goods. Restaurants purchase sesame seeds for use in ethnic dishes. In 1995, the United States imported 39,366 metric tons of sesame seeds.12 In the same year the member countries from the European Union (EU) imported 56,529 metric tons of sesame seeds, an increase of 100% by volume since 1988.12 The most significant sesame seed importers in the EU are Germany, the United Kingdom, and the Netherlands. The sesame species of greatest economic importance in the United States and the EU is Sesamum indicum. Despite the importance of sesame as a food allergen, the identification of its clinically relevant allergens is still incomplete. Very recently, one major allergen of sesame seeds, a 2S albumin seed storage protein, has been identified.13 Through the use of SDS-PAGE, 2-dimensional (2-D) proteomics, mass spectrometry, and internal sequencing, the present article reports 4 new sesame seed allergens.
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METHODS Preparation of sesame seed extract Sesame seeds (S indicum) were ground in a coffee grinder (Krups), followed by mortar and pestle, until a smooth paste was achieved. The paste was defatted by washing with at least 20 volumes (wt/vol) of cold acetone and dried overnight at 4°C. Dried acetone powder was stored at –80°C. Protein was extracted from the defatted, dried, powdered seed pulp by agitating with PBS and a protease inhibitor cocktail without EDTA (Roche) overnight at 4°C. After centrifugation at 2500g for 15 minutes at 4°C, supernatant was collected, filtered, and centrifuged at 12,000g for 3 minutes. Protein concentrations were determined by means of spectrophotometry (SmartSpec, Biorad) by using the Coomassie Plus Protein Assay (Pierce). All extracts were stored at –80°C.
SDS-PAGE analysis and 2-D proteomics mapping For SDS analysis, proteins were separated by means of SDSPAGE (Nu-Page MES Bis-Tris, Invitrogen), according to the protocol from the manufacturer. Ten micrograms of protein extract was loaded into each well. For 2-D gel electrophoresis, the crude extract was desalted and concentrated with Perfect Focus (GenoTechology Inc) and resuspended in rehydration buffer (9.8 mol/L urea, 4% chaps, 2 mmol/L tributylphosphine, 0.2% ampholytes, and 0.001% bromophenol blue; Biorad). The 50 µg of protein extract was applied to 7 cm of immobilized pH gradient strips (Biorad) for rehydration and focusing for 17 hours with the linear voltage slope of up to 20,000 volt-hours, followed by 2-D gel electrophoresis with Invitrogen Zoom Gels (Invitrogen). Before running the SDS-PAGE, immobilized pH gradient strips were equilibrated with urea, SDS, and glycerol containing TrisHCl buffer in the presence of 5 mmol/L tributylphosphine for 10 minutes and then 135 mmol/L iodoacetamide for 10 minutes. The proteins resolved by using both techniques were subsequently transferred to Immobilon-P membrane (Millipore) and then stained with 2.2% Coomassie blue for total protein analysis. For molecular weight determination, MultiMark Multi-Colored Standard (Invitrogen) was used.
Probing immunoblots with sera from patients with sesame seed allergy For detection of IgE binding to the separated sesame seed proteins, immunolabelling was performed with individual patient sera from 20 individuals with sesame seed allergy. Clinical history, age of the patients, and sesame seed–specific IgE levels detected with the Pharmacia FEIA-Cap System are shown in Table I. Normal human serum (Pharmacia) was used as a negative control. Patient and control sera diluted from 1:4 to 1:10, depending on the level of serum IgE, in PBS-Tween plus 1% BSA and 10% normal goat serum were incubated with immunoblots by means of gentle agitation at room temperature. After 1 hour, 2-D immunoblots were briefly rinsed with PBS, and phosphatase-labeled goat anti-human IgE (KPL) at a concentration of 1 µg/mL goat IgG per milliliter in PBS-Tween plus 1% BSA and 10% normal goat serum was applied. After agitation for 45 minutes at room temperature and washing with PBS, immunoblots were developed with phosphatase substrate 5-bromo-4-chloroindolyl-phosphatase/nitroblue tetrazolium (KPL). For SDS-PAGE analysis, immunoblots were rinsed after 2 hours with PBS, and
iodine 125–labeled goat anti-human IgE (DiaMed) diluted as per the manufacturer was applied. After agitation for 1 hour at room temperature and washing with PBS, immunoblots were mounted on filter paper and exposed to Kodak X-OMAT Imaging Film for 1 to 5 days. Protein images were analyzed with GelDoc 2000 (Biorad) by using Quantity One Quantitation Software.
MALDI-MS analysis and Edman sequencing Following the procedure described under 2-D proteomics mapping, the proteins were separated with 2-D PAGE, and the gels were stained with Coomassie blue. Proteins in spots of interest were analyzed at the Wistar Institute Protein Microchemistry/Mass Spectrometry Facility. Briefly, the proteins of interest were excised for in-gel trypsin digestion by using a sequencing-grade modified trypsin (Promega) and were alkylated with iodoacetamide (Sigma) as part of the digestion protocol. The resulting peptide mixture was separated by means of reverse-phase HPLC on a Zorbax 2.1 × 150–mm C-18 column (Agilent Technologies). The solvent gradient was delivered and controlled with System Gold HPLC (Beckman). Solvent A was 0.1% trifluoracetic acid in MilliQ water, and solvent B was 0.085% trifluoracetic acid in 95% acetonitrile. Selected HPLC fractions were then analyzed by means of MALDI-MS on a PerSeptive DE Pro, and Edman sequence analysis was completed on the Applied BioSystems Procise sequencer.
RESULTS Proteins were separated by means of SDS-PAGE, as well as 2-D gel electrophoresis, and either stained with Coomassie blue for total protein analysis or transferred to membranes and immunolabeled with sera from 20 individuals with sesame seed allergy to identify allergenic proteins of sesame seeds. Fig 1 shows the SDSPAGE and the results of the immunolabeling for all 20 patients and the control subject. IgE-binding proteins were identified at 78, 52, 45, 34, 32, 29, 25, 20, 9, and 7 kd. Fig 2 shows all sesame seed proteins separated by means of 2-D PAGE and the results of the immunolabeling for 4 representative patients and normal control serum. Four IgE-binding protein fractions at 7, 34, 45, and 78 kd that were recognized by 30%, 60%, 75%, and 50% of the patients, respectively, were chosen for further analysis by means of Edman sequencing (Figure 1 and 2). The internal sequences of these 4 proteins are shown in Table II. However, the protein at 78 kd that was recognized by half of the patients in 2-D gel electrophoresis showed weak binding in most patients and was not visible in the majority of patients in SDS-PAGE analysis. Analyzing internal sequences, the protein at 45 kd was found to be a 7S vicilin-type globulin, and the 7-kd protein was found to be a 2S albumin of S indicum (Table II). The 2 new sesame seed allergens were named Ses i 3* and Ses i 2.* 7S vicilin-type globulins or 2S albumins are known food allergens in several plants, including peanut, walnut, Brazil nut, and soybean. Looking at the entire sequence, the 2S albumin precursor of sesame seed (Ses i 2) has 38% homology to the walnut allergen Jug r *Allergen
data have been submitted to the World Health Organization/International Union of Immunological Societies Allergen Nomenclature SubCommittee for approval of the new name.
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Abbreviation used EU: European Union
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FIG 1. SDS-PAGE analysis for sesame seed proteins: lane 2, labeling with Coomassie blue stain for total protein analysis; lanes 4 to 23, immunolabeling with sera from 20 patients with sesame seed allergy; lane 24, labeling with control sera. Molecular weight standards (MW) are shown in lanes 1, 3, and 25.
Food and drug reactions and anaphylaxis
FIG 2. Two-dimensional proteomics map for sesame seed proteins. Shown is labeling with Coomassie blue for total protein analysis and immunolabeling of 4 representative patients and the control serum. Molecular weight standards (MW) are shown at left. Red circles indicate proteins recognized at 7, 34, 45, and 78 kd, for which Edman sequencing was performed.
1, 40% homology to the Brazil nut allergen Ber e 1, and 34% homology to the peanut allergen Ara h 2. The 7S vicilin-type globulin of sesame (Ses i 3) has 36% homology to the peanut allergen Ara h 1 and 41% homology to
the walnut allergen Jug r 2. It is of interest that one of the 22 known IgE-binding epitopes of Ara h 1 has 80% homology with the corresponding area of the sesame vicilin (Fig 3). The amino acids that differed were previ-
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FIG 3. Shown is the homology in amino acid sequences for the sesame seed 7S globulin and Ara h 1 in the area of an IgE-binding epitope of Ara h 1. Identical amino acids are shown in bold and underlined, and homologous amino acids are underlined only. Amino acids highlighted in gray are known to lead to a loss of IgE binding when altered.14
FIG 4. Shown are the amino acid sequences for the large subunits of the two 2S albumins of sesame seed. Identical amino acids are shown in bold and underlined, and homologous amino acids are underlined only. The two 2S albumins of sesame seed are, in general, only 35% identical and 47% homologous. Amino acids highlighted in light gray were found to be the sequence of an internal fragment of the 7-kd allergen in the present study, and amino acids are highlighted in dark gray in the sequence of an internal fragment of the allergen in the study by Pastorello et al.13 Adapted from Pastorello et al.13 Copyright 2001 with permission from Elsevier Science.
TABLE I. Shown are clinical reactions, age, and sesame seed–specific IgE levels for the 20 individuals with sesame seed allergy Patient
A B C D E F G I J K L M P Q R S T U V W
Clinical reaction
Angioedema, worsening of eczema Angioedema, urticaria, GI symptoms Angioedema, urticaria, rhinitis Angioedema, GI symptoms Mouth and throat itchiness GI symptoms, worsening of eczema Angioedema, urticaria Angioedema, respiratory symptoms Angioedema, GI symptoms Urticaria Angioedema Mouth and throat itchiness Angioedema, worsening of eczema Angioedema, GI symptoms Urticaria, angioedema, worsening of eczema Urticaria, angioedema, GI symptoms Urticaria GI symptoms GI symptoms, respiratory symptoms, throat tightness Urticaria, rhinoconjunctivitis
Age (y)
IgE (kU/L)
6 7 3 28 6 6 15 2 9 9 3 14 7 12 11 3 4 6 10 2
>100.00 >100.00 >100.00 >100.00 73.90 22.40 5.92 4.85 3.83 29.90 >100.00 64.70 37.50 >100.00 29.1 >100.00 6.65 8.78 13.10 8.23
ously shown not to be critical for IgE binding to Ara h 1 by using single-amino-acid substitution.14 In addition, the proteins at 78 and 34 kd were found to be homologous to the embryonic abundant protein and the seed maturation protein of soybeans, respectively (Table II).
DISCUSSION In the present study we identified 4 allergens for sesame seeds. Two of them, the 7S vicilin-type globulin
(Ses i 3) and the 2S albumin (Ses i 2), belong to the family of seed storage proteins. Sesame seed contains approximately 20% protein. 2S albumins are the major soluble part and constitute approximately 25% of the total sesame proteins.15 2S albumins are typically heterodimeric proteins with small and large subunits linked by disulfide bonds, both encoded as part of a large precursor polypeptide. Recently, one of the homologous genes encoding the 2S albumin precursor has been reported.16 Deduced amino acid sequences indicated that the corresponding 2S
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GI, Gastrointestinal.
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TABLE II. Shown are internal sequences for identified allergens (top) and sequences of the homologous proteins (bottom) Spot
7 kd
34 kd
45 kd
78 kd
Amino acid sequences
Homologous to:
1 108 1 108
QMQQEYGMEQX 10 QMQQEYGMEQE 118 QMQQEYGMEQEMQQMQQMMQYLPR 24 QMQQEYGMEQEMQQMQQMMQYLPR 131
1 127 1 116
IDILVNNAAEQYEASTVEEIDEPR 24 IDILVNNAAEQYECGTVEDIDEPR 150 VVEEVVNNYGR 11 VVDEVVSAYGC 126
1 336 1 565
SFSDEILEAAFNTR 14 SFSDEILEAAFNTR 350 SQQEEFFFK 9 SQQEEFFFK 574
1 435 1 16
EADQLTGQTFNDVGR 15 AADQIAGQTFNDVGR 449 TAADELSDVNR 11 VAAKELEQVNR 26
2S albumin precursor (sesame seed)
Seed maturation protein (glucose and ribitol dehydrogenases; soybean)
7S globulin (sesame seed)
Embryonic abundant protein (soybean)
Underlined amino acids are identical.
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albumin is a sulfur-rich protein.16 Even more recently, the same group cloned another cDNA encoding a second 2S albumin precursor. 17 Deduced amino acid sequences indicated that this 2S albumin is not sulfur rich.17 Comparison of the amino acid sequences of both 2S albumins of sesame seed showed only 47% homology and 35% identity. Previously, Pastorello et al13 identified a major allergen of sesame seed. The short amino acid sequence of their allergenic protein was reported to be homologous to the 2S albumin of Brazil nut, castor bean, and sunflower13 and was submitted to the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Sub-Committee, which designated it Ses i 1. Comparing the amino acid sequence of this allergen13 with both 2S albumins of sesame seed published by Tai et al,16,17 we were able to show that the sequence has high homology to the second, sulfur-poor form (Fig 3). In contrast to this, the 2S albumin allergen in the present study (Ses i 2) is identical with the first published, sulfur-rich form (Fig 4). The 2S albumin seed storage proteins from Castor bean, walnut, Brazil nut, and mustard seed have previously been identified as allergens. The pairwise homology between these proteins ranges from 16.6% to 49.3% (8.9%-38.8% identity). With our present study and the previous study by Pastorello et al,13 both 2S albumins of sesame could be identified as food allergens. Because the overall homology between these 2S albumins is moderate and they do not appear to be cross-reactive,18 they can be viewed as universal allergens. However, Ses i 2 is only recognized by 30% of the patients in the present study. In addition, only a minority of the 20 patients with systemic reactions in the present study recognized proteins in the molecular weight range of Ses i 1. This stands in striking contrast to the findings of Pastorello et al, in which Ses i 1 was recognized in 10 of 10 patients.13 There are some minor differences between the study groups: age (4-36
years [median, 10 years] in the Pastorello study vs 2-28 years [median, 6.5 years] in the present study) and specific IgE levels (0.8->100 kU/L [median, 10.4 kU/L] vs 3.83->100 kU/L [median, 33.7 kU/L], respectively). Further studies with recombinant Ses i 1 and Ses i 2 will be necessary to determine the importance of both 2S albumins in sesame seed allergy. We also identified the 7S vicilin-type globulin of sesame seed as another major food allergen and named it Ses i 3. In contrast to the 2S albumin, the 7S globulin appears only to be a minor constituent in sesame seeds and comprises a single polypeptide instead of 2 disulfidelinked subunits in its mature protein.17 Recently, a cDNA sequence encoding the 7S globulin was obtained.17 Similar to 2S albumins, 7S vicilin-type globulins have been described as food allergens in plants. Both the major peanut allergen Ara h 1 and the major walnut allergen Jug r 2 are 7S vicilin-type globulins. It is still not clear why some individuals have clinical reactions to only one nut or seed, whereas others reacts to several. The 7S globulin of sesame has only 36% homology with Ara h 1, but more interestingly, it seems to share an IgE-binding site with Ara h 1. It can be speculated that the recognition of this IgE-binding site in one patient might result in clinical reactivity to both peanut and sesame seed or that recognition of this binding site might explain the presence of measurable IgE levels to sesame without clinical reactivity in patients with peanut allergy. However, future studies will be necessary to identify Bcell epitopes on sesame seed allergenic proteins and then screen for similar IgE-binding sites with sera from patients with allergy to peanut, sesame seed, or both. The allergens at 34 and 78 kd were homologous to the seed maturation protein and the embryonic abundant protein of soybean, respectively. Neither protein is described as a food allergen in other plants. Future work will be necessary to obtain the full sequence of these proteins in sesame seed and to verify their role as sesame seed allergens.
In the present study we were able to identify 4 allergens in sesame seeds. Two of them belong to the group of seed storage proteins that are well-known food allergens. Interestingly, we could show sequence homology between an IgE-binding site of Ara h 1 and the 7S globulin of sesame seed that might explain cross-reactivity in certain patients. We thank Kaye Speicher and David Reim from the Wistar Institute Protein Microchemistry/Mass Spectrometry Facility for their great work and helpful discussions, as well as Dana Stalcup for technical assistance.
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