The 11S globulin Sin a 2 from yellow mustard seeds shows IgE cross-reactivity with homologous counterparts from tree nuts and peanut
© Sirvent et al.; licensee BioMed Central Ltd. 2012
Received: 1 October 2012
Accepted: 7 December 2012
Published: 11 December 2012
The 11S globulin Sin a 2 is a marker to predict severity of symptoms in mustard allergic patients. The potential implication of Sin a 2 in cross-reactivity with tree nuts and peanut has not been investigated so far. In this work, we studied at the IgG and IgE level the involvement of the 11S globulin Sin a 2 in cross-reactivity among mustard, tree nuts and peanut.
Eleven well-characterized mustard-allergic patients sensitized to Sin a 2 were included in the study. A specific anti-Sin a 2 serum was obtained in rabbit. Skin prick tests (SPT), enzyme-linked immunosorbent assay (ELISA), immunoblotting and IgG or IgE-inhibition immunoblotting experiments using purified Sin a 2, Sin a 1, Sin a 3, mustard, almond, hazelnut, pistachio, walnut or peanut extracts were performed.
The rabbit anti-Sin a 2 serum showed high affinity and specificity to Sin a 2, which allowed us to demonstrate that Sin a 2 shares IgG epitopes with allergenic 11S globulins from tree nuts (almond, hazelnut, pistachio and walnut) but not from peanut. All the patients included in the study had positive skin prick test to tree nuts and/or peanut and we subdivided them into two different groups according to their clinical symptoms after ingestion of such allergenic sources. We showed that 11S globulins contain conserved IgE epitopes involved in cross-reactivity among mustard, tree nuts and peanut as well as species-specific IgE epitopes.
The allergenic 11S globulin Sin a 2 from mustard is involved in cross-reactivity at the IgE level with tree nuts and peanut. Although the clinical relevance of the cross-reactive IgE epitopes present in 11S globulins needs to be investigated in further detail, our results contribute to improve the diagnosis and management of mustard allergic patients sensitized to Sin a 2.
KeywordsFood allergy Mustard allergy Tree nut allergy Peanut allergy Cross-reactivity 11S globulins Sin a 2 lgG/IgE epitopes
IgE-mediated food allergy is an important worldwide health problem of increasing prevalence affecting up to 2-10% of the population [1, 2]. More than 170 foods have been reported to induce allergic responses being peanuts, tree nuts, eggs, milk, fish, shellfish, wheat and soy responsible for the vast majority of reactions. Mustard is one of the most frequent spices causing IgE-mediated food allergy, and together with celery, sesame, lupine and shellfish is considered among the most significant allergenic sources in European countries . The ingestion of mustard seed flour or manufactured foods containing this spice has been frequently associated with the development of severe symptoms such as generalized urticaria, angioedema or anaphylaxis in hypersensitive patients [4–7]. Mustard is worldwide consumed in home-made meals and added as a hidden condiment in many sauces, salad dressings or manufactured and processed products, which makes difficult to avoid the ingestion of this spice and increases the risk of suffering unexpected life-threatening reactions . Therefore, mustard content must be declared according to the European Union directive for food labelling . Yellow mustard (Sinapis alba L.) is commonly used in Europe whereas oriental mustard (Brassica juncea) is the spice used in United States and Asia.
Four allergens from yellow mustard seeds have been identified, purified and characterized so far: i) 2S albumin Sin a 1 (14 kDa) [10, 11]; ii) 11S globulin Sin a 2 (51 kDa) [12, 13]; iii) LTP Sin a 3 (12 kDa) ; and iv) profilin Sin a 4 (13–14 kDa) . Sin a 1 and Sin a 3 but not Sin a 4 might act as genuine food allergens able to reach the gut immune system due to their high structural and immunological stability . The capacity of Sin a 2 to act as primary sensitizer at the intestinal mucosa has not been investigated so far. We also demonstrated that Sin a 1 is a diagnostic marker for sensitization to mustard, Sin a 2 is a marker to predict severity of symptoms, and Sin a 3 and Sin a 4 are allergens associated with sensitization to other plant-derived foods from the Rosaceae family and pollens . It has been reported that more than 50% of patients allergic to mustard present hypersensitivity to other different vegetable foods, mainly nuts and legumes, but whether this is due to cross reactivity and the allergens involved in such processes is an aspect that needs further investigations [16–18]. At this regard, 11S globulins might represent good candidates. Allergenic members of this protein family have been characterized from almond (Pru du 6) , hazelnut (Cor a 9) [20, 21], peanut (Ara h 3) , walnut (Jug r 4) , pistachio (Pis v 2) , soybean (glycinins G1-G2) , Brazil nut (Ber e 2) , cashew nut (Ana o 2) , sesame seeds (Ses i 6)  or pecan (Car i 4) . Some studies have previously shown that 11S globulins are involved in cross-reactivity between coconut and walnut , among buckwheat, poppy and hazelnut  or between peanut and other different seeds spices . In addition, two more detailed studies at the molecular level based on the three-dimensional (3D) structure of the allergens and identification of IgE-binding sites suggested that Jug r 4 shows cross-reactivity with Car i 4  or with Cor a 9 and other 11S globulins [33, 34]. Despite these reported data, there is still little evidence of IgE-cross reactivity involving 11S globulins, a family of proteins with an overall sequence identity under 40%.
In the present study, we sought to investigate the potential implication of the 11S globulin Sin a 2 in cross-reactivity involving mustard, tree nuts and peanut. Our results show that the allergenic 11S globulin Sin a 2 shares IgG epitopes with homologous counterparts from almond, hazelnut, pistachio and walnut but not from peanut and that Sin a 2 is involved in IgE cross-reactivity with tree nuts and peanut.
Patients allergic to mustard
Clinical characteristics of patients with mustard allergy sensitized to Sin a 2
Other food allergies
Tree nuts/ peanut Symptoms
Sin a 2
OAS, U, AE, D
OAS, AE, D
OAS, U, RC, D
r, l, m
OAS, U, TT, A, RC, D
OAS,U, AE, TT, A, RC, D
r, n, p
OAS, U, AE, TT, A, RC
OAS, RC, A
OAS, AE, A, RC
r, k, n, p, m
OAS, AE, TT
OAS, AE, CU, TT, A
r, k, n, p, m
OAS, AE, TT, RC
OAS, TT (local)
Specific IgE to mustard extract and purified mustard allergens by ELISA
Sin a 1
Sin a 2
Sin a 3
Sin a 4
Yellow mustard seeds extract, purified Sin a 2 and rabbit anti-Sin a 2 serum
Yellow mustard seeds, almond, hazelnut, pistachio, walnut and roasted peanut protein extracts were obtained as previously described for yellow mustard seeds . The allergenic 11S globulin Sin a 2 was purified from yellow mustard seeds extract as described .
The specific anti-Sin a 2 serum was prepared by immunizing a New Zealand white rabbit with purified Sin a 2 by weekly injection of the protein in complete Freund’s adjuvant. After 21 days of treatment the serum was obtained by centrifugation of the blood.
SPT were performed in all patients according to standard procedures . The panel of commercial food extracts included peach, chestnut, soy, sunflower seed, almond, hazelnut, peanut, walnut, pine nut, pistachio, chickpea, lentil and bean. SPT with kiwi and apple were performed by the prick-prick method as described . SPT with home-made mustard (Sinapis alba L.) extract (50 μg/ml) and with purified natural Sin a 2 (10 μg/ml) were performed. Histamine dihydrochloride (10 mg/ml) and physiologic saline solutions were used as the positive and negative controls, respectively. A wheal area <7 mm2 was considered as negative. We included almond as a nut (instead of a Rosaceae fruit), and considered peanut separately.
Electrophoresis and immunoblotting
SDS-PAGE was performed in 15% polyacrylamide gels. Proteins (0.5 μg/lane of purified proteins or 50 μg/lane of protein extracts) were visualized by Coomassie Blue or alternatively transferred to nitrocellulose membranes (Amersham, Buckinghamshire, United Kingdom). The protein concentration was determined using the method of bicinchoninic acid (Pierce Chemical Co, Rockford, Ill, USA).
Immunodetection of proteins in membranes was achieved as described  by using different pool of sera from patients allergic to mustard, (diluted 1/5), or rabbit specific anti-Sin a 2 serum (diluted 1/100000). The binding of human IgE was detected by mouse anti-human IgE antibodies, provided by ALK-Abelló (Madrid, Spain), diluted 1/5000, followed by horseradish peroxidase-labelled goat anti-mouse IgG (diluted 1/5000; Pierce). Reaction to anti-Sin a 2 serum was detected by horseradish peroxidase-labelled goat anti-rabbit IgG (diluted 1/3000; BioRad, Richmond, CA). The signal was developed by using the ECL-Western blotting reagent (Amersham). For the IgG and IgE-inhibition experiments in immunoblotting, the pools of sera (diluted 1/5) or the rabbit anti-Sin a 2 serum (diluted 1/100000) were preadsorbed with 1 mg/ml of yellow mustard seeds, almond, hazelnut, walnut, pistachio or peanut extracts or with 20 μg/ml of purified Sin a 2 for 2h prior to membrane incubation as described . Bovine serum albumin (BSA) was used as negative controls of inhibition. Volummograms of the reactive bands were analysed by scanning densitometry using the computer program Multigauge V3.0.
IgG quantitation was performed by ELISA in microtiter plates (Costar, Corning, NY, USA) coated with 100 μl/well of purified Sin a 2 (2 μg/ml) or yellow mustard seeds extract (20 μg/ml) . Plates were incubated with increasing dilutions of the rabbit anti-Sin a 2 serum for titration. Then, the plates were incubated with horseradish peroxidase-labelled goat anti-mouse IgG as describe above and peroxidase reaction was developed using fresh enzyme substrate and measuring optical density (OD) at 492 nm. Each value was calculated as the mean of 2 determinations after blank subtraction.
For IgG-inhibitions ELISA, after being coated with 100 μl of Sin a 2 (2 μg/ml) or yellow mustard seeds extract (20 μg/ml), the plates were incubated with the rabbit anti-Sin a 2 serum (diluted 1/100000) previously preadsorbed with increasing amounts of Sin a 2 and mustard extracts as inhibitors for 2 h . Then, the plates were incubated with horseradish peroxidase-labelled goat anti-mouse IgG and peroxidase reaction developed as described above. The percentage of inhibition was determined according to the formula: % Inhibition = (1- (OD492nm with inhibitor/OD492nm without inhibitor)) x 100. All the determinations were carried out as duplicates.
Sequence alignment and three-dimensional modelling
Multiple sequence alignments of Sin a 2, Pru du 6, Cor a 9, Pis v 2, Jug r 4 and Ara h 3 (accession numbers Q2TLW0, E3SH28, Q8W1C2, B7P073, Q2TPW5 and O82580, respectively) were performed with the Clustal Omega program . Amino acid sequences of all 11S globulins are shown without signal peptide. Location of the signal peptide cleavage sites were predicted using SignalP 4.0 Server . The 3D structure of Sin a 2 was modelled by using the services of the Swiss-Model Protein Modelling Server  and the structure of the soybean legumin Gly m 6 (PDB code 1od5A)  as template. The 3D structure of Ara h 3 correspond to PDB: 3c3v. Graphical processing of the three-dimensional structures was accomplished with PDB viewer PyMOL program.
Results and discussion
Clinical features of mustard-allergic patients sensitized to Sin a 2 suggest that 11S globulins might be involved in cross-reactivity with tree nuts and/or peanut
By linking the clinical features of 34 well-characterized patients allergic to mustard with component-resolved approaches we previously showed that 2S albumin Sin a 1 is a marker of genuine sensitization to mustard, 11S globulin Sin a 2 is associated to severe symptoms after mustard ingestion and LTP Sin a 3 and profilin Sin a 4, two well-known panallergens, are associated with sensitization to other plant-derived foods from the Rosaceae family and pollens . Although more than 50% of mustard allergic patients are also sensitized to tree nuts and/or peanut, significant clinical associations between sensitization to Sin a 2 and being allergic to other tree nuts or peanut were not found within our cohort of mustard allergic patients . However, considering that recent studies suggested that 11S globulins are implicated in cross-reactivity among taxonomically unrelated tree nuts and peanut [30–33] and that different members of this protein family have been reported as relevant allergens [19–24], we investigated at the IgG and IgE level whether the 11S globulin Sin a 2 might be implicated in cross-reactivity among mustard, tree nuts and peanut within mustard allergic patients specifically sensitized to Sin a 2. We included in the study the 11 patients allergic to mustard with the general clinical characteristics summarized in Table 1. All patients presented allergic reactions within the first 30 minutes after mustard consumption and the inclusion criteria was based on positive specific IgE to yellow mustard seed extract and Sin a 2 as determined by SPT and ELISA (Tables 1 and 2). In line with our previous data, 10 of the 11 patients sensitized to Sin a 2 developed immediate systemic reactions after the ingestion of mustard and 7 attended the emergency room, where epinephrine was administrated . All the patients included in the study had also positive SPT to tree nuts (almond or hazelnut or pistachio or walnut) or peanut and 6 of them (# 6, 7, 8, 9, 10 and 11) suffered from symptoms after ingestion of tree nuts (3 of them also with peanut, # 2, 4 and 5). Interestingly, only 3 patients showed positive specific IgE to the previously known cross-reactive mustard allergens Sin a 3 and 2 to Sin a 4 (Table 2), suggesting a potential role of Sin a 2, at least in the observed positive SPT to tree nuts and/or peanut. To further analyse the implication of Sin a 2 in cross-reactivity at the IgG and IgE level and to determine potential clinical relevance within the mustard allergic patients sensitized to Sin a 2, we obtained a rabbit anti-Sin a 2 serum and pooled patients’ sera into two different groups: i) Group 1, patients with positive SPT to tree nuts and/or peanut extracts without clinical symptoms to these allergenic sources; ii) Group 2, patients with positive SPT to tree nuts and/or peanut extracts with clinical symptoms to some of these allergenic sources (Table 1).
IgG epitopes of the 11S globulin Sin a 2 are present in almond, hazelnut, pistachio and walnut but not in peanut extracts
IgE cross-reactivity among allergenic 11S globulins from mustard, tree nuts and peanut
In this study we demonstrated at the molecular level that the 11S globulin Sin a 2, which is associated with severe reactions in mustard allergic patients, is involved in cross-reactivity among mustard, tree nuts and peanut. Sin a 2 shares IgG epitopes with allergenic 11S globulins from tree nuts (almond, hazelnut, pistachio and walnut) but not with peanut. At the IgE level, we showed that 11S globulins contain conserved IgE epitopes involved in cross-reactivity among mustard, tree nuts and peanut as well as specie-specific IgE epitopes. Due to the severity of symptoms associated to Sin a 2, the definitive demonstration of the clinical relevance and the involvement of the IgE cross-reactive epitopes of 11S globulins in triggering symptoms is an important issue that will require in depth clinical studies in future.
Enzyme-linked immunosorbent assay
Three dimensional modelling
The authors’ laboratories are supported by grants SAF2011-26716 from MINECO (Ministerio de Economía y Competitividad) and RD07/0064/0009 from FIS (Fondo de Investigación Sanitaria). O.P. is a Ramon y Cajal Scholar funded by MINECO and the European Social Fund.
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