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Biomolecules 2019, 9, 489; doi:10.3390/biom9090489 www.mdpi.com/journal/biomolecules
Article
Detection and identification of allergens from
Canadian mustard varieties of Sinapis alba and
Brassica juncea
Lamia L’Hocine *, Mélanie Pitre and Allaoua Achouri
Saint-Hyacinthe Research and Development Centre, Agriculture and Agri-Food Canada, 3600 Casavant Blvd.
W. Saint-Hyacinthe, QC, J2S 8E3, Canadamelanie.pitre@canada.ca (M.P.); allaoua.achouri@canada.ca (A.A.)
* Correspondence: lamia.lhocine@canada.ca; Tel.: +1-450-768-7944
Received: 23 July 2019; Accepted: 10 September 2019; Published: 14 September 2019
Abstract: Currently, information on the allergens profiles of different mustard varieties is rather
scarce. Therefore, the objective of this study was to assess protein profiles and immunoglobulin E
(IgE)-binding patterns of selected Canadian mustard varieties. Optimization of a non-denaturing
protein extraction from the seeds of selected mustard varieties was first undertaken, and the various
extracts were quantitatively and qualitatively analyzed by means of protein recovery determination
and protein profiling. The IgE-binding patterns of selected mustard seeds extracts were assessed by
immunoblotting using sera from mustard sensitized and allergic individuals. In addition to the
known mustard allergens—Sin a 2 (11S globulins), Sin a 1, and Bra j 1 (2S albumins)—the presence
of other new IgE-binding protein bands was revealed from both Sinapis alba and Brassica juncea
varieties. Mass spectrometry (MS) analysis of the in-gel digested IgE-reactive bands identified the
unknown ones as being oleosin, β-glucosidase, enolase, and glutathione-S transferase proteins. A
bioinformatic comparison of the amino acid sequence of the new IgE-binding mustard proteins with
those of know allergens revealed a number of strong homologies that are highly relevant for
potential allergic cross-reactivity. Moreover, it was found that Sin a 1, Bra j 1, and cruciferin
polypeptides exhibited a stronger IgE reactivity under non-reducing conditions in comparison to
reducing conditions, demonstrating the recognition of conformational epitopes. These results
further support the utilization of non-denaturing extraction and analysis conditions, as denaturing
conditions may lead to failure in the detection of important immunoreactive epitopes.
Keywords: mustard; Sinapis alba; Brassica juncea; allergens; IgE-binding; protein extraction; mass
spectrometry; immunoblotting; sequence alignment
1. Introduction
Mustard is one of the priority food allergens regulated by Canada, the European Union, and the
Gulf Cooperation Council (GCC), including the countries of Saudi Arabia, United Arab Emirates
(UAE), Kuwait, Bahrain, Oman, Qatar, and Yemen. The inclusion of mustard on the regulatory
allergen list of these countries was based on the view that mustard allergy poses a serious problem
because of its widespread use and high allergenic potency [1,2]. There are little data available on the
prevalence rates of mustard allergy, but it seems to vary around the world and appears to be more
common in Europe, accounting for 1–7% of food allergy based on estimated prevalence in France
[3,4]. Mustard allergy is also well documented in a number of published clinical studies reporting on
severe systemic reactions, including anaphylaxis following exposure to very small amounts of
mustard [5–8].
The international mustard market is led by Canada, which is the world’s second largest producer
and the first exporter of mustard seed, holding a 57% share of the market [9]. Canada produces food
Biomolecules 2019, 9, 489 2 of 25
grade mustard of three market classes, namely, yellow (Sinapis alba), oriental, and brown (Brassica
juncea) mustard. The different types of mustard vary in physical appearance and use. Of these, yellow
mustard seeds are larger, have higher protein (31.4%) and lower oil (30.4%) contents, and are milder
in pungency in comparison to brown and oriental seeds [10], which contain 27.1% and 26.1% of
protein and 38.5% and 42.4% of oil, respectively [11].
The major market for yellow mustard is the North American condiment industry, where it is
commonly used to produce dry milled flour (fine powder from dehulled seeds) for salad dressings,
mayonnaise, barbecue sauces, pickles and is also used as an excellent emulsifying agent and stabilizer
for processed meats. On the other hand, the wet milled mustard is mainly used for mustard paste
and the whole ground seeds as a seasoning. Brown mustard is primarily exported to Europe, where
it is used to produce condiments and specialty mustard such as Dijon mustard. Oriental mustard is
primarily grown for export to Asian countries, where it is used to produce condiments or spicy
cooking oil. Beyond these uses, there is a growing interest in mustard components including protein,
oil, and mucilage for a large spectrum of food, pharmaceutical, and industrial applications due to
their nutritional, biological, and functional properties. As a result, mustard utilization is expected to
increase in the future with an increasing risk of mustard being present as a hidden ingredient in many
prepared or prepackaged products.
To date, only four major allergens from yellow mustard (S. alba) have been identified, namely:
(a) Sin a 1, characterized as a seed storage protein napin and belonging to the 2S albumin family with
a molecular weight (MW) of 14 kDa [12–18]; (b) Sin a 2, belonging to the seed storage 11S globulin
with a MW of 51 kDa dissociated in two chains of 36 and 23 kDa [19,20]; (c) Sin a 3, a non-specific
lipid transfer protein/nsLTP lipid transfer protein, 12 kDa [21]; and (d) Sin a 4, profilin, 13–14 kDa
[21,22]. From the oriental mustard, Bra j 1, a seed storage protein from the 2S albumin family with a
MW of approximately 16 kDa has been also identified as a major mustard allergen [23,24]. Previous
studies revealed that Bra j 1 and Sin a 1 have a homologous epitope [23,24]. These findings imply that
individuals known to be sensitive to one species of mustard are likely to show sensitivity to other
species. However, information on mustard allergens from different mustard classes (types) and
varieties remains limited, which represents a major obstacle to effective risk management and
development of specific diagnostic tools and therapeutic approaches. The objective of this study was
therefore to use an optimized, non-denaturing protein extraction for the assessment of protein
profiles and Immunoglobulin E (IgE) binding patterns of major Canadian mustard varieties using
sera from sensitized/allergic mustard individuals. This research allows the detection and the
identification of new mustard allergens.
2. Materials and Methods
2.1. Preparation of Mustard Seed Flours
Seven different varieties of mustard were used in this study—two varieties of Sinapis alba (AC
Pennant and Andante) and five varieties of Brassica juncea (Duchess, Centennial Brown, AC Vulcan,
Cutlass, and Dahinda). All mustard seed samples were generously offered by Dr. Janitha
Wanasundara of the Saskatoon Research and Development Centre of Agriculture and Agri-Food
Canada (Saskatoon, SK, Canada). The seeds were frozen in liquid nitrogen, ground to a fine powder
using an analytical mill (IKA A11, IKA, Staufen, Germany), and defatted with hexane (1:5 w/v) under
constant magnetic stirring. The slurry was filtered using a Whatman No. 4 filter paper, and
extractions were repeated three times. Defatted samples were dried overnight (~10–12 h) in a fume
hood in order to remove all traces of residual solvent. Defatted flours were then homogenized for 30
s in a coffee grinder (Custom Grind Deluxe, Hamilton Beach, Washington, WA, USA) and stored in
screw-capped plastic tubes at −80 °C until further use. Protein content in the defatted flour samples
was determined by Dumas combustion (Leco FP-428, Leco Corporation, St Joseph, MI, USA). Percent
of protein was calculated from protein nitrogen using a conversion factor of 6.25.
2.2. Optimization of Mustard Seed Protein Extraction
Biomolecules 2019, 9, 489 3 of 25
Extractions were conducted at various pH values in order to evaluate the protein solubilization
and the extraction efficiency on mustard proteins from the defatted flours. A 3*7 full factorial
experimental design (63 combinations) was used to study the effects of three different extraction
buffers [phosphate-buffered saline (0.01 M, pH 7.4), borate-buffered saline (0.1 M, pH 8.45), and
carbonate buffer (0.05 M, pH 9.6)] on protein recovery of mustard varieties. Minitab Statistical
Software (version 16) (Minitab Inc., State College, PA, USA) was used to design the experiments.
Each extraction buffer was used to extract 0.5 g of defatted flour from each mustard variety at a
protein/buffer ratio of 1:250 (w/v). All extractions were conducted in 50 mL centrifuge tubes under
constant shaking at 45 rpm using a LabRoller Rotator (Labnet International, Woodbridge, NJ, USA)
at room temperature for 1 h. The crude extracts were transferred in 70 mL centrifuge bottles and
centrifuged in a Beckman JA-18 fixed angle rotor in a Beckman J2-21 centrifuge (Beckman Intruments,
Brea, CA, USA) at 16,000× g for 30 min at 4 °C . The supernatant was passed on a filter paper
(Whatman filter paper No. 4, Whatman International Ltd., Maidstone, UK) and further filtered on
0.45 µm filters. The pH of the extracts was measured at the beginning and the end of the extraction
time to verify its stability. The protein concentration of the mustard extracts was determined using
the Bradford protein assay [25]. Clarified extracts were transferred in 2 mL cryogenic vials and stored
at −80 °C until use.
All protein extraction experiments were performed in duplicate, and the present results are the
average values of four determinations (two experimental × two analytical replicates). Analysis of
variance (ANOVA) was carried out using XLSTAT version 2012.4.01 to compare data obtained from
different samples. Tukey multiple comparison was used to discriminate among the means of the
variables when necessary. Differences at p ≤ 0.05 were considered significant.
2.3. Protein Electrophoresis
Mustard seed extracts normalized to equal amounts of protein (10 µg) were subjected to SDS-
PAGE under reducing and non-reducing conditions using pre-cast Any KD TGX gels (Bio-Rad
Laboratories, Hercules, CA, USA) according to Laemmli [26]. The soluble extracts were mixed with
an equal volume of Laemmli sample buffer with 5% (v/v) of β-mercaptoethanol (β-ME) and boiled
for 5 min. Alternatively, electrophoresis was performed under non-reducing conditions by omitting
the addition of β-ME. The gels were run at a constant voltage of 150 V for 90 min using TGS buffer
(25 mM Tris, 192 mM glycine, and 0.1% SDS) in a Criterion cell (Bio-Rad). A molecular weight
standard (Precision Plus Protein Standard) was included on each gel. After electrophoresis, gels were
stained with Coomassie Brilliant Blue. Images were acquired by scanning stained gels using an Image
Scanner III (GE Healthcare, Salt Lake City, UT, USA) operated by LabScan 6.0 software (GE
Healtcare). For image and densitometry analysis, the Image Quant TL 7.0 Software (GE Healtcare)
was used.
2.4. Immunoblotting
Immunoblotting was carried out with human sera obtained from two different sources. Two
sera named P1 and P2 were from mustard sensitized and self-declared allergic donors and were
purchased from Plasma Lab International (Everett, WA, USA). A third serum named P3 was obtained
from a clinically confirmed mustard allergic patient of the Sainte-Justine University Hospital Center
(Montreal, QC, Canada). The three sera—P1, P2, and P3—showed a level of sensitization of Class III
to mustard with specific IgE antibody levels equal to 5.76, 3.76, and 6.3 [kilo units of antibody per
liter (kUA/L)], respectively, following measurement with the Pharmacia ImmunoCAP® system.
Control sera were obtained through Plasma Lab from patients with an allergic history to dust mites
but without food allergy. The study was approved by the Sainte-Justine’s Hospital Ethics Committee
and the Human Research Ethics Committee of Agriculture and Agri-Food Canada.
For western blots, 2 µ g of carbonate buffer protein extract from each mustard variety were
separated by SDS-PAGE (performed as described above); the separated proteins were then
transferred on a polyvinylidene fluoride (PVDF) membrane using a Mini Trans-Blot electrophoretic
transfer cell (Bio-Rad) at 100 V for 1 h at 4 °C according to Towbin [27]. The blotted membranes were
Biomolecules 2019, 9, 489 4 of 25
subsequently blocked in 5% (w/v) skim milk powder in phosphate-buffered saline with 0.1% Tween-
20 (PBS-T) for 1 h at room temperature. Membranes were then incubated overnight at 4 °C with 1:2
(v/v) dilutions of the three sera. IgE was detected by using a horseradish peroxidase (HRP) conjugated
mouse anti-human antibody (clone B3102E8, Southern Biotech, Birmingham, AL, USA).
Immunoreactive bands were visualized using amplified Opti-4CN reagents (Bio-Rad) following
manufacturer’s recommendations. The immunoblots were scanned and analyzed as previously
mentioned for the SDS-PAGE gels.
2.5. Indirect ELISA
High-binding 96-well microtiter plates (CostarTM, Corning, Tewksbury, MA, USA) were coated
with 0.25 µg/well of protein extracts from each variety of mustard in carbonate-bicarbonate buffer
(pH 9.6) and incubated overnight at 4 °C. Plates were blocked with 5% bovine serum albumin (BSA)
in PBS-T for 2 h at room temperature followed by washing and incubation with control and mustard
sensitive sera samples serially diluted in 1% BSA in PBS-T (dilution buffer) for another 2 h. For IgE
detection, plates were washed and incubated 1 h in a 1:1000 (v/v) dilution of mouse anti-human IgE-
HRP (clone B3102E8, Southern Biotech, AL, USA) prepared in dilution buffer. Bound peroxidase
activity was determined with 3,3’,5,5’-tetramethylbenzidine (TMB) (Sigma-Aldrich, St Louis, MO,
USA), the reaction was stopped by the addition of 1N sulfuric acid, and absorbance was measured at
450 nm. ELISA measurements were performed in duplicate.
2.6. Identification of Protein Bands as Allergens by LC-MS/MS
The protein in-gel digestion and the mass spectrometry experiments were performed by the
Proteomics platform of the Eastern Quebec Genomics Center, Quebec, Canada. Detailed
experimental parameters for the tryptic digestion, the mass spectrometry conditions, and the data
analysis were previously reported by Rioux et al. [28]. Scaffold (Scaffold_3_00_07, Proteome Software
Inc., Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications.
Peptide identifications were accepted if they could be established at greater than 95.0% probability,
as specified by the Peptide Prophet algorithm [29]. Protein identifications were accepted if they could
be established at greater than 95.0% probability and contained at least two identified peptides.
Protein probabilities were assigned by the Protein Prophet algorithm [30].
2.7. Protein Sequence Comparisons with Known and Putative Allergens
Each MS identified IgE-binding mustard protein sequence was compared to all proven and
putative protein allergens sequences included in the Food Allergy Research and Resource Program
(FARRP) AllergenOnline.org database version 19 (updated on 10 February 2019) [31]. This version
contains a comprehensive list of 2129 protein (amino acid) sequence entries that are categorized into
853 taxonomic-protein groups of unique proven or putative allergens (food, airway, venom/salivary,
and contact) from 384 species. All database entries are linked to sequences in the National Center for
Biotechnology Information (NCBI) of the National Institute of Health (NIH). Sequence comparison
was performed using the FASTA algorithm version 36 with a sliding window of 80 amino acid
segments of each protein to find identities greater than 35%, as recommended by the CODEX
Alimentarius guidelines [32]. The scoring matrix used on the AllergenOnline website is a BLOSUM
50 [33]. E-values and percent identities [(#identical residues/80 or more amino acids) * 100%)] were
evaluated to consider potential cross-reactivity.
Biomolecules 2019, 9, 489 5 of 25
3. Results and Discussion
3.1. Protein Content and Extractability from Different Mustard Varieties
The total seed protein content varied significantly (p < 0.0001) among the different mustard
varieties, ranging from 36.07–37.92% for the two varieties of Sinapis alba (AC Pennant and Andante)
and 31.38–37.22% for the five varieties of Brassica juncea (Duchess, AC Vulcan, Dahinda, Centennial
Brown, and Cutlass), accounting for about 7% difference across varieties (Figure 1A). These values
were generally higher than the mean protein values reported for Canadian mustard by the Canadian
grain commission [11].
Regardless of the variety, carbonate buffer was significantly (p < 0.0001) more efficient than
phosphate and borate buffers in solubilizing mustard proteins (Figure 1B). This result is in agreement
with a previous study on Brassicaceae oilseeds that showed that solubility varied between species and
varieties studied, while the highest value of N solubility was observed at pH 10 [34]. Similar results
were also observed for peanut and tree-nut proteins, where carbonate was found to be the most
efficient extracting buffer [35]. An alkaline medium allows more protein to be solubilized, and this
effect seems to be the result of electrostatic repulsion. The most abundant protein fraction in crucifers
is cruciferin [36], and since its isoelectric point (IP) is at 7.25 [37], this should explain why the
electrostatic repulsion reaches a higher value in the carbonate buffer, thus giving better protein
solubility.
Figure 1. Protein content and extractability from different mustard varieties. Box plots represent the
protein content of the studied Canadian mustard varieties (A), the effect of extraction buffer on
mustard protein recovery (B), and the effect of mustard variety on protein recovery (C). Buffers or
varieties with different superscripts are significantly different (Tukey multiple comparison of means,
p < 0.0001).
Protein extractability also showed some significant variation according to mustard varieties. As
presented in Figure 1C, Brassica juncea variety Dahinda exhibited the highest protein recovery (26.4%,
p < 0.0001). Dahinda is a Brassica juncea canola quality variety, an oilseed that was developed with a
low glucosinolate content but with oil equivalent to conventional canola species [38]. The breeding
of this variety has also resulted in a high cruciferin content [39]. The reported low surface
hydrophobicity of cruciferin at basic pH of 10 [40] might explain the higher solubility observed for
this variety. On the opposite end, the Sinapis alba variety Andante showed the poorest extractability
(19.7%, p < 0.0001), despite its high protein content (38%).
3.2. Protein Electrophoretic Profiles of Differents Mustard Varieties as a Function of Extraction Buffer
Polypeptide composition of the mustard varieties as a function of extraction buffer was resolved
by gel electrophoresis in both non-reducing (Figure 2A) and reducing (Figure 2B) conditions. These
figures revealed important qualitative differences in the protein profiles among the different mustard
types/classes. In the presence of mercaptoethanol, the polypeptide profiles of the Brassica and the
Biomolecules 2019, 9, 489 6 of 25
Sinapis varieties were similar to those obtained by Aluko et al. [41]. No major differences were found
in the extracts’ electrophoretic profiles between the varieties of the same mustard type and for the
same extraction buffer used (Figure 2A,B). A similar observation was made by Wanasundara et al.
[34] for the solubility profile of cruciferin and napin between pH 2 and 10. However, differences in
protein profiles were observed between the different buffer extracts for the same variety, suggesting
that the buffer type affected mustard protein extraction not only quantitatively but also qualitatively.
From the densitometry analysis of protein electrophoretic profile (Table 1), it was found that
phosphate buffer tended to enhance napin protein extractability, particularly for the B. juncea
varieties. Since napins have a high degree of polymorphism [36], it could be possible that some
isoforms would show different solubility according to pH. In the case of Brassica napus, it was shown
that napin was soluble at acidic, neutral, and basic pH, but that only a few isoforms were soluble at
a pH of 8.5 [40]. In addition, the polypeptide profiles of borate and carbonate buffers extracts revealed
the presence of protein bands at around 15 and 55 kDa, which were absent in the phosphate buffer
extract. Moreover, an additional band of about 17 kDa, which was previously identified as an oleosin
[42], was observed in borate and carbonate buffers extract under non-reducing conditions for the B.
juncea varieties.
Figure 2. Protein electrophoretic profiles of mustard seed varieties as a function of extraction buffer.
Proteins were separated under non-reducing (A) and reducing (B) conditions. 1: phosphate buffer; 2:
borate buffer; 3: carbonate buffer. Molecular weights (MWs) of the standards are indicated in the left
margin.
Biomolecules 2019, 9, 489 7 of 25
Table 1. Densitometry analysis of electrophoretic profiles of mustard protein extracts.
Band Intensity (1 × 106)
Sinapis alba
Brassica juncea
MW a
AC Pennant
Andante
MW a
Duchess
Centennial Brown
Vulcan
Cutlass
Dahinda
Band Name
kDa
1
2
3
1
2
3
Band Name
kDa
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Sin a 1 LC b
9.5
24.1
18.9
19.1
23.4
18.3
18.3
Bra j 1 LC b
9.5
23.7
15.0
19.6
23.7
13.0
15.7
22.4
18.1
19.9
27.2
16.7
21.2
26.4
23.1
22.5
Sin a 1 HC c
12
25.0
19.2
19.4
11.8
19.4
19.1
Bra j 1 HC c
12
19.2
11.1
14.1
17.7
10.0
18.0
13.9
12.9
14.7
19.6
10.8
15.0
16.1
13.9
14.6
Unknown
15.1
0.6
2.8
0.8
2.1
Unknown
14.6
2.4
1.2
3.3
1.1
2.8
1.1
3.7
0.8
Cruciferin
Cruciferin
16.8
7.1
6.8
5.5
5.4
7.3
8.2
6.8
6.0
3.5
10.5
9.9
β-polypeptides
18
13.3
14.8
15.0
11.8
14.2
10.6
β-polypeptides
18
11.3
10.3
8.7
12.7
11.8
10.9
13.9
12.3
9.7
14.8
12.5
11.8
13.7
14.3
13.3
11 S globulin
19.1
5.9
11.6
9.6
6.4
12.0
5.1
11 S globulin
19.1
5.7
6.7
5.2
5.7
6.4
5.3
6.6
7.9
5.9
5.7
6.5
4.7
6.7
8.2
7.0
Sin a 2
21.6
8.8
12.6
10.5
9.0
12.5
7.8
Sin a 2
20.9
6.3
6.1
5.2
7.5
7.1
6.5
7.2
6.5
5.2
7.2
6.4
5.4
7.9
7.7
6.7
Cruciferin
Cruciferin
23.7
1.4
1.3
2.3
2.2
1.7
2.2
1.9
1.6
2.2
1.9
1.6
2.6
2.7
2.7
α-polypeptides
26.8
6.2
7.0
6.5
4.4
4.2
4.7
α-polypeptides
25.3
10.5
10.4
10.4
13.0
12.6
12.1
13.7
12.5
10.7
14.1
12.9
11.9
11.1
10.4
10.0
11S globulin
28.4
10.6
13.2
11.7
11.7
14.1
12.3
11S globulin
28
2.6
2.3
1.6
2.8
3.1
2.5
2.7
3.0
2.1
2.8
2.8
2.1
3.7
3.5
3.1
Sin a 2
30.9
1.3
0.8
Sin a 2
30.5
9.6
9.1
7.3
12.0
10.6
9.8
10.2
8.7
7.2
10.2
8.7
7.2
6.2
5.6
5.0
32.9
10.5
11.0
9.4
10.0
10.4
9.3
11S fragment
55
1.7
1.5
1.9
1.6
11S fragment
52
1.0
0.5
1.1
1.4
1.3
0.8
1.0
0.9
1.1
1.2
1.0
1.8
2.4
1.3
Procruciferin
63.5
1.0
1.0
1.1
0.9
1.6
3.0
Procruciferin
63.6
2.9
1.1
1.6
1.5
1.3
1.5
1.7
1.7
1.3
1.2
2.0
3.0
1.0
66.8
2.0
1.5
2.0
2.7
65.9
3.8
2.6
2.3
1.5
1.1
1.1
1.3
1.2
69.4
3.4
5.9
5.2
2.0
3.2
2.0
69.9
2.0
1.8
1.2
1.1
1.9
2.4
2.0
1.2
73.9
2.7
2.5
1.7
2.3
3.3
72.5
2.7
1.0
1.3
2.8
2.3
2.4
1.4
3.4
1.2
a Based on reduced SDS-PAGE; b light chain; c heavy chain; 1: phosphate buffer; 2: borate buffer; 3: carbonate buffer.
Biomolecules 2019, 9, 489 8 of 25
Based on these results, carbonate was identified as the most favorable extracting buffer because
of its higher protein extraction capacity and more complete electrophoretic profile. Consequently,
carbonate protein extracts were retained for the remainder of the study.
3.3. Indirect ELISA for Serum IgE Response to Mustard Varieties
Indirect ELISA was performed with individual serum (P1, P2, and P3) to compare the IgE-
binding levels of the different S. alba and B. juncea varieties. As can be seen in Figure 3, all mustard
varieties exhibited the highest binding to IgEs from serum P1, followed by serum P3. IgE-binding to
serum P2 showed the lowest intensity. Differences in the intensity of mustard sensitive/allergic
individuals are common and were reported in previous studies [5,7,8]. Although not statistically
significant, the two sera P1 and P3 appeared to be less immunoreactive to S. alba varieties in
comparison to the B. juncea varieties. Dust mite sensitized control sera did not bind to any mustard
varieties used in this study (data not shown).
Figure 3. Indirect ELISA of specific Immunoglobulin E (IgE)-binding of different mustard protein to
sera from mustard sensitive/allergic individuals.
3.4. IgE-Binding Profiles of Select Canadian Mustard Varieties
In order to evaluate the varietal effect on the IgE binding profiles of mustard proteins,
immunoblotting with carbonate buffer extracts from the seven different mustard varieties was
performed using sera from three mustard sensitive/allergic persons individually (Figure 4). IgE-
immunoblotting was conducted for both non-reduced and reduced mustard proteins. The results
revealed important differences in protein profile, abundance, and IgE-binding intensity between the
S. alba and the B. juncea mustard types as well as in the IgE-reactivity profiles between the three sera.
According to Menendez-Arias et al. [13], Sin a 1 (2S albumin) is the major allergen of S. alba seeds and
Bra j1 of B. juncea seeds [23]. Under non-reduced conditions (Figure 4B), the 2S albumins showed
intense IgE-binding in the case of sera P1 and P3. However, under reduced electrophoretic conditions
(Figure 4A), these two sera only weakly bound the two napins bands. This observation suggests the
recognition of conformational epitopes. A previous study [14] identified two epitopes of Sin a 1—one
conformational and one linear. According to Monsalve et al. [24], it is the linear epitope that is
considered to be the antigenic determinant. Based on this epitope, an anti-epitope antibody for the
quantification of Sin a 1 by a non-competitive enzyme linked immunosorbent assay was developed
[43]. This same study also showed that the napin protein fraction of yellow mustard contained
proteins devoid of the linear epitope sequence, thereby not contributing to all cases of 2S allergenicity.
In the case of serum P2, there was no evidence of IgE-binding to napin proteins under both reduced
and non-reduced conditions. A previous study [44] also showed that, even though the majority of
Biomolecules 2019, 9, 489 9 of 25
mustard allergic persons reacted to Sin a 1, some patients’ sera did not bind to the 2S albumin. This
observation could be explained by the finding that Sin a 1 presents an important polymorphism [45],
resulting in significant variability in its allergenic potential.
IgE-binding pattern of the mustard sensitized sera revealed the presence of other reactive
protein bands from both S. alba and B. juncea varieties. For the latter, sera P1 and P3 exhibited IgE-
binding on the non-reduced immunoblots for polypeptide bands between 27 and 48 kDa and on
reduced ones for bands between 22 and 34 kDa. These regions corresponded to cruciferin (11S
globulin) and, in the case of S. alba seeds, to the allergen Sin a 2 [19]. To date, no reported allergen
has been recorded for the 11S of B. juncea seeds. However, a bioinformatics evaluation of the
cruciferin of B. juncea, B. napus, and S. alba showed that the cruciferin of B. juncea has a high similarity
to the one of S. alba [46]. As a result of this high homology, it would be possible for the 11S globulin
of B. juncea to present an allergic potential, but this still has to be clinically demonstrated. The IgE-
binding to the 11S globulin was less intense than for the 2S albumin in the case of sera P2 and P3.
Similar results were obtained by Menendez-Arias et al. [13]. In addition, for serum P1, intense IgE
binding was observed in B. juncea varieties to bands between 27 and 31 kDa on the non-reduced
immunoblot, also corresponding to the free polypeptide chains of the cruciferin. In the case of the
two S. alba varieties, binding was observed at a polypeptide band around 60 kDa. However, in both
cases, the binding to cruciferin was not observed on the reduced immunoblot. This is contrary to
what was previously published about the 11S cruciferin of S. alba reporting that, under reducing
conditions, the two subunits of the protein were able to bind IgE from the sera [19]. Such a difference
is probably due to the variability in the sensitization profile of the used sera; moreover, that study
involved the use of pooled sera, and a different result could have been obtained if the sera were used
individually. A strong IgE-binding was further observed on both the reduced and the non-reduced
immunoblots of serum P2 for a procruciferin band with molecular weight of about 75 kDa. It was
reported [36] that it is common to observe polypeptide bands that remain at apparent molecular
weight above 54 kDa, presumably from the precursor polypeptide or procruciferins of α-β, which
have not undergone regular in vivo processing [47,48]. Finally, sera P1 bound strongly to the B. juncea
protein around 17 kDa, while sera P2 and P3 showed binding to a band around 55 kDa on the reduced
immunoblot for the S. alba varieties. This last band was not observed on the electrophoretic profile
for the phosphate buffer extracts. However, it appeared on the profiles for the borate and the
carbonate extracts, thus confirming the importance of choosing a buffer that provides the most
complete protein profile so as to increase the probability of revealing as many IgE reactive bands as
possible. All IgE-reactive proteins were further subjected to LC-MS analysis for identification.
Biomolecules 2019, 9, 489 10 of 25
Figure 4. Protein and IgE- binding profiles of different mustard varieties. Carbonate mustard protein
extracts were separated under non-reducing (A) and reducing (B) conditions. 1: S. alba AC Pennant;
2: S. alba Andante; 3: B. juncea Duchess; 4: B. juncea AC Vulcan; 5: B. juncea Dahinda; 6: B. juncea
Centennial Brown; 7: B. juncea Cutlass; STD1 and STD2 are protein MW markers.
3.5. Identification of Mustard IgE-Binding Proteins by Mass Spectroscopy
To confirm the identity of the IgE-binding bands, electrophoretic analyses of mustard varieties
AC Pennant (S. alba) and AC Vulcan (B. juncea) were run again, and the immunoreactive bands were
excised and further analyzed by LC/ESI-MS/MS. Figure 5 represents the SDS-PAGE and the
immunoblot patterns of the two mustard varieties incubated with sera P1, P2, and P3. The list of MS
identified proteins is presented in Table 2. All the allergenic protein bands (as shown in Figure 5A,B)
were identified by MS/MS analysis as belonging to the Brassicaceae family. Excised bands S1, RS1, and
RS2 of S. alba and bands B1, RB1, and RB2 from B. juncea from both non-reduced and reduced gels
were identified as Sin a 1 (S. alba) and Braj 1 (B. juncea), respectively, thereby confirming their allergen
identity. As for B2 and RB4 bands, these were identified as oleosin proteins (OLES2_BRANA Oleosin
S2-2 and BRANA Oleosin S3-1) from the database (UniProtKB/Swiss-Prot and UniProtKB/TrEMBL;
www.uniprot.org). Although this is the first formal evidence of the allergenicity of such oil bodies-
associated proteins as potential allergens from mustard, future studies need to be performed to prove
the biological activity of these newly identified allergens. Recent work has shown that two oil body-
associated proteins [Oleosins, Ses i 4 (17 kDa) and Ses i 5 (15 kDa)] were found to be among the most
important sesame allergens [49]. Allergenic oleosins were also reported in peanuts, where five
different IgE-binding oleosins with a molecular weight from 14–18 kDa were identified [50–52], while
two oleosin isoforms of 17 and 14–16 kDa, now designated Cor a12 and Cor a13, were identified as
allergens in hazelnut [53]. Oleosin proteins were also identified from B1, B2, B3, and B4, suggesting
the presence of multiple isoforms of the proteins.
Biomolecules 2019, 9, 489 11 of 25
Figure 5. Identification of mustard IgE-binding protein bands for in gel-tryptic digestion and LC-
MS/MS analysis. Protein extracts from S. alba AC Pennant and B. juncea AC Vulcan were separated
under non-reducing (A) and reducing (B) conditions and immunoblotted using sera P1, P2, and P3.
STDs are protein MW markers.
Biomolecules 2019, 9, 489 12 of 25
Table 2. Identification of IgE-binding proteins from Sinapis alba and Brassicae juncea mustard by mass spectrometry analysis.
Band
Number a
MW on SDS-
PAGE (KDa)
Protein Name b
Protein Accession
Numbers
Protein MW
(KDa)
Exclusive Unique
Peptide Count
Percentage
Sequence Coverage
c
Sinapis alba (AC Pennant)
S1
16
ALL1_SINAL Allergen Sin a 1
[Sinapis alba (White mustard)]
P15322
16.4
5
23.40%
S2
28
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
16
39.40%
S3
30
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
16
39.40%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
13
30.10%
S4
44
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
19
44.30%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
14
29.70%
S5
75
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
18
44.30%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
7
23.40%
S6
107
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
21
46.50%
S7
223
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
20
39.40%
BRANA Beta-glucosidase [Brassica
napus (Rape)]
Q42618
56.5
20
34.40%
RS1
9.5
ALL1_SINAL Allergen Sin a 1
[Sinapis alba (White mustard)]
P15322
16.4
7
26.90%
RS2
12
ALL1_SINAL Allergen Sin a 1
[Sinapis alba (White mustard)]
P15322
16.4
9
57.90%
RS3
20
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
19
36.10%
BRANA Cruciferin (Fragment)
[Brassica napus (Rape)]
Q7XB53
51.3
8
23.20%
RS4
28
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
14
25.90%
Biomolecules 2019, 9, 489 13 of 25
CRU3_BRANA Cruciferin CRU1
[Brassica napus (Rape)]
P33525
56.5
7
27.50%
RS5
34
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
14
25.90%
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLV9
57.9
5
34.60%
RS6
44
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
18
46.70%
RS7
55
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
15
37.60%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
10
25.20%
RS8
62
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
20
45.10%
RS9
75
Q2TLW0_SINAL 11S globulin
[Sinapis alba (White mustard)]
Q2TLW0
56.5
16
40.20%
BRANA Beta-glucosidase [Brassica
napus (Rape)]
Q42618
58.5
12
20.80%
RS10
82
Q2TLW0_SINAL 11S globulin
[Sinapis alba (White mustard)]
Q2TLW0
56.5
14
37.10%
RS11
118
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
12
27.50%
RS12
140
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
9
24.50%
Brassicae juncea (AC Vulcan)
B1
15.5
Allergen Bra j 1-E [Brassica juncea
(Indian mustard)]
P80207
14.6
5
51.90%
OLES2_BRANA Oleosin S2-2
[Brassica napus (Rape)]
C3S7F1
19.9
15
46.80%
B2
17
BRANA Oleosin S3-1 [Brassica
napus (Rape)]
C3S7F8
19.6
10
41.70%
OLES2_BRANA Oleosin S2-2
[Brassica napus (Rape)]
C3S7F1
19.9
16
46.80%
B3
26
BRANA Caleosin CLO1-2 [Brassica
napus (Rape)]
C3S7H5
28.1
7
35.90%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
21
49.50%
Biomolecules 2019, 9, 489 14 of 25
B4
30
CRU3_BRANA Cruciferin CRU1
[Brassica napus (Rape)]
P33525
56.5
12
47.20%
OLES2_BRANA Oleosin S2-2
[Brassica napus (Rape)]
C3S7F1
19.9
11
46.30%
B5
29
BRANA Cruciferin (Fragment)
[Brassica napus (Rape)]
Q7XB53
51.3
4
8.80%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
8
24.30%
B6
34
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
7
20.80%
CRU3_BRANA Cruciferin CRU1
[Brassica napus (Rape)]
P33525
56.5
3
21.80%
B7
42.5
CRU3_BRANA Cruciferin CRU1
[Brassica napus (Rape)]
P33525
56.5
12
45.20%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
20
52.30%
B8
68
CRU3_BRANA Cruciferin CRU1
[Brassica napus]
P33525
56.5
14
52.80%
CRU4_BRANA Cruciferin CRU4
[Brassica napus]
P33522
51.4
13
40.90%
B9
200
Malate synthase. glyoxysomal
[Brassica napus (Rape)]
P13244
63.7
14
23.00%
BRANA Beta-glucosidase [Brassica
napus (Rape)]
Q42618
58.5
23
44.20%
RB1
9.5
Allergen Bra j 1-E [Brassica juncea
(Indian mustard)]
P80207
14.6
2
38.00%
RB2
12
Allergen Bra j 1-E [Brassica juncea
(Indian mustard)]
P80207
14.6
5
68.20%
ALL1_SINAL Allergen Sin a 1
[Sinapis alba (White mustard)]
P15322
16.5
4
35.20%
RB3
22
CRU3_BRANA Cruciferin CRU1
[Brassica napus (Rape)]
P33525
56.5
11
39.50%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
11
35.30%
RB4
27
BRANA Caleosin CLO1-2 [Brassica
napus (Rape)]
C3S7H5
28.1
8
37.60%
Biomolecules 2019, 9, 489 15 of 25
OLES2_BRANA Oleosin S2-2
[Brassica napus (Rape)]
C3S7F1
20.0
6
40.40%
BRAJU Glutathione S-transferase 3
[Brassica juncea]
Q7XZT2
24.1
7
46.00%
RB5
30
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
17
42.60%
RB6
30
CRU3_BRANA Cruciferin CRU1
[Brassica napus (Rape)]
P33525
56.5
12
45.20%
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
15
38.70%
RB7
34
CRU3_BRANA Cruciferin CRU1
[Brassica napus (Rape)]
P33525
56.5
8
28.90%
BRAJU Cysteine synthase [Brassica
juncea]
O23733
33.9
8
33.20%
RB8
47
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
25
54.00%
BRANA Cruciferin (Fragment)
[Brassica napus (Rape)]
Q7XB53
51.3
14
39.70%
RB9
52
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
16
45.40%
BRACM Enolase [Brassica
campestris (Field mustard)]
Q6W7E8
47.4
21
61.00%
RB10
62
BRANA Beta-glucosidase [Brassica
napus (Rape)]
Q42618
58.5
13
30.00%
RB11
76
CRU4_BRANA Cruciferin CRU4
[Brassica napus (Rape)]
P33522
51.4
8
30.10%
SINAL 11S globulin [Sinapis alba
(White mustard)]
Q2TLW0
56.5
5
15.30%
a Band numbers correspond to the IgE-binding bands detected in Figure 5; b only protein identifications with 100% probability were retained; c total percentage of
proteins amino acid sequence covered by the identified peptides in MS/MS analysis.
Biomolecules 2019, 9, 489 16 of 25
Subunit bands assigned as S4 to S7 and RS3–RS12 from AC Pennant (S. alba) in addition to B3–
B8 and RB3, RB5–RB9, and RB11 from AC Vulcan (B. juncea) mustard varieties were identified and
confirmed as cruciferin (11S globulin) fragments. The presence of several cruciferin (α-β)
polypeptides that form the subunits (protomers) of the 11S globulin molecule in mustard has been
reported [36]. However, not all of them have been characterized yet as potential mustard allergens
within the 11S globulin family. To date, the only 11S globulin storage protein that has been identified
as an important mustard allergen is Sin a 2 [19]. Future studies need to be performed to characterize
these newly identified allergens and name them in accordance with the World Health
Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature
Subcommittee [54].
Furthermore, the protein bands S7 and RS9 from S. alba as well as B9 and RB10 from B. juncea
were identified as β-glucosidase precursors (BRANA, accession No. Q42618) showing 44–50%
sequence coverage. Indeed, a β-glucosidase was previously purified from seeds of B. napus (oilseed
rape) as reported by Falk and Rask [55]. The 130 kDa native enzyme consisted of a disulfide linked
dimer of 64 kDa monomers. Evidence was previously reported about the potential allergenicity of a
β-glucosidase from wheat [56]. The protein band RB9 was also identified with 64% coverage and 21
unique peptides as an enolase (BRACM, accession No. Q6W7E8). Enolase is an essential glycolytic
enzyme that catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate [57]. It
has been recognized as an important allergen from various molds and some plants [58–60]. Finally,
in addition to oleosin, the RB4 band was also identified as a glutathione-S transferase (GST) (accession
No. Q7XZT2). Members of the GST family have been reported as relevant allergens in cockroach [61],
fungi [62], and wheat [63]. The allergenicity of these new identified IgE-binding proteins from
mustard would request further investigation and should be carefully evaluated not only by in vitro
IgE tests but also by in vivo and clinical tests.
3.6. Bioinformatic Assessment of Potential Cross-Reactivity of Identified Mustard IgE-Binding Proteins with
Known Allergens
The purpose of this analysis was to identify relevant homology in amino acid sequences between
the identified mustard IgE-binding proteins and proven or putative allergens, which could help
identify proteins that may share immunologic or allergic cross-reactivity. The Food Allergy Research
and Resource Program (FARRP) AllergenOnline.org database version 19 (updated on 10 February
2019; http://www.allergenonline.org/) was used for the primary comparisons to allergens. This public
database only shows sequences of proteins with sufficient published evidence of allergy at a
minimum-specific IgE binding from sera of subjects allergic to the source [31]. Based on the
recommendation of the CODEX Alimentarius guidelines [32], the FASTA3 algorithm with the criteria
of >35% identity over any segment of 80 or more amino acids as an indication of possible cross-
reactivity for allergens was used to compare all possible contiguous amino acid segments of each of
the identified mustard IgE-binding proteins against all sequences listed in the AllergenOnline
Database. Research has, however, shown that proteins with greater than 70% identical primary amino
acid sequences throughout the length of the protein are commonly cross-reactive, while those with
less than 50% identity are unlikely to be cross-reactive [64]. For increased confidence, only the best
scoring matches (>35% identity) with E-values smaller than 1e-7 are displayed in Table 3, as it has
been reported that larger E-values are unlikely to identify relevant matches, while matches with E-
values smaller than 1e-30 are much more likely to be cross-reactive in at least some allergic
individuals [65]. The complete search results are presented in a supplemental document (Appendix
1). The 80mer FASTA search confirmed the extensive homology and the high cross-reactivity between
the 2S albumins from Sinapis alba (Sin a 1) and Brassica species (Bra j 1) with a percentage of identity
(ID) over 87% and very small E-value. Interestingly, FASTA identified highly significant alignments
(>55% identity) of the IgE-binding 11S globulins (cruciferins) from both Sinapis and Brassica species
(accession No. Q2TLWO, Q7XB53, P33525, Q2TLV9) with 11S globulins allergens from tree-nut
species, most notably with black walnut (Juglans nigra), cashew (Anacardium occidentale), hazelnut
(Corylus avellana), almond (Prunus dulcis), and pecan (Carya illinoinensis), suggesting a strong
Biomolecules 2019, 9, 489 17 of 25
possibility of cross-reactivity, which would be worth testing using sera from individuals with clinical
reactivity to those species.
In addition, the search identified probable homology for 11S globulin proteins from Sinapis alba
(accession No. Q2TLWO and Q2TLV9) with high molecular weight (HMW) glutenin from wheat
(Triticum aestivum) based on 60% and 68% best identity and low E-values of 1.3e-12 and 3.5e-18,
respectively. Noteworthy, sequence alignments for the newly identified IgE-binding mustard
oleosins (accession No. C3S7F1 and C3S7F8) found highly significant matches with oleosin allergens
from hazelnut (Corylus avellana) with ID over 70% and E-values smaller than 1e-30. These results are
highly relevant for potential cross-reactivity. Significant scores were also found with oleosins from
peanut (Arachis hypogaea) and sesame (Sesamum indicum). Scoring results for the identified enolase
protein from Brassica juncea (accession No. Q6W7E8) resulted in the best alignments (over 80% ID
and very small E-value) to enolase allergens from latex tree (Hevea brasiliensis), yellowfin tuna
(Thunnus albacares), Atlantic salmon (Salmo salar), chicken (Gallus gallus), and fungi (Candida albicans
and Rhodotorula mucilaginosa). This finding adds to the existing knowledge that enolase is one of the
most conserved glycolytic enzyme protein across eukaryotes (animal, plant, and fungi) [58,59]. This
further suggests a strong possibility of cross-reactivity. Besides, the identified mustard Glutathione
S-transferase 3 (GST) enzyme (accession No. Q7XZT2) also significantly matched GST allergen Per a
5 from the insect American cockroach (Periplaneta americana), with 40% ID and an E-value of 1.2e-6.
Finally, other identified mustard proteins with accession No. Q42618 (β-glucosidase), O23733
(Cysteine synthase), P13244 (malate synthase, glyoxysomal), and C3S7H5 (caleosin) resulted in no
matches greater than 35% identity over 80 amino acids.
Biomolecules 2019, 9, 489 18 of 25
Table 3. Significant sequence alignment of the identified mustard IgE-binding proteins with allergens from the Allergen Online database.
Protein Identification a
Species
Best
%ID b
# Hits
>35%
Full Alignment
NCBI Links f
E-value c
%ID d
Length e
P15322: Allergen Sin a 1 [Sinapis alba (White mustard)]
gid|192|Allergen Allergen Sin a 1 precursor
Sinapis alba
100.00%
66 of 66
2.4e-34
100.0%
145
gi|51338758
gid|1172|Putative 2S storage protein
Brassica rapa
93.80%
66 of 66
2.5e-29
88.30%
145
gi|17697
gid|1142|Putative Napin-3
Brassica napus
91.20%
66 of 66
2.6e-17
82.60%
144
gi|75107016
gid|1170|Putative Allergen Bra j 1-E
Brassica juncea
87.50%
66 of 66
1.8e-17
79.90%
144
gi|32363444
P80207: Allergen Bra j 1-E [Brassica juncea (Indian mustard)]
gid|1170|Putative Allergen Bra j 1-E (Bra j 1)
Brassica juncea
100.00%
50 of 50
7.1e-23
100.0%
129
gi|32363444
gid|1142|Putative Napin-3 (Napin BnIII)
Brassica napus
92.50%
50 of 50
8.9e-20
89.10%
129
gi|75107016
gid|192|Allergen allergen sin a 1.0104
Sinapis alba
88.78%
50 of 50
2.5e-13
80.60%
144
gi|1009434
gid|1172|Putative 2S storage protein
Brassica rapa
85.00%
50 of 50
1e-11
77.10%
144
gi|17697
gid|386|Putative recombinant Ib pronapin
Brassica napus
55.00%
50 of 50
3.8e-6
50.00%
114
gi|26985163
Q2TLWO: 11S globulin [Sinapis alba (White mustard)]
gid|837|Allergen 11S globulin precursor
Sinapis alba
100.00%
431 of 431
0
100.0%
510
gi|62240390
gid|2066|Putative 11S legumin protein
Carya illinoinensis
62.51%
351 of 431
2.6e-61
43.20%
521
gi|158998782
gid|2597|Putative legumin
Juglans nigra
62.51%
362 of 431
2.7e-58
42.00%
528
gi|1126299828
gid|76|Allergen allergen Ana 0 2
Anacardium occidentale
60.04%
380 of 431
2.3e-70
44.80%
498
gi|25991543
gid|160|Allergen HMW glutenin
Triticum aestivum
60.00%
84 of 431
3.5e-18
37.60%
213
gi|288860106
gid|392|Allergen 11S globulin
Corylus avellana
58.00%
387 of 431
3.6e-61
45.70%
534
gi|18479082
gid|1572|Putative prunin 2 precursor
Prunus dulcis
57.80%
418 of 431
3e-61
45.60%
522
gi|307159114
gid|2650|Allergen 11S globulin-
Actinidia chinensis
57.50%
352 of 431
2.3e-68
41.40%
503
gi|82469930
gid|2274|Putative 11S globulin
Sesamum indicum
55.00%
342 of 431
3.7e-60
42.10%
470
gi|13183173
gid|1093|Putative 11S globulin
Pistacia vera
55.00%
356 of 431
6.5e-53
41.80%
505
gi|156001070
gid|531|Putative allergenic protein
Fagopyrum tataricum
55.00%
256 of 431
1.5e-43
38.10%
486
gi|113200131
gid|733|Putative glycinin subunit G3
Glycine max
53.10%
272 of 431
2e-38
36.20%
514
gi|18639
gid|347|Putative 11S globulin
Bertholletia excelsa
52.50%
318 of 431
5.8e-59
39.20%
510
gi|30313867
Q7XB53: Cruciferin (Fragment) [Brassica napus (Rape)]
gid|837|Allergen 11S globulin precursor
Sinapis alba
76.50%
387 of 387
3.5e-29
61.00%
472
gi|62240390
gid|392|Allergen Cor a 9 allergen
Corylus avellana
63.70%
353 of 387
9.4e-22
44.00%
509
gi|557792009
gid|1572|Putative Pru du 6 allergen
Prunus dulcis
62.51%
160 of 387
3.2e-10
48.20%
193
gi|523916668
Biomolecules 2019, 9, 489 19 of 25
gid|76|Allergen allergen Ana 0 2
Anacardium occidentale
61.30%
333 of 387
1.2e-22
44.70%
474
gi|25991543
gid|2597|Putative legumin
Juglans nigra
58.79%
298 of 387
3.8e-21
42.70%
503
gi|1126299828
gid|2066|Putative 11S legumin protein
Carya illinoinensis
58.79%
326 of 387
3.1e-21
43.50%
506
gi|158998782
gid|817|Putative seed storage protein
Juglans regia
57.52%
325 of 387
4.4e-21
42.90%
513
gi|56788031
gid|1093|Putative Pis v 2.0201 allergen 11S
Pistacia vera
56.30%
347 of 387
1.7e-33
41.60%
459
gi|110349085
gid|347|Putative 11S globulin
Bertholletia excelsa
53.80%
349 of 387
1.2e-24
43.30%
466
gi|30313867
gid|733|Putative glycinin subunit G3
Glycine max
53.80%
274 of 387
4.8e-22
38.00%
479
gi|18639
gid|2274|Putative 11S globulin
Sesamum indicum
52.46%
318 of 387
1.6e-24
40.80%
476
gi|13183173
gid|291|Allergen trypsin inhibitor
Arachis hypogaea
49.40%
59 of 387
1.9e-5
35.80%
204
gi|22135348
P33525: Cruciferin CRU4 [Brassica napus (Rape)
gid|837|Allergen 11S globulin precursor
Sinapis alba
98.80%
430 of 430
2.4e-210
91.60%
510
gi|62240390
gid|2066|Putative 11S legumin protein
Carya illinoinensis
65.00%
358 of 430
1.9e-61
44.70%
535
gi|158998782
gid|2597|Putative legumin
Juglans nigra
63.79%
373 of 430
4.1e-61
43.30%
533
gi|1126299828
gid|817|Putative seed storage protein
Juglans regia
63.79%
368 of 430
1e-63
44.50%
533
gi|56788031
gid|1572|Putative Pru du 6 allergen
Prunus dulcis
60.04%
168 of 430
4.6e-20
49.70%
195
gi|523916668
gid|76|Allergen allergen Ana 0 2
Anacardium occidentale
60.04%
379 of 430
7.9e-73
46.20%
487
gi|25991543
gid|392|Allergen Cor a 9 allergen
Corylus avellana
58.79%
391 of 430
4.6e-62
45.50%
528
gi|557792009
gid|160|Allergen glutenin
Triticum aestivum
56.26%
70 of 430
2.5e-11
33.30%
207
gi|736319
gid|1093|Putative Pis v 2.0101 11S globulin
Pistacia vera
55.00%
345 of 430
6.7e-51
41.10%
518
gi|110349083
gid|2274|Putative 11S globulin
Sesamum indicum
53.80%
343 of 430
4.8e-63
42.10%
478
gi|13183173
gid|531|Putative allergenic protein
Fagopyrum tataricum
53.80%
255 of 430
4.4e-44
35.60%
523
gi|113200131
gid|347|Putative 11S globulin
Bertholletia excelsa
52.50%
309 of 430
2.3e-59
39.70%
506
gi|30313867
gid|733|Putative glycinin subunit G3
Glycine max
51.20%
267 of 430
3e-39
36.00%
516
gi|18639
gid|291|Allergen allergen Arah3/Arah4
Arachis hypogaea
47.50%
219 of 430
2.1e-25
33.20%
548
gi|21314465
gid|574|Putative glycinin precursor
Glycine max
47.50%
116of430
4.9e-28
39.80%
191
gi|169971
Q2TLV9: 11S globulin [Sinapis alba (White mustard)]
gid|837|Allergen 11S globulin precursor
Sinapis alba
100.00%
444 of 444
0
100.00%
523
gi|62240392
gid|160|Allergen HMW glutenin
Triticum aestivum
68.80%
99 of 444
1.3e-12
38.60%
228
gi|288860106
gid|2066|Putative 11S legumin protein
Carya illinoinensis
63.79%
360 of 444
6.3e-41
41.90%
532
gi|158998782
gid|817|Putative seed storage protein
Juglans regia
62.51%
367 of 444
1.1e-41
41.70%
545
gi|56788031
gid|2597|Putative legumin
Juglans nigra
62.51%
364 of 444
2.5e-41
40.80%
539
gi|1126299828
gid|76|Allergen allergen Ana 0 2
Anacardium occidentale
61.30%
380 of 444
9.9e-70
42.70%
510
gi|25991543
gid|1572|Putative Chain A
Prunus dulcis
59.30%
417 of 444
6.5e-53
45.20%
524
gi|258588247
gid|392|Allergen Cor a 9 allergen
Corylus avellana
59.30%
390 of 444
3.4e-41
43.70%
545
gi|557792009
Biomolecules 2019, 9, 489 20 of 25
gid|1572|Putative prunin 1 precursor
Prunus dulcis
59.30%
436 of 444
2.6e-53
44.90%
543
gi|307159112
gid|160|Allergen HMW glutenin
Triticum aestivum
57.52%
76 of 444
4.2e-9
31.30%
307
gi|21751
gid|1572|Putative Pru du 6 allergen
Prunus dulcis
57.50%
168 of 444
4.8e-20
46.20%
208
gi|523916668
gid|2650|Allergen 11S globulin
Actinidia chinensis
56.30%
344 of 444
4.1e-67
39.70%
514
gi|82469930
gid|1093|Putative Pis v 2.0201 allergen 11S
Pistacia vera
56.30%
332 of 444
9.2e-53
39.50%
516
gi|110349085
gid|2274|Putative 11S globulin
Sesamum indicum
53.80%
345 of 444
2e-60
41.10%
482
gi|13183173
gid|574|Putative glycinin A3B4 subunit
Glycine max
53.80%
241 of 444
6.4e-27
32.40%
558
gi|10566449
gid|291|Allergen allergen Arah3/Arah4
Arachis hypogaea
47.50%
208 of 444
1.2e-20
32.40%
561
gi|21314465
C3S7F1: Oleosin S2-2 [Brassica napus (Rape)]
gid|2298|Allergen oleosin
Corylus avellana
70.01%
109 of 109
2.2e-42
52.50%
158
gi|49617323
gid|2283|Putative oleosin 1
Arachis hypogaea
64.98%
104 of 109
6.5e-39
46.20%
171
gi|113200509
gid|1893|Putative oleosin
Sesamum indicum
62.50%
103 of 109
2.2e-31
45.90%
157
gi|10834827
gid|389|Putative oleosin
Corylus avellana
50.00%
68 of 109
1.1e-21
41.00%
117
gi|29170509
gid|2285|Allergen oleosin 3
Arachis hypogaea
45.10%
77 of 109
9.1e-21
38.20%
144
gi|52001241
C3S7F8: Oleosin S3-1 [Brassica napus (Rape]
gid|1238|Putative 15 kDa oleosin
Sesamum indicum
76.20%
101 of 101
6e-31
63.20%
125
gi|5381321
gid|389|Putative oleosin
Corylus avellana
73.80%
98 of 101
1.1e-32
66.90%
121
gi|29170509
gid|2284|Putative oleosin 1
Arachis hypogaea
70.00%
94 of 101
1.2e-28
60.80%
125
gi|71040655
Q6W7E8: Enolase [Brassica campestris (Field mustard)]
gid|586|Putative Enolase
Hevea brasiliensis
98.80%
365 of 365
1.1e-170
89.60%
442
gi|14423687
gid|1955|Allergen Alpha-enolase
Thunnus albacares
88.70%
365 of 365
2.9e-129
69.50%
442
gi|576011129
gid|1959|Allergen enolase
Salmo salar
88.70%
365 of 365
5.1e-130
69.90%
442
gi|385145180
gid|2710|Allergen beta-enolase
Gallus gallus
83.80%
365 of 365
3.6e-119
65.10%
441
gi|46048765
gid|396|Allergen Enolase
Candida albicans
82.50%
365 of 365
1.5e-111
63.10%
444
gi|232054
gid|103|Putative enolase
Rhodotorula mucilaginosa
80.00%
365 of 365
9.5e-107
61.00%
441
gi|30314940
Q7XZT2-BRAJU Glutathione S-transferase 3 [Brassica juncea]
gid|856|Allergen glutathione S transferase c
Periplaneta americana
40.00%
14 of 134
1.2e-6
29.40%
163
gi|359326557
a gid: allergen group id number in the AllergenOnline database, which links to detailed information on the allergenicity references for the group, the type of allergen,
other sequences belonging to the same group, and more on the allergenonline.org website. b Highest scoring identity for FASTA3 alignments of every possible 80
amino acid segment. The Food and Agriculture Organization/World Health Organization (FAO/WHO) 2001 expert panel recommended using a criteria of >35%
identity over any segment of 80 or more amino acids as an indication of possible cross-reactivity for allergens, which was adopted by the Codex Alimentarius
Commission (2003). c The E-value (expectation value) is a calculated value that reflects the degree of similarity of the query protein to its corresponding matches.
The size of the E-value is inversely related to similarity of two proteins, meaning a very low E-value (e.g., 10e-30) indicates a high degree of similarity between the
query sequence and the matching sequence from the database, while a value of 1 or higher indicates the proteins are not likely to be related in evolution or structure.
Biomolecules 2019, 9, 489 21 of 25
d Overall percent identity (ID) (percentage of amino acids with a direct match in the alignment). e Length of amino sequence alignment. f Link to the unique assigned
protein identity (gi number) in the NCBI (National Centre for Biotechnology Information) Protein Database.
Biomolecules 2019, 9, 489 22 of 25
4. Conclusions
In this study, carbonate buffer was found as an efficient non-denaturing buffer for the extraction
of mustard seed proteins. Protein and IgE-binding patterns revealed important differences between
S. alba and B. juncea types of mustard, but no major differences were observed between the varieties
of the same mustard type. The presence of both napins (2S albumins) and cruciferins (11S globulins)
allergenic polypeptides under both non-reducing and reducing electrophoretic conditions was
confirmed. Sin a 1, Bra j 1, and cruciferin polypeptides exhibited a stronger IgE reactivity under non-
reducing conditions in comparison to reducing conditions, demonstrating the presence of
conformational allergenic epitopes in Sin a 1, Bra j 1, and other cruciferin subunits. Therefore, the use
of denaturing protein extraction and analysis conditions may lead to failure to detect important
immuno-reactive epitopes due to protein modification. Results also revealed the presence, in both S.
alba and B. juncea types of mustard, of a wide range of IgE binding cruciferin (11S globulin)
polypeptides/fragments with different molecular weight, indicating the existence of multiple
isoforms in all types of mustard seeds. We also reported, for the first time, new mustard IgE binding
polypeptides/proteins identified as oil body-associated proteins (oleosins) and enolase enzyme from
B. juncea type. A bioinformatics analysis to identify relevant homology in amino acid sequences
between the identified mustard IgE-binding proteins and known allergens revealed strong possible
cross-reactivity between mustard 11S globulin and equivalent allergen proteins from tree-nut species
and wheat. Strong cross-reactivity between mustard oleosins with those of hazelnut, peanut, and
sesame was also suggested. In addition, a highly significant homology between mustard enolase and
that of other eukaryotes (animal, plant, and fungi) was found, confirming the highly conserved
structure of this glycolytic enzyme and its high potential for allergic cross-reactions. The new putative
mustard allergens revealed in this study would request further biological and structural
characterization.
Author Contributions: Conceptualization, L.L.; methodology, data curation, writing, review and editing M.P.,
A.A., L.L.; supervision, project administration, L.L.; funding acquisition, L.L.
Funding: This research was funded by an A-Base grant from Agriculture and Agri-Food Canada.
Acknowledgments: The authors would like to gratefully thank Martin Blaquière (Sainte-Justine Hospital,
Montréal, QC, Canada) for his kind assistance in acquiring the ethical approval and patient consent for the study
and for the assistance in sera collection from mustard allergic patient. The authors are also grateful to Janitha
Wanasundara (AAFC, Saskatoon, SK, Canada) for her generous gift of the different mustard seeds samples used
in this study. The technical support of the Proteomics Platform of the Quebec Genomics Center (Québec, QC,
Canada) for the MS analysis is also acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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