Identification of oleosins as major allergens in sesame seed allergic patients

Abstract

The prevalence of sesame allergy is increasing in European countries. Cases of severe allergy lack any evidence of specific immunoglobulin (Ig)Es by prick tests and CAPSystem-FEIA. The reasons for this negativity are unknown.
In 32 patients displaying immediate symptoms such as anaphylactic shock, asthma, urticaria, angioedema, sesame allergy was diagnosed by double-blind placebo-controlled food challenge (DBPCFC) or convincing clinical history. However, 10 patients had negative prick tests and CapSystem-FEIA. The specificity of IgEs was further investigated by enzyme-linked immunosorbent assay (ELISA), isoelectrofocalisation (IEF)-blotting, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) blotting using total sesame extracts and purified fraction of oil bodies. Monospecific rabbit antibodies directed to two oleosin isoforms (15 and 17 kDa) were used.
By ELISA, white sesame seed extract allowed the detection of higher levels of IgE than brown sesame extract. In all sera, numerous bands binding IgEs were detected by IEF or SDS-PAGE. In reducing conditions, two bands (15-17 kDa), could be separated from 2S albumin. Oleosins, present in oil bodies fractions, were recognized by IgEs from all sera.
Oleosins are major allergens of sesame seeds and may be relevant to severe anaphylaxis. Falsely negative prick tests could be due to the lack of oleosins in presently available extracts, or to the fact that epitopes might be buried in the inner molecule. Detection tests currently used to identify sesame allergens based on sesame vicillins or other storage proteins could be insufficient for the detection of sesame seed contamination. Oleosins have been named Ses i 4 (17 kDa) and Ses i 5 (15 kDa), in accordance with the IUIS Nomenclature Committee.

Full text

Original article
Identification of oleosins as major allergens in sesame seed allergic
patients
Food allergy to sesame has been observed in children and
in adults in different countries including Israel, Japan,
and Europe (1–5). Anaphylaxis is often severe (6–8). The
prevalence is increasing in European countries and could
represent 2–4% of total food allergies (9, 10). Sesame is
among the 12 allergens requiring labelling on food
products (11).
The major allergen of sesame seeds has already been
described. Ses i 1 (9 kDa) is a member of 2S albumin
family (12) and recognized by all the patients studied
(n¼10). More recently, Beyer et al. (13) identified two
additional sesame allergens: Ses i 2 (7 kDa) and Ses i 3
(45 kDa), which are a subunit of 2S albumin and a 7S
vicilin-like globulin, respectively. A 14 kDa protein
belonging to the 2S albumin family was recognized by
22 of the 24 sera used (13). Numerous other allergens
have been observed by two-dimensional electrophoresis
followed by immunoblotting (14).
Cases of anaphylaxis have been reported despite
negative prick tests and absence of specific immuno-
globulin (Ig)Es (15, 16), where the authors suggested
that the anaphylaxis was IgG-mediated. Other data
have confirmed a potent immunogenicity of sesame
seeds eliciting a polyisotypic response, supporting this
assumption (17).
The aim of this study was to thoroughly investigate 32
sera from patients allergic to sesame, part of them
displaying no evidence of allergen-specific sensitization
by prick tests and CAP-FEIA. Enzyme-linked immuno-
sorbent (ELISA) tests, immunoblotting after isoelectrofo-
calisation (IEF), and sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) electro-
Background: The prevalence of sesame allergy is increasing in European coun-
tries. Cases of severe allergy lack any evidence of specific immunoglobulin (Ig)Es
by prick tests and CAPSystem-FEIA. The reasons for this negativity are un-
known.
Methods: In 32 patients displaying immediate symptoms such as anaphylactic
shock, asthma, urticaria, angioedema, sesame allergy was diagnosed by double-
blind placebo-controlled food challenge (DBPCFC) or convincing clinical his-
tory. However, 10 patients had negative prick tests and CapSystem-FEIA. The
specificity of IgEs was further investigated by enzyme-linked immunosorbent
assay (ELISA), isoelectrofocalisation (IEF)-blotting, and sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) blotting using total sesame
extracts and purified fraction of oil bodies. Monospecific rabbit antibodies
directed to two oleosin isoforms (15 and 17 kDa) were used.
Results: By ELISA, white sesame seed extract allowed the detection of higher
levels of IgE than brown sesame extract. In all sera, numerous bands binding
IgEs were detected by IEF or SDS-PAGE. In reducing conditions, two bands
(15–17 kDa), could be separated from 2S albumin. Oleosins, present in oil
bodies fractions, were recognized by IgEs from all sera.
Conclusion: Oleosins are major allergens of sesame seeds and may be relevant to
severe anaphylaxis. Falsely negative prick tests could be due to the lack of
oleosins in presently available extracts, or to the fact that epitopes might be
buried in the inner molecule. Detection tests currently used to identify sesame
allergens based on sesame vicillins or other storage proteins could be insufficient
for the detection of sesame seed contamination. Oleosins have been named Ses i
4 (17 kDa) and Ses i 5 (15 kDa), in accordance with the IUIS Nomenclature
Committee.
V. Leduc
1
, D. A. Moneret-Vautrin
2
,
J. T. C. Tzen
3
, M. Morisset
2
,
L. Guerin
1
, G. Kanny
2
1
Allerbio Laboratory (R and D), Varennes en
Argonne, France;
2
Department of Internal Medicine,
Clinical Immunology and Allergology, University
Hospital, Nancy Cedex, France;
3
Graduate Institute
of Biotechnology, National Chung-Hsing University,
Taichung, Taiwan
Key words: food allergy; oil bodies; oleosins; sesame
allergens.
Denise-Anne Moneret-Vautrin MD, Pr
Department of Internal Medicine, Clinical
Immunology and Allergology
University Hospital
29 avenue du Marchal de Lattre de Tassigny
54035 Nancy Cedex
France
Accepted for publication 22 September 2005
Abbreviations: IEF, isoelectrofocalisation; pNPP, p-nitrophenyl-
phosphate; CNBr, cyanogen bromide; NC, nitrocellulose; SPT, skin
prick test.
Allergy 2006: 61: 349–356 Copyright Blackwell Munksgaard 2006
ALLERGY
DOI: 10.1111/j.1398-9995.2006.01013.x
349

phoresis were carried out with a white sesame (WS) seed
extract and purified oil bodies containing the two
isoforms of oleosins.
Specific IgEs to the oleosin fraction were detected in
almost all sera from subjects allergic to sesame with
positive or negative prick tests and CAP-FEIA. These
results indicate that oleosins represent a new class of
sesame major allergens.
Materials and methods
Patients
Patients (n¼32) including 15 children and 17 adults were selected
and determined to exhibit sesame allergy as evidenced by double-
blind placebo-controlled food challenge (DBPCFC), labial test or
clinical history (Table 1). Patients were ranged according to the
tests performed: 23 patients were positive after food challenge (la-
bial test or DBPCFC), four patients did not react to 965 mg and
7 g, and five patients were not tested. Among the 23 patients, six
patients were negative by skin prick tests (SPT) to three varieties of
sesame seeds and to the commercial extract, as well as with the
CAP-FEIA, which was performed twice for each case. One of them
(patient 1) experienced a prelethal shock after the consumption of
an industrial dish containing an artificial flavoring in a matrix of
Mexican sesame oil. Allergy to sesame was very likely in cases 26
and 27, because of anaphylactic episodes related to successive
ingestions of sesame seeds or of food products that may have
contained sesame seeds or sesame oil.
Skin prick tests
The SPT were carried out with three varieties of natural sesame
seeds (white, brown, and black), crushed in saline according to the
technique of prick-in-prick (18) and compared with a commercial
extract (Allerbio, Varennes en Argonne, France). The positive and
negative controls were codein phosphate 9% and saline buffer,
respectively. Criteria for positivity was a wheal diameter equal to
75% of the positive control wheal.
CAP-FEIA
The patientsÕspecific IgE levels were determined by using the
CAPSystem IgE to sesame (f10 Pharmacia, Uppsala, Sweden). Levels
of specific IgE of >0.35 kU/l (‡classe 1) were considered positive.
Food challenge
The DBPCFC was performed as previously described (19). Briefly,
the progression increased up to a cumulative dose (CD) of 965 mg.
The vehicle used was a stewed apple and the placebo material was
crushed brown-dried toast.
The DBPCFC using cold-pressed sesame oil was tested in nine
patients, successively using 1, 5, and 10 ml volumes. The placebo
was paraffin oil (mineral oil) (20).
Sesame seed extracts
Brown and white sesame (WS) seeds were ground and extracted
(1 : 5, w/v) overnight in sodium bicarbonate 4&at 4C. After
centrifugation, extracts were filtered on a 0.2 lm filter. Protein
content was measured according to the Bradford method with ser-
um albumin as standard. Protein concentrations were 5.5 mg/ml for
brown sesame (BS) and 2.95 mg/ml for WS extract.
Oil bodies were extracted from sesame seeds and subjected to
further purification using the protocol developed by Tzen et al. (21)
including two-layer flotation by centrifugation, detergent washing,
ionic elution, treatment of chaotropic agent (urea 9 M), and
integrity testing with hexane.
Direct ELISA
Specific IgE were assessed by direct ELISA using white and BS
seed extracts. The ELISA plates (Immulon 2; Thermo Lab sys-
tems, Franklin, MA, USA) were coated with white or BS seed
extracts (10 lg/ml in carbonate buffer, pH 9.6) at 4C overnight.
Plates were washed with phosphate-buffered saline (PBS) con-
taining 0.05% (v/v) Tween 20 (PBS-T) and blocked with 0.5% (w/
v) gelatin in PBS-T at 37C for 1 h. Plates were incubated at 37C
for 2 h with 100 ll per well of patient sera diluted 1 : 5 in blocking
buffer. After three washes, an alkaline phosphatase-coupled affin-
ity purified goat antihuman IgE antibody (KPL, Gaithesburg,
MD, USA) diluted 1 : 1000 in blocking buffer was used to detect
bound IgE. After 2-h incubation at 37C, plates were washed three
times and the color reaction was started with p-nitrophenylphos-
phate (pNPP) in diethanolamin buffer (pH 9.5). The absorbance
was determined at 410 nm (A410 nm). All determinations were
carried out in duplicates. Positive absorbance was defined as the
mean background absorbance ± 3 SD. A Dactylis glomerata
pollen extract and a grass pollen-sensitized human serum (CAP-
FEIA g3 500 kU/l) were used as the standard.
Isoelectric focusing
Isoelectric focusing (IEF) was performed on 1.5% agarose gel with
2.6% ampholytes 3-10 (Serva, Heidelberg, Germany). Samples of
160 lg/cm were applied on the anodic part of the gel. After focus-
ing, the gel was submitted to a 10 min pressure blot onto cyanogen
bromide (CNBr)-activated nitrocellulose (NC; 22).
SDS-PAGE
The SDS-PAGE was performed on a 12% acrylamide gel with a 6%
stacking gel in a Tris-Tricine buffer (23). Extracts were diluted in
20 mM Tris-HCl, pH 6.8, containing 2% (w/v) SDS and brom-
phenol blue. In reducing conditions, 1% (w/v) dithiothreitol (DTT)
was added to the sample buffer and samples were incubated for
10 min at 95C before application on the gel. Proteins of 20 lg/cm
were applied for blotting and 2 lg per lane for silver staining.
Electrophoresis was performed at 15C at 40 mA for 1 h. After
electrophoresis, the gel was electroblotted onto CNBr-activated NC
or silver-stained according to Rabilloud et al. (24).
Immunodetection
The IEF or SDS-PAGE strips were blocked in 2% polyvinyl-
pyrrolidone (PVP) in Tris-buffered saline (TBS) containing 0.1%
(v/v) Tween 20 (TBS-T) for 1 h. Blots were then incubated at
room temperature overnight with individual serum diluted 1 : 5 in
blocking buffer. After three washes in TBS-T, blots were incu-
bated with alkaline phosphatase-labeled antihuman IgE in
blocking buffer (1/2000) for 6 h. After three washes in TBS-T,
IgE-binding was revealed using NBT/BCIP (KPL). For the
detection of oleosins, NC strips were incubated with polyclonal
Leduc et al.
350

monospecific rabbit antisesame oleosin (15 or 17 kDa) antiserum
(1 : 2000 dilution; 25). Rabbit IgG were detected by antirabbit
IgG conjugated with alkaline phosphatase (1/10 000 dilution;
Sigma-Aldrich, St Louis, MO, USA) in blocking buffer. After
washing, color development was performed by incubating the
strips in Tris, NaCl, MgCl
2
buffer containing 0.33 mg/ml nitro-
blue tetrazolium and 0.165 mg/ml 5-bromo-4-chloro-indolyl
phosphate at room temperature in the dark until a sufficient
coloring occurred. The reaction was stopped by rinsing the
membrane in water.
Table 1. Clinical characteristics of 32 patients with sesame allergy
ID Sex
Age
(years) Symptoms
Prick test
IgE
CAP-RAST
(kU/l)
DBPCFC/LT to sesame seeds (S) and/or oil (O)
CE Varieties DBPCFC LT (grade)
1 M 18 RAS Neg B: neg/W: neg/b: neg <0.35 O (15 ml): rash, asthma/S (100 mg): urticaria
2 F 63 RAS to 162 mg ND B: neg/W: neg/b: neg <0.35 S (965 mg): systemic reaction
3 F 9 nf AS Neg B: neg/W: neg/B: neg <0.35 O (8 ml): late onset erythema
4 M 44 AS Neg B: neg/W: neg/B: neg <0.35 O (5 ml): AS/S (200 mg): flush, facial erythema
5 M 54 AS Neg B: neg/W: neg/b: neg <0.35 O (0.7 ml): generalized erythema, abdominal pain
6 M 33 SR Neg B: neg/W: neg/b: neg <0.35 S (7 g): urticaria
7 M 23 AS Neg B: neg/W: neg/b: neg 5.11 O (1 ml): AS/S (265 mg): urticaria, angioedema
8 F 25 Asth Neg B: neg/W: neg/b: neg 0.51 O (20 ml): palpebral erythema, abdominal
pain/S (965 mg): erythema, abdominal pain
9 F 4.5 Asth 11 mm B: 20 mm/W: 7 mm
/b: 11.5 mm
44 S: Grade 3
10 F 17 SR ND B: neg/W: 2 mm/b: 1 mm 4.3 O (16 ml): negative/S (965 mg): urticaria
11 M 3 Asth ND B: 17.5 mm/W: 4 mm
/b: 11.5 mm
76.2 S: Grade 3
12 M 6 Asth ND B: 0.5 mm/W: 2 mm
/b: 2 mm
9.01 S (7 g): abdominal pain, cough and wheezing
13 M 36 AS 1.5 mm B: 5 mm/W: 6.5 mm
/b: nd
<0.35 S (7 g): generalized prurit is erythema S: Grade 1
14 F 3 AD ND B: 13 mm/W: 8 mm
/b: 11 mm
8.26 S (965 mg): exacerbation of AD
15 M 13 SR ND B: 8.5 mm/W: 17 mm
/b: 15.5 mm
13.1 S: Grade 2
16 M 4 Asth ND B: 10 mm/W: 8 mm
/b: 9.5 mm
40 S: Grade 3
17 M 32 AS ND B: 10 mm/W: nd/b: nd <0.35 O: Grade 3
18 F 10 AO 8 mm B: 12.5 mm/W: 5.5 mm
/b:13.5 mm
16.5 S (965 mg): urticaria, wheezing, and vomiting
19 M 4 SR ND B: 8.5 mm/W: 18 mm
/b: 10 mm
4.59 O (6 ml): negative
20 M 47 AS ND B: 9 mm/W: 6 mm
/b: 6 mm
ND S (6 mg): generalized pruritis, erythema on
the neck
21 M 11 Asth ND B: 8.5 mm/W: 6 mm
/b: 4.5 mm
20.5 S (2 g): asthma
22 M 22 Asth 3.5 mm B: 3 mm/W: 4 mm/b: nd 13.2 S (10 g): asthma, erythema
23 M 7 Asth ND B: 13 mm/W: 11 mm
/b: 12 mm
>100 S (7 g): abdominal pain, vomiting, conjunctivitis,
eczema
S: Grade 2
24 M 4 Vom ND B:12.5 mm/W: 14.5 mm
/b:7 mm
15.8 S (7 g): negative
25 M 4 SR ND B: 3 mm/W: 12.5 mm
/b:15 mm
>100 ND
26 F 70 AO ND B: neg/W: neg/b: neg <0.35 ND
27 F 31 SR Neg B: neg/W: neg/b: neg <0.35 ND
28 F 26 RAS ND B: neg/W: neg/B : neg <0.35 S (965 mg): negative
29 M 4 SR ND B: neg/W: neg/b: neg <0.35 S (7 g): negative
30 M 12 AO ND B: 3.5 mm/W: 5.5 mm
/b: 5.5 mm
0.41 S (7 g): negative
31 M 21 AO 6.5 mm B:13.5 mm/W: 15.5 mm
/b:14 mm
4.58 ND
32 M 32 AO, U ND B: 7 mm/W: 10 mm
/b: 9 mm
<0.35 ND
RAS, recurrent anaphylactic shock; nf, near fatal; AS, anaphylactic shock; SR, systemic reaction; Asth, asthma; AD, atopic dermatitis; AO, angioedema; Vom, vomiting; LT, labial
test; B, brown; W, white; b, black; M, male; F, female; Neg, negative; ND, not detectable; DBPCFC, double-blind placebo-controlled food challenge; IgE, immunoglubulin E.
Sesame oleosins are relevant major allergens
351

Results
Patients
Clinical symptoms were of immediate type – anaphylactic
shock: seven, recurrent anaphylactic shock: three, systemic
reaction: seven, asthma: eight, angioedema: five, and
vomiting: one. A single patient had atopic dermatitis.
Twenty-seven patients had DBPCFC or labial tests. Four-
teen of 32 DBPCFC to seeds were positive and six of 14 to
oil. Labial tests to seeds were positive in five of five patients
and one of one to oil. Five patients were not challenged
according to the severity of the clinical symptoms. Ten of
32 patients (with positive labial test or DBPCFC in seven
cases), showed negative SPTs to all sesame extracts and no
detectable specific IgE by CAP-FEIA, confirming the
difficulty of sesame seed allergy diagnosis.
Specific IgE
Direct ELISAs were performed to assess the level of
patient-specific IgE to sesame seed by comparing WS and
BS extracts. Whereas ImmunoCAP were found negative
in 13 cases, nine patient sera displayed IgE-binding to WS
and BS extracts by ELISA. White sesame seed extract was
used for the following studies.
IEF immunoblotting. The IEF separation of WS extract
followed by immunoblotting showed numerous bands
recognized by serum IgE of all patients, even from those
with negative CAP-FEIA System (Fig. 1). The isoelectric
points of the bands ranged from 4.5 to 8.5.
SDS-PAGE immunoblotting. White sesame extract was
separated by SDS-PAGE under nonreducing (Fig. 2) or
reducing conditions (Fig. 2). Under nonreducing condi-
tions, allergens were identified migrating between 43 and
67 kDa and between 15 and 17 kDa (Fig. 2). Under
reducing conditions, the silver staining revealed that the
15–17 kDa band, observed in the nonreducing condi-
tions, dissociated into a smaller molecule of 9 kDa,
corresponding to the 2S albumin large subunit (Fig. 3).
However, a doublet located at 15 and 17 kDa was still
observed after immunoblotting (Fig. 3), which was pre-
sumably oleosin isoforms (Ole).
To prove that oleosins are major allergens, SDS-PAGE
immunoblotting of purified oil body fraction (Fig. 4) was
performed. The results showed three major bands
between 15 and 17 kDa corresponding to oleosins
isoforms, as detected by rabbit monospecific anti-
15 kDa and 17 kDa oleosin.
Immunoblots showed that 29 over 32 sera have specific
IgE to the 15.5 and 17 kDa oleosins. The IgE-binding to
both oleosin isoforms was much stronger in case 1 who
exhibited a prelethal shock to a mixture of flavoring
containing a Mexican sesame oil.
Discussion
The IgE-dependent sensitization to foods may not neces-
sarily coincide with positive prick tests to commercial
extracts, because a maximum of diagnostic sensitivity (i.e.
100%) is difficult to achieve. However, the possibility of
Figure 1. Specificity of immunoglobulin (Ig)E of the 32 patient sera on sesame extract separated by isoelectrofocalisation (IEF)
followed by immunoblotting. On the left, isoelectric point markers and sesame extract stained by Coomassie Brilliant Blue. All sera
were diluted 1 : 5 in blocking buffer (C: control with blocking buffer).
Leduc et al.
352

falsely negative SPT is often linked to the nature of the
food, and is characteristic of aqueous fruit and vegetables
(26, 27). Extracts of seeds are currently the most efficient
reagents as they are directly related to the concentration
of proteins in the seeds. With this in mind, the fact of no
evidence of positivity of SPT to three natural varieties of
sesame seeds is rather surprising. As sesame seeds are
crushed in a saline solution, we raised the hypothesis of
the presence of hydrophobic allergens that are insoluble
in saline.
Specific IgEs were detected by ELISA and binding of
IgE to numerous proteins were demonstrated after
immunoblotting of IEF gels. Applying both techniques,
the sesame seed proteins were not denatured. Conform-
ational epitopes may be detected, which might otherwise
escape recognition by the CAP-FEIA, where the coupling
procedure could alter these epitopes.
The comparison of the protein profiles on SDS-PAGE
in both reducing and nonreducing conditions revealed
several groups of allergens. The first group consisted of
Figure 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of sesame seed extract. Silver-
stained gel in nonreducing conditions and specificity of immunoglobulin (Ig)E of the 32 patient sera (1–32). All sera were diluted 1 : 5
in blocking buffer (C: control with blocking buffer).
Figure 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of sesame seed extract. Silver-
stained gel in reducing conditions and specificity of immunoglobulin (Ig)E of the 32 patient sera (1–32). All sera were diluted 1 : 5 in
blocking buffer (C: control with blocking buffer).
Sesame oleosins are relevant major allergens
353

11S globulins that represent 60–70% of the total seed
proteins (28). Each of the 11S globulin isoforms consists
of an acidic subunit (30–40 kDa) and a basic subunit (20–
25 kDa) linked by a disulfide bond (29). Group 2 is the
major soluble protein, 2S albumin, constituting approxi-
mately 15–25% of the total sesame proteins (30). The two
2S albumin isoforms consist of a small subunit (4 kDa)
and a large subunit (9 kDa), linked by a disulfide bond.
Under nonreducing conditions, oleosins migrate at the
same molecular weight as 2S albumins (15–17 kDa). The
third group of two 7S globulin isoforms of 55–60 kDa,
represents 1–2% of the total proteins: they have been
identified as minor constituents in protein bodies (31). In
contrast to 11S globulin and 2S albumin, 7S globulin is a
single polypeptide, recognized by nearly all the patientÕ
sera.
Oil bodies are discrete spherical organelles that are also
named lipid bodies, or oleosomes and their storage lipids
are mainly composed of triacylglycerols (TAGs) in most
seeds. The abundant protein referred to as oleosins, which
represent 80–90% of total oil body proteins, corresponds
to only 1–2% of total seed proteins. Three different
oleosins, 17, 15.5 and 15 kDa have been described (32),
and 17 and 15 kDa oleosins have been sequenced (Fig. 5).
These non glycosylated proteins are present at the surface
of oil bodies and play a structural role to stabilize the
organelles during desiccation of the seed by preventing
coalescence of the oil. In this study, we were able to
identify oleosins in oil bodies that were recognized by
IgEs from all patient sera. The intensity of antibody
binding is striking in case 1 (anaphylactic shock after
ingestion of sesame oil), leading us to suspect that the
majority of oleosins remains residually present in oil and
is probably not denatured, as sesame oil is only cold-
pressed. The specific risk of sesame oil in allergic
responses has been pointed out previously (20, 33).
Indeed, patients can react by anaphylactic shock to only
a few milliliters of sesame oil (20). Other oils such as
peanut or soybean oil do not exhibit such severe
reactions, even in highly sensitized subjects allergic to
peanuts or to soybeans. Interestingly, the presence of
oleosin in peanut oil has been shown and the allergenicity
of a recombinant peanut oleosin has been established
(34).
Figure 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting of purified sesame seed oil bodies.
Silver-stained gel and immunoblot revealed with immunoglobulin (Ig)E of patient sera (1–32). Lanes: (A) monospecific polyclonal
rabbit antibody (Ab) to 15-kDa oleosin; (B) monospecific polyclonal rabbit Ab to 17-kDa oleosin; (C) control with blocking buffer.
Figure 5. Amino acid sequences of the 17 and 15 kDa sesame oleosins, respectively Ses i 4 and Ses i 5. The single-letter amino acid
code is used. A dash indicates a gap introduced for the purposes of alignment. Ô*Õmeans that the residues in the column are identical in
both sequences in the alignment; Ô:Õmeans that conserved substitutions have been observed.
Leduc et al.
354

In conclusion, we have identified oleosins as new
sesame seed major allergens, present in seeds and
presumably in oil. The IgEs from all sesame allergic
patients studied consistently bound to oleosins. To
explain the negativity of SPT to natural sesame, a first
hypothesis could be that, in these six patients, the
amount of specific IgEs might be predominantly directed
to oleosins. The negativity of SPT to natural varieties of
seeds could indicate that oleosins are not solubilized in
saline, or, alternatively that their epitopes are hindered
by the association of oleosins to the TAG, or in the
inner part of the molecules, masked by folding of the
tertiary structure. If such is the case, the negativity of
SPT to natural varieties crushed in saline could imply
that these patients have IgEs directed toward epitopes of
oleosins buried in the inner part of the molecules.
However, the results of ELISA do not support this
hypothesis. Neither the level of specific IgE to oil bodies,
nor the ratio of IgE to oil bodies/total sesame seed
differs in patients with negative or positive PT to sesame
seeds (data not shown). This issue cannot be elucidated
at present.
Two oleosin (17 and 15 kDa) sequences are known
(Fig. 5). According to the IUIS Nomenclature, we
submitted these oleosins as Ses i 4 and Ses i 5, respect-
ively. They may characterize severe anaphylaxis without
evidence of specific IgEs by the present methods of
diagnosis.
Some homology between oleosins of different species
has been found for a Chinese spice shiso (Perilla
frutescens, 75% identity) and for carrot oleosin (64% of
identity). Sequence comparison (BLAST) with peanut
and soybean oleosins showed lower levels: 56% and 51%.
Higher levels of identity can be reached if the sequence is
restricted to the central part of oleosins. This domain
whose sequence is conserved, is highly hydrophobic and
interacts with the lipids. Moreover, this central domain
does not present any trypsin cleavage site.
This study supports the obvious need to improve the
quality of extracts of sesame for diagnosis. Moreover, it is
noteworthy that detection tests for masked sesame
allergens are based on vicilins (35). The fact that oleosins
are major allergens supports the proposition of oleosins
as new markers of masked allergens.
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