- Top of page
- Materials and methods
Background: 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.
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 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.
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.
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 (n = 10). 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 immunoglobulin (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 immunosorbent (ELISA) tests, immunoblotting after isoelectrofocalisation (IEF), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis 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.
- Top of page
- Materials and methods
The IgE-dependent sensitization to foods may not necessarily 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 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. Conformational 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 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 approximately 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 patient’ 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 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. ‘*’ means that the residues in the column are identical in both sequences in the alignment; ‘:’ means that conserved substitutions have been observed.
Download figure to PowerPoint
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, respectively. 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.