Sofía Sirvent

Isolation and identification of an 11S globulin as a new major allergen in mustard seeds

BACKGROUND Although mustard seed allergy has been largely reported during the preceding 20 years, currently only 2 allergens, Sin a 1 and Bra j 1, have been identified. OBJECTIVE To improve the characterization of the allergenic profile of yellow mustard seeds by reporting the identification and biochemical characterization of an 11S globulin as a new major allergen. METHODS Mustard seed proteins were separated using size exclusion and ion-exchange chromatographic columns, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 2-dimensional polyacrylamide gel electrophoresis. Separation of different polypeptide chains was achieved by reverse-phase high-performance liquid chromatography. Mass spectrometry after tryptic digestion and Edman degradation were used to determine amino acid sequences of peptides. IgE binding assays were performed with 13 serum samples from mustard allergic patients in immunoblotting and enzyme-linked immunosorbent inhibition assays. RESULTS A protein of 51 kDa was recognized as a major allergen by patients allergic to mustard and called Sin a 2. The allergen was dissociated in 2 chains of 36 and 23 kDa, which also bound IgE. N-terminal end and internal amino acid sequences allowed identification of the new allergen as a seed storage 11S globulin belonging to the Cupin super family. Purified allergen was able to inhibit the IgE binding of sera from allergic patients to mustard seeds extract in up to 55% of the responses. CONCLUSIONS An 11S globulin storage protein has been isolated and identified as a novel major allergen of mustard seeds.

Plant Lipid Transfer Protein Allergens: No Cross-Reactivity between Those from Foods and Olive and Parietaria Pollen

Background: Cross-reactivity among plant food allergens belonging to the nonspecific lipid transfer protein (LTP) family is well known. In contrast, the relationship among these allergens and their putative homologs from olive (Ole e 7) and Parietaria (Par j 1) pollen has not been clarified. Methods: Sera with specific IgE to LTP allergens were obtained from peach-, mustard- and olive pollen-allergic patients. Purified LTP allergens from foods (peach, apple, mustard and wheat) and pollens (olive, mugwort and Parietaria) were tested by ELISA and ELISA-inhibition assays. Results: Plant food LTP-allergic patients showed a significantly higher number of sera (89–100 vs. 33–64%) with specific IgE and mean specific IgE levels (0.30–1.56 vs. 0.21–0.34 OD units) to the 4 food LTP allergens tested than to olive Ole e 7 and Parietaria Par j 1 pollen. ELISA-inhibition assays indicated cross-inhibition between food LTP allergens but no cross-reactivity between these allergens and Ole e 7 and Par j 1, or, even more, between the LTP allergens from olive and Parietaria pollen. Conclusions: LTP allergens from olive and Parietaria pollen cross-react neither with allergenic LTPs from plant foods nor between themselves. Therefore, both pollens do not seem to be related with the LTP syndrome.

nsLTP and profilin are allergens in mustard seeds: cloning, sequencing and recombinant production of Sin a 3 and Sin a 4.

Background Patients allergic to mustard are frequently sensitized to peach.

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

Detailed characterization of Act d 12 and Act d 13 from kiwi seeds: implication in IgE cross-reactivity with peanut and tree nuts

Act d 12 (11S globulin) and Act d 13 (2S albumin) are two novel relevant allergens from kiwi seeds that might be useful to improve the diagnostic sensitivity and the management of kiwifruit‐allergic patients.

Act d 12 and Act d 13: Two novel, masked, relevant allergens in kiwifruit seeds

To the Editor: Kiwifruit (Actinidia deliciosa) is a common cause of IgE-mediated food allergy. Kiwifruit allergy has been associated with pollen, latex, and occupational baker’s allergy, and the main clinical manifestations range from local oral allergy syndrome to severe systemic reactions. Diagnosis of kiwifruit allergy has significantly improved during the last years. However, the main limitations in diagnosis are the low specificity of prick-to-prick tests and the low sensitivity of in vivo skin prick tests (SPTs) and in vitro serum specific IgE determinations. A current limitation of kiwifruit allergen–based in vitro diagnostics is that 35% of patients with kiwifruit allergy are overlooked as false-negative responders. To date, 11 kiwifruit allergens have been described according to the International Union of Immunological Societies allergen nomenclature subcommittee (www.allergen.org). Considering that all the currently known kiwifruit allergens, except Act d 10 (the lipid transfer protein), have been exclusively described in the pulp of kiwifruit, together with the fact that seeds are commonly ingested with the pulp, we hypothesized that novel key allergens in kiwi seeds might have been masked in the context of kiwifruit allergy. The aim of this study was to identify new relevant allergens in kiwi seeds that might help to increase diagnostic sensitivity. Fifty-five patients with kiwifruit allergy given diagnoses according to previously reported criteria were recruited from the Allergy Service of 3 different centers in Spain: Hospital Carlos Haya (M alaga) andHospitals Fundaci on Jim enez D ıaz and Infanta Leonor (Madrid). The study was approved by the ethics committees of the 3 hospitals, and written informed consent was obtained from all subjects. Fully detailed methods are described in the Methods section in this article’s Online Repository at www.jacionline.org. The general clinical characteristics of the 55 patients with kiwifruit allergy included in this study are summarized in Table E1 in this article’s Online Repository at www.jacionline.org. Themedian age of the patientswas 32.0 years (ranging from 16.0 to 71.0 years), with a predominance of female subjects (39 vs 16). Approximately 62% of the patients referred exclusively local symptoms, and 38.2% had immediate systemic reactions after ingestion of kiwifruit. Themost frequently reported symptoms were oral allergy syndrome (61.8%), anaphylaxis (16.3%), and angioedema (14.5%). Approximately 22% of the patients were only allergic to kiwifruit, and 78.2% had symptoms with other plant foods, such as members of the Rosaceae family (50.1%), tree nuts (41.8%), or peanut (30.9%). Only 50.9% and 43.6% of the patients had positive SPT and ELISA results with kiwifruit extract, respectively, which is in accordance with previous reports. To identify novel allergens in kiwi seeds, we grouped the patients with kiwifruit allergy according to positive (group 1) or negative (group 2) SPT responses to kiwifruit extract (see Table E1).We assayed in immunoblotting a pool of sera from each group against homemade total kiwifruit (pulp and seeds), pulp kiwifruit, and kiwi seed extracts prepared as previously described for each specific tissue (see the Methods section in this article’s Online Repository). The protein content of total and pulp kiwifruit extracts was very similar (Fig 1, A, lane CBS). Two main protein bands of approximately 51 and 12 kDa detected in kiwi seeds but not in total and pulp kiwifruit extracts were recognized by the 2 serum pools (Fig 1, A). The serum pool from group 2 exclusively reacted against the allergens contained in kiwi seed extract, whereas the pool from group 1 also reacted to different protein bands contained in total and pulp kiwifruit but not in kiwi seed extract. The IgE-reactive protein bands of 51 and 12 kDa contained in kiwi seeds were separated by means of SDS-PAGE (Fig 1, B, inset), excised from the gel, and digested with trypsin. The obtained peptides were analyzed by using Edman degradation, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MS), and tandem mass spectrometry (MS/MS). The identified peptides from each protein are indicated in the MS profile (Fig 1,B). The comparison of these peptides with those from databases (see Fig E1 in this article’s Online Repository at www.jacionline.org) allowed the identification of the proteins of 51 and 12 kDa as an 11S globulin and a 2S albumin, respectively, which were named Act d 12 (UniProt accession no. C0HJF9) and Act d 13 (UniProt accession no. C0HJG0) according to the International Union of Immunological Societies Allergen Nomenclature Subcommittee. 11S globulins and 2S albumins have been previously described as potent food allergens able to induce primary sensitization at the gastrointestinal level. We tested all the sera against kiwi seed extract (Fig 1, C) and total kiwifruit extract (see Fig E2 in this article’s Online Repository at www.jacionline.org). Thirty-nine (70.9%) of the 55 patients reacted to the 51-kDa allergen (Act d 12) and 10 (18.2%) of the patients reacted to the 12-kDa allergen (Act d 13) from kiwi seeds (Fig 1, C). Approximately 71% of the patients from group 1 and only 18.5% from group 2 reacted to at least 1 allergen in total kiwifruit extract (see Fig E2). Act d 12 and Act d 13 were not detected in total or pulp kiwifruit extract, likely because of the poor extraction of such proteins when using conventional protocols to prepare aqueous extracts from fruits, which might well justify why patients from group 2 did not have positive SPT responses. We purified both allergens to homogeneity from kiwi seeds (Fig 2, A) according to conventional chromatographic procedures (see the Methods section in this article’s Online Repository). Purified Act d 12 and Act d 13 were recognized in ELISA by 65.5% and 25.5% of the patients, respectively (see Table E1). Patients 25, 30, and 44 had positive responses to purified Act d 12 in immunoblotting (data not shown) but not in ELISAs, suggesting the existence of potential internal epitopes. Patients 30, 34, 41, and 42 reacted to purified Act d 13 in ELISAs but not in immunoblotting, suggesting the existence of potential conformational epitopes. Act d 12 and Act d 13 demonstrated in vivo biological activity. Patients with kiwifruit allergy, but not healthy control subjects, had positive SPT responses to kiwi seed extract (4/4), purified Act d 12 (4/4), and purified Act d 13 (3/4; Fig 2, B). A representative figure of the forearm from a patient with kiwifruit allergy with a positive SPT response after challenge with the indicated allergens is shown (Fig 2, C). Considering that approximately 85% of the patients included in this study had positive responses to purified Act d 12 or Act d 13, the inclusion of whole kiwi seed extracts for in vitro clinical

Analysis of the structural and immunological stability of 2S albumin, nonspecific lipid transfer protein, and profilin allergens from mustard seeds.

This work investigates the resistance to proteolysis and heating of the yellow mustard (Sinapis alba L.) allergens Sin a 1 (2S albumin), Sin a 3 (nonspecific lipid transfer protein, LTP), and Sin a 4 (profilin) to explain their potential capability to induce primary sensitization at the gastrointestinal level. Sin a 1 and Sin a 3 resisted gastric digestion showing no reduction of the IgE reactivity. Intestinal digestion of Sin a 1 and Sin a 3 produced a limited proteolysis but retained significant IgE-binding reactivity. Sin a 1 was stable after heating, and although Sin a 3 was modified, most of its structure was recovered after cooling back. These two allergens would be therefore able to sensitize by ingestion. Sin a 4 was completely digested by gastric treatment and its conformational structure markedly modified at 85 °C. Thus, this allergen can be described as a nonsensitizing mustard allergen.

The 11S globulin Sin a 2 from yellow mustard seeds shows IgE cross-reactivity with homologous counterparts from tree nuts and peanut

BackgroundThe 11S globulin Sin a 2 is a marker to predict severity of symptoms in mustard allergic patients. The potential implication of Sin a 2 in cross-reactivity with tree nuts and peanut has not been investigated so far. In this work, we studied at the IgG and IgE level the involvement of the 11S globulin Sin a 2 in cross-reactivity among mustard, tree nuts and peanut.MethodsEleven well-characterized mustard-allergic patients sensitized to Sin a 2 were included in the study. A specific anti-Sin a 2 serum was obtained in rabbit. Skin prick tests (SPT), enzyme-linked immunosorbent assay (ELISA), immunoblotting and IgG or IgE-inhibition immunoblotting experiments using purified Sin a 2, Sin a 1, Sin a 3, mustard, almond, hazelnut, pistachio, walnut or peanut extracts were performed.ResultsThe rabbit anti-Sin a 2 serum showed high affinity and specificity to Sin a 2, which allowed us to demonstrate that Sin a 2 shares IgG epitopes with allergenic 11S globulins from tree nuts (almond, hazelnut, pistachio and walnut) but not from peanut. All the patients included in the study had positive skin prick test to tree nuts and/or peanut and we subdivided them into two different groups according to their clinical symptoms after ingestion of such allergenic sources. We showed that 11S globulins contain conserved IgE epitopes involved in cross-reactivity among mustard, tree nuts and peanut as well as species-specific IgE epitopes.ConclusionsThe allergenic 11S globulin Sin a 2 from mustard is involved in cross-reactivity at the IgE level with tree nuts and peanut. Although the clinical relevance of the cross-reactive IgE epitopes present in 11S globulins needs to be investigated in further detail, our results contribute to improve the diagnosis and management of mustard allergic patients sensitized to Sin a 2.

Pollen and plant food profilin allergens show equivalent IgE reactivity

BACKGROUND Profilins are commonly involved in polysensitization of allergic patients; therefore, appropriate markers should be used in component-resolved diagnosis. OBJECTIVE To evaluate the immunological equivalence between profilins from pollens and plant-derived foods, to be used in component-resolved diagnosis. METHODS Specific immunoglobulin (Ig) G antibodies against pollen and fruit profilins, as well as sera from patients allergic to mustard, melon, or olive pollen, were used. Purified profilins from mustard seeds, fruit melon, and chenopod and birch pollen were assayed in immunoblotting, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition assays. RESULTS Significant correlation was found in the response of purified profilins by ELISA and immunoblotting for both specific IgG and IgE. The highest levels of IgE binding were obtained for olive pollen-allergic patients, which could be related to the route of sensitization. The responses of individual patients to profilins were also similar and independent of the sensitizing source. The inhibition between pairs of allergens was generally higher than 70%, indicating that profilins share most of the IgE epitopes. Modeling of mimotopes in the conformational structure of the implicated profilins supports their strong cross-reactivity obtained experimentally. CONCLUSIONS No correlation exists between the level of IgE response of individual patients to specific profilins and the corresponding theoretical sensitizing source, suggesting that the sensitization could be attributable to any profilin present in the environment of the patients. This would bear out the use of most profilins as a common marker for polysensitization in component-resolved diagnosis and for therapeutic approaches.

Structural studies of novel glycoconjugates from polymerized allergens (allergoids) and mannans as allergy vaccines.

Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy. Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells. Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde. The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (non-oxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and 1H-13C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition.