Analysis of the Structural and Immunological Stability of 2S Albumin, Nonspecific Lipid Transfer Protein, and Profilin Allergens from Mustard Seeds.
ABSTRACT 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.
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ABSTRACT: In this study, we used a mass spectrometry-based quantification approach employing isotopic (ICPL) and isobaric (iTRAQ) labeling to investigate the pattern of protein deposition during castor oil seed (Ricinus communis L.) development, including that of proteins involved in fatty acid metabolism, seed storage proteins, toxins and allergens. Additionally, we have used an off-line Hydrophilic Interaction Chromatography (HILIC) as a step of peptide fractionation preceding the Reverse Phase nanoLC coupled with a LTQ Orbitrap. We were able to identify a total of 1875 proteins and from these 1748 could be mapped to extant castor gene models, expanding considerably the number proteins so far identified from developing castor seeds. Cluster validation and statistical analysis resulted in 975 protein trend patterns and the relative abundance of 618 proteins. The results presented in this work give important insights into certain aspects of the biology of castor oil seed development such as carbon flow, anabolism and catabolism of fatty acid and the pattern of deposition of reserve proteins, toxins and allergens such as ricin and 2S albumins. We also found, for the first time, some genes of reserve proteins that are differentially expressed during seed development.Journal of Proteome Research 10/2013; · 5.06 Impact Factor
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ABSTRACT: Background 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.Objective: To perform a comprehensive structural and immunological characterization of purified Act d 12 and Act d 13 from kiwi seeds.Methods Sera from 55 well-defined kiwifruit allergic patients were used. Act d 12 and Act d 13 were purified by chromatographic procedures. Circular dichroism, mass spectrometry, concanavalinA detection, immunoblotting, enzyme-linked immunosorbent assays, basophil activation tests and IgE-inhibition experiments were used.ResultsAct d 12 and Act d 13 were purified from kiwi seeds to homogeneity by combining size-exclusion, ion-exchange and RP-HPLC chromatographies. Both purified allergens preserve the structural integrity and display typical features of their homologous counterparts from the 11S globulin and 2S albumin protein families, respectively. These allergens are released from kiwi seeds after oral and gastric digestion of whole kiwifruit, demonstrating their bioavailability after ingestion. The allergens retain the capacity to bind serum IgE from kiwifruit allergic patients, induce IgE cross-linking in effector circulating basophils and display in vitro IgE cross-reactivity with homologous counterparts from peanut and tree nuts.Conclusion Purified Act d 12 and Act d 13 from kiwi seeds are well-defined molecules involved in in vitro IgE cross-reactivity with peanut and tree nuts. Their inclusion in component-resolved diagnosis of kiwifruit allergy might well contribute to improve the diagnostic sensitivity and the management of kiwifruit allergic patients.This article is protected by copyright. All rights reserved.Allergy 07/2014; · 5.88 Impact Factor
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ABSTRACT: The paper presents a duplex real-time PCR assay for the simultaneous detection of three potentially allergenic mustard species commonly used in food: white mustard (Sinapis alba), black mustard (Brassica nigra) and brown mustard (Brassica juncea). White mustard is detected in the “green” and black/brown mustard in the “yellow” channel. The duplex real-time PCR assay does not show cross-reactivity with other Brassicaceae species including broccoli, cauliflower, radish and rapeseed. Low cross-reactivities (difference in the Ct value ⩾11.91 compared with the positive control) were obtained with cumin, fenugreek, ginger, rye and turmeric. When applying 500 ng DNA per PCR tube, the duplex real-time PCR assay allowed the detection of white, black and brown mustard in brewed model sausages down to a concentration of 5 mg/kg in 10 out of 10 replicates. The duplex real-time PCR assay was applied to verify correct labelling of commercial foodstuffs.Food Chemistry 01/2014; 153:66–73. · 3.33 Impact Factor