Nitride, Chiara (2013) PROTEOMIC AND IMMUNOCHEMICAL CHARACTERIZATION OF FOOD ALLERGENS OF PLANT ORIGIN: THE HAZELNUT (Corylus avellana) CASE OF STUDY. [Tesi di dottorato]

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Abstract

The application of proteomics strategies to identify and characterize food allergens has been defined ‘allergenomics’ in 2005 by the Division of Medical Devices, National Institute of Health Science in Japan. The main purposes are to achieve more detailed and comprehensive characterization of food allergens, to define markers for “hidden allergens” monitoring in complex food matrices and to develop more specific and standardized protocols for clinical diagnosis and immunotherapy. Hazelnut (Corylus avellana) is one of the most common causes of lifetime lasting IgE-mediated food allergy. Immune reactions to hazelnut range from mild oral allergy syndromes to severe life-threatening anaphylaxis. Hazelnut allergy is more frequent in infancy than in adulthood and its prevalence varies among countries. In the years different studies aimed to identify the hazelnut allergic determinant(s) have been carried out. Several important hazelnut seeds storage proteins have been identified and characterized as allergens such as Cor a 1.04, major allergen in the Northern Europe related to 65 patients sensitized to birch (Bet v 1, Betula verrucosa); 11S globulin (Cor a 9, with 30–40 kDa acidic and 20–25 kDa basic chain bounds together via disulfide bridge), described as the major non-pollen related allergen in United States; 48 kDa-glycoprotein (Cor a 11); ns-LTP (Cor a 8), supposed to be the major allergen in Southern Europe, and 2S albumin (Cor a 14). The predominance of the specific allergens appears to be associated to the geographical origin of allergic subjects. The complexity of the hazelnut allergen pattern challenges a clear definition of both the allergens and the allergenic determinants (epitopes). Mass spectrometry-based strategies, combining two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography (LC) techniques for proteins resolving, were developed with the aim of identifying and characterizing proteins that can function as markers for hazelnut allergens. These achievements made possible the identification of still not described hazelnut proteins has been possible by homology comparison with gene products arising from other seeds, even with a relatively low sequence coverage; the complete characterization of the both oligosaccharide chains of the 48 kDa glycoprotein, Cor a 11 allergen; the improvement of the strategy for ns-lipid transfer protein, Cor a 8 allergen, purification. Immunochemical assays were exploited to investigate the pattern of the immunological response of hazelnut allergic children from Campania region. Fifteen out fifteen children’s sera were immunoreactive to a protein that has not been annotated in database so far. The mass spectrometry-based characterization, also including the “de novo” sequencing of tryptic peptides, provided evidence of the high homology degree between the unknown IgE-binding polypeptide and the 11S globulin-like storage proteins that are expressed in several other seeds. In spite of a low sequence similarity, the new allergen, share structural traits with the hazelnut 11S globulin-like proteins (Cor a 9). The simulation of in vitro gastrointestinal digestion of the pure isoform of 11S globulin-like and the dot-blot analysis with sera of allergic patients showed that the total peptide digest of crude proteins and pure 55 kDa IgE binding proteins retained IgE-reactivity. These finding suggest the presence of linear IgE epitope(s). Further proteomic investigations are required to identify IgE-binding domains of putative hazelnut allergen(s) resistant to digestion.

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