title: Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6; characterization of intermediate- and end products.
abstract: Conglutins, the major peanut allergens, Ara h 2 and Ara h 6, are highly structured proteins stabilized by multiple disulfide bridges and are stable towards heat-denaturation and digestion. We sought a way to reduce their potent allergenicity in view of the development of immunotherapy for peanut allergy. Isoforms of conglutin were purified, reduced with dithiothreitol and subsequently alkylated with iodoacetamide. The effect of this modification was assessed on protein folding and IgE-binding. We found that all disulfide bridges were reduced and alkylated. As a result, the secondary structure lost α-helix and gained some β-structure content, and the tertiary structure stability was reduced. On a functional level, the modification led to a strongly decreased IgE-binding. Using conditions for limited reduction and alkylation, partially reduced and alkylated proteins were found with rearranged disulfide bridges and, in some cases, intermolecular cross-links were found. Peptide mass finger printing was applied to control progress of the modification reaction and to map novel disulfide bonds. There was no preference for the order in which disulfides were reduced, and disulfide rearrangement occurred in a non-specific way. Only minor differences in kinetics of reduction and alkylation were found between the different conglutin isoforms. We conclude that the peanut conglutins Ara h 2 and Ara h 6 can be chemically modified by reduction and alkylation, such that they substantially unfold and that their allergenic potency decreases.
authors.affiliation: HAL Allergy B.V., J.H. Oortweg 15-17, 2333 CH Leiden, The Netherlands; Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11 000 Belgrade, Serbia.
authors.forename: Danijela
authors.initials: D
authors.lastname: Apostolovic
authors.forename: Dion
authors.initials: D
authors.lastname: Luykx
authors.forename: Hans
authors.initials: H
authors.lastname: Warmenhoven
authors.forename: Dennis
authors.initials: D
authors.lastname: Verbart
authors.forename: Dragana
authors.initials: D
authors.lastname: Stanic-Vucinic
authors.forename: Govardus A H
authors.initials: GA
authors.lastname: de Jong
authors.forename: Tanja Cirkovic
authors.initials: TC
authors.lastname: Velickovic
authors.forename: Stef J
authors.initials: SJ
authors.lastname: Koppelman
mesh_terms: D055516:2S Albumins, Plant / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000478:Alkylation; D052179:Antigens, Plant / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry* / Q000276:immunology; D008297:Male; D010084:Oxidation-Reduction; D021183:Peanut Hypersensitivity / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017433:Protein Structure, Secondary
chemical_list: D055516:2S Albumins, Plant; D052179:Antigens, Plant; C500890:Ara h 2 allergen, Arachis hypogaea; C492737:Ara h 6 allergen, Arachis hypogaea; D006023:Glycoproteins; D010940:Plant Proteins; D020033:Protein Isoforms; D007073:Immunoglobulin E
keywords: Allergen; Immunochemistry; Mass spectrometry (MS); Peanut; Protein structure; Spectroscopy