TY  - JOUR
AU  - Apostolović, Danijela
AU  - Luykx, Dion
AU  - Warmenhoven, Hans
AU  - Verbart, Dennis
AU  - Stanić-Vučinić, Dragana
AU  - de Jong, Govardus A. H.
AU  - Ćirković-Veličković, Tanja
AU  - Koppelman, Stef J.
PY  - 2013
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/1463
AB  - 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 allcylated 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 a-helix and gained some beta-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.
PB  - Elsevier Science Bv, Amsterdam
T2  - Biochimica et Biophysica Acta: Proteins and Proteomics
T1  - Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6 characterization of intermediate- and end products
VL  - 1834
IS  - 12
SP  - 2832
EP  - 2842
DO  - 10.1016/j.bbapap.2013.10.004
ER  - 

@article{
author = "Apostolović, Danijela and Luykx, Dion and Warmenhoven, Hans and Verbart, Dennis and Stanić-Vučinić, Dragana and de Jong, Govardus A. H. and Ćirković-Veličković, Tanja and Koppelman, Stef J.",
year = "2013",
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 allcylated 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 a-helix and gained some beta-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.",
publisher = "Elsevier Science Bv, Amsterdam",
journal = "Biochimica et Biophysica Acta: Proteins and Proteomics",
title = "Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6 characterization of intermediate- and end products",
volume = "1834",
number = "12",
pages = "2832-2842",
doi = "10.1016/j.bbapap.2013.10.004"
}

Apostolović, D., Luykx, D., Warmenhoven, H., Verbart, D., Stanić-Vučinić, D., de Jong, G. A. H., Ćirković-Veličković, T.,& Koppelman, S. J.. (2013). Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6 characterization of intermediate- and end products. in Biochimica et Biophysica Acta: Proteins and Proteomics
Elsevier Science Bv, Amsterdam., 1834(12), 2832-2842.
https://doi.org/10.1016/j.bbapap.2013.10.004

Apostolović D, Luykx D, Warmenhoven H, Verbart D, Stanić-Vučinić D, de Jong GAH, Ćirković-Veličković T, Koppelman SJ. Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6 characterization of intermediate- and end products. in Biochimica et Biophysica Acta: Proteins and Proteomics. 2013;1834(12):2832-2842.
doi:10.1016/j.bbapap.2013.10.004 .

Apostolović, Danijela, Luykx, Dion, Warmenhoven, Hans, Verbart, Dennis, Stanić-Vučinić, Dragana, de Jong, Govardus A. H., Ćirković-Veličković, Tanja, Koppelman, Stef J., "Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6 characterization of intermediate- and end products" in Biochimica et Biophysica Acta: Proteins and Proteomics, 1834, no. 12 (2013):2832-2842,
https://doi.org/10.1016/j.bbapap.2013.10.004 . .