555 Identification and characterization of IgE binding epitopes of patatin, a major food allergen of potato (original) (raw)
Related papers
Journal of Agricultural and Food Chemistry, 2005
The influence of thermal processing and nonenzymatic browning reactions on the IgE-binding activity of rAra h 2 was studied and compared to findings recently reported for the allergen's natural counterpart. ELISA experiments as well as inhibition assays revealed that thermal treatment of rAra h 2 in the presence of reactive carbohydrates and carbohydrate breakdown products induces a strong increase of the IgE-binding activity, thus collaborating with the data reported for the natural protein isolated from peanuts. To localize the Ara h 2 sequences responsible for the formation of highly IgE-affine glycation sites, model peptides have been synthesized mimicking sequences which contain possible targets for glycation as well as the immunodominant epitopes. Immunological evaluation of these peptides heated in the absence or presence of reducing sugars and carbonyls, respectively, revealed that neither the two lysine residues of Ara h 2 nor its N-terminus are involved in the formation of IgE-affine structures by Maillard reaction. Also, the cysteine-containing major epitope 3 (aa 27-36) was found to lose its IgE-binding capacity upon heating. By contrast, the overlapping major epitopes 6 and 7, which do not contain any lysine or arginine moieties, showed a distinct higher level of IgE binding when subjected to Maillard reaction, thus giving the first evidence that nonbasic amino acids might be accessible for nonenzymatic glycation reactions and that these posttranslational modifications might induce increased IgE binding of the glycated Ara h 2. Analogous experiments were performed with peanut agglutinin, considered in the literature as a minor allergen. ELISA experiments revealed that the majority of tested sera samples from peanut-sensitive patients showed a high level of IgE binding to the lectin even after heat treatment. In contradiction to published data, nonenzymatic browning reactions seem to deteriorate the IgE affinity of the lectin.
Structural and immunologic characterization of Ara h 1, a major peanut allergen
Journal of Biological Chemistry, 2011
Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170 -586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated.
Molecular Immunology, 2009
Eight distinct sequential IgE-binding epitopes were identified along the amino acid sequence of Ara h 3 using the Spot technology. They essentially correspond to preferencially electropositive regions exposed on the molecular surface of the protein. A few IgE-binding epitopes are coalescent to create more extended IgE-binding regions exposed on the surface of the allergen. Ara h 3 contains a core region corresponding to the cupin motifs and predicted to be preserved upon the trypsin and chymotrypsin attack in the gastro-intestinal tract. Some of the identified IgE-binding epitopes should remain unaltered in the core region to subsequently interact with the local immune system. They most probably account for the strong allergenic potency of Ara h 3. Most of the identified IgE-binding epitopes of Ara h 3 readily differ from the corresponding regions of other legume and tree-nut legumin allergens except for epitope #1 and #7 which are rather conserved essentially in other allergens. These structurally related epitopes could account for some cross-reactions occurring between Ara h 3 and other legumin allergens.
Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity
Molecular Nutrition & Food Research, 2011
Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1. Methods and results: Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (1001C 15 min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, Hand G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein. Conclusion: Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.
Degradation 1 May Protect IgE-Binding Epitopes from Structure of the Major Peanut Allergen Ara h
2000
In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.