Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.

BACKGROUND: Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy. METHODS: Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods. RESULTS: AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE. CONCLUSIONS: AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2.

Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts.

BACKGROUND: Cross-reactivity between peanuts and tree nuts implies that similar immunoglobulin E (IgE) epitopes are present in their proteins. OBJECTIVE: To determine whether walnut sequences similar to known peanut IgE-binding sequences, according to the property distance (PD) scale implemented in the Structural Database of Allergenic Proteins, react with IgE from sera of patients with allergy to walnut and/or peanut. METHODS: Patient sera were characterized by western blotting for IgE binding to nut protein extracts and to peptides from walnut and peanut allergens, similar to known peanut epitopes as defined by low PD values, synthesized on membranes. Competitive enzyme-linked immunosorbent assay (ELISA) was used to show that peanut and predicted walnut epitope sequences compete with purified Ara h 2 for binding to IgE in serum from a cross-reactive patient. RESULTS: Sequences from the vicilin walnut allergen Jug r 2, which had low PD values to epitopes of the peanut allergen Ara h 2, a 2S albumin, bound to IgE in sera from five patients who reacted to either walnut or peanut or both. A walnut epitope recognized by sera from six patients mapped to a surface-exposed region on a model of the N-terminal pro-region of Jug r 2. This predicted walnut epitope competed for IgE binding to Ara h 2 in serum as well as the known IgE epitope from Ara h 2. CONCLUSIONS: Sequences with low PD value (< 8.5) to known IgE epitopes could contribute to cross-reactivity between allergens. This further validates the PD scoring method for predicting cross-reactive epitopes in allergens.

The effects of roasting on the allergenic properties of peanut proteins.

BACKGROUND: Because of the widespread use of peanut products, peanut allergenicity is a major health concern in the United States. The effect or effects of thermal processing (roasting) on the allergenic properties of peanut proteins have rarely been addressed. OBJECTIVE: We sought to assess the biochemical effects of roasting on the allergenic properties of peanut proteins. METHODS: Competitive inhibition ELISA was used to compare the IgE-binding properties of roasted and raw peanut extracts. A well-characterized in vitro model was used to test whether the Maillard reaction contributes to the allergenic properties of peanut proteins. The allergic properties were measured by using ELISA, digestion by gastric secretions, and stability of the proteins to heat and degradation. RESULTS: Here we report that roasted peanuts from two different sources bound IgE from patients with peanut allergy at approximately 90-fold higher levels than the raw peanuts from the same peanut cultivars. The purified major allergens Ara h 1 and Ara h 2 were subjected to the Maillard reaction in vitro and compared with corresponding unreacted samples for allergenic properties. Ara h 1 and Ara h 2 bound higher levels of IgE and were more resistant to heat and digestion by gastrointestinal enzymes once they had undergone the Maillard reaction. CONCLUSIONS: The data presented here indicate that thermal processing may play an important role in enhancing the allergenic properties of peanuts and that the protein modifications made by the Maillard reaction contribute to this effect.

Structure of the major peanut allergen Ara h 1 may protect IgE-binding epitopes from degradation.

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.

Biochemical and structural analysis of the IgE binding sites on ara h1, an abundant and highly allergenic peanut protein.

Allergy to peanut is a significant IgE-mediated health problem because of the high prevalence, potential severity, and chronicity of the reaction. Ara h1, an abundant peanut protein, is recognized by serum IgE from >90% of peanut-sensitive individuals. It has been shown to belong to the vicilin family of seed storage proteins and to contain 23 linear IgE binding epitopes. In this communication, we have determined the critical amino acids within each of the IgE binding epitopes of Ara h1 that are important for immunoglobulin binding. Surprisingly, substitution of a single amino acid within each of the epitopes led to loss of IgE binding. In addition, hydrophobic residues appeared to be most critical for IgE binding. The position of each of the IgE binding epitopes on a homology-based molecular model of Ara h1 showed that they were clustered into two main regions, despite their more even distribution in the primary sequence. Finally, we have shown that Ara h1 forms a stable trimer by the use of a reproducible fluorescence assay. This information will be important in studies designed to reduce the risk of peanut-induced anaphylaxis by lowering the IgE binding capacity of the allergen.

MyoD-E12 heterodimers and MyoD-MyoD homodimers are equally stable.

Muscle development is controlled by the MyoD family of basic helix-loop-helix (bHLH) DNA-binding proteins. These proteins dimerize with ubiquitous products of the E2A gene (E12 and E47) and bind in a sequence-specific manner to enhancer regions of muscle-specific genes activating their expression. In this study, fluorescence anisotropy has been utilized to characterize the interactions of recombinant MyoD and E12 in solution in the absence of DNA. The Gibb's free energies of dissociation (deltaG) and the equilibrium dissociation constants (K(D)) for the protein-protein interactions are reported. The deltaG for the MyoD homodimers in 100 mM KCl was 8.7 kcal/mol (K(D) = 340 nM), and increasing the salt concentration resulted in destabilization of the dimer. From titrations of MyoD-dansyl with E12 at 100 mM KCl, a free energy of heterodimerization of 8.7 (+0.4/-2.4) kcal/mol was recovered using rigorous confidence limit testing. The titrations of E12-dansyl with MyoD yielded a free energy of 8.3 kcal/mol with tighter confidence limits, +0.5/-0.8 kcal/mol. Thus, in the absence of DNA, both MyoD homodimers and MyoD-E12 heterodimers are relatively weak complexes of approximately the same stability. E12 does not form stable homo-oligomeric complexes; remaining monomeric at concentrations as high as 20 microM. Based on these results and the apparent binding constants reported previously for DNA binding, DNA is likely to facilitate the dimerization of MyoD and E12. Furthermore, higher affinity interactions of MyoD-E12 heterodimers versus MyoD homodimers with DNA binding sites is not due to preferential heterodimerization.

High-level expression and purification of MyoD, myogenin, and E12.

The myogenic regulatory factors (MRFs), MyoD, myogenin, Myf-5, and MRF4, function as transcriptional activators of muscle-specific gene expression by forming heterodimers with the ubiquitously expressed products of the E2A gene, E12 and E47. To enable quantitative biochemical and biophysical analyses of the wild-type proteins, as well as mutants designed to reveal structure-function relationships, we developed protocols for the high-level expression and rapid purification of milligram quantities of MyoD, myogenin, and E12 using conventional biochemical techniques. T7 expression systems were used to direct expression of cDNA encoded proteins in Escherichia coli. Whereas MyoD and E12 were expressed well without alteration, high-level expression of myogenin required changing several rare arginine codons by in vitro mutagenesis to a commonly used E. coli arginine codon. Presumably, inefficient translation of the rare arginine codons inhibited high-level expression of myogenin in the original expression plasmid. Purification protocols are described which involve a simple strategy of cell lysis, ammonium sulfate precipitation, and ion-exchange chromatography. Using this approach, 20 to 50 mg of MyoD, myogenin, or E12 can be purified to 90-95% homogeneity from induced cell pellets in 1 day's time.