Engineering, characterization and in vitro efficacy of the major peanut allergens for use in immunotherapy.

BACKGROUND: Numerous strategies have been proposed for the treatment of peanut allergies, but despite the steady advancement in our understanding of atopic immune responses and the increasing number of deaths each year from peanut anaphylaxis, there is still no safe, effective, specific therapy for the peanut-sensitive individual. Immunotherapy would be safer and more effective if the allergens could be altered to reduce their ability to initiate an allergic reaction without altering their ability to desensitize the allergic patient. METHODS: The cDNA clones for three major peanut allergens, Ara h 1, Ara h 2, and Ara h 3, have been cloned and characterized. The IgE-binding epitopes of each of these allergens have been determined and amino acids critical to each epitope identified. Site-directed mutagenesis of the allergen cDNA clones, followed by recombinant production of the modified allergen, provided the reagents necessary to test our hypothesis that hypoallergenic proteins are effective immunotherapeutic reagents for treating peanut-sensitive patients. Modified peanut allergens were subjected to immunoblot analysis using peanut-positive patient sera IgE, T cell proliferation assays, and tested in a murine model of peanut anaphylaxis. RESULTS: In general, the modified allergens were poor competitors for binding of peanut-specific IgE when compared to their wild-type counterpart. The modified allergens demonstrated a greatly reduced IgE-binding capacity when individual patient serum IgE was compared to the binding capacity of the wild-type allergens. In addition, while there was considerable variability between patients, the modified allergens retained the ability to stimulate T cell proliferation. CONCLUSIONS: These modified allergen genes and proteins should provide a safe immunotherapeutic agent for the treatment of peanut allergy.

Carboxyl residues in the active site of human phenol sulfotransferase (SULT1A1).

The carboxyl-specific amino acid modification reagent, Woodward's reagent K (WK), was utilized to characterize carboxyl residues (Asp and Glu) in the active site of human phenol sulfotransferase (SULT1A1). SULT1A1 was purified using the pMAL-c2 expression system in E. coli. WK inactivated SULT1A1 activity in a time- and concentration-dependent manner. The inactivation followed first-order kinetics relative to both SULT1A1 and WK. Both phenolic substrates and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) protected against the inactivation, which suggests the carboxyl residue modification causing the inactivation took place within the active site of the enzyme. With partially inactivated SULT1A1, both V(max) and K(m) changed for PAPS, while for phenolic substrates, V(max) decreased and K(m) did not change significantly. A computer model of the three-dimensional structure of SULT1A1 was constructed based on the mouse estrogen sulfotransferase (mSULT1E1) X-ray crystal structure. According to the model, Glu83, Asp134, Glu246, and Asp263 are the residues likely responsible for the inactivation of SULT1A1 by WK. According to these results, five SULT1A1 mutants, E83A, D134A, E246A, D263A, and E151A, were generated (E151A as control mutant). Specific activity determination of the mutants demonstrated that E83A and D134A lost almost 100% of the catalytic activity. E246A and D263A also decreased SULT1A1 activity, while E151A did not change SULT1A1 catalytic activity significantly. This work demonstrates that carboxyl residues are present in the active site and are important for SULT1A1 catalytic activity. Glu83 and E134 are essential amino acids for SULT1A1 catalytic activity.

Molecular cloning and epitope analysis of the peanut allergen Ara h 3.

Peanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. Following our characterization of the two peanut allergens Ara h 1 and Ara h 2, we have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from approximately 45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3-allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions.