Cathie Connaughton

Identification of a major peanut allergen, Ara h I, in patients with atopic dermatitis and positive peanut challenges

Peanuts are among the most common causes of immediate hypersensitivity reactions to foods. Serum from nine patients with atopic dermatitis and a positive double-blind, placebo-controlled, food challenge to peanut were used to begin the process of identification and purification of the major peanut allergens. Identification of a major peanut allergen was accomplished by use of anion-exchange column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ELISA, thin-layer isoelectric focusing, and IgE-specific immunoblotting. Anion-exchange chromatography revealed several fractions that bound IgE from the serum of the challenge-positive patient pool. By measuring antipeanut-specific IgE in the ELISA and in IgE-specific immunoblotting, we identified an allergenic component with two Coomassie brilliant blue staining bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a mean molecular weight of 63.5 kd. Examination of this fraction by the IgE antipeanut ELISA with individual serum and by the ELISA-inhibition assay with pooled serum, we identified this fraction as a major allergen. Thin-layer isoelectric focusing and immunoblotting of this 63.5 kd fraction revealed it to have an isoelectric point of 4.55. Based on allergen nomenclature of the IUIS Subcommittee for Allergen Nomenclature, this allergen is designated, Ara h I (Arachis hypogaea).

Identification and characterization of a second major peanut allergen, Ara h II, with use of the sera of patients with atopic dermatitis and positive peanut challenge

Peanuts are frequently a cause of food hypersensitivity reactions in children. Serum from nine patients with atopic dermatitis and a positive double-blind, placebo-controlled, food challenge to peanut were used in the process of identification and purification of the peanut allergens. Identification of a second major peanut allergen was accomplished with use of various biochemical and molecular techniques. Anion exchange chromatography of the crude peanut extract produced several fractions that bound IgE from the serum of the patient pool with positive challenges. By measuring antipeanut specific IgE and by IgE-specific immunoblotting we have identified an allergic component that has two closely migrating bands with a mean molecular weight of 17 kd. Two-dimensional gel electrophoresis of this fraction revealed it to have a mean isoelectric point of 5.2. According to allergen nomenclature of the IUIS Subcommittee for Allergen Nomenclature this allergen is designated, Ara h II (Arachis hypogaea).

Allergenicity of peanut and soybean extracts altered by chemical or thermal denaturation in patients with atopic dermatitis and positive food challenges

Peanuts and soybeans are two of the six most common foods to cause food hypersensitivity reactions in children. We used the serum of 10 patients with atopic dermatitis and positive double-blind, placebo-controlled, food challenges to peanut and two patients with atopic dermatitis and positive double-blind, placebo-controlled, food challenges to soybean to investigate the change in IgE-specific and IgG-specific binding to these proteins altered by either chemical or thermal denaturation. We used IgE- and IgG-specific ELISA-inhibition analyses to compare these effects on the crude peanut and crude soy extracts, as well as on the major allergenic fractions of both proteins. Heating the soy proteins at various temperatures and time intervals did not significantly change the IgE- or IgG-specific binding of the soy positive pooled serum. When the peanut proteins were subjected to similar heating experiments, the IgE- and IgG-specific binding did not change. When these same proteins were treated with enzymes in the immobilized digestive enzyme assay system used to mimic human digestion, the binding of IgE to the crude peanut and crude soy extracts was reduced; 100-fold for peanut and 10-fold for soybean. Therefore it appears that thermal denaturation of peanut and soybean protein extracts does not enhance or reduce IgE- and IgG-specific binding activity. Chemical denaturation appears to minimally reduce the binding of these proteins.

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.

A soybean G2 glycinin allergen. 2. Epitope mapping and three-dimensional modeling.

Background: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, has been shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 22 kD. These findings suggested that this unique protein fraction from soybean might be responsible, in part, for soybean allergic reactivity. The objective of the present study was to characterize specific B cell epitopes, to determine if any amino acid was critical to IgE binding and to model the 22-kD G2 soybean allergen to the three-dimensional (3-D) phaseolin molecule. Methods: B cell epitopes were identified using SPOTs peptide analysis. Structural orientation of the IgE-binding regions was mapped to the 3-D phaseolin molecule using molecular modeling of the protein tertiary structure. Results: Eleven linear epitopes, representing 15 amino acid peptide sequences, bound to IgE in the glycinin molecule. These epitopes were predicted to be distributed asymmetrically on the surface of G2 trimers. Conclusions: Only 1 epitope could be rendered non-IgE binding by alanine substitutions in the peptide. The nonrandom distribution of the IgE binding sites provides new insight into their organization in trimers in 11S complexes of the G2 glycinin allergen.

Identification of Peanut Agglutinin and Soybean Trypsin Inhibitor as Minor Legume Allergens

Peanuts and soybeans are frequent causes of food hypersensitivity reactions in children. Sera from 12 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to peanut and sera from 5 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to soybean were used to identify and characterize specific legume allergens. Identification of a minor allergen from peanut and a minor allergen from soybean was accomplished using various physicochemical techniques. The peanut fraction, peanut agglutinin, isolated by anion-exchange chromatography and electrolution and confirmed by amino acid sequencing, bound IgE in only 50% of the peanut challenge positive patients. The soybean fraction, soybean trypsin inhibitor, identified by gel filtration and electroelution and confirmed by amino acid sequencing, bound IgE in only 20% of the soy challenge positive patients. The identification of these two known legume proteins as minor allergens should allow further immunologic and structural investigations to compare the major and minor legume allergens.

Mutational analysis of the IgE-binding epitopes of P34/Gly m Bd 30K

BACKGROUND Peanuts and soybeans are 2 foods that have been shown to be responsible for many atopic disorders. Because of their nutritional benefit, soybean proteins are now being used increasingly in a number of food products. Previous studies have documented multiple allergens in soybean extracts, including glycinin, beta-conglycinin, and the P34/Gly m Bd 30K protein. OBJECTIVE Our overall goal was to identify soybean-specific allergens to begin to understand molecular and immunochemical characteristics of legume proteins. The specific aim of the current investigation was to identify the essential amino acid residues necessary for IgE binding in the 5 distinct immunodominant epitopes of P34/Gly m Bd 30K. METHODS Serum IgE from 6 clinically sensitive soybean-allergic individuals was used to identify P34/Gly m Bd 30K in the native and single amino acid substituted peptides with use of the SPOTS peptide synthesis technique to determine critical amino acids required for IgE binding. RESULTS The intensity of IgE binding and epitope recognition by serum IgE from the individuals varied substantially. With use of serum from 6 clinically soybean-sensitive individuals, 2 of the 5 immunodominant epitopes could be mutagenized to non-IgE binding peptides. CONCLUSIONS Single-site amino acid substitution of the 5 immunodominant epitopes of Gly m Bd 30K with alanine revealed that IgE binding could be reduced or eliminated in epitopes 6 and 16 in the serum obtained from 6 soybean-sensitive patients.

A soybean G2 glycinin allergen. 1. Identification and characterization.

Background: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, was shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 21 kD. These findings suggested that unique proteins in soybeans might be responsible for soybean allergic reactivity. The objective of the present study was to identify unique proteins in soybean extracts that bind to specific IgE from soybean-sensitive individuals, and to characterize the allergen using physicochemical methods and IgE binding. Methods: Two-dimensional and preparative SDS-PAGE/IgE immunoblot analysis was used to identify a 22-kD soybean-specific allergen from crude soybean extracts. N-terminal sequence analysis was used to determine the identification of the protein binding IgE from soybean-sensitive individuals. Results: IgE immunoblot and amino acid sequence analysis identified the 22-kD protein as a member of the G2 glycinin soybean protein family. Further investigation revealed that the IgEs reacted with basic chains from each member of the glycinin family of soybean storage proteins. Conclusions: Each of the subunits from glycinin, the storage protein that is the most prevalent component of soybean, are major allergens.

Modification of peanut allergen Ara h 3: effects on IgE binding and T cell stimulation.

Background: Peanut allergy is a major health concern due to the increased prevalence, potential severity, and chronicity of the reaction. The cDNA encoding a third peanut allergen, Ara h 3, has been previously cloned and characterized. Mutational analysis of the Ara h 3 IgE-binding epitopes with synthetic peptides revealed that single amino acid changes at critical residues could diminish IgE binding. Methods: Specific oligonucleotides were used in polymerase chain reactions to modify the cDNA encoding Ara h 3 at critical IgE binding sites. Four point mutations were introduced into the Ara h 3 cDNA at codons encoding critical amino acids in epitopes 1, 2, 3 and 4. Recombinant modified proteins were used in SDS-PAGE/Western IgE immunoblot, SDS-PAGE/Western IgE immunoblot inhibition and T cell proliferation assays to determine the effects of these changes on in vitro clinical indicators of peanut hypersensitivity. Results: Higher amounts of modified Ara h 3 were required to compete with the wild-type allergen for peanut-specific serum IgE. Immunoblot analysis with individual serum IgE from Ara-h-3-allergic patients showed that IgE binding to the modified protein decreased ∼35–85% in comparison to IgE binding to wild-type Ara h 3. Also, the modified Ara h 3 retained the ability to stimulate T cell activation in PBMCs donated by Ara-h-3-allergic patients. Conclusions: The engineered hypoallergenic Ara h 3 variant displays two characteristics essential for recombinant allergen immunotherapy; it has a reduced binding capacity for serum IgE from peanut-hypersensitive patients and it can stimulate T-cell proliferation and activation.

Antibody response to milk proteins in patients with milk-protein intolerance documented by challenge

To evaluate the role of specific antibody response to milk proteins in patients with milk-protein intolerance, allergen-specific IGE, IgG, and IgG4 to these proteins were measured by ELISA. Bovine casein, gamma globulin (GG), beta-lactoglobulin, and lactalbumin were the milk proteins used. Antibody production to these proteins were analyzed in 18 patients who underwent milk-protein challenges (eight positive and 10 negative) and in five normal children used in the analysis. ELISA results for specific IgE, IgG, and IgG4 to these specific proteins demonstrated no statistically different response to the four milk proteins among the three patient groups by multivariate analysis. When the specific antibody results from the positive challenge group, the negative challenge group, and the normal group were combined, the IgE and IgG4 to GG and the IgG to casein were significantly higher (p less than 0.01) than the corresponding specific antibody to the other proteins tested. The IgG or IgG4 to GG would differentiate the positive from the negative challenge group (p less than 0.05) but were not significantly different from the normal control group. Contrary to previously published studies, these results indicate IgG specific for the milk proteins are not increased in patients with milk-protein intolerance. The data also support the concept that IgE and IgG4 are not elevated in these patients. Therefore, there appears to be no pathogenic role for these specific immunoglobulins in milk-protein intolerance.