University of Arkansas

Other

Mount Sinai School of Medicine

Gary A Bannon

A Wesley Burks, Jr

Ricki M Helm

J Steven Stanley

Hugh A Sampson

Alan D Elbein

Gael Cockrell

Nina E King

Patrick A Rabjohn

It has been determined that allergens, which are characterized by both humoral (IgE) and cellular (T cell) binding sites, can be modified to be less allergenic by modifying the IgE binding sites. The IgE binding sites can be converted to non-IgE binding sites by masking the site with a compound that prevents IgE binding or by altering as little as a single amino acid within the protein, most typically a hydrophobic residue towards the center of the IgE-binding epitope, to eliminate IgE binding. The method allows the protein to be altered as minimally as possible, other than-within the IgE-binding sites, while retaining the ability of the protein to activate T cells, and, in some embodiments by not significantly altering or decreasing IgG binding capacity The examples use peanut allergens to demonstrate alteration of IgE binding sites. The critical amino acids within each of the IgE binding epitopes of the peanut protein that are important to immunoglobulin binding have been determined. Substitution of even a single amino acid within each of the epitopes led to loss of IgE binding. Although the epitopes shared no common amino acid sequence motif, the hydrophobic residues located in the center of the epitope appeared to be most critical to IgE binding.

Methods and reagents for decreasing clinical reaction to allergy

It has been determined that allergens, which are characterized by both humoral (IgE) and cellular (T cell) binding sites, can be modified to be less allergenic by modifying the IgE binding sites. The IgE binding sites can be converted to non-IgE binding sites by masking the site with a compound that prevents IgE binding or by altering as little as a single amino acid within the protein, most typically a hydrophobic residue towards the center of the IgE binding epitope, to eliminate IgE binding. The method allows the protein to be altered as minimally as possible, other than within the IgE-binding sites, while retaining the ability of the protein to activate T cells, and, in some embodiments by not significantly altering or decreasing IgG binding capacity. The examples use peanut allergens to demonstrate alteration of IgE binding sites. The critical amino acids within each of the IgE binding epitopes of the peanut protein that are important to immunoglobulin binding have been determined. Substitution of even a single amino acid within each of the epitopes led to loss of IgE binding. Although the epitopes shared no common amino acid sequence motif, the hydrophobic residues located in the center of the epitope appeared to be most critical to IgE binding.

Nucleic acids encoding ara h 3 polypeptides

The present invention provides a purified, homogeneous plant enzyme that adds a &#x3b2;-1,2-linked xylose to the &#x3b2;-linked mannose on the N-linked oligosaccharides of storage glycoproteins. This &#x3b2;1,2-xylosyltransferase was purified from the microsomal fraction of soybean cells approximately 51,000-fold. Also provided is an antibody recognizing this &#x3b2;1,2-xylosyltransferase.

Antibody directed against purified &#x3b2; 1,2-xylosyltransferase

One of the major peanut allergens, Ara h I, was selected from cDNA expression library clones using Ara h I specific oligo-nucleotides and polymerase chain reaction technology. The Ara h I clone identified a 2.3 kb mRNA species on a Northern blot containing peanut poly A&plus;RNA. DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants. The isolation of the Ara h I clones allowed the synthesis of this protein in cells and subsequent recognition of this. recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity.

Peanut allergens and methods

The present invention provides a purified, homogeneous plant enzyme that adds a &bgr;-1,2-linked xylose to the &bgr;-linked mannose on the N-linked oligosaccharides of storage glycoproteins. This &bgr;1,2-xylosyltransferase was purified from the microsomal fraction of soybean cells approximately 51,000-fold. Also provided is polyclonal antiserum recognizing this &bgr;1,2-xylosyltransferase enzyme and uses thereof.

Purified &bgr;1,2-xylosyltransferase and uses thereof

Peanuts are a common cause of food hypersensitivity reactions. The sera of 10 patients who had atopic dermatitis and a positive double-blind placebo-controlled food challenge to peanut were used to investigate the major allergens of peanut. Crude Florunner extracts were fractionated by anion-exchange chromatography using a step gradient (limit buffer, 0.05M BisTris/1.5M NaCl). A protein peak (OD 280) which eluted at 10% NaCl and demonstrated intense IgE-binding was further analyzed by two-dimensional SDS-PAGE/immunoblot analysis. The majority of this fraction is a protein which has a molecular weight of 17 kD and a pI of 5.2. Sequencing data from the N-terminus revealed the following initial 9 amino acids: (*)-Q-Q-(*)-E-L-Q-D-L. Based on IgE-binding activity and no known amino acid sequence identity to other allergens, this allergen is designated Ara h II. Ara h II may be used to detect and quantify peanut allergens in foodstuffs. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens.

The present invention provides a purified, homogeneous plant enzyme that adds a &bgr;-1,2-linked xylose to the &bgr;-linked mannose on the N-linked oligosaccharides of storage glycoproteins. This &bgr;1,2-xylosyltransferase was purified from the microsomal fraction of soybean cells approximately 51,000-fold. Also provided is polyclonal antiserum recognizing this &dgr;1,2-xylosyltransferase enzyme and uses thereof.

Growing community of inventors

Little Rock, AR, United States of America

Gary A Bannon