Analysis of the composition of an immunoglobulin E reactive high molecular weight protein complex of peanut extract containing Ara h 1 and Ara h 3/4.
Peanuts (Arachis hypogaea) contain some of the most potent food allergens. In recent years an increasing prevalence of peanut allergies both in children and adults has been observed in the USA and in Europe. In vitro identification and characterization of allergens including those from peanut have been frequently performed by Western blotting. However this method may alter the immunoglobulin E (IgE) antibody reactivity since the proteins are denatured by detergent treatment and/or reduction of disulfide bonds by reducing reagents and does not answer the question how peanut allergens interact with the human digestive apparatus and immune system. Size exclusion chromatography of peanut extract shows that approximately 90% of the total protein content is eluted as one peak in the exclusion volume with a molecular mass of over 200 kDa. The proteins of this fraction were analyzed by blue-native polyacrylamide gel electrophoresis (PAGE), immunoblotting, two-dimensional PAGE and Western blotting. A complex of Ara h 1 (Acc. no. P43237), Ara h 3/4 (AAM46958), Ara h 3 (AAC63045), Ara h 4 (AF086821), Gly 1 (AAG01363) and iso-Ara h 3 (AAT39430) was identified using patients' IgE and allergen-specific monoclonal antibodies; N-terminal sequencing and matrix-assisted laser desorption/ionisation-time of flight analysis verified these findings. A comparison of the peanut allergen sequences of Ara h 3/4, Ara h 3, Ara h 4 and peanut trypsin inhibitor (AF487543) and the proteins Gly 1 and iso-Ara h 3, not yet described as allergens, leads to the conclusion that these proteins are isoallergens of each other. It was shown that these isoallergens are post-translationally cleaved and held together by disulfide bonds in accordance to the 11S plant seed storage proteins signature.
Boldt
Andrea
A
Research Center Borstel, Borstel, Germany.
Fortunato
Donatella
D
Conti
Amedeo
A
Petersen
Arnd
A
Ballmer-Weber
Barbara
B
Lepp
Ute
U
Reese
Gerald
G
Becker
Wolf-Meinhard
WM
D000485:Allergens / Q000032:analysis* / Q000276:immunology; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D010367:Arachis; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010936:Plant Extracts / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000032:analysis* / Q000276:immunology; D055314:Seed Storage Proteins; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D052179:Antigens, Plant; D006023:Glycoproteins; D007073:Immunoglobulin E; D008565:Membrane Proteins; D010936:Plant Extracts; D010940:Plant Proteins; D055314:Seed Storage Proteins; C500889:Ara h 1 protein, Arachis hypogaea; C118207:allergen Ara h3