RESUMO
Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric alpha-amylase inhibitor as well as major allergens to baker's asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying the penetration characteristics of the CM proteins through intestinal mucosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni2+-chelating column. The recombinant proteins were radioiodinated and admministered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.
Assuntos
Alérgenos/metabolismo , Alérgenos/química , Alérgenos/genética , Animais , Sequência de Bases , Células CACO-2 , DNA Complementar/genética , DNA de Plantas/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Humanos , Absorção Intestinal , Masculino , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Subunidades Proteicas , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Hipersensibilidade a Trigo/imunologia , alfa-Amilases/antagonistas & inibidoresRESUMO
The purpose of the present study was to determine whether wheat alpha-amylase inhibitor subunits bind IgE sera from Japanese with atopic dermatitis (AD) related to wheat. IgE in sera from eight out of the 11 patients with RAST value of 3 or 4 against wheat-bound salt-soluble proteins of wheat and the alpha-amylase inhibitor proteins separated by gel filtration chromatography. The subunits of tetrameric alpha-amylase inhibitor, CM2, CM3 and CM16 (molecular weight approximately 14 kd), known to be major allergens for baker's asthma, were purified. Then the binding activity to IgE sera from the AD patients was examined by immunoblotting. The IgE sera bound only to CM3, not to CM2 and CM16. These results suggest that CM3 may be involved in both atopic dermatitis and baker's asthma.
