Studies on the carbohydrate moieties of the timothy grass pollen allergen Phl p I

Timothy grass pollen was investigated in order to determine the carbohydrate moieties of its major grass group I (Phl p I) and to study its impact on allergenicity. Based on computer calculations one N-glycosylation site was deduced from the cDNA data of Phl p I. After two-dimensional polyacrylamide gel electrophoresis, followed by blotting of pollen extract and by use of the monoclonal antibody IG 12 we identified at least six isoallergens of Phl p I with the main spots at a molecular mass of 35-37 kDa and a pI range of 6.5-7.3. Deglycosylation by trifluoromethanesulfonic acid resulted in a decrease of about 2 kDa. Treatment with N-glycosidase A resulted in a partial deglycosylation, while N-glycosidase F and O-glycosidase had no effect. Ten lectins were investigated for their binding to Phl p I components: Aleuria aurantia agglutinin showed strong reactivity (indicating fucose residues), while Galanthus nivalis agglutinin (indicating mannose residues) and concanavalin A (indicating mannose, glucose or N-acetylglucosamine residues) showed weak binding. By neutral sugar analysis we determined similar contents of the monosaccharides in the isoallergens. In order to study the influence of the carbohydrate structures of Phl p I on IgE reactivity we tested some patient sera for their reactivity with intact and deglycosylated Phl p I. Even though most of the IgE antibodies bind at the protein core, we detected one serum that recognized carbohydrate moieties on the Phl p I.