STUDIES ON THE CARBOHYDRATE MOIETIES OF THE TIMOTHY GRASS-POLLEN ALLERGEN PHL-P-I

Timothy grass pollen was investigated in order to determine the carboh ydrate moieties of its major grass group I (Phl p I) and to study its impact on allergenicity. Based on computer calculations one N-glycosyl ation site was deduced from the cDNA data of Phl p I. After two-dimens ional polyacrylamide gel electrophoresis, followed by blotting of poll en extract and by use of the monoclonal antibody IG 12 we identified a t 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. DegIycosylation by trifl uoromethanesulfonic acid resulted in a decrease of about 2 kDa. Treatm ent with N-glycosidase A resulted in a partial deglycosylation, while N-glycosidase F and O-glycosidase had no effect. Ten lectins were inve stigated for their binding to Phl p I components: Aleuria aurantia agg lutinin showed strong reactivity (indicating fucose residues), while G alanthus nivalis agglutinin (indicating mannose residues) and concanav alin A (indicating mannose, glucose or N-acetylglucosamine residues) s howed weak binding. By neutral sugar analysis we determined similar co ntents of the monosaccharides in the isoallergens. In order to study t he influence of the carbohydrate structures of Phl p I on IgE reactivi ty we tested some patient sera for their reactivity with intact and de glycosylated Phl p I. Even though most of the IgE antibodies bind at t he protein core, we detected one serum that recognized carbohydrate mo ieties on the Phl p I.