b-Conglycinin is a functional glycoprotein and one of the most important soybean allergens. The aim of the present research was to investigate the role of the N-glycans moieties of b-conglycinin on its in vitro immunoreactivity. The soy allergen was obtained by isoelectric precipitation from commercial soy protein isolate and was enzymatically deglycosylated by PNGase F (Peptide N-Glycosidase F EC 3.5.1.52). In order to optimize deglycosylation conditions different reaction times and allergen concentrations were tested. The extent of deglycosylation was estimated by SDS-PAGE, CZE, RP-HPLC, and MALDI-TOF MS analyses, which provided information related to changes in protein structure. The antigenicity of both native b-conglycinin and its deglycosylated form was evaluated by western-blotting and indirect ELISA employing polyclonal rabbit anti-soybean sera and horseradish peroxidase-labeled goat anti-rabbit IgG while the in vitro allergenicity was assessed by means of indirect competitive inhibition ELISA employing human sera (IgE) of soy allergics. b-Conglycinin was effectively deglycosylated by PNGase F. Data on immunological tests suggested that glycosyl moieties forming this glycoprotein might be involved in its immunoreactivity.
Carbohydrate moieties on the in vitro immunoreactivity of soy b-conglycinin
Pasquale Ferranti;
2009
Abstract
b-Conglycinin is a functional glycoprotein and one of the most important soybean allergens. The aim of the present research was to investigate the role of the N-glycans moieties of b-conglycinin on its in vitro immunoreactivity. The soy allergen was obtained by isoelectric precipitation from commercial soy protein isolate and was enzymatically deglycosylated by PNGase F (Peptide N-Glycosidase F EC 3.5.1.52). In order to optimize deglycosylation conditions different reaction times and allergen concentrations were tested. The extent of deglycosylation was estimated by SDS-PAGE, CZE, RP-HPLC, and MALDI-TOF MS analyses, which provided information related to changes in protein structure. The antigenicity of both native b-conglycinin and its deglycosylated form was evaluated by western-blotting and indirect ELISA employing polyclonal rabbit anti-soybean sera and horseradish peroxidase-labeled goat anti-rabbit IgG while the in vitro allergenicity was assessed by means of indirect competitive inhibition ELISA employing human sera (IgE) of soy allergics. b-Conglycinin was effectively deglycosylated by PNGase F. Data on immunological tests suggested that glycosyl moieties forming this glycoprotein might be involved in its immunoreactivity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
