Carbohydrate moieties on the in vitro immunoreactivity of soy b-conglycinin

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.