N-glycans Other Than MUXF3 Are Targets for Specific IgE Antibodies Against Juniperus Ashei Pollen Allergens

J. ashei pollen is a frequent cause of respiratory allergy. Allergen extracts from this source show high concentration of glycoproteins. We hypothesize a significant allergenic role of glycans bound to its allergens. J. ashei pollen extract was chemically deglycosylated for removing N- and O-glycans. The resulting product was analyzed by SDS-PAGE and IgE-Immunoblotting. Jun a 1 was detected by a polyclonal antibody. N-glycans were excised from J. ashei proteins by PNGase A deglycosylation and purified by ultrafiltration. The remained glycoproteins were then incubated with NaBH4/NaOH for separating O-glycans, and isolated by filtration. IgE-binding capacity of N-glycans, O-glycans and MUXF3 to J. ashei pollen allergens was investigated by Immunoblot inhibition assays. Chemically deglycosylated J. ashei extract showed several bands in a range between 11 and 54 kDa but none of them had IgE-binding capacity. The presence of Jun a 1 was confirmed in the deglycosylated extract. Purified N-glycans produced a dose-dependent inhibition of IgE-binding to J. ashei allergens. O-glycans and MUXF3 did not show IgE-binding inhibition. The results demonstrate that IgE is mainly directed against glucidic component of J. ashei allergens, specifically against N-glycans, and these N-glycans play a crucial role in allergic sensitization to J. ashei.