Correlation Between N-Glycan GnGnXF3 and the Allergic Immune Response Against Juniperus ashei Pollen

Background

Cupressaceae pollen increasingly causes respiratory allergies worldwide. Carbohydrates are abundant in extracts of these pollens, and the associated allergens are highly glycosylated. However, the contribution of saccharides to the allergenicity of these species remains unknown.

Methods

Juniperus ashei pollen extract was deglycosylated and characterised using SDS-PAGE and immunoblotting. Additionally, N- and O-glycans were purified from the extract, identified, used as inhibitors in IgE-immunoblotting and further analysed via basophil activation tests. The interactions between IgE and J. ashei glycans were analysed using a glycan array. Purified Jun a 1 was treated with β-N-acetylglucosaminidase S and analysed using immunoblotting. The native pollen extract was used to immunise rabbits, and the IgG response was analysed using ELISA and glycan array.

Results

Deglycosylation of J. ashei proteins abolished the interaction between IgE and allergens. This effect primarily depends on N-glycans. Purified N-glycans triggered basophil activation in some patients. A biantennary N-glycan with terminal GlcNAc, β-1,2 xylose and core α-1,3 fucose (GnGnXF3) was the most abundant glycan identified. The glycan array confirmed its interaction with IgE. The contribution of terminal N-acetylglucosamines (GlcNAc) to IgE–Jun a 1 interaction was validated. Moreover, effective immunisation of rabbits with the native extract confirmed the immunogenicity of their N-glycans.

Conclusions

The IgE–J. ashei allergen interaction is broadly controlled through N-glycans different from MUXF3. GnGnXF3 exerts an immunogenic effect in humans and rabbits; terminal GlcNAc residues influence its recognition by IgE. These discoveries reinforce the role of N-glycans in the allergic response to J. ashei.