Freezing influences, the exposure of IgG glycans in sera from multiple sclerosis patients

Abstract

N-glycan residues attached to Asn297 of the immunoglobulin IgG molecule are responsible for changing­ its structural conformation and are used as markers of many inflammatory diseases. Freezing stabilizes protein structure, while recent solution NMR data showed greatly altered IgG glycan mobility at different­ temperatures. The aim of the current work was to investigate whether freezing sera samples from multiple sclerosis (MS) patients and normal healthy donors (NHD) influences exposure of IgG glycans. The developed lectin immunosorbent assay was used to evaluate exposure of native IgG glycans with fucose-binding AAL lectin and sialic acid-binding SNA lectin. Sera samples were divided and either immediately frozen at -20 °C or stored at 4 °C. Lectin exposure was compared between 5 MS patient groups (n = 75) vs NHD (n = 23) and in paired samples with and without freezing. A significant increase in the exposure of fucose residues on IgG glycans in MS patients, compared to NHD, was observed. This increase was only observed if sera were frozen before analysis. The exposure of sialic acid was decreased in MS vs NHD samples after freezing sera samples. The exposure of core fucose residues and terminal sialic residues differed significantly in paired sera samples after freezing. Combined parameters of fucose and sialic acid exposure on native IgG glycans using frozen sera samples serve as a discriminative marker between MS and NHD. For AAL exposure, the discrimination of MS was characterized by AUROC of 0.906, sensitivity of 76.7% and specificity of 59.0% (P < 0.0001).

Keywords

• freezing
• glycosylation
• igg
• inflammation
• multiple sclerosis