N-linked glycosylation is a protein post-trasductional modification that results in the covalent attachment of an oligosaccharide onto a given asparagine residue at the polypeptide chains . It is involved in protein folding, trafficking, and stability, and regulates many cellular activities, especially of extracellular nature . The αsubunit of the IgE high affinity receptor (FcɛRIα) is a transmembrane protein with 7 N-glycosylation sites on the extracellular domain. The glycosylation is key for secretion and stability of the protein .
Herein we have analysed the glycoprofile of the FcɛRIαexpressed in human HEK 293T cells, identifying the presence and the relative abundance of specific glycan epitopes, using Nuclear Magnetic Resonance Spectroscopy.
The anomeric region of the 1H,13C-HSQC spectrum shows a high degree of heterogeneity in terms of glycosylation. The deduced structures include the presence of high-mannose (23%), hybrid (35%), and bi- tri- and tetra-antennary complex type (32%) N-glycans with different degrees of fucosylation and sialylation (Fig 1).
The estimation of the solvent accessible surface area (SASA) of FcɛRIαrevealed that Asn132 is the most protected residue. For that reason, we guessed that the high mannose glycan chain is attached to this site. Molecular modelling protocols permitted to guess that the glycan is packed within the two immunoglobulin domains, thus contributing to the stability and structure of the glycoprotein. This hypothesis has been evaluated by expressing the Asn132Ala mutant. The analysis of the secondary structure of the mutant protein using circular dichroism along with the quantification of the 1H,13C-HSQC spectrum has permitted to describe the existing differences in glycan composition between the WT and mutant poroteins.
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