Pairing of high-mannose glycans in human IgE-Fc: implications for secretion and stability

Immunoglobulin E (IgE) is the least abundant antibody class in serum, but plays a central role in type I allergic responses. The Fc region of human IgE (IgE-Fc) contains four potential N-glycosylation sites: Asn265 and Asn371 are modified with complex-type glycans; Asn394 predominantly carries a high-mannose glycan; and Asn383 remains unmodified. Despite the recognized importance of glycosylation in antibody function, the structure-function relationships of the individual IgE-Fc glycans remain poorly understood. This study aimed to elucidate the structural and functional significance of N-glycans on IgE-Fc, particularly the high-mannose glycan attached to Asn394.

The expression and secretion of recombinant human IgE-Fc constructs including wild-type IgE-Fc, a high-mannose-deficient mutant (N394Q), and a triple mutant lacking complex-type glycans (N265Q/N371Q/N383Q) was assessed in mammalian Expi293F cells. In addition, the thermal stability of wild-type IgE-Fc treated with Endo H or PNGase F was evaluated by a thermal shift assay. Glycan compositions and pairing patterns in IgE-Fc were characterized by intact mass spectrometry (MS) and liquid chromatography-MS analysis of released glycans.

Wild-type IgE-Fc and the triple mutant were stably expressed, whereas secretion of the high-mannose-deficient mutant (N394Q) was markedly impaired, demonstrating that the Asn394 glycan is essential for proper folding and efficient secretion. Endo H treatment significantly decreased the melting temperature of wild-type IgE-Fc, indicating that high-mannose glycans contribute to structural stability. Integration of MS data on the intact protein and LC-MS data on the enzyme-released N-glycans revealed that high-mannose glycans at Asn394 associate in a non-selective manner between heavy chains.

These findings establish that the high-mannose glycan at Asn394 is required for both the secretion and structural stability of IgE-Fc. Furthermore, the observation of non-selective glycan pairing between heavy chains provides insight into the temporal coordination of disulfide bond formation and N-glycan processing during IgE biosynthesis, thereby advancing our understanding of the molecular mechanisms underlying IgE structure and function.