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Background <p>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 <i>N</i>-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 <i>N</i>-glycans on IgE-Fc, particularly the high-mannose glycan attached to Asn394.</p> Methods <p>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.</p> Results <p>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 <i>N</i>-glycans revealed that high-mannose glycans at Asn394 associate in a non-selective manner between heavy chains.</p> Conclusions <p>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 <i>N</i>-glycan processing during IgE biosynthesis, thereby advancing our understanding of the molecular mechanisms underlying IgE structure and function.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月15日

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  • Sakurako Nomura,
  • Tatsuro Iwabuchi,
  • Haruka Sawataishi,
  • Kizashi Kamo,
  • Noriyoshi Manabe,
  • Ryohei Uematsu,
  • Izumi Sakamoto,
  • Kenji Hirose,
  • Eunsang Kwon,
  • Yoshiki Yamaguchi

摘要

Background

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.

Methods

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.

Results

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.

Conclusions

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.