Each N-glycan on human IgA and J-chain uniquely affects oligomericity and stability

Abstract

Background: Immunoglobulin A (IgA) plays a pivotal role in various immune responses, especially that of mucosal immunity. IgA is usually assembled into dimers with the contribution of J-chains. There are two N-glycosylation sites in human IgA1-Fc and one in the J-chain. There is no consensus as yet on the functional role of the N-glycosylation.

Methods: To gain a better understanding of their role, we designed a series of IgA1-Fc mutants, which were expressed in the absence or presence of the J-chain.

Results: IgA1-Fc without the J-chain, was predominantly expressed as a monomer, and in its presence dimers and some polymers appeared. N263 (Fc Cα2), N459 (Fc tailpiece) and N49 (J-chain) were shown to be site-specifically modified with N-glycans by mass spectrometry analysis. Mutant IgA1-Fc N459Q failed to form a proper dimer in the presence of the J-chain, instead higher-order aggregates appeared. Fluorescence experiments suggest that the N459-glycans cover a hydrophobic surface at the Fc tailpiece that prevents other Fc molecules from approaching the dimeric IgA. A thermofluor assay revealed that the N-glycans at N263 (Fc) and N49 (J-chain) both contribute in different ways to the thermal stability of the Fc-J-chain complex. NMR analysis of ¹³C-labeled Fc suggests that the N459-glycan is relatively flexible while the N263-glycan is more rigid.

Conclusions: We conclude that the N459-glycan of IgA1-Fc is essential for dimer formation and prevention of higher-order aggregates while those at N263 (Fc) and N49 (J-chain) stabilize the Fc-J-chain complex.

General significance: Site-specific role for N-glycan in molecular assembly is addressed.

Keywords: Fc; Immunoglobulin A (IgA); J-chain; N-linked glycosylation; Protein assembly; Protein stability.