Intricate Proteoform Heterogeneity in the Tailpiece of IgA within and across Its Circulatory and Secretory Configurations

Human immunoglobulin A (IgA) naturally occurs in multiple molecular forms─monomers, dimers, and even higher-order polymers─that are differentially distributed across serum, breast milk, and mucosal surfaces. The C-terminal tailpiece of IgA is essential to its polymerization mediated by the joining chain (J-chain) and thus also an important determinant of its extensive structural heterogeneity, yet its features remain somewhat poorly defined. Here, we used HILIC-LC-MS/MS-based glycopeptide analysis to characterize the N-glycan structures, disulfide linkages, and C-terminal amino acid sequence variants within the IgA tailpiece across the different molecular configurations of endogenous IgA. We find extensive correlation between these post-translational modifications; i.e., variations in tailpiece Asn459 N-glycosylation, redox status and disulfide connectivity of Cys471, and the presence or absence of C-terminal Tyr472 are intrinsically interconnected. When Cys471 was disulfide-bound to the J-chain, Asn459 glycans showed broader structural heterogeneity and retained more high-mannose structures, whereas free Cys471 tailpieces mainly carried mature, sialylated complex-type glycans. Serum IgA and milk SIgA displayed distinct tailpiece signatures, including differences in N-glycan maturation and processing of the ultimate C-terminal Tyr472 residue. Together, these findings define biofluid-specific molecular forms of IgA and highlight the remarkable structural diversity of the human IgA tailpiece. Overall, our results advance the understanding of the IgA structure and could offer a much-needed molecular basis to assist future bioengineering of IgA-based therapeutics.