Secretory IgA N- and O-Glycans Provide a Link between the Innate and Adaptive Immune Systems

Abstract

Secretory IgA (SIgA) is a multi-polypeptide complex consisting of a secretory component (SC) covalently attached to dimeric IgA containing one joining (J) chain. We present the analysis of both the N-and O-glycans on the individual peptides from this complex. Based on these data, we have constructed a molecular model of SIgA1 with all its glycans, in which the Fab arms form a T shape and the SC is wrapped around the heavy chains. The O-glycan regions on the heavy (H) chains and the SC N-glycans have adhesin-binding glycan epitopes including galactose-linked ␤1-4 and ␤1-3 to GlcNAc, fucoselinked ␣1-3 and ␣1-4 to GlcNAc and ␣1-2 to galactose, and ␣2-3 and ␣2-6-linked sialic acids. These glycan epitopes provide SIgA with further bacteria-binding sites in addition to the four Fab-binding sites, thus enabling SIgA to participate in both innate and adaptive immunity. We also show that the N-glycans on the H chains of both SIgA1 and SIgA2 present terminal GlcNAc and mannose residues that are normally masked by SC, but that can be unmasked and recognized by mannosebinding lectin, by disrupting the SC-H chain noncovalent interactions.

Recognition of Gram-positive Intestinal Bacteria by Hybridoma- and Colostrum-derived Secretory Immunoglobulin A Is Mediated by Carbohydrates

“…We found that carbohydrates present on the ␣ chain could not be removed following exposure to N-glycosidase F. This observation is consistent with the masking of N-glycan residues by SC in the context of the whole SIgA molecule (20,51). It is plausible that these noncleavable carbohydrates would be responsible for the various residual interaction observed upon incubation of Gram-positive bacteria with SIgAdg.…”

“…Because SIgA and SC are highly glycosylated proteins (19,20), we performed deglycosylation cleaving all types of N-branched glycan residues bound to asparagine. Contrary to most of other methods, N-glycosidase F removes carbohydrate residues by preserving the conformational structure of the protein (23).…”

“…In secretions, SC is bound covalently, as well as noncovalently, to IgA, and is found also as free SC (18). Both polymeric IgA (pIgA) and SC are heavily glycosylated (19,20); remarkably, N-and O-carbohydrate residues have already been implicated in the interaction with bacteria, participating in the protection against enteric pathogens (20 -25) either as adhesion competitors or in anchoring SIgA in the mucus to ensure optimal biological function. In face of the increasing involvement of glycans in the function of SIgA, we sought to determine their potential role in commensal binding (25,26).…”

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

“…As previously reported, IgA1 with truncated O-glycans in the hinge region is prone to self-aggregation and forms immune complexes with the IgG antibody against the hinge region of IgA1. 37 As such, the O-linked glycans at the hinge region may affect the stability and function of Fc fusion proteins. 37 In the CTLA4-Ig fusion proteins, we detected the O-linked glycans on T8-9 (SSDKTHTSPPSPA-PELLGGSSVFLF PPKPK).…”

“…37 As such, the O-linked glycans at the hinge region may affect the stability and function of Fc fusion proteins. 37 In the CTLA4-Ig fusion proteins, we detected the O-linked glycans on T8-9 (SSDKTHTSPPSPA-PELLGGSSVFLF PPKPK). As shown in Figure 3D, at the lower (6v) MS level, we confirmed the peptide sequence utilizing collision-induced dissociation (CID) for glycopeptide fragmentation.…”