IgA1 hinge-region clustered glycan fidelity is established early during semi-ordered glycosylation by GalNAc-T2

Abstract

GalNAc-type O-glycans are often added to proteins post-translationally in a clustered manner in repeat regions of proteins, such as mucins and IgA1. Observed IgA1 glycosylation patterns show that glycans occur at similar sites with similar structures. It is not clear how the sites and number of glycans added to IgA1, or other proteins, can follow a conservative process. GalNActransferases initiate GalNAc-type glycosylation. In IgA nephropathy, an autoimmune disease, the sites and O-glycan structures of IgA1 hinge-region are altered, giving rise to a glycan autoantigen. To better understand how GalNAc-transferases determine sites and densities of clustered O-glycans, we used IgA1 hinge-region (HR) segment as a probe. Using LC-MS, we demonstrated a semi-ordered process of glycosylation by GalNAc-T2 towards the IgA1 HR. The catalytic domain was responsible for selection of four initial sites based on amino-acid sequence recognition. Both catalytic and lectin domains were involved in multiple second site-selections, each dependent on initial site-selection. Our data demonstrated that multiple start-sites and follow-up pathways were key to increasing the number of glycans added. The lectin domain predominately enhanced IgA1 HR glycan density by increasing synthesis pathway exploration by GalNAc-T2. Our data indicated a link between site-specific glycan addition and clustered glycan density that defines a mechanism of how conserved clustered O-glycosylation patterns and glycoform populations of IgA1 can be controlled by GalNAc-T2. Together, these findings characterized a correlation between glycosylation pathway diversity and glycosylation density, revealing mechanisms by which a single GalNAc-T isozyme can limit and define glycan heterogeneity in a disease-relevant context.

TLR7 in B cells promotes renal inflammation and Gd-IgA1 synthesis in IgA nephropathy

“…The hinge region of IgA1 contained more exposed GalNAc residues in patients with IgAN than in healthy individuals (73,74), which may be explained by the relative insufficiency of Gal residues compared with GalNAc residues. Recently, Stewart et al have shown that GALNT2 is a critical determinant of the number and pattern of O-glycans added to the hinge region of IgA1 molecules (75). Therefore, the ratio of C1GALT1/GALNT2 likely determines the O-glycan composition of IgA1 molecules, and our results support this hypothesis.…”

Racial heterogeneity of IgA1 hinge-region O-glycoforms in patients with IgA nephropathy

“…Humans express 20 isoforms of ppGalNAc-Ts, among which ppGalNAc-T1, T2, T3, T4, T6, and T9 are expressed in IgA1-producing B cells ( Gerken et al., 2013 ; Iwasaki et al., 2003 ). Our in vitro studies demonstrated that pre-existing glycans affect sites of GalNAc attachment by GalNAc-Ts, as well as subsequent activity of other glycosyltransferases ( Stewart et al., 2019 , 2020 ). This study suggested that the expression or activity of ppGalNAc-Ts may be altered in IgA1-producing cells of patients with IgAN, resulting in secretion of IgA1 with under-glycosylated HR.…”

Mucinomics as the Next Frontier of Mass Spectrometry

“…Follow-up studies have focused on the sequential addition of glycosites to the mucin domain by various glycosyltransferases including GalNAc-T2, GalNAc-T14, core 1 β1,3-galactosyltransferase1 (C1GalT1), ST6GalNAc2, and ST3Gal1. While this work was not necessarily focused on the site-specific analysis of the hinge region, the authors were able to identify eight sites of GalNAcylation in a time and enzyme resolved manner. , Finally, Takahashi and colleagues investigated an O-glycanase, which removes GalNAc-Gal glycans, in the analysis of glycopeptides in the IgA hinge region. In two separate analyses, the authors digested with (a) sialidase and trypsin or (b) an IgA protease, sialidase, O-glycanase, and trypsin .…”