Enzymatic sialylation of IgA1 O-glycans: implications for studies of IgA nephropathy.

Patients with IgA nephropathy (IgAN) have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal)-deficient N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc). We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale) myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1) of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal) than α2,6-sialylated O-glycans (NeuAc attached to GalNAc). As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc), we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale) myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA) is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a GalNAc-specific lectin is modulated by sialylation of GalNAc as well as Gal in the clustered IgA1 O-glycans. Thus, enzymatic sialylation offers a useful model to test the role of NeuAc in reactivities of the clustered O-glycans with lectins.

Aberrant O-glycosylation and anti-glycan antibodies in an autoimmune disease IgA nephropathy and breast adenocarcinoma.

Glycosylation abnormalities have been observed in autoimmune diseases and cancer. Here, we compare mechanisms of aberrant O-glycosylation, i.e., formation of Tn and sialyl-Tn structures, on MUC1 in breast cancer, and on IgA1 in an autoimmune disease, IgA nephropathy. The pathways of aberrant O-glycosylation, although different for MUC1 and IgA1, include dysregulation in glycosyltransferase expression, stability, and/or intracellular localization. Moreover, these aberrant glycoproteins are recognized by antibodies, although with different consequences. In breast cancer, elevated levels of antibodies recognizing aberrant MUC1 are associated with better outcome, whereas in IgA nephropathy, the antibodies recognizing aberrant IgA1 are part of the pathogenetic process.