Analysis of IgA1 O-glycans in IgA nephropathy by fluorophore-assisted carbohydrate electrophoresis

Abnormal O-glycosylation of IgA1 may contribute to pathogenic mechanisms in IgA nephropathy (IgAN). Observations of altered lectin binding to IgA1 in IgAN suggest that the O-glycan chains may be undergalactosylated, but preci se structural definition of the defect has proved technically difficult, an d it remains unconfirmed. This is the first study using fluorophore-assiste d carbohydrate electrophoresis (FACE) to analyze IgA1 O-glycans in IgAN and controls. IgA1 was purified from serum, and the intact O-glycans were rele ased by hydrazinolysis at 60 degrees C. After re-N-acetylation, the glycans were fluorophore-labeled and separated by polyacrylamide gel electrophores is. Sequential exoglycosidase digestions of IgA1 allowed identification of the different O-glycan bands on FACE gels, and their relative frequencies i n IgA1 samples were measured by ultraviolet densitometry. Lectin binding of the IgA1 samples was also measured. In some patients with IgAN, FACE analy sis demonstrated a significant increase in the percentage of IgA1 O-glycan chains consisting of single N-acetyl galactosamine (GalNAc) units rather th an the more usual galactosylated and sialylated forms. This finding was con firmed using both desialylated IgA1 and enzymatically released O-glycans. G ood correlation was also found between O-glycan agalactosylation on FACE an alysis and IgA1 lectin binding in IgAN, supporting the value of lectins as tools for detection of this abnormality. This is the first study in which a ll of the predicted O-glycan forms of IgA1 have been analyzed simultaneousl y, and demonstrates that in IgAN, the IgA1 O-glycan chains are truncated, w ith increased terminal GalNAc. This abnormality has the potential to signif icantly affect IgA1 behavior and handling with pathogenic consequences in I gAN.