Methodological approaches to the analysis of IgA1 O-glycosylation in IgA nephropathy

IgA nephropathy (IgAN) is a common form of glomerulonephritis in which IgAl molecules deposit in the renal mesangium, leading to progressive glomerula r inflammatory injury in a significant proportion of patients. The mechanis ms underlying the pathogenesis of IgAN remain poorly understood, but altere d O-glycosylation, a physicochemical abnormality of IgAl observed in these patients, may be a contributory faster. Although many studies have reported aberrant IgAl O-glycosylation in IgAN, the precise structural nature of th e defect remains to be fully characterised, and analysis of IgAl O-glycans has proved technically challenging. Three main strategies have been employe d: lectin binding to the O-glycans in situ on the whole IgAl molecule; mass spectroscopy of isolated, O-glycosylated glycopeptides; and size/charge se paration of free O-glycans released from IgAl. In this review, the basic pr inciples, strengths and weaknesses of each of these methodological approach es are considered, together with a summary of the data obtained from their use. One of the common criticisms of many studies of IgAl O-glycosylation i s the method of IgAl purification employed, and therefore, this issue is al so critically discussed.