CHARACTERIZATION OF THE KEY AUTOANTIGEN, ABERRANTLY GLCOSYLATED IGA1, IN IGA NEPHROPATHY
Jocelyne Fadiga1, Jan Novak2.
1University of California, Merced, Merced, CA, 2The University of Alabama at Birmingham, Birmingham, AL.
IgA nephropathy (IgAN) is a chronic kidney disease with up to 40% of patients progressing to end-stage renal disease (ESRD). IgAN is characterized by IgA1-containing immunodeposits in the mesangium. These immunodeposits, enriched for galactose-deficient IgA1 (Gd-IgA1), likely originate from the circulating immune complexes consisting of Gd-IgA1 bound by antiglycan autoantibodies. Gd-IgA1 is produced by IgA1-producing cells due to abnormal expression of key glycosylation enzymes. Serum levels of Gd-IgA1 are associated with disease progression. Serum IgA1 is predominantly monomeric, with only about 10% in the polymeric form as dimers or higher polymers. To better characterize the autoantigen, we analyzed the O-glycosylation of polymeric and monomeric IgA1 from serum and secreted by IgA1-producing cells. IgA1 in serum and media of immortalized cells derived from healthy controls and patients with IgAN, and the mono/polymeric forms were isolated by affinity and size-exclusion chromatography. The molecular form of IgA1 was assessed by nonreducing SDS-PAGE, and the degree of galactose deficiency was determined by enzyme linked immunosorbent assay (ELISA) with lectin from Helix aspersa (HAA) and mass spectrometry. We tested the separation of polymeric and monomeric IgA1 using several matrices at low pressure vs. standard high-pressure liquid chromatography. We hypothesize that the lectin-reactive serum IgA1 (Gd-IgA1) is predominantly in immune complexes and free polymeric form and that most Gd-IgA1 produced by IgA1-secreting cells is a polymer. In IgAN, polymeric IgA1 forms are more reactive with Gd-IgA1-specific lectin. These findings will need to be validated using a larger cohort of subjects and analyses expanded by mass spectrometry profiling.