Methods: We established protocols for enzymatic α2,6-sialylation (ST6GalNAc-I or II) or α2,3-sialylation (ST3Gal1; adds NeuAc to galactose) of IgA1 O-glycans of an asialo-IgA1 myeloma protein (Ale) that mimics the Gal-deficient IgA1 in IgAN patients. The products of sialyltransferase reactions were assessed by high-resolution
mass spectrometry and ELISA with the GalNAc-specific lectin from Helix aspersa (HAA). Results: Changes in SDS-PAGE mobility of the IgA1 heavy chain indicated that both enzymes were active. Enzymatic sialylation of the myeloma protein generated sialylated IgA1 that mimics the circulating nephritogenic IgA1 in IgAN patients, characterized by α2,6-sialylated GalNAc, or the IgA1 typical for healthy controls, characterized by an α2,3-sialylated Ibrutinib datasheet Gal attached to GalNAc. Lectin ELISA was used to assess binding to Deforolimus datasheet the IgA1 before and after the enzymatic reactions. α2,6- as well as α2,3-sialylation of IgA1 markedly decreased reactivity with the HAA lectin. Neuraminidase treatment (to remove sialic acid) completely restored the level of lectin reactivity. Thus, lectin binding to GalNAc decreased after sialylation of Gal on
a nearby glycan in the cluster of O-glycans of the IgA1 HR. Conclusion: Neuraminidase should be used to remove sialic acid from serum IgA1 before a lectin assay to assess the total content of HR Gal-deficient GalNAc. Our in vitro enzymatic sialylation model will be useful to study the biological roles of NeuAc in the IgA1 HR in the pathogenesis of IgAN. SUZUKI HITOSHI1, YANAGAWA HIROYUKI1, SUZUKI YUSUKE1, KIRYLUK KRZYSZTOF2, GHARAVI ALI G2, MATSUOKA JOE3, MAKITA YUKO1, JULIAN BRUCE A4,5, NOVAK JAN5, TOMINO YASUHIKO1 1Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine; 2Department of Medicine, Columbia University; 3Clinical Research Center, Juntendo University Faculty of Medicine; 4Departments of Medicine, University of BCKDHB Alabama at Birmingham; 5Departments of Microbiology, University of Alabama at Birmingham
Introduction: IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. There is increasing evidence that galactose-deficient IgA1 (Gd-IgA1) and Gd-IgA1-containing immune complexes are important players in the pathogenesis of IgA nephropathy (IgAN). Moreover, serum levels of Gd-IgA1-specific antibodies (IgG and IgA), responsible for the formation of immune complexes with Gd-IgA1, are also elevated in IgAN. In the present study, we assessed a novel noninvasive approach using multi-biomarkers combined with analysis of clinical data by a logistic model as a diagnostic test for IgAN. Methods: We compared serum levels of IgA, IgG, Gd-IgA1, Gd-IgA1-specific IgG and Gd-IgA1-specific IgA in 135 IgAN patients, 79 patients with non-IgAN chronic kidney disease (CKD) controls and 106 healthy controls.