Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity
Hitoshi Suzuki, … , Jiri Mestecky, Jan Novak
Hitoshi Suzuki, … , Jiri Mestecky, Jan Novak
Published May 26, 2009
Citation Information: J Clin Invest. 2009;119(6):1668-1677. https://doi.org/10.1172/JCI38468.
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Research Article

Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity

Abstract

IgA nephropathy (IgAN) is characterized by circulating immune complexes composed of galactose-deficient IgA1 and a glycan-specific IgG antibody. These immune complexes deposit in the glomerular mesangium and induce the mesangioproliferative glomerulonephritis characteristic of IgAN. To define the precise specificities and molecular properties of the IgG antibodies, we generated EBV-immortalized IgG-secreting lymphocytes from patients with IgAN and found that the secreted IgG formed complexes with galactose-deficient IgA1 in a glycan-dependent manner. We cloned and sequenced the heavy- and light-chain antigen-binding domains of IgG specific for galactose-deficient IgA1 and identified an A to S substitution in the complementarity-determining region 3 of the variable region of the gene encoding the IgG heavy chain in IgAN patients. Furthermore, site-directed mutagenesis that reverted the residue to alanine reduced the binding of recombinant IgG to galactose-deficient IgA1. Finally, we developed a dot-blot assay for the glycan-specific IgG antibody that differentiated patients with IgAN from healthy and disease controls with 88% specificity and 95% sensitivity and found that elevated levels of this antibody in the sera of patients with IgAN correlated with proteinuria. Collectively, these findings indicate that glycan-specific antibodies are associated with the development of IgAN and may represent a disease-specific marker and potential therapeutic target.

Authors

Hitoshi Suzuki, Run Fan, Zhixin Zhang, Rhubell Brown, Stacy Hall, Bruce A. Julian, W. Winn Chatham, Yusuke Suzuki, Robert J. Wyatt, Zina Moldoveanu, Jeannette Y. Lee, James Robinson, Milan Tomana, Yasuhiko Tomino, Jiri Mestecky, Jan Novak

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Figure 1

Serum IgG from IgAN patients exhibits specificity for GalNAc, binding to Gal-deficient and desialylated IgA1.

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Serum IgG from IgAN patients exhibits specificity for GalNAc, binding to...
(A) Western blot analysis with Gal-deficient IgA1 (Mce) as antigen demonstrated binding of serum IgG from 2 IgAN patients but only minimal binding of IgG from 2 healthy controls to the IgA1 heavy chain. After removal of sialic acid, IgG binding increased, as it did for binding to HAA. N+, treated with neuraminidase; N-, not treated with neuraminidase. (B) To test glycan-specific IgG binding to GalNAc, these IgA1 proteins were used: lane 1, Gal-deficient IgA1 (Mce); lane 2, dd-IgA1; lane 3, enzymatically regalactosylated dd-IgA1; and lane 4, enzymatically resialylated dd-IgA1. dd-IgA1 bound the greatest amount of HAA, with enzymatically galactosylated or sialylated dd-IgA1 binding very little. IgG from an IgAN patient bound to these antigens in a fashion similar to that for HAA. (C and D) Component chains of Gal-deficient IgA1 (Mce) were separated by SDS-PAGE under reducing conditions and electroblotted. The membrane was then treated with HAA to assess whether blockade with this GalNAc-specific lectin can inhibit IgG binding. The intensity of each band was quantified by densitometry. The binding of serum IgG from an IgAN patient to Gal-deficient IgA1 was reduced by 66% after treatment with HAA. Conversely, blocking with serum IgG from an IgAN patient reduced the binding of HAA to Gal-deficient IgA1 by 60%. Binding of anti-human IgA (heavy-chain specific) confirmed equivalent loading. Representative results from 3 experiments are shown in A–C; lanes were run on the same gel but were noncontiguous.