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 5

Serum levels of IgG specific for Gal-deficient IgA1 are elevated in patients with IgAN.

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Serum levels of IgG specific for Gal-deficient IgA1 are elevated in pati...
(A) Gal-deficient IgA1 (Ale) placed in 96-well plates with PVDF membranes was incubated with normalized concentrations of serum IgG from IgAN patients, disease controls, and healthy controls; a representative example from 3 experiments is shown (20 samples from each group). The rIgG from an IgAN patient served as a positive control. Serum IgG from IgAN patients bound more to Gal-deficient IgA1 compared with the IgG from disease controls or healthy controls. (B) The intensity of signal in each well was measured by densitometry; the intensity of rIgG bound to Gal-deficient IgA was assigned a value of 100%. Serum IgG from IgAN patients has significantly higher reactivity to Gal-deficient IgA1 compared with that from healthy (P < 0.0001) and disease controls (P < 0.0001). Serum IgG from 54 of the 60 patients with IgAN showed values greater than the 90th percentile of the values for healthy controls. Wilcoxon’s rank-sum test was used for 2-sample comparison. Data are shown as individual values and the mean ± SD. (C) ROC for serum IgG binding to Gal-deficient IgA1. The area under the curve is 0.9644. These data indicate a sensitivity of 88.3% and a specificity of 95.0% (P < 0.0001; 95% CI, 0.928-1.00). The value of specificity is plotted as 1-specificity on the x axis. (D) The intensity of IgG binding to Gal-deficient IgA1 correlated with the UP/Cr ratio (P < 0.0001) as well as with urinary IgA-IgG immune complexes (E) (P = 0.0082) in contemporaneously collected urine samples. UIgA-IgG IC/Cr, urinary excretion of IgA-IgG immune complexes/creatinine ratio.