Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity
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
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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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 4

Importance of the A to S substitution in YCAR/K sequence of CDR3 in the binding of IgG to Gal-deficient IgA1.

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Importance of the A to S substitution in YCAR/K sequence of CDR3 in the ...
(A) Western blot analysis using Gal-deficient IgA1 (Ale poly) as antigen demonstrated binding of rIgG cloned from an IgAN patient (subject 1123) but only marginal binding of rIgG from a healthy control (subject 9017). (B) The reduced Gal-deficient IgA1 (Mce1) (lane 1); enzymatically desialylated Gal-deficient IgA1 (lane 2); and desialylated and degalactosylated Gal-deficient IgA1 (lane 3) were incubated with rIgG after SDS-PAGE/Western blotting. Removal of sialic acid and Gal in the IgA1 hinge region increased the binding, suggesting that the rIgG bound specifically to GalNAc. (C) The aa sequence (YCSK) in the CDR3 of VH of IgG from an IgAN patient (subject 1123) was reverted to the healthy control germline counterpart sequence (YCAK) using an overlap PCR strategy. Conversely, the CDR3 of IgG from a healthy control (subject 9017) was mutated to generate YCSR. (D) After the S to A substitution was introduced in CDR3 of VH of IgG of the cells from an IgAN patient (subject 1123), rIgG binding to Gal-deficient IgA1 was reduced by 72%. Conversely, the A to S substitution in CDR3 of IgG of the cells from a healthy control (subject 9017) increased binding to Gal-deficient IgA1. Anti-human IgA (heavy-chain specific) Western blotting was used as load control. Results were evaluated densitometrically. Representative results from 2 experiments are shown in A–D; lanes were run on the same gel but were noncontiguous.