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.

Figure 1. Serum IgG from IgAN patients exhibits specificity for GalNAc, binding to Gal-deficient and desialylated IgA1.

(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.

Figure 2. Characterization of antibodies specific for Gal-deficient IgA1 secreted by cloned cell lines.

The levels of antigen-specific IgG produced by IgG-secreting cell lines were measured by capture ELISA. The results are expressed as OD measured at 490 nm. Levels of IgG directed against dd-IgA1 (A) and Fab-IgA1 (B) were higher in IgAN patients than in controls. Each group, n = 16. **P < 0.0001; data are shown as individual values and mean ± SD. (C) IgG secreted by cell lines from IgAN patients and healthy controls (each group, n = 10) was tested for binding with a hinge-region glycopeptide (HR-GalNAc-BSA) or HR-BSA, with or without HAA blockade. IgG produced by cell lines from IgAN patients bound to HR-GalNAc in an HAA-inhibitable fashion. *P < 0.001; data are shown as the mean ± SD. P values were generated using 2-tailed Student’s t test. The experiments were repeated 3 times with similar results.

Figure 3. Characterization of immune-complex formation.

(A) Size-exclusion chromatography and ELISA analysis of immune complexes formed in vitro with monomeric Gal-deficient IgA1 (50 μg) and glycan-specific IgG (50 μg) from 3 patients with IgAN (filled circles) or 3 healthy controls (open circles). IgG and monomeric (m) and dimeric (d) IgA1 standards were used to calibrate the column. Glycan-specific IgG from IgAN patients exhibited more binding to Gal-deficient IgA1 as compared with the binding of IgG from healthy controls. Immune complexes likely contained 1 or 2 molecules of IgA1 bound to 1 molecule of IgG. Data are shown as mean ± SD. (B) Dot-blot analysis showed that IgG secreted by cell lines from 5 of the 6 IgAN patients exhibited high binding to Gal-deficient IgA1; cell line no. 3081 from an IgAN patient and cells from 5 of the 6 healthy controls exhibited low binding. (C) Findings shown in Table 1 were confirmed by densitometrical analysis. P < 0.01; P values were generated using the 2-tailed Student’s t test. Data are shown as individual values and mean ± SD. Experiments were repeated 3 times with similar results.

Figure 4. Importance of the A to S substitution in YCAR/K sequence of CDR3 in the binding of IgG to Gal-deficient IgA1.

(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 V_H 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 V_H 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.

Figure 5. Serum levels of IgG specific for Gal-deficient IgA1 are elevated in patients with IgAN.

(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.