IgA Nephropathy Caused by Unusual Polymerization of IgA1 with Aberrant N-Glycosylation in a Patient with Monoclonal Immunoglobulin Deposition Disease

<div><p>Immunoglobulin A nephropathy (IgAN) is a form of chronic glomerulonephritis characterized by the deposition of IgA immune complexes in the glomerular region. The cause of IgAN is unknown, but multiple mechanisms have been suggested. We previously reported a rare case of mesangioproliferative glomerulonephritis in a patient with monoclonal immunoglobulin deposition disease associated with monoclonal IgA1. In this study, we performed the detailed analyses of serum IgA1 from this patient in comparison with those from patients with mIgA plasma cell disorder without renal involvement and healthy volunteers. We found unusual polymerization of IgA1 with additional <i>N</i>-glycosylation distinctive in this patient, which was different from known etiologies. Glycan profiling of IgA1 by the lectin microarray revealed an intense signal for <i>Wisteria floribunda</i> agglutinin (WFA). This signal was reduced by disrupting the native conformation of IgA1, suggesting that the distinct glycan profile was reflecting the conformational alteration of IgA1, including the glycan conformation detected as additional <i>N</i>-glycans on both the heavy and light chains. This unusually polymerized state of IgA1 would cause an increase of the binding avidity for lectins. WFA specifically recognized highly polymerized and glycosylated IgA1. Our results of analysis in the rare case of a patient with monoclonal immunoglobulin deposition disease suggest that the formation of unusually polymerized IgA1 is caused by divergent mechanisms including multiple structural alterations of glycans, which contributes to IgA1 deposition and mesangium proliferation.</p></div

Sandwich lectin ELISA of sequential deglycosylated IgA1 in mIgA-MIDD.

<p>IgA1 purified from mIgA-MIDD serum was digested with neuraminidase, and then β-galactosidase or β1,4-galactosidase. Digested and undigested samples were subjected to a sandwich lectin ELISA with HPA (A), VVA (B), PNA (C), and WFA (D) as described in the Methods. The relative intensity of each lectin was normalized to the IgA1 concentration. HPA, <i>Helix pomatia</i> agglutinin; VVA, <i>Vicia villosa</i> lectin; PNA, peanut agglutinin.</p

Differential glycan profiles of purified IgA1.

<p>IgA1 purified from sera of three HVs, two MPCD patients, and one mIgA-MIDD patient was subjected to lectin microarray. IgA1-binding signals on the lectin microarray were detected with a biotinylated anti-IgA1 mAb. The relative intensity of each lectin was normalized to the maximum fluorescence intensity. mIgA-MIDD, monoclonal immunoglobulin deposition disease associated with monoclonal IgA; MPCD, monoclonal IgA plasma cell disorder; HV, healthy volunteers.</p

SDS-agarose gel electrophoresis of purified IgA1.

<p>IgA1 purified from sera of two HV, two MPCD patients, and an mIgA-MIDD patient was incubated in SDS buffer and then subjected to agarose gel electrophoresis analysis.</p

Time course of PNGase F treatment of IgA1.

<p>IgA1 purified from the sera of the mIgA-MIDD patient (A) and an MPCD patient (B) was digested partially with PNGase F for the indicated times (lanes 1–5, 7–10). Complete digestion of <i>N</i>-glycans was performed by incubation with PNGase F for 120 min (lanes 6, 11). Arrowheads indicate the position of PNGase F.</p

Differential glycan profiles of purified IgA1 after trypsin digestion.

<p>IgA1 purified from sera of three HVs, two MPCD patients, and one mIgA-MIDD patient was incubated with trypsin. Each tryptic digest was labeled with Cy3-SE and subjected to the lectin microarray. The relative intensities of lectins were normalized to the maximum fluorescence intensity.</p

Characterization of Binding Epitopes of CA125 Monoclonal Antibodies

The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics of the recognized epitopes and the role played by glycosylation has remained elusive. Here a comprehensive set of recombinant MUC16 tandem repeats (TRs) expressed in glycoengineered mammalian cells and <i>E. coli</i>, together with overlapping peptides, was used to probe antigen-binding epitopes. We present a complete analysis of N- and O-glycosylation sites of a MUC16 TR expressed in CHO cells and demonstrate that neither N- nor O-glycosylation appear to substantially influence binding of OC125 and M11 mAbs. A series of successive N- and C-terminal truncations of a MUC16 TR construct expressed in <i>E. coli</i> narrowed down the epitopes for OC125 and M11 to a segment containing parts of two consecutive SEA domains with a linker. Thus, a complete SEA domain is not required. These findings suggest that binding epitopes of mAbs OC125 and M11 are dependent on conformation but not on glycosylation. The availability of recombinant TR constructs with and without aberrant glycosylation now opens the way for vaccine studies

DataSheet_1_Role of N-Glycosylation in FcγRIIIa interaction with IgG.pdf

Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.</p

Table_1_Role of N-Glycosylation in FcγRIIIa interaction with IgG.xlsx

Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.</p