Naturally Occurring Structural Isomers in Serum IgA1 <i>O</i>-Glycosylation

IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of <i>O</i>-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr²²⁵, Thr²²⁸, Ser²³⁰, Ser²³² and Thr²³⁶ have been identified as the predominant sites of <i>O</i>-glycan attachment. The range and distribution of <i>O</i>-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 <i>O</i>-glycan heterogeneity by use of high resolution LC–MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 <i>O</i>-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the <i>O</i>-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable <i>O</i>-glycan chains attached to Ser²³⁰, Thr²³³ or Thr²³⁶ produce the predominant positional isomers, including <i>O</i>-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 <i>O</i>-glycoforms, define the single-site heterogeneity for all <i>O</i>-glycan sites in a single sample, and have implications for defining epitopes based on clustered <i>O</i>-glycan variability

Serum galactose-deficient-IgA1 and IgG autoantibodies correlate in patients with IgA nephropathy

<div><p>IgA nephropathy is an autoimmune disease characterized by IgA1-containing glomerular immune deposits. We previously proposed a multi-hit pathogenesis model in which patients with IgA nephropathy have elevated levels of circulatory IgA1 with some <i>O</i>-glycans deficient in galactose (Gd-IgA1, autoantigen). Gd-IgA1 is recognized by anti-glycan IgG and/or IgA autoantibodies, resulting in formation of pathogenic immune complexes. Some of these immune complexes deposit in the kidney, activate mesangial cells, and incite glomerular injury leading to clinical presentation of IgA nephropathy. Several studies have demonstrated that elevated circulatory levels of either Gd-IgA1 or the corresponding autoantibodies predict progressive loss of renal clearance function. In this study we assessed a possible association between serum levels of Gd-IgA1 and IgG or IgA autoantibodies specific for Gd-IgA1 in serum samples from 135 patients with biopsy-proven IgA nephropathy, 76 patients with other renal diseases, and 106 healthy controls. Our analyses revealed a correlation between the concentrations of the autoantigen and the corresponding IgG autoantibodies in sera of patients with IgA nephropathy, but not of disease or healthy controls. Moreover, our data suggest that IgG is the predominant isotype of Gd-IgA1-specific autoantibodies in IgA nephropathy. This work highlights the importance of both initial hits in the pathogenesis of IgA nephropathy.</p></div

Serum levels of Gd-IgA1-specific IgG in IgAN patients with high-Gd-IgA1 or normal-Gd-IgA1 in comparison to CKD and healthy controls.

<p>We divided IgAN patients into two subgroups: patients with serum levels of Gd-IgA1 ≥ the 90^th percentile for healthy controls (high-Gd-IgA1 group; n = 56) and patients with levels Gd-IgA1 < the 90^th percentile for healthy controls (normal-Gd-IgA1 group; n = 79). Although serum levels of Gd-IgA1-specific IgG were significantly higher in IgAN patients with high Gd-IgA1 levels (<i>vs.</i> CKD controls; *P<0.0001, <i>vs.</i> healthy controls; **P<0.0001), IgAN patients with normal Gd-IgA1 levels also had elevated Gd-IgA1-specific IgG (<i>vs.</i> CKD controls; *P<0.0001, <i>vs.</i> healthy controls; **P<0.0001).</p

IgAN patients predominantly display increased Gd-IgA1-specific IgG.

<p>Plot of the Gd-IgA1-specific IgG versus IgA autoantibodies for the healthy controls (red, H) and IgAN patients (blue, I). Cutoffs were set at 2-standard deviations from the mean values for the healthy controls (1.536 and 0.763 units for IgG and IgA, respectively). Percent occupancy in each quadrant is depicted where occupancy was observed.</p

Serum levels of Gd-IgA1-specific IgG autoantibodies correlate with serum levels of Gd-IgA1 only in patients with IgAN.

<p>Plots of Gd-IgA1 versus Gd-IgA1-specific IgG (A-D) or Gd-IgA1-specific IgA (E-H) in serum samples of patients with IgAN (blue circles; A, E, D, H), CKD controls (red squares; B, F, D, H), or healthy controls (purple triangles; C, G, D, H). Overlays of A-C and E-G are presented in D and H, respectively. Correlation is observed only for Gd-IgA1-specific IgG in IgAN patients (blue circles, r = 0.491) which is depicted by the blue line in panels A and D. No such correlation was observed in either CKD (red) or healthy controls (purple). No correlation was observed for Gd-IgA1-specific IgA autoantibodies. Pearson correlation (r) values and significance (P) are shown for panels A-C and E-G.</p

Statistics summarized data; Discrimination between IgAN versus healthy and CKD controls for serum Gd-IgA1 and Gd-IgA1-specific IgG levels.

<p>* CKD non-immune-mediated renal disease includes diabetic nephropathy, nephrosclerosis, interstitial nephritis and Fabry's disease.</p><p>** CKD immune-mediated renal disease includes lupus nephritis, membranous nephropathy, minimal change disease, membranoproliferative glomerulonephritis, other types of non-IgAN glomerulonephritis.</p><p>***AIC: Akaike's Information Criterion.</p

Statistics summarized data; Discrimination between IgAN versus immune-mediated CKD and non-immune-mediated CKD controls for serum Gd-IgA1-specific IgG levels.

<p>PPV, Positive Predictive Value; NPV, Negative Predictive Value.</p

Correlation between biomarkers, histological findings and clinical findings.

<p>The strength of correlation between biomarkers, histological findings and clinical findings was measured by the Spearman's correlation coefficient. The serum level of Gd-IgA1-specific IgA correlated with the amount of mesangial IgA deposits (A). Histological prognostic stage (Clinical Guidelines for IgA Nephropathy in Japan, second version) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098081#pone.0098081-Tomino1" target="_blank">[17]</a> correlated with the urinary protein/creatinine ratio (B), and percentage of glomeruli with a crescent (C).</p

Distribution of serum levels of (A) Gd-IgA1, (B) Gd-IgA1-specific IgG and (C) Gd-IgA1-specific IgA in patients with IgAN (n = 135), CKD controls (n = 79) and healthy controls (n = 106).

<p>Each biomarker was measured by capture ELISA. The serum levels of Gd-IgA, Gd-IgA1-specific IgG and Gd-IgA1-specific IgA were higher in IgAN patients compared with those of the CKD controls (*P<0.001) and healthy controls (**P<0.0001).</p

Characteristics of patients with IgAN and CKD controls.

<p>Values are mean ±SD.</p><p>eGFR, estimated glomerular filtration rate; P/C, protein/creatinine ratio; ND, not determined.</p><p>Hematuria: Assessed by assigning scores according to number of red blood cells per high-power field (RBC/HPF).</p><p>≤5 RBC/HPF  = 0, 6–10 RBC/HPF  = 1, 11–15 RBC/HPF  = 2, 16–20 RBC/HPF  = 3, 21–25 RBC/HPF  = 4, 26–30 RBC/HPF  = 5, >30 RBC/HPF  = 6.</p