Abstract
Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis worldwide and is histologically characterized by the deposition of IgA1 and consequent inflammation in the glomerular mesangium. Prior studies suggested that serum IgA1 from IgAN patients contains aberrant, undergalactosylated O-glycans, for example, Tn antigen and its sialylated version, SialylTn (STn), but the mechanisms underlying aberrant O-glycosylation are not well understood. Here we have used serial lectin separation technologies, Western blot, enzymatic modifications, and mass spectrometry to explore whether there are different glycoforms of IgA1 in plasma from patients with IgAN and healthy individuals. Although total plasma IgA in IgAN patients was elevated ∼ 1.6-fold compared with that in healthy donors, IgA1 in all samples was unexpectedly separable into two distinct glycoforms: one with core 1 based O-glycans, and the other exclusively containing Tn/STn structures. Importantly, Tn antigen present on IgA1 from IgAN patients and controls was convertible into the core 1 structure in vitro by recombinant T-synthase. Our results demonstrate that undergalactosylation of O-glycans in IgA1 is not restricted to IgAN and suggest that in vivo inefficiency of T-synthase toward IgA1 in a subpopulation of B or plasma cells, as well as overall elevation of IgA, may contribute to IgAN pathogenesis.
Highlights
Immunoglobulin A (IgA) nephropathy (IgAN)1, called Berger’s Disease, was first described by Jean Berger in 1968
Plasma Concentration of IgA is Increased from Immunoglobulin A nephropathy (IgAN) Patients, and O-Glycans on Plasma IgA1 From Both IgAN Patients and Control Donors Contain Tn and STn Structures—A common feature reported in IgAN patients is an increase of plasma IgA as compared with healthy controls [42]
We determined the concentration of plasma IgA by ELISA, and found that, consistent with earlier studies, IgA was present at an average of 4.77 mg/ml (Ϯ 1.09 mg/ml) from 14 IgAN patients, whereas in control plasma from 14 healthy donors we found 2.97 mg/ml (Ϯ 0.70 mg/ml) IgA, representing a low, but significant ϳ1.6 fold elevation of IgA in patients with IgAN (Fig. 2A)
Summary
Plasma Samples and Cell Culture—Blood samples from both biopsy-proven patients with IgAN and healthy controls were obtained from the Emory Clinic under the approved IRB protocol (IRB00008410). The plasma, erythrocytes and leukocytes were separated using Lymphoprep (StemcellTM Technologies, Vancouver, Canada) following the manufacturer’s protocol. All plasma samples were aliquotted and stored at Ϫ80 °C, or Ϫ20 °C during experiments. ELISA Assays—Flat-bottomed 96-well ELISA Microplates (Greiner bio-one, Frickenhausen, Germany) were coated overnight at 4 °C with 50 l of 1 g/ml F(Ab)’ fragment of goat IgG anti-human IgA (Jackson Immuno-Research Labs, West Grove, PA) in 0.05 M carbonate/bicarbonate pH 9.6 buffer. Coated plates were blocked with 1% BSA in phosphate-buffered saline (PBS)-0.05%, Tween 20 (PBS-T), 1h at room temperature (RT) and washed three times with PBS-T. Plasma samples diluted 1:10,000 in PBS-T were incubated in dupli-
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