Propensity of IgA to self-aggregate via tailpiece cysteine-471 and treatment of IgA nephropathy using cysteamine

IgA nephropathy is caused by deposition of circulatory IgA1 in the kidney. Hypogalactosylated IgA1 has the propensity to form poly-IgA aggregates that are prone to deposition. Herein, we purified poly-IgA from the plasma of patients with IgA nephropathy and showed that the complex is susceptible to reducing conditions, suggesting intermolecular disulfide connections between IgA units. We sought to find the cysteine residue(s) that form intermolecular disulfide. Naturally assembled dimeric IgA, also known as secretory IgA, involves a J chain subunit connected with 2 IgA1 molecules via their penultimate cysteine-471 residue on a “tailpiece” segment of IgA heavy chain. It is plausible that, with the absence of J chain, the cysteine residue of mono-IgA1 might aberrantly form a disulfide bond in poly-IgA formation. Mutagenesis confirmed that cysteine-471 is capable of promoting IgA aggregation. These discoveries prompted us to test thiol-based drugs for stabilizing cysteine. Specifically, the cystine-reducing drug cysteamine used for treatment of cystinosis showed a remarkable potency in preventing self-aggregation of IgA. When administrated to rat and mouse models of IgA nephropathy, cysteamine significantly reduced glomerular IgA deposition. Collectively, our results reveal a potentially novel molecular mechanism for aberrant formation of IgA aggregates, to which the repurposed cystinosis drug cysteamine was efficacious in preventing renal IgA deposition.


IgA purification from plasma
IgAN patients who donated plasma samples had provided informed consent in writing before study inclusion and their diagnoses were based on kidney biopsy. IgA1 from IgAN patient sera was purified by Jacalin (Thermo Scientific, USA)-directed affinity chromatography. Poly-IgA1 contents were further enriched via running Superdex 200 Increase gel filtration molecular sieve (GE Biosciences) and collecting highmolecular weight fractions from an AKTA protein purification system (GE Biosciences).

SDS-PAGE and Western blotting
Proteins in sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) with or without TCEP or 2mercaptoethanol (for reducing or nonreducing condition, respectively) were resolved by 4−12% SDS-PAGE (Bio-Rad Laboratories). rIgA bands were subsequently visualized either by staining with GelCode Blue (Thermo Fisher Scientific), or by Western blotting on PVDF membrane. For Western blotting, 5% non-fat milk was used in blocking for one hour at room temperature. The membrane was then incubated with mouse anti-Histag antibody (Thermo Scientific), or HRP-conjugated goat anti-rat IgA α-chain antibody (Cat. ab97185, Abcam, UK), or goat anti-human IgA HRP antibody (Cat:2050-05, SouthernBiotech) or goat anti-human IgA1 HRP antibody (Cat:9130-05, SouthernBiotech) for detecting rat or human IgA (Fc) or human IgA1 (Fc), respectively. The membrane was developed using the Clarity™ ECL substrate (Bio-Rad Laboratories, CA, USA).

TEM analyses
Structural characterizations of purified mono-rIgA and poly-rIgA were performed with transmission electron microscopy (TEM) as described previously(1). TEM analyses of the structures of poly-rIgA and mono-rIgA were conducted following a standard negative staining protocol. In brief, purified poly-rIgA or mono-rIgA was diluted in PBS to a concentration of 100 μg/ml. A 10 μl droplet was applied to a glow-discharged carboncoated copper grid and allowed to sit for 1 min. The grid was washed by dipping in two separate drops 3 in water followed by two drops in 2% uranyl acetate (Electron Microscopy Sciences). Grids were examined at the Northwestern Electron Probe Instrumentation Center (EPIC) using Hitachi HT-7700 Biological S/TEM

Immunofluorescence staining
Animal studies were approved by Northwestern University IACUC committee (Protocol: IS00009990).

1.
Booth DS, Avila-Sakar A, and Cheng Y. Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition. J Vis Exp. 2011(58  These immunofluorescence images were from the same study as in Figure 6, in which rats or mice incurred IgA deposition in the kidney following injections with either recombinant rat rIgA1 (to rats) or native human IgA extracted from human plasma (to mice). (A). Prominent rIgA deposition in glomerular mesangium (arrowheads) was in control rats treated with buffer, in contrast to weaker IgA signals in glomeruli of cysteamine-treated rats. (B). Prominent IgA1-deposition to the glomerular were found in control-treatment group of mice. Pretreatment of mice with cysteamine greatly reduced IgA1-deposits in the glomerulus. (C). Plasma human IgA1 levels of mice at 0.5h, 1h and 2h after IgA1 injection did not show significant differences between control group and cysteamine-treated group. NS, not significant. Recombinant rat IgA was resolved by SEC. Poly-rIgA (~600 kDa) and mono-rIgA (~50 kDa) complexes were separately collected for i.v. injection studies, as well as recombinant rIgAC471S mutation. Rats (n=3 in each group) were injected with daily doses of either poly-IgA (A) or mono-rIgA (B) or rIgAC471S (C) for 5 consecutive days. Representative immunofluorescence staining images of the glomeruli are shown with IgA in green, Col4A1 in red. Scale bar 50μm.