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  • Loss of surface transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies.

Loss of surface transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies.

Life science alliance (2022-12-23)
Annika Möller-Kerutt, Birgit Schönhoff, Yvonne Rellmann, Britta George, Daniela Anne Braun, Hermann Pavenstädt, Thomas Weide
ABSTRACT

Crumbs2 (CRB2) is a central component of the renal filtration barrier and part of the slit diaphragm, a unique cell contact formed by glomerular podocytes. Some CRB2 variants cause recessive inherited forms of steroid-resistant nephrotic syndrome. However, the disease-causing potential of numerous CRB2 variants remains unknown. Here, we report the establishment of a live-cell imaging-based assay, allowing a quantitative evaluation of the pathogenic potential of so far non-categorized CRB2 variants. Based on in silico data analysis and protein prediction software, putative disease-associated CRB2 missense variants were selected, expressed as CRB2-GFP fusion proteins, and analyzed in reporter cell lines with BFP-labeled plasma membrane. We found that in comparison with PM-localized WT, disease-associated CRB2 variants remained predominantly at the ER. Accumulation at the ER was also present for several non-characterized CRB2 variants and variants in which putative disulfide bridge-forming cysteines were replaced. Strikingly, WT CRB2 retained inside the ER in cells lacking protein disulfide isomerase A3, indicating that posttranslational modification, especially the formation of disulfide bridges, is a crucial step for the CRB2 PM transport.

MATERIALS
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Product Description

Sigma-Aldrich
Anti-PDIA3 antibody produced in rabbit, Ab2, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution