Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin
Peter S. Topham, Hiroshi Kawachi, Samir A. Haydar, Sumant Chugh, Theresa A. Addona, Kathryn B. Charron, Lawrence B. Holzman, Michael Shia, Fujio Shimizu, David J. Salant
Peter S. Topham, Hiroshi Kawachi, Samir A. Haydar, Sumant Chugh, Theresa A. Addona, Kathryn B. Charron, Lawrence B. Holzman, Michael Shia, Fujio Shimizu, David J. Salant
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Article

Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin

Abstract

mAb 5-1-6 identifies an antigen on rat podocyte slit-diaphragms and induces severe proteinuria when injected into rats. Nephrin, an Ig-like transmembrane protein that is mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the slit-diaphragm on human podocytes. Here we document that the mAb 5-1-6 antigen is rat nephrin. After incubation of rat glomeruli with this mAb, the antibody/antigen complex was chemically cross-linked, extracted, and immunoprecipitated, prior to Western analysis. By mass spectrometry and 2D gel electrophoresis, we identified several peptides with complete identity to human nephrin. In addition, the 185-kDa protein immunoprecipitated by mAb 5-1-6 from rat glomerular extracts reacts with a rabbit anti-mouse nephrin antibody. Finally, nephrin and the mAb 5-1-6 antigen have identical glomerular localization patterns on immunofluorescence of rat kidney. These results demonstrate that the nephritogenic mAb 5-1-6 identifies the extracellular domain of nephrin, thereby documenting the importance of the slit-diaphragm and its component, nephrin, in the regulation of glomerular permselectivity.

Authors

Peter S. Topham, Hiroshi Kawachi, Samir A. Haydar, Sumant Chugh, Theresa A. Addona, Kathryn B. Charron, Lawrence B. Holzman, Michael Shia, Fujio Shimizu, David J. Salant

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Schematic representation of nephrin demonstrating the origin of the pept...
Schematic representation of nephrin demonstrating the origin of the peptide sequences identified by MS/MS in immunoprecipitates of rat glomeruli. The sequence above nephrin was obtained from the cross-linked immunoprecipitate, and the 2 below were identified from the 185-kDa 2D-gel spot. The corresponding human sequence is shown in italics. The arrow highlights the single amino acid mismatch between the MS/MS–derived sequence and human sequence. In the nephrin schematic, numbered black regions indicate the Ig-like domains; FN, the fibronectin type III–like domain; M, the transmembrane domain; and C, the cytoplasmic domain.