nephrin and 3-3--dithiobis(sulfosuccinimidyl-propionate)

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.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens; Cross-Linking Reagents; Electrophoresis, Gel, Two-Dimensional; Fluorescent Antibody Technique; Humans; Kidney Glomerulus; Mass Spectrometry; Membrane Proteins; Molecular Sequence Data; Nephrotic Syndrome; Precipitin Tests; Proteins; Proteinuria; Rats; Rats, Sprague-Dawley; Sequence Alignment; Succinimides