Abstract
The NPHS2 gene encoding the podocin protein was causally linked to the autosomal recessive type of steroid-resistant nephrotic syndrome. In this study, we investigated the consequence of the R138Q mutation of podocin, one of the most common missense mutations in the NPHS2 gene, by examining the expression of the wild-type and R138Q mutant podocins in mammalian cells. Either myc- or FLAG-tagged wild-type podocin was strongly stained in plasma membrane, particularly in the fine processes wherein the protein was colocalized with actin stress fibers. On the other hand, the R138Q mutant podocin was completely retained intracellularly and colocalized with the endoplasmic reticulum (ER) marker, calnexin. These results suggest that the R138Q mutation affected podocin protein folding, thereby interfering with the mutant protein's departure from the ER. To determine if the ER retention of R138Q mutant is correctable, cells were incubated with the chemical chaperones glycerol, trimethylamine-N-oxide, and DMSO. Using these two methods, namely, cell surface labeling with sulfo-NHS-S-S-biotin and Alexa 488-streptavidin, and immunostaining to detect the podocin protein close to the plasma membrane, we confirmed that these chemical chaperone treatments elicit a cellular redistribution of R138Q podocin. Our results reveal defective cellular processing of the mutant podocin, and provide evidence for pharmacological correction of the processing defect.
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Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC, Niaudet P, Antignac C (2000) NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 24:349–354
Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS, Welch WJ (1996) Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones 1:117–125
Bückig A, Tikkanen R, Herzog V, Schmitz A (2002) Cytosolic and nuclear aggregation of the amyloid precursor peptide following its expression in the endoplasmic reticulum. Histochem Cell Biol 118:353–360
Caridi G, Bertelli R, Carrea A, Di Duca M, Catarsi P, Artero M, Carraro M, Zennaro C, Candiano G, Musante L, Seri M, Ginevri F, Perfumo F, Ghiggeri GM (2001) Prevalence, genetics, and clinical features of patients carrying podocin mutations in steroid-resistant nonfamilial focal segmental glomerulosclerosis. J Am Soc Nephrol 12:2742–2746
Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, Welsh MJ (1992) Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358:761–764
Fassio A, Sitia R (2002) Formation, isomerization and reduction of disulphide bonds during protein quality control in the endoplasmic reticulum. Histochem Cell Biol 117:151–157
Frishberg Y, Rinat C, Megged O, Shapira E, Feinstein S, Raas-Rothschild A (2002) Mutations in NPHS2 encoding podocin are a prevalent cause of steroid-resistant nephrotic syndrome among Israeli–Arab children. J Am Soc Nephrol 13:400–405
Gekko K, Timasheff SN (1981) Mechanism of protein stabilization by glycerol: preferential hydration in glycerol–water mixtures. Biochemistry 20:4667–4676
Hirano K, Roth J, Zuber C, Ziak M (2002) Expression of a mutant ER-retained polytope membrane protein in rat hepatocytes results in Mallory body formation. Histochem Cell Biol 117:45–53
Huang M, Gu G, Ferguson EL, Chalfie M (1995) A stomatin-like protein necessary for mechanosensation in C. elegans. Nature 378:292–295
Huber TB, Kottgen M, Schilling B, Walz G, Benzing T (2001) Interaction with podocin facilitates nephrin signaling. J Biol Chem 276:41543–41546
Karle SM, Uetz B, Ronner V, Glaeser L, Hildebrandt F, Fuchshuber A (2002) Novel mutations in NPHS2 detected in both familial and sporadic steroid-resistant nephrotic syndrome. J Am Soc Nephrol 13:388–393
Roth J, Zuber C, Guhl B, Fan J-Y, Ziak M (2002) The importance of trimming reactions on asparagine-linked oligosaccharides for protein quality control. Histochem Cell Biol 117:159–169
Saleem MA, Ni L, Witherden I, Tryggvason K, Ruotsalainen V, Mundel P, Mathieson PW (2002) Co-localization of nephrin, podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation. Am J Pathol 161:1459–1466
Salzer U, Ahorn H, Prohaska R (1993) Identification of the phosphorylation site on human erythrocyte band 7 integral membrane protein: implications for a monotopic protein structure. Biochim Biophys Acta 1151:149–152
Sato S, Ward CL, Krouse ME, Wine JJ, Kopito RR (1996) Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem 271:635–638
Schmitz A, Tikkanen R, Kirfel G, Herzog V (2002) The biological role of the Alzheimer amyloid precursor protein in epithelial cells. Histochem Cell Biol 117:171–180
Schwarz K, Simons M, Reiser J, Saleem MA, Faul C, Kriz W, Shaw AS, Holzman LB, Mundel P (2001) Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest 108:1621–1629
Tamarappoo BK, Verkman AS (1998) Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones. J Clin Invest 101:2257–2267
Tamarappoo BK, Yang B, Verkman AS (1999) Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus. J Biol Chem 274:34825–34831
Tsukaguchi H, Sudhakar A, Le TC, Nguyen T, Yao J, Schwimmer JA, Schachter AD, Poch E, Abreu PF, Appel GB, Pereira AB, Kalluri R, Pollak MR (2002) NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele. J Clin Invest 110:1659–1666
Tsutsui T, Yumura W, Nitta K, Nihei H (1997) A stable transfected line of human glomerular epithelial cells. In Vitro Cell Dev Biol 33:489–491
Welsh MJ, Smith AE (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73:1251–1254
Winn MP (2002) Not all in the family: mutations of podocin in sporadic steroid-resistant nephrotic syndrome. J Am Soc Nephrol 13:577–579
Yokota S, Kamijo K, Oda T (2000) Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins. Quality control of organelle destined proteins by the endoplasmic reticulum. Histochem Cell Biol 114:433–446
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This study was supported in part by Grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Ohashi, T., Uchida, K., Uchida, S. et al. Intracellular mislocalization of mutant podocin and correction by chemical chaperones. Histochem Cell Biol 119, 257–264 (2003). https://doi.org/10.1007/s00418-003-0511-x
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DOI: https://doi.org/10.1007/s00418-003-0511-x