A missense mutation in the nephrin gene impairs membrane targeting
| Autor: | Junya Shimizu, Shigeru Ito, Kunihiko Aya, Yoshikazu Sado, Yoshiki Seino, Hiroyuki Tanaka |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Cytoplasm
Nephrotic Syndrome Recombinant Fusion Proteins Blotting Western Genetic Vectors Green Fluorescent Proteins Mutation Missense Protein Sorting Signals Kidney Transfection urologic and male genital diseases medicine.disease_cause Cell Line Green fluorescent protein Nephrin medicine Humans Missense mutation Genetics Mutation biology urogenital system Point mutation Cell Membrane HEK 293 cells Membrane Proteins Proteins Fusion protein Molecular biology female genital diseases and pregnancy complications Luminescent Proteins Nephrology Protein Biosynthesis biology.protein |
| Zdroj: | American Journal of Kidney Diseases. 40:697-703 |
| ISSN: | 0272-6386 |
| Popis: | Background: NPHS1, which encodes nephrin, recently has been identified as the gene in which mutations cause congenital nephrotic syndrome of the Finnish type (CNF). We previously reported novel missense mutations of NPHS1 in a Japanese patient with CNF. However, the mechanism by which these missense mutations cause the disorder remains to be clarified. Methods: Wild-type nephrin and mutated nephrin complementary DNA were each tagged by the green fluorescence protein (GFP) gene; the expressing vectors of the fusion protein were each transfected to human embryonic kidney 293 cells. We compared intracellular localization of mutated nephrin with that of wild-type nephrin by using GFP and immunostaining examination. Results: In both wild-type and mutated nephrin (Glu 447 Lys), GFP and immunostaining resulted in a colocalized microgranular pattern along the cell membrane that indicated these recombinant proteins were located at the cell surface. Conversely, in mutated nephrin (Asp 819 Val), GFP aggregation was observed in the cytoplasm, and no fluorescence was observed at the cell membrane, indicating that recombinant mutated nephrin (Asp 819 Val) could not be distributed at the cell membrane and instead was retained in cytoplasm. Conclusion: We confirmed that the missense mutation GAC-to-GTC transversion leading to an Asp 819 Val caused the disorder. The present study analyzes in vitro distribution of nephrin with a missense point mutation. The analysis uses a new convenient method, construction of a nephrin-GFP fusion protein. © 2002 by the National Kidney Foundation, Inc. |
| Databáze: | OpenAIRE |
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