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|Title:||Large-scale identification of calcium oxalate monohydrate crystal-binding proteins on apical membrane of distal renal tubular epithelial cells|
Shui Tein Chen
Genomics Research Center, Academia Sinica
|Keywords:||Biochemistry, Genetics and Molecular Biology;Chemistry|
|Citation:||Journal of Proteome Research. Vol.10, No.10 (2011), 4463-4477|
|Abstract:||Adhesion of calcium oxalate monohydrate (COM) crystals onto apical surface of renal tubular epithelial cells is a crucial mechanism for crystal retention, leading to kidney stone formation. Various proteins on apical membrane may bind to COM crystals; however, these crystal-binding proteins remained unidentified. The present study therefore aimed to identify COM crystal-binding proteins on apical membrane of distal renal tubular epithelial cells. Madin-Darby Canine Kidney (MDCK) cells were cultivated to be polarized epithelial cells and apical membrane was isolated from these cells using a peeling method established recently. Enrichment and purity of isolated apical membrane were confirmed by Western blot analysis for specific markers of apical (gp135) and basolateral (Na + /K + -ATPase) membranes. Proteins derived from the isolated apical membrane were then resuspended in artificial urine and incubated with COM crystals. The bound proteins were eluted, resolved by SDS-PAGE, and analyzed by Q-TOF MS and MS/MS, which identified 96 proteins. Among these, expression and localization of annexin II on apical surface of MDCK cells were confirmed by Western blot analysis, immunofluorescence staining, and laser-scanning confocal microscopic examination. Finally, the function of annexin II as the COM crystal-binding protein was successfully validated by COM crystal-binding assay. This large data set offers many opportunities for further investigations of kidney stone disease and may lead to the development of new therapeutic targets. © 2011 American Chemical Society.|
|Appears in Collections:||Scopus 2011-2015|
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