Morphological changes in diabetic kidney are associated with increased O-GlcNAcylation of cytoskeletal proteins including α-actinin 4.
Morphological changes in diabetic kidney are associated with increased O-GlcNAcylation of cytoskeletal proteins including α-actinin 4.
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DOI：
10.1186/1559-0275-8-15
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发表时间：
2011-09-21
影响因子：
3.8
通讯作者：
Kawakami H
中科院分区：
文献类型：
--
作者：
Akimoto Y;Miura Y;Toda T;Wolfert MA;Wells L;Boons GJ;Hart GW;Endo T;Kawakami H
本研究的目的是确定糖尿病模型Goto-Kakizaki(GK)大鼠肾脏中O-连接N-乙酰氨基葡萄糖(O-GlcN-Ac)修饰程度的变化，并探讨O-GlcN-Ac修饰与糖尿病病理状态的关系。O-GlcNacylated蛋白通过双向凝胶电泳、免疫印迹和多肽质量指纹图谱进行鉴定。用免疫沉淀法、免疫印迹法和原位近距离连接分析法检测这些蛋白的O-GlcN酰化水平。O-GlcN酰化程度发生显著变化的蛋白质被鉴定为细胞骨架蛋白(α-肌动蛋白、α-微管蛋白、α-肌动蛋白4、肌球蛋白)和线粒体蛋白(β、丙酮酸羧基酶)。糖尿病大鼠肾脏中上述蛋白的O-GlcN酰化程度增加。免疫荧光和原位聚乳酸研究显示，糖尿病大鼠肾小球和近曲小管中肌动蛋白、α-肌动蛋白4和肌球蛋白的O-GlcN酰化水平显著增加。免疫电子显微镜显示肾小球足突和近曲小管微绒毛中α-Actinin4的免疫标记被干扰和增强。这些结果表明，细胞骨架蛋白O-GlcN酰化的变化与糖尿病肾小球足突和糖尿病肾脏近端小管微绒毛的形态变化密切相关。这是首次报道α-肌动蛋白4是O-GlcN酰化的。α-Actinin4有望成为研究O-GlcN酰化与糖尿病肾病关系的良好标志性蛋白。
The objective of the present study is to identify proteins that change in the extent of the modification with O-linked N-acetylglucosamine (O-GlcNAcylation) in the kidney from diabetic model Goto-Kakizaki (GK) rats, and to discuss the relation between O-GlcNAcylation and the pathological condition in diabetes. O-GlcNAcylated proteins were identified by two-dimensional gel electrophoresis, immunoblotting and peptide mass fingerprinting. The level of O-GlcNAcylation of these proteins was examined by immunoprecipitation, immunoblotting and in situ Proximity Ligation Assay (PLA). O-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (α-actin, α-tubulin, α-actinin 4, myosin) and mitochondrial proteins (ATP synthase β, pyruvate carboxylase). The extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunofluorescence and in situ PLA studies revealed that the levels of O-GlcNAcylation of actin, α-actinin 4 and myosin were significantly increased in the glomerulus and the proximal tubule of the diabetic kidney. Immunoelectron microscopy revealed that immunolabeling of α-actinin 4 is disturbed and increased in the foot process of podocytes of glomerulus and in the microvilli of proximal tubules. These results suggest that changes in the O-GlcNAcylation of cytoskeletal proteins are closely associated with the morphological changes in the podocyte foot processes in the glomerulus and in microvilli of proximal tubules in the diabetic kidney. This is the first report to show that α-actinin 4 is O-GlcNAcylated. α-Actinin 4 will be a good marker protein to examine the relation between O-GlcNAcylation and diabetic nephropathy.