Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Clin Proteomics. 2011 Sep 21;8(1):15. doi: 10.1186/1559-0275-8-15.
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.
本研究旨在鉴定糖尿病模型 Goto-Kakizaki（GK）大鼠肾脏中 O-连接 N-乙酰葡萄糖胺（O-GlcNAc）修饰程度改变的蛋白质，并探讨 O-GlcNAc 化与糖尿病病理状况的关系。
通过二维凝胶电泳、免疫印迹和肽质量指纹图谱鉴定 O-GlcNAc 化蛋白。通过免疫沉淀、免疫印迹和原位邻近连接分析（PLA）检测这些蛋白质的 O-GlcNAc 化水平。
鉴定出显著改变 O-GlcNAc 化程度的 O-GlcNAc 化蛋白为细胞骨架蛋白（α-肌动蛋白、α-微管蛋白、α-辅肌动蛋白 4、肌球蛋白）和线粒体蛋白（ATP 合酶β、丙酮酸羧化酶）。这些蛋白的 O-GlcNAc 化程度在糖尿病肾脏中增加。免疫荧光和原位 PLA 研究表明，糖尿病肾脏肾小球和近端肾小管中肌动蛋白、α-辅肌动蛋白 4 和肌球蛋白的 O-GlcNAc 化水平显著升高。免疫电镜显示，α-辅肌动蛋白 4 的免疫标记在肾小球足突和近端肾小管微绒毛中受到干扰和增加。
这些结果表明，细胞骨架蛋白的 O-GlcNAc 化变化与糖尿病肾脏中肾小球足突和近端肾小管微绒毛中 podocyte 形态变化密切相关。这是首次报道α-辅肌动蛋白 4 被 O-GlcNAc 化。α-辅肌动蛋白 4 将是研究 O-GlcNAc 化与糖尿病肾病关系的一个很好的标记蛋白。
