Abstract
PurposeThe 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.MethodsO-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).ResultsO-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.ConclusionThese 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.
Highlights
O-linked N-acetyl-b-D-glucosamine, termed O-GlcNAc, is a post-translational modification involved in modulation of signaling and transcription in response to cellular nutrients or stress by interplay with O-phosphorylation [1,2,3]
We examined the localization of O-GlcNAcylated cytoskeletal proteins and quantified their signals
In this study using proteomic analysis, we identified several O-GlcNAcylated proteins including cytoskeletal proteins, actin, a-actinin 4, a-tubulin, and myosin and we demonstrated that their extent of O-GlcNAcylation was elevated in the diabetic kidney
Summary
O-linked N-acetyl-b-D-glucosamine, termed O-GlcNAc, is a post-translational modification involved in modulation of signaling and transcription in response to cellular nutrients or stress by interplay with O-phosphorylation [1,2,3]. Elevated O-GlcNAc modification (O-GlcNAcylation) of proteins by increased flux through the hexosamine biosynthetic pathway has been implicated in the development of insulin resistance and diabetic complications and in the up-regulated gene expression of transforming growth factor-beta, plasminogen activator inhibitor 1, and upstream stimulatory factor proteins in mesangial cells, leading to mesangial matrix expansion and diabetic glomerulosclerosis [2,4,5,6,7,8,9]. In this study we carried out proteomic analysis, especially focused on the proteins with remarkable change of the O-GlcNAc level in the kidney from GK rats, and suggested the potential of O-GlcNAcylation as a biomarker of diabetic nephropathy. Immunoprecipitation and immunoblot findings demonstrated that O-GlcNAcylation of these identified proteins was increased in the diabetic rats. The localization and quantity of these O-GlcNAcylated proteins were further examined by performing the in situ Proximity Ligation Assay (PLA), which was developed to examine protein-to-protein interaction and post-translational modification of proteins [13,14]
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