Modification (O-GlcNAcylation) of proteins by O-linked N-acetylglucosamine occurs in the nucleus and in the cytoplasm. O-GlcNAcylation is particularly relevant to chronic human diseases including diabetes, cancer, neurodegenerative disorders, and cardiovascular disease.
Increased flux through the hexosamine biosynthesis pathway promotes the O-GlcNAcylation, and has been implicated in the development of insulin resistance and diabetes complications. In our previous immunohistochemical study, we demonstrated that the O-GlcNAcylation level increased in various tissues including kidney from diabetic GK rats, which is an animal model of type 2 diabetes.
To identify marker proteins that change in their extent of O-GlcNAcylation in the diabetic kidney from GK rats, we separated total kidney proteins by two-dimensional gel electrophoresis. O-GlcNAcylated proteins were detected by the immunoblot using anti-O-GlcNAc antibody. Selected proteins that changed markedly in the O-GlcNAc level were identified by Mass Spectrometry analysis. The localization and the quantity of these O-GlcNAcylated-proteins were analyzed by 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, pyruvate carboxylase). Results of immunoprecipitation and immunoblot studies, as well as in situ PLA demonstrated that the extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunoelectron microscopy revealed that α-actinin 4 increased in the foot process of podocytes and the proximal tubules. To further examine the changes of the O-GlcNAcylation of glomerular proteins accompanied with diabetic nephropathy, we isolated glomerulus from kidney and performed proteomic analysis.
It was revealed that some glomerulus-specific proteins including synaptopodin were O-GlcNAcylated. To elucidate the role of O-GlcNAcylation of glomerular proteins in the diabetic nephropathy the morphological changes of the glomerular epithelial cells were examined under the various conditions in vitro.
References
1) Hart, G.W., Housley, M.P., Slawson, C.: Nature 2007, 446:1017-1022
2) Degrell P, Cseh J, Mohás M, et al.: Life Sci 2009, 84:389-393
3) Akimoto, Y., Miura, Y., Toda, T., et al.: Clin Proteomics 2011, 8:15
This study was supported in part by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (C-24590260 to YA), from Japan Diabetes Foundation (to YA), from Kyorin University School of Medicine, Kyorin Medical Research Award 2013 (to YA) and by NIH R01 DK61671 (to GWH).