Characterization of tools to detect and enrich human and mouse O-GlcNAcase

Abstract

O-linked ß-N-acetylglucosamine (O-GlcNAc) is an essential regulatory post-translational modification of thousands of nuclear, cytoplasmic and mitochondrial proteins. O-GlcNAc is dynamically added and removed from proteins by the O-GlcNAc transferase and the O-GlcNAcase (OGA), respectively. Dysregulation of O-GlcNAc-cycling is implicated in the etiology of numerous diseases including tumorigenesis, metabolic dysfunction and neurodegeneration. To facilitate studies focused on the role of O-GlcNAc and OGA in disease, we sought to identify commercially available antibodies that enable the enrichment of full-length OGA (fOGA) from lysates of mouse and human origin. Here, we report that antibodies from Abcam and Bethyl Laboratories can be used to immunoprecipitate OGA to near-saturation from human and mouse cell lysates. However, western blotting analysis indicates that both antibodies, as well as three noncommercially available antibodies (345, 346, 352), detect fOGA and numerous cross-reacting proteins. These nonspecific signals migrate similarly to fOGA and are detected robustly, suggesting that the use of appropriate controls is essential to avoid the misidentification of OGA.