O-GIcNAc

Determining the biophysical consequences of the O-GlcNAc protein modification

O-linked beta-N-acetylglucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification that regulates the function of thousands of proteins. GlcNAc is added to proteins at the serine and threonine hydroxyl group in the cytoplasm and nucleoplasm by the activity of one enzyme, O-GlcNAc transferase (OGT). The long-term goal of this project is to investigate the effects of the O-GlcNAc modification on proteins. The hypothesis is that the O-GlcNAc modification affects the structure, solubility, and stability of the enzymes it regulates. In order to investigate the structure of O-GlcNAc-modified proteins, we aimed to develop an E. coli expression system to produce sufficient quantities of glycoprotein for analysis by co-expressing human OGT with a substrate protein of interest. During development, we discovered that NagZ, a cytosolic E. coli beta-N-acetylglucosaminidase, removes O-GlcNAc from modified proteins. With our collaborator, Dr. William Doerrler, Department of Biological Sciences, Louisiana State University, we made a nagZ knockout strain.

Related publications

1. O. Y. Goodwin, M. M. Sweeney, M. S. Thomasson, A. J. Lin, and M. A. Macnaughtan, E. coli sabotages the in vivo production of O-linked beta-N-acetyglucosamine-modified proteins. Journal of Biotechnology, 2013, 168(4), 315-323.
2. M. Schimpl, X. W. Zheng, V. S. Borodkin, D. E. Blair, A. T. Ferenbach, A. W. Schüttelkopf, I. Navratilova, T. Aristotelous, O. Albarbarawi, D. A. Robinson, M. A. Macnaughtan, and D. M. F. van Aalten,* O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis. Nature Chemical Biology, 2012, 8(12), 969-974., PDB ID: 4AY5 "Human O-GlcNAc transferase (OGT) in complex with UDP and glycopeptide", PDB ID: 4AY6 "Human O-GlcNAc transferase (OGT) in complex with UDP-5S-GlcNAc and substrate peptide"