Versatile Human O-β-N-acetyl Glucosaminyl Transferase(OGT) Assay In Saccharomyces Cerevisiae

N-acetylglucosamine(O-Glc NAc) modification on protein is a post-translational modification. Similar with the protein phosphorylation, it regulates many biological processes in high eukaryotic cells. It has demonstrated that O-Glc NAc plays important roles in some human diseases, such as cancers, diabetes and neurodegenerative diseases. Unlike the protein phosphorylation, O-Glc NAcylation cycle is regulated only by a pair of enzymes, β-N-acetyl glucosaminyl transferase(OGT) and β-D-N-acetylglucosaminidase(OGA). More than thousand proteins have been found to have O-Glc NAc modification in mammalian cell. Its regulation mechanism has been widely concerned in the field of life science research, but so far it has not been a breakthrough. On the other hand, yeast cell conserves a similar basic biological processes including their molecular mechanisms with mammalian cells, and is able to provide a suitable environment for expressing and even studying the biological function of human derived proteins. Taking the advantage of the absence of O-Glc NAc modification in yeast cell, we have tried to construct an ectopic mammalian O-Glc NAcylation system by transforming the human OGT and OGA genes into Saccharomyces cerevisiae cell. So that, we can study the regulation mechanism of OGT enzyme activity in a blank background without the interference from the complex physiological process in mammalian cells, and contributes to understanding the pathogenesis-related diseases and the evolution of their processes.OGT comprises two distinct regions: an N-terminal region consisting of a series of tetratricopeptiderepeat(TPR) units, involved in substrate recognition, and a C-terminal catalytic domain. There are three isoforms of OGT have been found in mammalian cell. They are nucleoplasmic OGT(nc OGT), mitochondrial OGT(m OGT) and shorter OGT(s OGT). The main difference among OGT isoforms is the number of TPRs. nc OGT and s OGT contains 13.5 and 2.5 TPRs, respectively. In this study, we demonstrated that ectopic expression of s OGT causes a severe growth defect in S. cerevisiae in the O-Glc NAc transferase activity dependent manner; such a growth defect was not observed by expression of an enzymatically inactive mutant, s OGTH127 A. O-Glc NAcylated proteins were detected s OGT, but not s OGTH127 A, expressing cells. Furthermore, we found that co-expression of OGA could rescue the growth defect in s OGT expressing cells and, in these cells, amount of O-Glc NAcylated proteins are decreased. Expression of nc OGT also caused a growth defect in yeast cells, though the growth defect is not due to the O-Glc NAc transferase activity. In fact, similar growth defect was observed by expression of either catalytically inactive nc OGTH498 A mutant or the TPR domain alone. Thus, toxicity of nc OGT is largely attributed to the TPR domain. It is intriguing, however, that co-expression of OGA could also rescue the growth defect in nc OGT expressing cells. This is probably because of physical interaction between nc OGT and OGA through the TPR domain, so that toxicity of the TPR domain is alleviated.In conclusion, we have successfully constructed two customized yeast strains for studying the regulation system of human OGT activity.(1) The growth defect phenomenon of s OGT expressed cell can be used to screen genes from human c DNA library, which encode proteins involved in regulation system of OGT activity;(2) The nc OGT expressed strain can be utilized to filter proteins, which interact with TPR domain of OGT, for studying the substrate recognition mechanism of human nc OGT.Using these humanized yeast strains, a chemical compounds screening can be performed for searching inhibitors of O-Glc NAc glycosylation in human cells.