Detecting and Imaging O-GlcNAc Sites Using Glycosyltransferases: A Systematic Approach to Study O-GlcNAc

Abstract

O-GlcNAcylation is a reversible serine/threonine glycosylation for regulating protein activity and availability inside of cells. In a given protein, O-GlcNAcylated and unoccupied O-GlcNAc sites are referred as closed and open sites, respectively. The balance between open and closed sites is believed to be dynamically regulated. In this report, closed sites are detected using in vitro incorporation of GalNAz by B3GALNT2, and open sites are detected by in vitro incorporation of GlcNAz by OGT, via click chemistry. For assessing total O-GlcNAc sites, a sample is OGlcNAcylated in vitro by OGT before detecting by B3GALNT2. The methods are demonstrated on purified protein, cellular extracts, and whole cells. Through O-GlcNAc imaging, the modification degree of O-GlcNAc in nuclei of CHO cells was estimated. The detection and imaging of both open and closed O-GlcNAc sites provide a systematic approach to study this important post-translational modification.