Use of Galactosyltransferase to Assess the Biological Function of O-linked N-Acetyl-[formula omitted]-Glucosamine: A Potential Role for O-GlcNAc during Cell Division

Abstract

Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl-d-glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes β1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation.