Use of galactosyltransferase to assess the biological function of O-linked N-acetyl-d-glucosamine: a potential role for O-GlcNAc during cell division.
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 beta1-4-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.
Fang
B
B
Department of Biological Sciences, Wright State University, Dayton, Ohio, 45435-0001, USA.
Miller
M W
MW
D000117:Acetylglucosamine / Q000378:metabolism*; D000818:Animals; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002474:Cell-Free System; D005700:Galactosyltransferases / Q000378:metabolism / Q000494:pharmacology*; D008845:Microinjections; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D011374:Progesterone / Q000494:pharmacology; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism; D014982:Xenopus laevis
D011374:Progesterone; D005700:Galactosyltransferases; D000117:Acetylglucosamine