Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry
Vanessa Dehennaut1, Xavier Hanoulle, Jean-François Bodart
1UMR-CNRS 8576, UGSF, USTL, IFR 147, 59655 Villeneuve d'Ascq, France.
Abstract:
In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads to a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.
Insights
O-linked N-acetylglucosaminylation (O-GlcNAc) is crucial for cell cycle progression. Increased levels of the O-GlcNAc transferase (OGT) enzyme accelerate germinal vesicle breakdown (GVBD) in Xenopus oocytes, highlighting OGT
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- O-linked N-acetylglucosaminylation (O-GlcNAc) is a dynamic post-translational modification.
- O-GlcNAc plays a role in regulating various cellular processes, including the cell cycle.
Purpose of the Study:
- To investigate the role of O-GlcNAc transferase (OGT) isoforms in regulating cell cycle progression.
- To determine the impact of OGT on Xenopus laevis oocyte germinal vesicle breakdown (GVBD).
Main Methods:
- Microinjection of OGT isoforms (ncOGT and sOGT) into Xenopus oocytes.
- Assessment of O-GlcNAc levels and activity of cell cycle regulators (MPF and MAPK).
- Inhibition of OGT using anti-OGT antibodies.
Main Results:
- Increased levels of the long OGT isoform (ncOGT) accelerated Xenopus oocyte GVBD.
- A truncated OGT isoform (sOGT) lacking N-terminal repeats had no effect on GVBD.
- ncOGT microinjection enhanced O-GlcNAc addition, leading to faster MPF and MAPK activation and accelerated GVBD.
- Anti-OGT antibody injection delayed GVBD kinetics.
Conclusions:
- OGT is a key regulator of timely cell cycle progression in Xenopus oocytes.
- The N-terminal tetratrico-peptide repeats of OGT are important for its function in GVBD.
- O-GlcNAc modification mediated by OGT is essential for regulating the speed of cell cycle progression.
