Characterizing the Role of Protein O-GlcNAcylation in Human Embryonic Stem Cell Development

The addition of a single monossacharide, O-linked b-N-acetylglucosamine (O-GlcNAc), to the hydroxy side chain of Ser or Thr residues has recently emerged as an important post-translational modification in eukaryotes. This modification is rapid and reversible, and can occur on phosphorylation sites known to be phosphorylated. Although much is known about the crucial role of O-GlcNAcylation in cellular processes, this modification has yet to be studied in the context of stem cell biology. The broad objective of this project is to understand how O-GlcNAc regulates human embryonic stem cell (hESC) viability, self-renewal and differentiation. This goal will be accomplished using a method known as metabolic labeling, where unnatural monosaccharides derivatized with an azide functionality can be metabolized by cells and incorporated into glycans in place of natural monosacharides, along with state-of-the-art mass spectrometry and biochemical analysis. Currently, I have been able to maintain and differentiate hESCs into neurons. Preliminary data also demonstrates that metabolic labeling can be used to label O-GlcNAc in hESCs. Proteins unique to azide treated fractions were identified in H1 hESCs, some of which are known to be glycosylated. Further optimization is needed to achieve a complete enrichment of nuclear and cytoplasmic fractions. Importantly, azido sugar treatment had no discernible effect on hESC’s morphology or expression of stem cell markers. Taken together, the results of the proposed project will provide the first comprehensive, functional characterization of protein O-GlcNAcylation in hESCs and will form a foundation for the manipulation of these processes for research and therapeutic benefit.