O-linked β-N-acetylglucosamine (O-GlcNAc) is a vital post-translational modification involving the transfer of β-N-acetylglucosamine (GlcNAc) onto serine and threonine residue of over 7000 nucleocytoplasmic proteins. O-GlcNAcylation is involved in the regulation of many biochemical, metabolic processes and its misregulation is a hallmark of disease states including cancer, diabetes, and neurodegeneration.  Each glycosite regulates protein behavior through a variety of mechanisms including stability, inducing conformational changes, or affecting protein-protein interactions. An understudied mechanism of O-GlcNAc biology is how specific O-GlcNAcylation drives protein-protein interactions (PPIs). Many sites of O-GlcNAcylation are well characterized, but protein specific O-GlcNAcylation driven interactomes remain unknown. In this work, we analyzed the PPI changes of site-specific glycosylation events on the transcription factor SOX2. This was tested using two methods. One, design and expression of a fusion construct of Sox2 with a promiscuous biotin ligase. For this fusion construct, mutants containing Ser/Thr or Ala (non-glcosylated) mutant were expressed in cellulo and differences in the resulting interactome analyzed by pulldown proteomics. Two, design and synthesis of rationally designed glycopeptide mimics based on bioactive O-GlcNAc sites. We validated our strategy through known O-GlcNAc driven SOX2 binding interactions both for in vivo (fusion protein) and in vitro (Glycopeptide pulldowns). From this we determined currently synthesized glycopeptide tools were unable to recapitulate PPIs based on sequence length. From this study we were able to expand the knowledge of SOX2 O-GlcNAc specific interactome changes.

Design and synthesis of labeling tools for analysis of O-GlcNAc-specific protein interactions

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ACS Fall 2022

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