Characterization of an O‐GlcNAc‐binding protein (1007.3)

Abstract

O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification of nuclear, cytoplasmic, and mitochondrial proteins. While O‐GlcNAc has been demonstrated to modulate numerous cellular processes, the molecular mechanisms by which O‐GlcNAc mediates protein function are not well defined. One mechanism by which O‐GlcNAc may regulate protein function is by promoting protein‐protein interactions. In this study we have investigated whether the intracellular hyaluronan‐binding protein p32 binds proteins in an O‐GlcNAc‐dependent manner. Using ELISA and dot blotting techniques we have demonstrated that p32 binds GlcNAc‐conjugates preferentially to other sugar‐conjugates in vitro. Suggesting that p32 interacts with O‐GlcNAc in vivo, p32 enriches O‐GlcNAc‐modified proteins similar to the O‐GlcNAc‐specific antibody RL2. Finally, our data demonstrate that overexpression of p32 fused to the biotin ligase BirA results in a biotinylation pattern that can be modulated by manipulating O‐GlcNAc levels. Preliminary studies suggest that one candidate protein bound by p32 in an O‐GlcNAc‐dependent manner is the O‐GlcNAc transferase (OGT), the enzyme responsible for the addition of O‐GlcNAc. Taken together, these data suggest that p32 may contain an O‐GlcNAc‐binding motif that dictates its ability to interact with a subset of intracellular proteins such as OGT. Grant Funding Source: Supported by the National Heart, Lung, and Blood Institute (P01HL107153)