Enhanced transfer of a photocross-linking N-acetylglucosamine (GlcNAc) analog by an O-GlcNAc transferase mutant with converted substrate specificity.
O-Linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins in multicellular organisms. O-GlcNAc modification is catalyzed by the O-GlcNAc transferase (OGT), which transfers N-acetylglucosamine (GlcNAc) from the nucleotide sugar donor UDP-GlcNAc to serine or threonine residues of protein substrates. Recently, we reported a novel metabolic labeling method to introduce the diazirine photocross-linking functional group onto O-GlcNAc residues in mammalian cells. In this method, cells are engineered to produce diazirine-modified UDP-GlcNAc (UDP-GlcNDAz), and the diazirine-modified GlcNAc analog (GlcNDAz) is transferred to substrate proteins by endogenous OGT, producing O-GlcNDAz. O-GlcNDAz-modified proteins can be covalently cross-linked to their binding partners, providing information about O-GlcNAc-dependent interactions. The utility of the method was demonstrated by cross-linking highly O-GlcNAc-modified nucleoporins to proteins involved in nuclear transport. For practical application of this method to a broader range of O-GlcNAc-modified proteins, efficient O-GlcNDAz production is critical. Here we examined the ability of OGT to transfer GlcNDAz and found that the wild-type enzyme (wtOGT) prefers the natural substrate, UDP-GlcNAc, over the unnatural UDP-GlcNDAz. This competition limits O-GlcNDAz production in cells and the extent of O-GlcNDAz-dependent cross-linking. Here we identified an OGT mutant, OGT(C917A), that efficiently transfers GlcNDAz and, surprisingly, has altered substrate specificity, preferring to transfer GlcNDAz rather than GlcNAc to protein substrates. We confirmed the reversed substrate preference by determining the Michaelis-Menten parameters describing the activity of wtOGT and OGT(C917A) with both UDP-GlcNAc and UDP-GlcNDAz. Use of OGT(C917A) enhances O-GlcNDAz production, yielding improved cross-linking of O-GlcNDAz-modified molecules both in vitro and in cells.
['Rodriguez|Andrea C|AC|', 'Yu|Seok-Ho|SH|', 'Li|Bin|B|', 'Zegzouti|Hicham|H|', 'Kohler|Jennifer J|JJ|http://orcid.org/0000-0001-5373-3329']
D000117:Acetylglucosamine D019943:Amino Acid Substitution D006367:HeLa Cells D006801:Humans D020014:K562 Cells D020125:Mutation, Missense D017351:N-Acetylglucosaminyltransferases D013379:Substrate Specificity
O-linked N-acetylglucosamine (O-GlcNAc) O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) glycosyltransferase nuclear pore photoaffinity labeling protein cross-linking