Enhanced Transfer of a Photocross-linking N-Acetylglucosamine (GlcNAc) Analog by an O-GlcNAc Transferase Mutant with Converted Substrate Specificity

Andrea C. Rodriguez; Seok-Ho Yu; Bin Li; Hicham Zegzouti; Jennifer J. Kohler

doi:10.1074/jbc.M115.667006

Enhanced Transfer of a Photocross-linking N-Acetylglucosamine (GlcNAc) Analog by an O-GlcNAc Transferase Mutant with Converted Substrate Specificity*

From the ^‡Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390 and
^§Promega Corp., Madison, Wisconsin 53711

↵3 To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390. Tel.: 214-648-0535; E-mail: jennifer.kohler{at}utsouthwestern.edu.

↵1 Both authors contributed equally to this work.

Background: Photocross-linking O-GlcNAc (O-GlcNDAz) can be used to capture O-GlcNAc-mediated protein-protein interactions.

Results: Mutagenesis of the UDP-GlcNAc binding pocket of OGT enhances transfer of a photoreactive GlcNAc analog (GlcNDAz).

Conclusion: OGT(C917A) catalyzes increased enzymatic incorporation of GlcNDAz at sites of protein O-GlcNAc modification in vitro and in cells.

Significance: Enabling identification of O-GlcNAc-mediated protein interactions will provide insights into molecular mechanisms of O-GlcNAc function.

Abstract

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.

Footnotes

↵2 Supported by training grants from the National Institutes of Health (Grant T32GM007062) and the Graduate Programs Initiative from the University of Texas System.
↵* This work was supported, in whole or in part, by National Institutes of Health Grant R01GM090271. This work was also supported by Welch Foundation Grant I-1686. H. Z. is an employee of Promega Corp., which sells the UDP-Glo^TM glycosyltransferase assay. The remaining authors declare that they have no conflicts of interest with the contents of this article.