Mutational Analysis of the Catalytic Domain of O-Linked N-Acetylglucosaminyl Transferase

Brooke D Lazarus,Mark D Roos,John A Hanover

doi:10.1074/jbc.m504948200

Abstract

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.

Highlights

O-Linked N-acetylglucosaminyltransferase (OGT)3 is a nucleocytoplasmic enzyme that catalyzes the addition of a single GlcNAc residue, in an O-glycosidic linkage to serine or threonine residues of target proteins [1, 2]
Our aim was to perform a mutational analysis of residues in the C-terminal catalytic domain that may be important for UDP-GlcNAc sensing, providing information regarding the catalytic roles of CD I and CD II
To identify potentially important amino acid residues that could be targeted for mutagenesis, sequence comparisons were made with OGT homologues, as well as with other glycosyltransferase families for which a mechanism has been proposed for catalytic function (38, 40 – 44)

Summary

Introduction

O-Linked N-acetylglucosaminyltransferase (OGT) is a nucleocytoplasmic enzyme that catalyzes the addition of a single GlcNAc residue, in an O-glycosidic linkage to serine or threonine residues of target proteins [1, 2] This dynamic form of post-translational modification is analogous to that of phosphorylation [3, 4], suggesting a role in cell signaling and cellular regulation. Mutagenesis of O-GlcNAc Transferase superfamily suggests that they may be either in or close to the active site of the enzyme These results provide important new insights into the structure/function of this enzyme and may help understand the mechanism by which OGT recognizes UDP-GlcNAc and catalyzes transfer to substrate proteins

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