OGT Catalyzes Three Distinct Post-Translational Modifications via Glycosylation

Abstract

It is estimated that the human genome encodes on the order of 20,000 genes, yet the number of proteins in the human proteome has been estimated to include more than one million proteins. One major source of diversity stems from protein post-translational modifications (PTMs). In metazoans, β-O-GlcNAcylation, the transfer of N-acetylglucosamine to ser- ine and threonine (Ser/Thr) residues of numerous nuclear and cytoplasmic proteins, is a common PTM. Notably, all O-GlcNAc modifications are installed by a single essential pro- tein, O-linked-β-N-acetylglucosamine transferase, OGT. Since its discovery over 30 years ago, scientists have been trying to understand the role of OGT in metazoan biology, with the vast majority of studies focused on the functional consequences of β-O-GlcNAcylation. This thesis elucidates the mechanisms of two other OGT-catalyzed modifications, amide backbone cleavage and isomerization of aspartate to isoaspartate, and lays the groundwork for addressing their physiological roles. Host cell factor-1 (HCF-1) is a cell cycle regulatory protein that contains six 26-amino acid proteolytic repeats that must be cleaved for the protein to function properly. In 2011, OGT was implicated in HCF-1 cleavage, but it was unclear whether the process was auto- catalytic and resulted from conformational changes related to Ser/Thr glycosylation or was directly catalyzed by OGT. Chapter two describes work demonstrating that OGT directly catalyzes HCF-1 cleavage using the same active site employed for Ser/Thr glycosylation. This study also showed that cleavage occurs between Cys9 and Glu10 of the proteolytic repeat and resulted in a C-terminal product bearing a pyroglutamate residue. Chapter three establishes the mechanism of HCF-1 cleavage using an OGT mutant that accumulates reaction intermediates. Cleavage proceeds via glutamate glycosylation followed by on-enzyme formation of an internal pyroglutamate, which hydrolyzes spontaneously after release from OGT. Chapter four describes studies showing that aspartyl-containing model peptides can also be glycosylated by OGT, resulting in a peptide backbone rearrangement to produce isoaspartate through the intermediacy of a succinimide. Aspartate-to-isoaspartate iso- merization in proteins occurs in cells, but was previously thought to be exclusively non- enzymatic. These studies suggest that enzymes capable of aspartate glycosylation, which include PARPs, may catalyze this transformation as well.