title: The active site of O-GlcNAc transferase imposes constraints on substrate sequence.
abstract: O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the -3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Shalini
authors.initials: S
authors.lastname: Pathak
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Jana
authors.initials: J
authors.lastname: Alonso
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Marianne
authors.initials: M
authors.lastname: Schimpl
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Karim
authors.initials: K
authors.lastname: Rafie
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: David E
authors.initials: DE
authors.lastname: Blair
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Vladimir S
authors.initials: VS
authors.lastname: Borodkin
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Osama
authors.initials: O
authors.lastname: Albarbarawi
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.|Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Daan M F
authors.initials: DMF
authors.lastname: van Aalten
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase