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Meek RW, Blaza JN, Busmann JA, Alteen MG, Vocadlo DJ, Davies GJ. Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT. Nature communications 2021 12(1) 34764280
Abstract:
The O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.
O-GlcNAc proteins:
TAB1
Species: Homo sapiens
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Escobar EE, King DT, Serrano-Negrón JE, Alteen MG, Vocadlo DJ, Brodbelt JS. Precision Mapping of O-Linked N-Acetylglucosamine Sites in Proteins Using Ultraviolet Photodissociation Mass Spectrometry. Journal of the American Chemical Society 2020 142(26) 32510947
Abstract:
Despite its central importance as a regulator of cellular physiology, identification and precise mapping of O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (PTM) sites in proteins by mass spectrometry (MS) remains a considerable technical challenge. This is due in part to cleavage of the glycosidic bond occurring prior to the peptide backbone during collisionally activated dissociation (CAD), which leads to generation of characteristic oxocarbenium ions and impairs glycosite localization. Herein, we leverage CAD-induced oxocarbenium ion generation to trigger ultraviolet photodissociation (UVPD), an alternate high-energy deposition method that offers extensive fragmentation of peptides while leaving the glycosite intact. Upon activation using UV laser pulses, efficient photodissociation of glycopeptides is achieved with production of multiple sequence ions that enable robust and precise localization of O-GlcNAc sites. Application of this method to tryptic peptides originating from O-GlcNAcylated proteins TAB1 and Polyhomeotic confirmed previously reported O-GlcNAc sites in TAB1 (S395 and S396) and uncovered new sites within both proteins. We expect this strategy will complement existing MS/MS methods and be broadly useful for mapping O-GlcNAcylated residues of both proteins and proteomes.
O-GlcNAc proteins:
PHP, TAB1
Alteen MG, Gros C, Meek RW, Cardoso DA, Busmann JA, Sangouard G, Deen MC, Tan HY, Shen DL, Russell CC, Davies GJ, Robinson PJ, McCluskey A, Vocadlo DJ. A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High-Throughput Screening: Application to O-GlcNAc Transferase. Angewandte Chemie (International ed. in English) 2020 59(24) 32092778
Abstract:
Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead-based strategy to measure the group-transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O-GlcNAc transferase (OGT) as a model system through detailed Michaelis-Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group-transfer enzymes.
O-GlcNAc proteins:
HCFC1, CSK21, TAB1
Species: Homo sapiens