Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.
Daniel H. Ramirez,Chanat Aonbangkhen,Hung-Yi Wu,Jeffrey A. Naftaly,Stephanie Tang,Timothy R. O’Meara,Christina M. Woo +6 more
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TLDR
These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future.Abstract:
O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.read more
Citations
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A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry-based Glycoproteomics.
TL;DR: Common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles are reviewed.
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Role of O-Linked N-Acetylglucosamine Protein Modification in Cellular (Patho)Physiology.
TL;DR: The current understanding of the processes involved in regulating O- GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O- GloverNAc cycling contributes to pathophysiological processes are outlined.
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Deciphering protein post-translational modifications using chemical biology tools
TL;DR: Chemical biology tools provide access to proteins bearing site-specific post-translational modifications, helping to decipher their roles in health and disease, with selected recent examples of how they have been applied.
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Analytical and Biochemical Perspectives of Protein O-GlcNAcylation.
Junfeng Ma,Ci Wu,Gerald W. Hart +2 more
TL;DR: A comprehensive review of protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification from analytical and biochemical perspectives is presented in this article.
Journal ArticleDOI
Exploring cellular biochemistry with nanobodies
Ross W. Cheloha,Ross W. Cheloha,Thibault J. Harmand,Thibault J. Harmand,Charlotte Wijne,Charlotte Wijne,Thomas U. Schwartz,Hidde L. Ploegh,Hidde L. Ploegh +8 more
TL;DR: Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible.
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