Selective protein O-GlcNAcylation in cells by a proximity-directed O-GlcNAc transferase

Abstract

O-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Yet, the study of post-translational modifications like O-GlcNAc has been limited by the lack of strategies to induce O-GlcNAcylation on a target protein in cells. Here, we report a generalizable genetic strategy to induce O-GlcNAc to specific target proteins in cells using a nanobody as a proximitydirecting 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(4), a truncated form of OGT, yielded a nanobody-OGT(4) construct that selectively delivered O-GlcNAc to the target protein (e.g., JunB, cJun, Nup62) and reduced alteration of global O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the selective 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. Finally, we demonstrate the ability to selectively target endogenous ⍺-synuclein for glycosylation in HEK293T cells. Thus, the use of nanobodies to redirect OGT substrate selection is a versatile strategy to induce glycosylation of desired target proteins in cells that will facilitate discovery of O-GlcNAc functions and provide a mechanism to engineer O-GlcNAc signaling. The proximity-directed OGT approach for protein-selective O-GlcNAcylation is readily translated to additional protein targets and nanobodies that may constitute a generalizable strategy to control post-translational modifications in cells. Significance StatementNature uses post-translational modifications (PTMs) like glycosylation as a mechanism to alter protein signaling and function. However, the study of these modified proteins in cells is confined to loss-of-function strategies, such as mutagenic elimination of the modification site. Here, we report a generalizable strategy for induction of O-GlcNAc to a protein target in cells. The O-GlcNAc modification is installed by O-GlcNAc transferase (OGT) to thousands of nucleocytoplasmic proteins. Fusion of a nanobody to OGT enables the selective increase of O-GlcNAc levels on a series of target proteins. The described approach will facilitate direct studies of O-GlcNAc and its regulatory enzymes and drive new approaches to engineer protein signaling via a strategy that may be conceptually translatable to additional PTMs.

Engineered kinases as a tool for phosphorylation of selected targets in vivo

“…Directed protein manipulation in specific cell types has multiple applications, from developmental studies to designing synthetic biology systems and eventually to therapeutic uses. A similar nanobody-based paradigm has been recently applied to induce glycosylation and deglycosylation of desired target proteins in cells ( Ramirez et al, 2019 ; Ge et al, 2021 ). Furthermore, in the elegant work by Karginov et al (2014b) , a rapamycin-regulated engineered Src kinase was used in cultured cells to dissect the effect of Src-dependent phosphorylation of its two substrates, FAK and p130Cas, on cell morphology and filopodia formation.…”

In vivoregulation of fluorescent fusion proteins by engineered kinases

“…Protein binder-based approaches for directing a given enzymatic function to a specific, single target open new avenues for unravelling protein-protein interactions in a given cell type and for manipulating them in a desired way, both for developmental studies, for designing synthetic biology systems or eventually for therapeutic goals. Similar nanobody-based paradigm has been recently applied to induce glycosylation and deglycosylation of desired target proteins in cells 93,94 . Furthermore, in the elegant work by Karginov et al, a rapamycin-regulated engineered Src kinase was used in cultured cell to dissect the effect of Src-dependent phosphorylation of its two substrates, FAK and p130Cas (containing a modified rapamycin– binding domain insertion), on cell morphology and filopodia formation 95 .…”