Engineering a Proximity-Directed O‑GlcNAc Transferase for Selective Protein O‑GlcNAcylation in Cells

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.