Dataset Information

Targeted deglycosylation with a nanobody-directed split OGA in living cells

ABSTRACT: O-GlcNAc is an essential and dynamic post-translational modification present on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a target protein in cells is critical yet challenging without disturbing O-GlcNAc globally or after extensive glycosite mapping. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as a targeted O-GlcNAc eraser for protein-selective O-GlcNAc removal in cells. Through systematic screening, we identified the essential domains of OGA and afforded a split OGA with limited activity, which selectively reinstated deglycosylation activity on the target protein upon fusion of a nanobody in living cells. We demonstrate the generality of the O-GlcNAc eraser against a series of target proteins and reveal that O-GlcNAc stabilizes the transcription factor c-Jun and promotes its interaction with c-Fos. Thus, nanobody-directed split OGA speeds the selective removal and functional evaluation of O-GlcNAc on individual proteins via an approach that is generalizable to a broader array of post-translational modifications.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christina Woo

LAB HEAD: Christina Woo

PROVIDER: PXD022347 | Pride | 2022-02-16

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	201006L_SAM07386_BY81_YG280_TF_pH1_new.raw	Raw
	201006L_SAM07386_BY81_YG280_TF_pH2.mzML	Mzml
	201006L_SAM07386_BY81_YG280_TF_pH2.raw	Raw
	201006L_SAM07386_BY81_YG280_TF_pH3.mzML	Mzml
	201006L_SAM07386_BY81_YG280_TF_pH3.raw	Raw

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Publications

Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase.

Ge Yun Y Ramirez Daniel H DH Yang Bo B D'Souza Alexandria K AK Aonbangkhen Chanat C Wong Stephanie S Woo Christina M CM

Nature chemical biology 20210308 5

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inhe ...[more]

PMID: 33686291

Similar Datasets

Project description:Drosophila development is a complex and dynamic process regulated, in part, by members of the Polycomb (Pc), Trithorax (Trx) and Compass chromatin modifier complexes. O-GlcNAc Transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers N-acetyl-D-glucosamine (GlcNAc) onto hydroxyl groups of serine or threonine residues of key transcriptional regulators using the nutrient-derived UDP-GlcNAc as a substrate, which is dynamically removed by O-GlcNAcase (OGA). We performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells and found that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. To examine the in vivo consequences of disturbed O-GlcNAc cycling in the whole animal, we produced a null allele of oga (ogadel.1) in Drosophila. Epigenetic activators including Trx group members Trithorax (Trx), Absent small or homeotic discs 1 (Ash1) and Compass member Set1 histone methyltransferases are O-GlcNAc modified in ogadel.1 mutants. ogadel.1 mutants displayed altered expression of a distinct set of cell cycle related genes in ovaries. Our results suggest that the loss of OGA could affect epigenetic machinery by accumulating O-GlcNAc on numerous chromatin factors including Trx, Ash1 and Set1 in Drosophila. We performed affymetrix tilingarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells to find if that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), ------------------------------- This represents the gene expression component only