Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential human glycosyltransferase that adds O-GlcNAc modifications on numerous proteins. However, little is known about how OGT recognizes various protein substrates. Here we report GlcNAc electrophilic probes (GEPs) to expedite the characterization of OGT-substrate recognition. Data from mass spectrometry, X-ray crystallization, and biochemical and radiolabeled kinetic assays support the application of GEPs to rapidly report the impacts of OGT mutations on protein substrate or sugar binding and to discover OGT residues crucial for protein recognition. Interestingly, we found that the same residues on the inner surface of the N-terminal domain contribute to OGT interactions with different protein substrates. By tuning reaction conditions, a GEP enables crosslinking of OGT with acceptor substrates in situ, affording a unique method to discover genuine substrates that weakly or transiently interact with OGT. Hence, GEPs provide new strategies to dissect OGT-substrate binding and recognition.

Molecular mechanisms regulating O-linked N-acetylglucosamine (O-GlcNAc)–processing enzymes

“…An OGT mutant construct consisting of alanine in place of these 5 asparagine residues (5N5A) retained activity toward short peptides but impaired modification of proteins [10], supporting the importance of these residues in indiscriminate recognition of the polypeptide backbone of proteins distant from the site of glycosylation. In a different approach, the Jiang group developed a GlcNAc electrophilic probe (GEP) that consists of a UDP-GlcNAc analog containing an allyl chloride electrophile extending as an N-acyl group from the 2-amino position [12]. This GEP was used to discern whether mutations in OGT affected sugar binding or protein acceptor binding and glycosylation by examining its relative partitioning to OGT (C917) or the acceptor protein.…”

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O‐GlcNAc‐Cycling Enzymes

“…In combination with structureguidedm utagenesis, this new probe can rapidly assesst he role of specific OGT residues in bindings ugar donor compared to acceptorsubstrates (Figure 3). [69] GEP1 contains an allyl chloride electrophile that extends from the N-acetyl group of UDP-GlcNAc. In the presence of protein substrates, this probe can be used as ar egular sugar donor to conduct glycosylation by OGT.I nt he absence of acceptor substrates, this probe can efficiently label OGT active-site residue C917 to generateacovalent modification on OGT.T he binding ability of OGT toward sugar and protein substrates can be evaluated by varying the level of probe modified protein and OGT.T his can be reported by using click-chemistry conjugation of an alkyne fluorophore with the 6'-azido group on am odified GEP1 probe "GEP1A"i n af luorescence assay (Figure 3).…”

“…Following brief preincubation with OGT,G EP1 wasa ble to in situ crosslink OGT with over 100 proteins in cell lysates (Figure 3). [69] Most of thesec rosslinked proteins were reported to be O-GlcNAcylated, and dozens of possible new OGT substrate proteins were also identified. The crosslinking assay of GEP1 can facilitate the identification of novel OGT substrates and could be optimized for characterizing the binding mode of particular substrates with OGT.I ts hould be noted that this probe is not currently applicable in cells, and the electrophiles and assay conditions might requireo ptimization for different substrates.…”

“…A UDP‐GlcNAc analogue called GlcNAc electrophilic probe 1 (GEP1) that addresses this limitation was recently reported. In combination with structure‐guided mutagenesis, this new probe can rapidly assess the role of specific OGT residues in binding sugar donor compared to acceptor substrates (Figure ) . GEP1 contains an allyl chloride electrophile that extends from the N ‐acetyl group of UDP‐GlcNAc.…”

“…GEP1 can also potentially stabilize OGT–protein substrate complexes to facilitate investigation of their interactions. Following brief preincubation with OGT, GEP1 was able to in situ crosslink OGT with over 100 proteins in cell lysates (Figure ) . Most of these crosslinked proteins were reported to be O‐GlcNAcylated, and dozens of possible new OGT substrate proteins were also identified.…”