Photocrosslinking O‐GlcNAcylated Proteins to Neighboring Biomolecules

Abstract

This protocol enables identification of the interaction partners of O-GlcNAcylated proteins. The method involves the introduction of the diazirine photocrosslinker onto the O-GlcNAc modification within living cells. The photocrosslinker is activated by UV light to yield covalent crosslinking between O-GlcNAcylated proteins and neighboring molecules. The binding partners can be further characterized by immunoblot or proteomics mass spectrometry methods. The benefits of using the photocrosslinker include the capacity to trap lowaffinity binding interactions and the ability to selectively target the interaction partners of the O-GlcNAcylated form of the protein of interest.

Synthesis of Cell-Permeable N-Acetylhexosamine 1-Phosphates

“…Colo205 cells stably expressing UAP1 F383G were prepared by lentiviral infection using pSin4 UAP1 F383G (Addgene ID 169891) as reported previously . Wild-type Colo205 cells, or Colo205 cells stably expressing UAP1 F383G, were cultured in RPMI-1640.…”

“…To mask the phosphate, we used S -acetyl-2-thioethyl (Ac-SATE) protecting groups, which have been widely used to improve delivery of nucleoside phosphate and phosphonate prodrugs. , Intracellular esterases act on Ac-SATE groups to produce an unstable O -2-mercaptoethyl substituent, which has been proposed to further cyclize to form a thiirane and reveal the unprotected phosphate (Figure B) . Using this protecting group strategy, we successfully delivered diazirine-modified GlcNAc-1-phosphate [GlcNDAz­(2me)-1-P] to a variety of mammalian cell lines. ,, This compound can be transformed to the corresponding UDP-GlcNDAz­(2me) by the action of the F383G mutant of human UAP1. UDP-GlcNDAz­(2me) is a substrate for the O -GlcNAc transferase (OGT), allowing for intracellular production of diazirine-modified O -GlcNAc modifications ( O -GlcNDAz) .…”

“…We also evaluated the ability of compounds 21 and 28 to support photo-cross-linking of an O -GlcNAcylated protein, following our previously described protocol . The wt or UAP1 F383G Colo205 cells were cultured with 21 or 28 and then subjected to 365 nm irradiation to induce cross-linking.…”

“…4 Using this protecting group strategy, we successfully delivered diazirine-modified GlcNAc-1-phosphate [GlcNDAz(2me)-1-P] to a variety of mammalian cell lines. 2,6,7 This compound can be transformed to the corresponding UDP-GlcNDAz(2me) by the action of the F383G mutant of human UAP1. UDP-GlcNDAz(2me) is a substrate for the O-GlcNAc transferase (OGT), allowing for intracellular production of diazirine-modified O-GlcNAc modifications (O-GlcNDAz).…”

“…We also evaluated the ability of compounds 21 and 28 to support photo-cross-linking of an O-GlcNAcylated protein, following our previously described protocol. 7 The wt or UAP1 F383G Colo205 cells were cultured with 21 or 28 and then subjected to 365 nm irradiation to induce cross-linking. Cells were lysed, and lysates were analyzed by immunoblotting using an antibody that recognizes NUP153, a heavily O-GlcNAcylated protein.…”