Chemical approaches to understanding O-GlcNAc glycosylation in the brain

Abstract

O-GlcNAc glycosylation is a unique, dynamic form of glycosylation found on intracellular proteins of all multicellular organisms. Studies suggest that O-GlcNAc represents a key regulatory modification in the brain, contributing to transcriptional regulation, neuronal communication and neurodegenerative disease. Recently, several new chemical tools have been developed to detect and study the modification, including chemoenzymatic tagging methods, quantitative proteomics strategies and small-molecule inhibitors of O-GlcNAc enzymes. Here we highlight some of the emerging roles for O-GlcNAc in the nervous system and describe how chemical tools have significantly advanced our understanding of the scope, functional significance and cellular dynamics of this modification.

Figure 1

O-GlcNAc glycosylation is the addition of β-N-acetylglucosamine to serine or threonine residues of proteins. OGT, O-GlcNAc transferase; OGA, β-N-acetylglucosaminidase.

Figure 2

O-GlcNAc proteome from rodent brain tissue. Approximately 31% of the known O-GlcNAc proteins are involved in neuronal communication and signaling, 24% participate in transcriptional regulation, and 21% are associated with cytoskeletal structures. Proteins were classified according to categories described by Schoof et al.

Figure 3

Strategy for chemically tagging O-GlcNAc proteins. (a) An engineered mutant GalT enzyme transfers the UDP-ketogalactose substrate onto O-GlcNAc proteins. The ketone functionality is then reacted with an aminooxy biotin derivative. Tagged O-GlcNAc glycoproteins are then detected by chemiluminescence or isolated by streptavidin affinity chromatography. Adapted from ref. . (b) Detection of αA-crystallin using the ketogalactose-biotin tagging approach and comparison with other methods. The chemical tagging approach provides significantly improved sensitivity relative to antibodies and lectins. 0.75 μg of αA-crystallin was used for the ketogalactose labeling method; 5 μg of protein was used for the lectins and antibodies. (c) The UDP-ketogalactose substrate can be replaced with the UDP-azidogalactose substrate shown, and an azide-alkyne [3+2] cycloaddition reaction can be used to attach biotin or fluorescent dyes.

Figure 4

The BEMAD strategy for mapping glycosylation sites. In this approach, the GlcNAc sugar undergoes a β-elimination reaction. Michael addition with dithiothreitol produces a sulfide adduct that is stable to MS/MS analysis.

Figure 5

Small-molecule inhibitors of O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (OGA). (a) OGT inhibitors (1–3) identified by screening a 64,416-member library of compounds. The benzoxazolinone compound 1 inhibits OGT activity in oocytes and prevents meiotic progression. (b) Representative OGA inhibitors (4–8) with enhanced selectivity for OGA over β-hexosaminidase.

Figure 6

Chemical tools for monitoring O-GlcNAc dynamics. (a) A fluorescence resonance energy transfer (FRET)-based sensor designed to detect the dynamics of O-GlcNAc glycosylation in living cells. Upon glycosylation, binding of the GafD lectin to the O-GlcNAc moiety induces a conformational change and produces a stronger FRET signal. CFP, enhanced cyan fluorescent protein; YFP, yellow fluorescent protein. (b) The quantitative isotopic and chemoenzymatic tagging (QUIC-Tag) approach for quantitative proteomics. Proteins from two different cell states are tagged with a ketogalactose-biotin group as shown in Figure 3a. After proteolytic digestion, the peptides are isotopically labeled and combined. The biotinylated O-GlcNAc peptides are captured using avidin chromatography, and quantified and sequenced by mass spectrometry. This approach enables the identification of O-GlcNAc proteins undergoing changes in glycosylation in response to cellular stimuli and allows for those changes to be monitored at specific specific sites within proteins. Adapted from ref. .