O-GlcNAcylated peptides and proteins for structural and functional studies

Abstract

O-GlcNAcylation is an enzymatic post-translational modification occurring in hundreds of protein substrates. This modification occurs through the addition of the monosaccharide N-acetylglucosamine to serine and threonine residues on intracellular proteins in the cytosol, nucleus, and mitochondria. As a highly dynamic form of modification, changes in O-GlcNAc levels coincide with alterations in metabolic state, the presence of stressors, and cellular health. At the protein level, the consequences of the sugar modification can vary, thus necessitating biochemical investigations on protein-specific and site-specific effects. To this end, enzymatic and chemical methods to 'encode' the modification have been developed and the utilization of these synthetic glycopeptides and glycoproteins has since been instrumental in the discovery of the mechanisms by which O-GlcNAcylation can affect a diverse array of biological processes.

Figure 1.. O-GlcNAc modification and methods to prepare site-specifically modified proteins.

a) O-GlcNAc modification is the dynamic addition of N-acetylglucosamine to serine/threonine side-chains of intracellular proteins. b) Posttranslational mutagenesis can be used to transform a cysteine residue into an S-linked analog of O-GlcNAc. c) Solid-phase peptide synthesis used alone or in combination with protein ligation techniques can be used to prepare O-GlcNAc modified peptides and proteins.

Figure 2.. Structural characterization of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA).

Synthetic (glyco)peptides played key roles in helping determine the biochemistry of OGT and OGA using structural biology.

Figure 3.. Protein synthesis for studying O-GlcNAc biochemistry.

a) Posttranslational mutagenesis was used to install O-GlcNAc analogs onto the the histone proteins H2A and H2B, allowing the effects of site-specific O-GlcNAc on nucleosome biology. b) Protein ligation was used to generate O-GlcNAc modified versions of α-synuclein, showing that these glycans have site-specific effects on amyloid formation. The synthesis of α-synuclein with O-GlcNAc at threonine 72 is shown as an example.

Figure 4.. Determining O-GlcNAc stoichiometry using mass shifting.

Synthetic, homogeneously O-GlcNAc modified ubiquitin was used to optimize conditions for chemoenzymatic mass-shifting of O-GlcNAc modified proteins.