GLCNAC-PROBE · Novel chemical and enzymatic strategies for probing O-GlcNAc glycosylation

Novel chemical and enzymatic strategies for probing O-GlcNAc glycosylation

O-GlcNAc glycosylation, the modification of serine and threonine residues of nuclear and cytoplasmic proteins by the covalent attachment of N-acetylglucosamine (GlcNAc), is an unusual form of protein glycosylation that occurs on intracellular proteins. Also known as O-GlcNAcylation, this single-sugar modification is not further elongated into complex glycans, and is highly dynamic, through enzymatic addition and removal by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) respectively. Importantly, it is involved in extensive crosstalk with other post-translational modifications, especially phosphorylation, and plays a relevant role in regulating cellular functions in response to nutrients and stress. In fact, as a nutrient sensor underlies fundamental mechanisms of chronic diseases of aging such as diabetes, neurodegeneration and cancer. Despite its abundance on most of the cell’s regulatory proteins, progress in understanding the molecular roles of O-GlcNAc in the cell has been slow due largely to the lack of methods to study this modification. The major hurdles have been the lack of sensitive and facile tools that could enable a direct, fast, and selective identification of O-GlcNAc proteins. Therefore, there is a clear gap for the detection of O-GlcNAcylation by current methods and novel, improved strategies are much needed. Thus, the primary objective of the project was the development of a novel chemoenzymatic strategy for detection of O-GlcNAcylation. The first step in the proposed chemoenzymatic approach relied on the use of an enzyme to convert the natural GlcNAc-nucleotide into an unnatural analogue bearing a chemical handle. Initially, several rounds of protein expression and purification were carried out to obtain the required, pure enzyme. Next efforts focused on getting consistent, reliable, and scalable results with this enzymatic reaction to obtain considerable amounts of this modified sugar-nucleotide. After some optimization, including parallel exploration of alternative synthetic chemistry strategies, both of these tasks were successfully accomplished, securing a practical and scalable enzymatic synthesis of such unnatural derivative. With good amounts of the modified GlcNAc-nucleotide in hand, the next task was to explore the ability of OGT, the enzyme that transfers GlcNAc from native UDP-GlcNAc to protein substrates, to do so using the unnatural analogue. First, initial OGT reactions were performed with a model peptide known to be an OGT substrate. Gratifyingly, after confirming enzymatic activity with the natural sugar-nucleotide, the corresponding modified variant was also recognized by the OGT enzyme and transfered to the peptide substrate, as monitored by LC-MS. Following some optimization, conversion of this OGT enzymatic reaction could be increased up to ca. 50% to give the corresponding glycopeptide bearing the unnatural GlcNAc incorporating the chemical handle. Next efforts focused on using bioorthogonal chemistry, including inverse electron demand Diels Alder as well as thiol-ene reactions, on the above sugar tag for the attachment of relevant probes, such as a biotin moiety. While the initial chemoselective reactions on the glycopeptide structure or even on the unnatural UDP-sugar itself did not lead to the desired modification, the use of the simplest model system consisting of the single monosaccharide synthesized with the chemical handle provided the corresponding construct bearing the biotin affinity probe when reacted with a synthetic biotin-PEG-thiol derivative under thiol-ene UV conditions. These results open the door to the eventual development of this bioorthogonal chemistry into our real system of interest. The enzymatic transfer of the unnatural sugar by OGT was then assessed at the protein level, by trying the enzymatic glycosylation of the corresponding protein derived from the peptide above. While the activity of the enzyme was first confirmed using the natural UDP-sugar and despite using different protein substrates and conditions, transfer of the unnatural sugar to the relevant proteins could not be detected using Western Blot or mass spectrometry methods. Nonetheless, the successful results obtained at the peptide level give some hope for the prospective translation of the OGT glycosylation reaction to the protein context upon further exploration and optimization. Subsequent bioorthogonal labelling of the modified protein with appropriate probes such as the biotin affinity tag described above would then set the stage for the use of this novel chemoenzymatic method for the prospective identification of O-GlcNAc glycosylated proteins.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз