Genetics: Enzyme and Glycosylated Protein Labeling Essay

Submitted By AlexKreisman1
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* Slide 1 * The emerging method I chose to focus on was the identification of O-GlcNac modifications through the utilization of a newly constructed chemoenzymatic approach developed by the Hsieh-Wilson Lab at Cal Tech. * Slide 2 * The O-GlcNac modification is a posttranslational modification analogous to phosphorylation * OGT and OGA are the two enzymes that catalyse the reversible addition and removal of O-GlcNAc on proteins [As shown in this figure) * Numerous publications have implicated O-GlcNAc modifications as regulators with respect to enzyme activity, DNA binding, phosphorylation binding and many other biochemical processes. * Slide 3 * Prior to the construction of the development chemoenzymatic approach by the Hsieh-Wilson lab, the common method utilized for glycosylated protein labeling included the utilization of antibodies, which lacks with respect to binging affinity and selectivity. * Another method commonly utilized to identify O-GlcNAc modifications has been the utilization of radioactive labeling * In which a galactose ring with a labeling tritium is transferred to the GlcNAc group with the help of the GalT enzyme * The flaws associated with this method are price, utilization of radioactive material, and long exposure time. * The Hsieh-Wilson lab choose to adapt this radioactive labeling through utilizing GalT to transfer a galactose ring that contained chemically functionally and eliminate the use of radioactive regents. * Slide 4 * So they constructed UDP analogue 1 that posses a galactose ring with a functional ketone moiety. * The transfer of this galactose ring onto GlcNAc will be done with Y289L GalT, a GalT mutant that has been shown to posses a larger binding pocket that increases catalytic activity with respect to larger substrates. * The ketone group on the galactose group can subsequently undergo oxime ligation with the amine terminal of amooxy biotion label. * Slide 5 * Oxime ligation is selective with respect to other macromolecules in the cell. * Focusing on the ketone on the UDP 1 * First there is a nucleophilic addition * Followed by protanantion and subsequent dehydration of the addition product. * Which can then be probed with streptivin for chemiluminescence visualization and protein isolation. * Slide 6 * So once the group strategized their chemoenzymatic approach they employed it on a known glycosylated CREB peptide. * LC-MS was utilized to monitor the progression of the reaction * Initially we observe glycosylated peptide, confirmed via mass * After incubation of CREB peptide with GalT mutant, and UDP analogue 1 we observe complete keto-sugar transfer. * A subsequent biotin labeling can be observed in the lower chromatogram * Slide 7 * After demonstrating the ability to label a single peptide, the group wanted to demonstrate the selectivity of their method with respect to the entire CREB protein, known to be highly glycosylated. * Point out band observed in lane one when all reagents were utilized. * And lane 4 utilized glycosylated CREB, demonstrating this method is specific to GlcNAc modifications. * Slide 8 * As a result of demonstrating the selectivity of their labeling method utilizing CREB protein, the group wanted to test the sensitivity of their method utilization alpha crystillan protein * Prior publications have
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