Investigation of Neural Pathogenic Mechanisms Associated with Congenital Disorders of Glycosylation

PROJECT SUMMARY/ABSTRACT Congenital Disorders of Glycosylation (CDG) are a group of rare genetic disorders that affect enzymes involved in glycosylation, leading to altered or incomplete glycan structures. These enzymes also affect multiple pathways in cells and tissues often leading to multisystem disorders that include neurological symptoms. Although several studies have been performed to understand CDG at a molecular level, we still lack a clear understanding of how inherited alterations in glycosylation result in the profound neurological features observed in CDG patients. To answer this question, our multidisciplinary research teams will collaborate with each other and will generate animal and cellular models that represent PMM2-CDG and PIGA-CDG including zebrafish models (Project 1), drosophila models (Project 2) and 3D human cortical organoids as models (Project 3). The products of PMM2 and PIGA genes catalyze early steps in N-linked glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis, respectively. The Proteomics/Glycoproteomics Core under the supervision of Dr. Akhilesh Pandey at Mayo Clinic will perform in-depth quantitative proteomics and N-glycoproteomics analysis of these models using latest generation mass spectrometers and optimized protocols for enrichment, fractionation and peptide/glycopeptide fragmentation to evaluate the effects of these CDG on cellular proteome and glycosylation. Notably, the Proteomics/Glycoproteomics Core is equipped with several automated sample preparation platforms for robust and reproducible analysis. For proteomics analysis, a multiplexed quantitation approach will be deployed using tandem mass tag (TMT)-labeling of peptides coupled to deep fractionation for optimal quantitation and comprehensive coverage. This is made possible by combining real-time search functionality with synchronous precursor selection and MS3-based quantitation of reporter ions on Orbitrap Ascend Tribrid mass spectrometers. Highly innovative and relevant to this proposal are the deployment of size exclusion chromatography (SEC) and mixed-mode strong anion exchange (MAX)-based enrichment for glycopeptides and stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) for more complete fragmentation of N-glycopeptides and glycans along with site localization. The proteomics and glycoproteomics data will be analyzed by publicly available and in- house developed scripts. The combined proteomics and glycoproteomics studies will be used to evaluate glycan composition, glycan heterogeneity, their site of attachment to the proteins and site occupancy of proteins. We believe that this approach has the potential to provide us the better molecular understanding of development of neurological symptoms in CDG. Finally, the staff of the Proteomics/Glycoproteomics Core are highly experienced and have previously collaborated and co-published with several other participating investigators of this Program Project Grant ensuring the likelihood of success of this proposal.