Structure and Function in alpha-Dystroglycan Glycosylation

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PROJECT SUMMARY/ABSTRACT The significance of O-mannosyl-based protein glycosylation in higher animals was elucidated in studies primar- ily on α-dystroglycan (α-DG) that established a role for the glycans in anchoring cells to the extracellular matrix (ECM).  α-DG hypoglycosylation is associated with a subset of muscular dystrophies, often accompanied by cognitive or other abnormalities. Searching for a molecular basis of these pathologies revealed new biosynthet- ic pathways involving previously unrecognized enzymes and glycans whose defects impacted α-DG glycosyla- tion, adding to the list of congenital disorders in glycosylation. During the course of evaluating the O- mannosylation pathway, it was discovered this pathway is also involved in mediating certain viral infections and metastasis. The full structure of arguably the most functionally critical of the O-Man glycan classes on α-DG was only established in 2016 through work by us and others. Additional protein O-mannosylation pathways have also recently emerged. Our goal here is to further understand the role and regulation of O-mannosyl- based protein glycosylation as well as the elaboration and distribution of the repeating disaccharide polymer matriglycan, first identified on the α-DG M3 core trisaccharide. Matriglycan is responsible for the functional in- teractions with laminin-G domains in ECM proteins. The first two aims address outstanding questions on the interplay and consequences of the alternative options in extending the initial O-Man sites. The critical decision point in glycan extension comes in the ER from the linkage formed when POMGNT2 adds a GlcNAc on the O- Man modified α-DG. The M3 glycan core that ensues is currently known only to be at two sites on one protein, α-DG. We have already identified some protein sequence elements controlling the sites, but we will develop a comprehensive understanding of the high degree of specificity to address whether there are other potential re- ceptive sites for this glycan on proteins, and whether sites can be engineered into other proteins with therapeu- tic prospects for rescuing phenotypes. We will use a combination of approaches to understand POMGNT2 specificity. In contrast, POMGNT1 can act in the Golgi on the remaining O-Man sites inserting a GlcNAc with a different linkage generating the other major classes of O-Man glycans, particularly M1. The explicit function of these glycans is not known, but in the absence of M1, the M3 glycan is not fully extended and α-DG is func- tionality compromised. We will define where the M3 glycan elaboration is disrupted, what M1 interacts with, and better understand POMGNT1 function. The third aim addresses regulation of the matriglycan length and its relationship to function at biochemical and cellular levels. We will also explore the function of matriglycan on non-O-Man glycoproteins and independent of protein. Taken as a whole, the completion of the proposed aims will establish the rules at a molecular level for functional O-Mannosylation that can be taken advantage of in designing therapeutic approaches.