SUMMARY Dystroglycanopathies, a subset of congenital muscular dystrophies, impact more than 1:100,000 individuals and encompass a range of disorders with patient sequalae ranging from mild muscular dystrophy with near normal lifespan to severe muscular dystrophy with major neural and ocular defects leading to death in the first few years of life. The vast majority of genetically-defined dystroglycanopathies are a result of mutations in genes encoding enzymes of the O-mannosylation pathway, specifically the so-called M3 pathway. To date, the M3 pathway, which consists of approximately a dozen enzymes, has only been found to elaborate O-mannose glycans on a small subset of threonine residues on the peripheral membrane protein alpha-dystroglycan that is involved in a complex that bridges the extracellular matrix to the actin cytoskeleton across the plasma membrane. In the prior funding period, we and others elucidated the M3 structure that terminates in a repeating disaccharide referred to as matriglycan. We have recently demonstrated that matriglycan alone, absent the underlying M3 glycan and alpha-dystrolgycan, is both necessary and sufficient to bind LG domain-containing proteins as well as facilitate binding and infection by certain arenaviruses, such as Lassa virus. Here, we seek to address key remaining basic science issues especially those that would facilitate early stage clinical investigations, including substrate enhancement therapy, AAV gene transfer therapy, and novel gene identification. Thus, we will test the specificity of M3 enzymes including testing whether M3 glycans can be built on other non-O-Man glycan structures (A1). Further testing specificity and to address the evolutionary quandary of building an entire M3 pathway for one protein substrate, we will test whether other proteins contain M3 glycan structures, which could be involved in the phenotypes of certain dystroglycanopathies (A1). Driven by our desire to understand the structure-function relationship of M3 enzymes as well as to inform clinical collaborators, we will investigate the impact of missense variants on M3 enzymes with regards to stability and activity using our established protocols (A2). Further, given the high carrier frequency (1:150 in Northern Europe) of the L276I FKTN variant, we will investigate whether heterozygous and homozygous cell lines harboring this variant are less susceptible to matriglycan-dependent pseudovirus infection (A2). Finally, given that ~1/3 of all dystroglycanopathies are of unknown genetic etiology, we will conduct an unbiased CRISPR/Cas9 screen in relevant human cell lines to identify novel gene products that are required for functional glycosylation of alpha-dystroglycan (A3). Coupled to this unbiased approach, we will also investigate the pathway for CDP-ribitol synthesis, that is necessary for M3 glycan synthesis, to identify gene products involved that may be responsible for a subset of the unknown cases of dystroglycanopathy (A3). Completion of the proposed work will provide a more comprehensive understanding of the M3 pathway enzymes and protein targets, the role of variants in disease and infection, as well as inform the work of clinical collaborators.

PROJECT NARRATIVE This proposal is focused on a glycosylation pathway that when defective leads to congenital muscular dystrophy. This work will elucidate the specificity of the enzymes in the pathway, the impact of disease- causing variants, and uncover novel gene products involved in the pathway that are potentially associated with disease.