Role of B3GNT4 mutations in alpha-dystroglycanopathy

About This Grant

This project focuses on defining the biochemical and cellular functions of B3GNT4 in support of the long-term goal of learning about its role in alpha-dystroglycanopathy and other human diseases. B3GNT4 is an understudied protein. It is highly expressed in the brain and the limited biochemical analysis that has been performed is consistent with it functioning as a Golgi-resident beta-1-3-N-acetylglucosaminyltransferase (beta- 1-3-GlcNAc-transferase) that participates in the biosynthesis of poly-N-acetyllactosamine (polyLacNAc) chains. A recent report described biallelic mutations in the B3GNT4 gene in a patient with progressive muscular weakening and brain atrophy but no mutations in genes known to be associated with muscular dystrophy. However, the mechanism by which B3GNT4 mutation could cause an alpha-dystroglycanopathy was not investigated. In this R03 project, we will conduct cell culture experiments and in vitro biochemical assays to obtain fundamental information about B3GNT4 in effort to elucidate possible mechanisms for its role in alpha- dystroglycanopathy phenotypes. We will establish neuronal and skeletal muscle cell lines with genetic knockout of B3GNT4, as well as cell lines with rescued overexpression of wild-type B3GNT4 or the disease-associated G160W mutant. We will also prepare purified, recombinant B3GNT4 catalytic domain as well as the corresponding G160W mutant. These reagents will be important tools for our aims and will be available to the research community. In the first aim, we will use these reagents to identify molecules glycosylated by B3GNT4. Specifically, we will determine whether alpha-dystroglycan is glycosylated by B3GNT4 and also identify any additional proteins glycosylated by B3GNT4. We will define the glycan structure that serves as a B3GNT4 acceptor as well the glycan structures that are produced by B3GNT4 activity. In the second aim, we define the impact of the G160W disease-associated mutation on B3GNT4. Using recombinant proteins, we will measure how G160W impacts B3GNT4 enzyme kinetics. Using cell culture experiments, we will evaluate how G160W affects B3GNT4 localization and binding partners. Studying the biochemical function of B3GNT4 will lead to new insights into (1) the neurological phenotypes of alpha-dystroglycanopathies and (2) the roles of B3GNT4 in other diseases.