We are interested in understanding the structural biology of lysosomal storage diseases. Lysosomal storage diseases are caused by defects in enzymes normally found in the lysosome. The defects result in the loss of enzyme activity in affected individuals, which leads to the accumulation of substrate in the tissues, ultimately leading to the development of disease symptoms. The severity of the lysosomal storage disease depends on the amount, location, and toxicity of the substrates that accumulate. We propose to study the structural biology of Fabry disease by x-ray crystallographic methods. Fabry disease is caused by defects in the human a-galactosidase (a-GAL) gene, and is one of the best studied members of the lysosomal storage disease family. Over three hundred different mutations in a-GAL (most of which are single point mutations) have been identified in patients. These mutations lead to the loss of a-GAL enzyme activity and the subsequent development of Fabry disease. We have recently determined the structure of the human a-GAL glycoprotein by x-ray crystallography, opening the door to understanding the molecular basis for defects leading to Fabry disease. The structure produced surprising results, including that the mutations responsible for Fabry disease were not localized to the active site of the enzyme, but were distributed throughout the structure. We seek to extend our initial structural observations on human a-GAL to learn more about Fabry disease specifically and about lysosomal storage diseases generally.
Our specific aims are as follows: 1. To determine the catalytic mechanism of a-GAL The details of how the enzyme catalyzes its reaction are now accessible to crystallographic studies. 2. To study specific defects responsible for the loss of enzyme activity in Fabry disease patients. We will choose a series of point mutants in a-GAL to determine how they ultimately lead to Fabry disease. We will study the biochemical and biophysical properties of the mutant molecules to understand how they lead to a loss of enzyme activity. 3. To define the molecular basis for pharmocolgical chaperone therapy in Fabry patients Small molecule ligands are currently candidates in clinical trials for Fabry and other diseases. We will use a-GAL as a model system for understanding the molecular basis for chemical chaperone therapies. 4. To investigate the ligand specificities of a-GAL and a-NAGAL a-GAL and the related a-N-acetylgalactosaminidase (a-NAGAL) enzymes have different substrate specificities, yet the active sites differ only by two residues. We will map the substrate specificities and interconvert the enzymes by site directed mutagenesis.
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