Gaucher disease (GD) is a unique autosomal recessively inherited disorder. It is the most prevalent Iysosomal storage disease and it has remarkable phenotypic heterogeneity. Efficacious enzyme (acid beta-glucosidase, beta-Glc) therapy is available. Progress also has been made in delineating disease alleles, genotype/phenotype correlations, and beta-Glc's basic enzymology. Because of this, GD has become a prototype for molecular medicine's emphasis on predictive testing and gene-based therapies. However, insufficient basic understanding of beta-Glc's structure and function and GD pathophysiology are limiting continued progress in this prototype inherited metabolic disease. The applicant will focus on two hypotheses: 1) a fuller understanding of structure/function relationships of this enzyme will provide insight into the genotype/phenotype correlations and the design of improved therapeutic strategies; and 2) the in vivo level of tissue enzyme activity is a major, albeit not exclusive, determinant of GD pathogenesis. Mutational analysis of GD alleles, phylogenetic conservation of sequences, and direct analyses of function and structure will identify regions and/or domains important to the enzyme's catalytic mechanism, the disulfide structure (i.e., ES-MS), and intracellular targeting signals. Purified human enzymes from a baculovirus expression system will provide sufficient normal and specifically mutagenized enzymes (e.g., high activity mutants) for systematic analyses of active-site function, phosphatylserine and saposin C activation, and x-ray diffraction of crystals. Selectively mutated and truncated beta-Glcs, in MFG-based retroviral vectors, will be used to identify the oligosaccharide independent trafficking signals for this Iysosomal enzyme. Low copy (1-3) integration and use of CRIMneg GD fibroblasts will avoid the potential problems of intracellular receptor overload and/or competition. The pathophysiology of Gaucher disease will be studied using transgenic mice containing regulatable beta-Glc constructs. Based on preliminary studies showing control of beta-Glc expression, they are adapting the tetracycline transactivator (tTA)-based system to transgenic mice. The tTA system uses tetracycline as a """"""""rheostat"""""""" to up or down beta-Glc. Once bred into newly developed GD """"""""knock-out"""""""" mice, the pre- and post- natal temporal and spatial biology of GD will be systemically evaluated by selectively timing the turn-off or -on of enzyme expression. Immunofluorescence, western blot, enzyme, and glycosphingolipid analyses will quantitatively delineate the effects and reversibility of GD. The results of these investigations should provide a deeper understanding of beta-GIc's function and the pathophysiology of GD as a basis for improved therapies for this and other inborn errors of metabolism.
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