The overall objective of the proposed research is to investigate the molecular and cellular mechanisms of two genetically related, but functionally different, lysosomal glycosidases, alpha-galactosidase A (alpha-Gal A) and alpha-N-acetylgalactosaminidase (alpha-Gal B) by characterization of their respective structural domains, enzyme complexes, and deficiency diseases. To facilitate these studies, we 1) isolated and characterized the full-length human and murine cDNAs and the complete human genomic sequences encoding alpha-Gal A and alpha-Gal B, 2) developed methods for their high-level eukaryotic expression and purification, 3) identified a variety of mutations that result in their respective lysosomal disorders, Fabry and Schindler diseases. We propose to determine the structures of these two remarkably homologous glycosidases whose genes presumably evolved by duplication and diverged to have different substrate specificities, stabilities and N-linked oligosaccharide chains. Large amounts of recombinant alpha-Gal A and alpha-Gal B will be produced and purified for crystallization and solution of their 3D structures, and for the biochemical and computer-assisted analyses of their respective structural domains, including N-glycosylation sites and the oligosaccharide structures of each, galactose recognition and catalytic residues in active site domains, cysteines in disulfide bridges, and subunit association domains. To investigate the metabolic and cellular pathologies of these inborn errors of glycolipid and glycoprotein metabolism, alpha-Gal A or alpha-Gal B deficient mice will be generated using murine genomic constructs altered for homologous recombination in ES cells followed by blastocyst implantation. Characterization of the biochemical, morphologic and pathophysiologic alterations in the animal models may provide insights into the pathogenesis of their respective human lysosomal disease. Finally, the role of alpha-Gal B in neuroaxonal transport will be explored by determining the alpha-Gal B distribution in neural tissues, characterizing the effect of alpha-Gal B inhibition on axonal transport in sciatic nerve preparations, and studies of alternative alpha-Gal B substrate specificities (e.g., alpha-N-acetylgalactosaminylphosphate-linked residues involved in neural-specific cell-cell adhesion. These studies should provide fundamental understanding of the molecular and cellular mechanisms of these two related glycosidases in health and disease.
|Effective start/end date||1/05/92 → 30/09/05|
- National Institute of Diabetes and Digestive and Kidney Diseases: $310,092.00
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