Alpha-galactosidase A is a lysosomal enzyme that catalyzes the conversion of ceramide trihexoside to lactosylceramide and galactose. This enzyme activity is found in many tissues of normal individuals but its absence leads to a metabolic block in the lysosomal degradation of glycoshpingolipids. The physiological role of alpha-galactosidase A in lysosomal degradation of certain proteins is further defined by the fact that individuals with Fabry's disease lack alpha-galactosidase A activity in their visceral tissues. Due to the fact that alpha-galactosidase A is found in very low quantities in normal human tissues and it is fairly labile, its biochemical and molecular properties are not well defined. For example, the alpha-galactosidase A protein has a molecular weight range ot 49,000 - 101,000 and no accurate amino acid sequence data is available. Alpha-galactosidase A is synthesized in many tissues in vivo, but it is found to represent approximately 0.02% of the total liver proteins. Using an affinity purified rabbit polyclonal antibody to human alpha-galactosidase A, we have demonstrated its monospecificity and screened a human liver cDNA library constructed using the expression vector gt-11. A putative human alpha-galactosidase A clone was identified after screening 2 million recombinants in the library. The authenticity of the cDNA clone was verified by demonstrating the corresponding human chromosomal gene maps to the x chromosome, which is the known locus for Fabry's disease in man. This was accomplished by using the putative alpha--galactosidase A cDNA clone to probe both gene dosage and somatic cell hybrid DNA panels. Having isolated a human alpha-galactosidase A cDNA clone, we propose to perform detailed biochemical and molecular characterization of human alpha-galactosidase A. A full-length cDNA clone will be isolated and its nucleotide sequence determined, from which the amino acid sequence of the protein will be deduced. The chromosomal alpha-galactosidase A gene will be isolated and its molecular structure will be defined. Mutant alpha-galactosidase A genes will also be isolated from Fabry's patients and sequenced. These studies will define the molecular basis of Fabry's disease, and should provide the foundation for development of analytical methodologies for prenatal diagnosis and carrier detection of the herediatry disorder by gene mapping.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Molecular Biology Study Section (MBY)
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University of Alabama Birmingham
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