I plan to pursue several aspects of the work in my laboratory that have been supported by this project grant in the area of the biochemistry and molecular genetics of lysosomal acid hydrolases. Two genes coding for lysosomal proteins are to be studied; GM1 beta-galactosidase and the sphingolipid activator proteins (SAPS). Abnormalities in these genes result in distinct neurological disorders, -- GM1-gangliosidosis/ Morquio B disease (acid beta-galactosidase deficiency), metachromatic leukodystrophy-like and Gaucher-like syndromes-(SAP-1 and SAP-2 deficiencies respectively), and a unique pleomorphic syndrome (total SAP deficiency). Human cDNAs and the genes coding for other proteins in the above list have been cloned. In addition, we have also cloned the cDNA and the gene for the mouse GM1 beta-galactosidase. Research projects proposed include: (1) Cloning and characterization of mouse cDNA and the gene coding for the sphingolipid activator proteins (SAPs), (2) Functions of the translation products of the SAP gene, (3) Exploration of structure/function relationship of SAPs and GM1 beta-galactosidase, (4) Transgenic production of a mouse model of total SAP deficiency and GM1- gangliosidosis (GM1 beta-galactosidase deficiency) by the homologous recombination technology, and (5) Continuing identification of mutations in patients who suffer from the disorders due to defects in the above listed genes, and attempts at genotype/phenotype correlations through naturally-occurring mutations, site-directed mutagenesis, computer- assisted molecular modeling and expression systems. Standard methodologies include cDNA and genomic library construction, screening, the polymerase chain reaction, sequencing, allele-specific oligonucleotide screening, and transient functional expression in COS I cells. Isolation and characterization of the cDNA and the gene coding for the mouse SAPS will be accomplished through the expected homology between the human and mouse genes, as we have successfully done for cloning the mouse acid beta-galactosidase. Physiological functions of the SAP-gene products will be evaluated with metabolic loading of potential substrates in vivo and also in culture systems. The normal clones will be over-expressed and purified so that we can obtain milligram quantities of these proteins for subsequent biochemical experiments. The studies of the structure/function relationship of these proteins will extensively utilize site-directed mutagenesis based on the knowledge gained from naturally-occurring mutations and their enzymological properties coupled with information from computerized molecular modeling. The transgenic lines of mice will be generated by the homologous recombination technology using appropriate constructs of the mouse beta-galactosidase and SAP genes. Both naturally-occurring and mutagenesis-generated mutants will be evaluated functionally in the COS I cell and baculovirus systems. The new knowledge to be generated by this series of studies should contribute to our eventual understanding of the molecular pathogenesis of these serious genetic neurological disorders and to future attempts at the gene therapy of these disorders.
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