GM2 gangliosidosis is a heterogeneous group of genetic disorders due to defective catabolism of ganglioside GM2. This severe and nearly always fatal neurological disorder results when any one of the three tissue constituents required for normal degradation of GM2 ganglioside is genetically abnormal. Abnormality in the enzyme hexosaminidase alpha subunit leads to a disease clinically known as Tay Sachs disease, whereas abnormality in the subunit is identified as Sandhoff disease. A deficiency of the GM2 activator protein is termed """"""""AB variant"""""""". cDNA's for all three proteins and genomic clones for the first two forms are now available for analyses of the genetic abnormalities at the nucleic acid level. During the past three years, the applicants' laboratory has identified seven of the thirteen known mutations in Tay Sachs disease, and the only known specific mutation in Sandhoff disease, and has successfully cloned cDNA's coding for the activator protein. This application proposes to extend their work to further characterize naturally occurring mutations in the three genes among patients with genetic GM2 gangliosidoses. Identification of naturally occurring mutations will be accomplished largely by standard molecular biological techniques, including cDNA and genomic library construction, screening, the polymerase chain reaction, sequencing, allele specific oligonucleotide screening, and transient functional expression in COS I cells. The second specific aim is to obtain and characterize full length cDNA's and the gene coding for the GM2 activator protein and to search for the structural requirement for the mannose phosphate recognition marker, taking advantage of the small size and only one glycosylation site of the protein. Characterization of the cDNA and the genomic structure of the GM2 activator proteins will also be done with standard recombinant DNA technologies, since they already have full length cDNA clones and are on their way to obtain genomic fragments. The third specific aim is to produce and purify milligram quantities of the three proteins using the new baculovirus system. The proteins will be used for subsequent experiments such as to produce antibodies. The fourth specific aim is to explore the structure/function relationship within the different domains of each of the constituents for the enzymatic reaction, the two subunits, the activator protein, and the substrate GM2 ganglioside, and interactions among the three proteins. These studies will extensively utilize sitedirected mutagenesis based on the knowledge gained from naturally occurring mutations and their enzymological properties coupled with information from computerized molecular modeling. 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 may contribute to eventual understanding of the molecular pathogenesis of these genetic neurological disorders and may be useful to future attempts at the gene therapy.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Neurology C Study Section (NEUC)
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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