Tay-Sachs disease is a form of the GM2 gangliosidoses caused by mutations in the HEXA gene. We have disrupted the HEXA gene in the murine embryonic stem cell line, J-1, by gene targeting via homologous recombination. Two targeted cell lines were used to derive chimeric mice that transmitted the mutated allele to their offspring. Heterozygous mice were intercrossed to produce mice homozygous for the disrupted gene. The homozygous mice exhibit a complete deficiency of beta-hexosaminidase A, the enzyme that is absent in Tay-Sachs disease. Beta-Hexosaminidase exists as two predominant isozymes, A, a heterodimer of alpha and beta chains and B, a homodimer of B chains. Each subunit carries a different active giving rise to a preferred spectrum of substrates degraded by the respective isozyme. For example, only the heterodimer is able to degrade GM2 ganglioside. We have constructed chimeric subunits composed of proteins of alpha and beta sequences in order to identify the regions responsible for substrate preference. By this strategy we have determined that the alpha subunit substrate recognition site is composed of discontinuous stretches of amino acid sequence from both the N-terminal and C-terminal portions of the polypeptide. This work may lead to the creation of a homodimeric enzyme that can degrade CM2 ganglioside and which may be useful for enzyme replacement and/or gene therapy in Tay-Sachs disease.

Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
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State
Country
United States
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