The Molecular Basis of Canavan Disease: Aspartoacylase Enzyme Characteristics

Mutations in the gene for aspartoacylase EC 3.5.1.15 ASPA, which catalyzes deacetylation of N-acetyl-L-aspartate NAA, correlate with Canavan Disease CD, a neurodegenerative disorder usually fatal during childhood. Defective ASPA activity has been linked to characteristically elevated NAA levels in the urine of CD patients, and ASPA knockout mice and ASPA deletion rats display CD-like symptoms. While efforts have focused on treating CD, there is limited evidence to support ASPA protein regulation. The ASPA enzyme is thought to be cytoplasmic. In this dissertation, we used immunohistochemistry to show ASPA within nuclei of rat brain oligodendrocytes, in rat kidney proximal tubule cells, and in cultured mouse and rat oligodendrocytes. Subcellular fractionation analysis from wild-type rats revealed low enzyme activity against NAA in nuclear fractions. While two recent reports have indicated that ASPA is a dimer, size-exclusion chromatography of both nuclear and cytoplasmic fractions showed ASPA is an active monomer. Since ASPA is small enough to passively diffuse through the nuclear pore complex, we constructed, expressed, and detected in COS-7 cells a green fluorescent protein-human ASPA fusion protein. The mixed nuclear-cytoplasmic localization of GFP-hASPA demonstrated that the subcellular localization of ASPA is regulated. We then investigated regulation of the ASPA protein at the structural level. A recent alignment study identified ASPA as a member of the carboxypeptidase A CPA family. Therefore, we developed and tested a three-dimensional homology model of ASPA based on CPA. Mutations of the putative zinc-binding residues H21G, E24D, and H116G, the general proton donor E178A, and mutants designed to switch the order of the zinc-binding residues H21EE24H and E24HH116E were created and expressed in COS-7 cells. Each mutation yielded wild-type ASPA protein levels, but undetectable ASPA activity. Finally, the analysis of several CD-associated ASPA mi