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Aspartoacylase is a regulated nuclear-cytoplasmic enzyme

Jeremy R. Hershfield^*, Chikkathur N. Madhavarao^*, John R. Moffett^*, Joyce A. Benjamins, James Y. Garbern^, and Aryan Namboodiri^*,1

^* Department of Anatomy, Physiology, and Genetics, The Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA;

Department of Neurology, Wayne State University School of Medicine, Detroit, Michigan, USA;

Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA

¹Correspondence: Dept. of Anatomy Physiology and Genetics, USUHS, 4301 Jones Bridge Rd, Bethesda, MD 20814 USA. E-mail: anamboodiri{at}usuhs.mil

ABSTRACT

Mutations in the gene for aspartoacylase (ASPA), which catalyzes deacetylation of N-acetyl-L-aspartate in the central nervous system (CNS), result in Canavan Disease, a fatal dysmyelinating disease. Consistent with its role in supplying acetate for myelin lipid synthesis, ASPA is thought to be cytoplasmic. Here we describe the occurrence of ASPA within nuclei of rat brain and kidney, and in cultured rodent oligodendrocytes. Immunohistochemistry showed cytoplasmic and nuclear ASPA staining, the specificity of which was demonstrated by its absence from tissues of the Tremor rat, an ASPA-null mutant. Subcellular fractionation analysis revealed low enzyme activity against NAA in nuclear fractions from normal rats. Whereas two recent reports have indicated that ASPA exists as a dimer, size-exclusion chromatography of subcellular fractions showed ASPA is an active monomer in both subcellular fractions. Western blotting detected ASPA as a single 38 kD band. Because ASPA is small enough to passively diffuse into the nucleus, we constructed, expressed, and detected in COS-7 cells a green fluorescent protein-human ASPA (GFP-hASPA) fusion protein larger than the permissible size for the nuclear pore complex. GFP-hASPA was enzymatically active and showed mixed nuclear-cytoplasmic distribution. We conclude that ASPA is a regulated nuclear-cytoplasmic protein that may have distinct functional roles in the two cellular compartments.—Hershfield, J. R., Madhavarao, C. N., Moffett, J. R., Benjamins, J. A., Garbern, J. Y., Namboodiri, A. Aspartoacylase is a regulated nuclear-cytoplasmic enzyme.

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