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* 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
SPECIFIC AIMS
Mutations in the gene for the aspartoacylase (ASPA) enzyme, which catalyzes deacetylation of N-acetyl-L-aspartate (NAA) in the central nervous system (CNS), result in Canavan Disease (CD), a fatal dysmyelinating disease. The aims of this study were to explore the subcellular localization of ASPA in rodent tissues and cells, analyze biochemical properties of nuclear vs. cytoplasmic ASPA, and determine whether ASPA is actively imported into the nucleus.
PRINCIPAL FINDINGS
1. ASPA is an active nuclear-cytoplasmic enzyme in cultured rodent oligodendrocytes and in rat brain and kidney
We recently reported ASPA staining throughout the rat brain, primarily in oligodendrocytes. Our investigations raised the possibility that ASPA might be expressed in the nucleus in addition to the cytoplasm. An online program predicts that human ASPA is 34.8% cytoplasmic and 17.4% nuclear. Now we show using indirect immunofluorescence of cultured primary rat and mouse oligodendrocytes that ASPA merges with 4',6'-diamidino-2-phenylindole (DAPI) in representative cells (Fig. 1
). We utilized newly generated polyclonal antibodies against a conserved ASPA peptide (pepASPA) as well as against full-length recombinant murine ASPA (
ASPA) to substantiate antibody (Ab) specificity for ASPA in the nucleus. We confirm previous reports that ASPA is largely confined to mature oligodendrocytes by using an Ab against myelin oligodendrocyte glycoprotein (MOG) (Fig. 1A
) as well as CC1 (Fig. 1B
). In a representative mature oligodendrocyte cultured from rat brain, MOG was confined to the peripheral cytoplasm and cell processes, while ASPA stained throughout cytoplasm and the nucleus (Fig. 1A
). This phenomenon was confirmed in murine oligodendrocytes using CC1 (Fig. 1B
, top panel). ASPA staining that does not colocalize with cytoplasmic CC1 can be described as nuclear by colocalization with DAPI (Fig. 1B
, bottom panel).
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We confirmed that ASPA staining in Fig. 1
represented true nuclear localization rather than cytoplasmic overlay by using confocal microscopy Z-series capturing multiple depths of a rat brain section stained for ASPA. The Z-series indicated that ASPA can be found in three distinct configurations within oligodendrocytes: predominantly nuclear, mixed nuclear-cytoplasmic, and predominantly cytoplasmic. We next confirmed that nuclear staining was due to the ASPA gene by performing peroxidase immunohistochemistry on brain and kidney from wild-type (WT) and ASPA –/– Tremor rats. We also present the first localization results of ASPA within kidney: ASPA was selectively localized to kidney cortex, where it was strongly expressed only in proximal tubule cells. There was strong ASPA staining in these proximal tubule cells.
In fractionation studies, ASPA activity has previously been found to be associated with cytoplasm and membrane fractions. We therefore investigated whether ASPA was present and functional in sucrose density gradient-purified nuclei from adult rat brain and kidney. LDH, a cytoplasmic marker, showed specific activity in the nuclear extract that was virtually negligible compared to cytoplasmic extract (
2%). Using a highly sensitive radiometric ASPA assay, we determined that ASPA activity was 10-fold higher in kidney than in brain. Further, we showed that ASPA nuclear activity relative to cytoplasmic activity was 3% in kidney and 6% in brain extracts.
2. Active ASPA in nuclear and cytoplasmic fractions is a monomer with differential ionic binding properties in each location
Previous immunoblotting studies from other laboratories using different ASPA antibodies have identified an ASPA monomer and a putative ASPA dimer. Western blot analysis of subcellular extracts with both ASPA and pepASPA detected ASPA in nuclear fractions at a lower level than in cytoplasmic fractions. To differentiate from two cross-reactive, homologous aminoacylases also present in kidney, we noted that the ASPA monomer band at 38 kD was the only band missing from Tremor rat extracts. The putative ASPA dimer band also appeared in Western blots of Tremor rat extracts, indicating it was the result of cross-reactivity rather than covalent dimerization.
To assess the biochemical nature of ASPA within cytoplasm and within nuclei, we partially purified ASPA from subfractionated kidney extracts and compared column elution properties. Diethylaminoethyl (DEAE) cellulose chromatography was used as the first chromatographic step for enzyme purification. For cytoplasmic extracts, as much as 70% of ASPA activity was bound to the column and eluted with 150 mM NaCl medium. For nuclear extracts, less than 50% of total enzyme activity was bound to the column and eluted with 150 mM NaCl buffer. This indicates that nuclear ASPA displays weaker anionic behavior than cytoplasmic ASPA.
DEAE cellulose chromatographed fractions containing the majority of cytoplasmic enzyme activity were pooled and concentrated for further purification by HPLC size-exclusion chromatography. Maximum specific and total activities were observed in a size range (29–43 kD) containing the ASPA monomer (Fig. 2
A). The MW values calculated from a log plot using MW standard proteins corresponded well (35–38 kD) to the size of the ASPA monomer. Nuclear protein samples were found to follow a similar pattern, with peak activity corresponding to the ASPA monomer (Fig. 2B
). Western blot analysis of size-exclusion fractions complimented both activity elution profiles.
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3. ASPA is actively imported into the nucleus
We tagged 38 kD ASPA with GFP, and the 69 kD size of the resulting fusion protein (GFP-hASPA) exceeded the 40–60 kD size cutoff for passive diffusion through the nuclear pore complex. The distribution of transiently transfected GFP-hASPA in formalin-fixed COS-7 cells was mostly nuclear or mixed-to-mostly cytoplasmic. The ratio of nuclear to cytoplasmic GFP-hASPA was determined by merging GFP and DAPI images from several fields of transfected COS-7 cells to assess the extent of nuclear, mixed, and cytoplasmic staining. GFP-hASPA’s distribution was 22.6% nuclear, 41.5% mixed, and 35.8% cytoplasmic. Finally, we used indirect immunofluorescence with pepASPA to verify that transfected native ASPA is also a mixed nuclear-cytoplasmic protein, as visualized at high magnification by merger with DAPI.
CONCLUSIONS AND SIGNIFICANCE
Whereas ASPA has been assumed to be a soluble, cytoplasmic enzyme with some partial membrane association, our recent immunohistochemical localization study raised the possibility that ASPA may also be found in the nucleus. In our current report we explored this possibility and have established that ASPA is a nuclear-cytoplasmic enzyme. Additionally, we have shown that ASPA in the nucleus of rat kidney and brain cells retains low levels of catalytic activity toward NAA. Analysis of GFP-hASPA, which was larger than the size cutoff for passive diffusion through the NPC, demonstrated active import of ASPA into the nucleus. We now consider ASPA to be a member of burgeoning families of previously assumed cytoplasmic proteins that are small enough to passively diffuse through the NPC but have been shown to be actively imported.
The functions served by ASPA in the CNS have been fairly well studied; however, the physiological role of ASPA within kidney has yet to be examined closely. The fundamental diagnostic marker of children with CD is elevated urine NAA, which raises the possibility that kidney ASPA serves a crucial role in deacetylating NAA retained from the circulation. In CD patients, dysfunctional proximal tubule ASPA could result in impaired ability to deacetylate retained NAA, resulting in its accumulation in urine where it is easily detectable.
At least two possibilities pertain to the low level of ASPA activity in the nucleus. First, a small pool of ASPA is targeted to the nucleus by a hereto unknown regulatory signal and retains catalytic activity against NAA. Second, when ASPA is targeted to nuclei, either the nuclear environment or some modification to the enzyme causes it to lose most of its specificity toward NAA. In addition to possibly being active against other N-acetylated amino acids, nuclear ASPA may also be active against N-acetylated proteins such as actin, which in its active form has an acetylated aspartate as its N-terminal residue and is also found in the nucleus.
Our current study is the first to examine native ASPA from animal tissue by size-exclusion chromatography, allowing us to observe that native ASPA is active as a monomer. ASPA is thus distinguished from aminoacylase III, which, through similar means, was demonstrated to be active as a tetramer. These data, however, do not rule out the possibility that ASPA also exists as an inactive dimer.
In summary, we have shown that ASPA is localized to both cytoplasm and the nucleus in specific cell types in rodent brain and kidney and that there is less nuclear activity against the only known substrate for the cytoplasmic form of the enzyme. Concurrently, we have shown that ASPA is active as a monomer and that nuclear import is not due to simple diffusion. The biological role of nuclear ASPA remains to be established. Regulation of ASPA’s nuclear-cytoplasmic shuttling could be highly significant in understanding both the molecular basis for, and the potential treatment of, CD.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5358fje
This article has been cited by other articles:
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  E. Bitto, C. A. Bingman, G. E. Wesenberg, J. G. McCoy, and G. N. Phillips Jr. From the Cover: Structure of aspartoacylase, the brain enzyme impaired in Canavan disease PNAS, January 9, 2007; 104(2): 456 - 461. [Abstract] [Full Text] [PDF]
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