Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II

Abstract

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length. In situ hybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.

Figure 1

Comparison of rat cDNAs. Six rat cDNAs are aligned with the full-length human cell line and rat brain NAALADase cDNAs. The partial clones are comprised for the following bases relative to the full rat sequence: W6, 921-2347; R1–2, 2720951; R11, 1–433; R70, 24–951; R72, 24–2347; and R104, 77–2286.

Figure 2

Transfection of R72 confers NAALADase activity and immunoreactivity. (Upper) Samples of R72-transfected and pcDNA3CAT-transfected (control) cell lysates were assayed for NAALADase activity; activity is expressed as total [³H]Glu liberated from N-Ac-Asp-[³H]Glu ([³H]NAAG) after subtraction of protein-free blank. SEM is contained within the outlines of the bars in the graph. NAALADase inhibitors quisqualate (1, 20) and 2-(phosphonomethyl)glutaric acid (PMG) (16) were included at concentrations of 10 and 100 times their reported Kis. Expressed in terms of percent inhibition, the data translate as follows: 20 μM quisqualate, 91.5%; 200 μM quisqualate, 98.5%; 2.75 nM PMG, 80%; 27.5 nM PMG, 97.4%. Control cell lysates demonstrated no NAAG hydrolytic activity. (Lower) Twenty micrograms of total protein from control or R72-transfected cells was immunoblotted with anti-NAALADase antiserum. A specific immunoreactive band with an apparent molecular mass of approximately 100 kDa is observed.

Figure 3

Full sequence of rat brain NAALADase cDNA (2,347 bp). The cDNA contains a 2,256-base ORF; its translation appears under the cDNA sequence. Numerical index for the cDNA sequence is in roman type; index for the amino acid sequences is in italics. Start and stop codons are indicated by bold lettering. The putative transmembrane domain is underlined. Glycosylvation consensus sites are indicated by + (weak, NXT or NXS for X = D, W, or P) or ∗ (strong, NXT or NXS for X ≠ D, W, or P).

Figure 4

Northern hybridization to the R72 cDNA. Electroblots of rat brain RNA (10 μg total RNA), rat kidney RNA [1 μg of poly(A) RNA], and rat liver RNA [1 μg of poly(A) RNA] were hybridized to a radiolabeled R72 cDNA probe. The same three transcripts of approximately 3.9, 3.0, and 2.8 kb in length are observed in both NAALADase-positive tissues (brain and kidney) whereas no hybridization is seen to RNA from the liver, which is NAALADase-negative. Hybridization to the poly(A) RNA (kidney) shows that the 3.9-kb band does not represent artifactual hybridization to 28S ribosomal RNA.

Figure 5

In situ hybridization histochemistry for NAALADase-like mRNAs and colocalization with GFAP. Expression of NAALADase-like mRNA visualized with cRNA probes appears to be widespread in the brain, with midbrain-brainstem structures generally exhibiting higher hybridization than forebrain areas, except for the olfactory bulb, which shows a relatively high level of expression (a). Note particularly intense labeling of a narrow band near the Purkinje cell layer of the cerebellum. The colocalization of NAALADase-like mRNA to GFAP-positive astroglia in a field of rat hippocampal cells in a coronal brain section (arrows indicate examples of dual-labeled cells in registry. (b) Labeling with a NAALADase cRNA probe shows that small cell bodies in the hippocampus are encircled with dark staining for NAALADase-like message. (d) Immunostaining of this same group of hippocampal cells for the astrocyte-specific marker GFAP is shown, indicated by fluorescent fluorescein isothiocyanate-labeling of fibers within the cell processes. (c) A double-exposure photomicrograph shows that the two signals colocalize to the same cells, with fluorescent GFAP-positive processes extending from dark perinuclear halos of NAALADase labeling. (e) Bright-field photomicrograph of an adjacent section of tissue to which a control sense NAALADase cRNA was applied shows no labeling. [Bars = 50 μm (a) and 5 μm (b–e).]