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Keywords:

  • glutamate carboxypeptidase II;
  • glutamate carboxypeptidase III;
  • metabotropic glutamate receptors;
  • mGluR3;
  • NAAG peptidase;
  • N-acetylaspartylglutamate

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar Vmax and Km values (1.4 µm and 54 pmol/min/mg; 3.5 µm and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.

Abbreviations used
CHO

Chinese hamster ovary

GCPII

glutamate carboxypeptidase II

mGluR3

glutamate carboxypeptidase III

NAAG

N -acetylaspartylglutamate

The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) (reviewed in Neale et al. 2000) is colocalized with a spectrum of small amine transmitters, including glutamate and GABA. This peptide activates presynaptic mGluR3 receptors to inhibit transmitter release (Zhao et al. 2001; Garrido Sanabria et al. 2004) and may have a role in moderating some pathophysiological consequences of glutamatergic transmission (Slusher et al. 1999; Yamamoto et al. 2001a,b; Ghadge et al. 2003; Hirasawa et al. 2003).

NAAG is hydrolyzed to form glutamate and N-acetylaspartate by extracellular brain peptidase activity that was first reported by Riveros and Orrego (1984). The membrane-bound NAAG peptidase activity in rat brain was characterized (Robinson et al. 1987; Serval et al. 1990), purified (Slusher et al. 1990) and partially sequenced. These sequence data led to the discovery (Carter et al. 1996) that the enzyme had been cloned previously from a human prostatic carcinoma cell line and identified as prostate specific membrane antigen (Israeli et al. 1993). This NAAG peptidase subsequently was cloned from rat brain (Bzdega et al. 1997; Luthi-Carter et al. 1998a). The enzyme, which was referred to as NAALADase and NAAG peptidase, has been designated glutamate carboxypeptidase II (GCPII). The pharmacological characteristics of GCPII are similar to those of the total membrane bound brain enzyme activity, leading to the tentative conclusion that GCPII is the sole enzyme that inactivates NAAG. However, a strain of mice in which GCPII had been deleted continued to express low levels of NAAG peptidase activity in the kidney and nervous system, suggesting the possibility that other enzymes may contribute to the inactivation of NAAG (Bacich et al. 2002).

A series of NAAG peptidase inhibitors (Jackson et al. 1996; Nan et al. 2000; Kozikowski et al. 2001; Majer et al. 2003) have been developed that increase the efficacy of synaptically released NAAG. The applications of these inhibitors to models of excitotoxicity and inflammatory and neuropathic pain have supported the contention that NAAG peptidase is a significant therapeutic target (Slusher et al. 1999; Yamamoto et al. 2001a,b; Chen et al. 2002; Ghadge et al. 2003; Hirasawa et al. 2003).

Pangalos et al. (1999) identified an EST from a human carcinoma that possessed 67% identity and 80% homology with human GCPII. Both sequences retain the highly conserved zinc binding domain that is common to zinc peptidases. When expressed in COS cells, this cloned human cDNA had both dipeptidyl peptidase IV and some NAAG peptidase activity. While this peptidase was not detected in nervous system by northern analysis, its expression in brain was suggested by RT-PCR data. The aim of the present study was to clone the homologous enzyme using mouse ESTs, to characterize this enzyme, glutamate carboxypeptidase III (GCPIII), with respect to its expression in the nervous system and to compare its activity profile with rat GCPII. Additionally, we tested the hypothesis that this enzyme was up-regulated in a strain of mice in which the GCPII gene had been deleted and compared GCPIII with the residual enzyme activity in the brains of GCPII null mutants.

Cloning of glutamate carboxypeptidase III

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

We performed a BLAST search of the GenBank mouse EST database using human NAALADase II (accession number AJ012370) as a query sequence. Several ESTs containing partial coding sequence of putative mouse GCPIII were found. Four EST clones were purchased: AU018710 (mouse embryo), vk75g08 (mouse embryo) and vi75a08 (mouse testis) from ATCC and 6193175 (mouse brain) from Research Genetics. Each clone was sequenced using Applied Biosystems Prism 3100 sequencer. Clones AU018710 and vk75g08 contained over 50% of the coding sequence including a stop codon, while vi75a08 and 6193175 both contained 5′ UTR followed by an initiation codon and a short stretch of coding sequence. Based on these sequences, we designed PCR primers to amplify the entire 5′ end of the coding sequence. Primer sequences were: 5′-TGCAGTTGCAGTCAGAGGTACT-3′, and 5′-TTTGTCTGGTGGAGCAGCT-3′. RT-PCR was performed on mouse brain total RNA as a template, using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and AccuTaq DNA polymerase (Sigma, St Louis, MO, USA). The product of this amplification was subcloned into PCR-Script plasmid (Stratagene, La Jolla, CA, USA) predigested with Srf I, and subsequently cut out with EcoRI and BsrGI restriction enzymes. Clone AU018710 was cut out from pSPORT 1 vector (Invitrogen) with BsrGI and SphI, and then joined to the amplification product at the BsrGI site. The product of this ligation was subcloned into the Litmus 38 cloning vector (New England Biolabs, Beverly, MA, USA) digested with EcoRI and SphI restriction enzymes.

The construct was then sequenced in both directions and the sequence was compared with that found in the mouse genome database.

Expression of GCPIII

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

The GCPIII insert was subcloned into the pCI-neo mammalian expression vector (Promega, Madison, WI, USA) with EcoRI and SalI restriction enzymes. Chinese hamster ovary (CHO) cells were transfected with the construct or with an unmodified pCI-neo vector as a control. Stably transfected cell lines were selected with the antibiotic G418.

Enzymatic activity assays

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Transfected cells were harvested in 50 mm Tris-HCl buffer, pH 7.5, frozen at −80°C, thawed and sonicated. Membranes were sedimented in a refrigerated microcentrifuge for 15 min, washed once, and resuspended in the same buffer. Protein concentrations were determined by the bicinchoninic acid method (Pierce, Rockford, IL, USA). NAAG peptidase activity was determined using 4 µm NAAG as a substrate unless otherwise stated. Trace amounts of 3H-NAAG (Perkin Elmer/NEN) were added to the reaction, and substrate and product were separated by ion exchange chromatography as previously described (Fuhrman et al. 1994). In all experiments, membranes from cells transfected with pCI-neo vector were used as controls. The background values obtained from these vector controls were subtracted from GCPIII stable transfect activity values.

In order to compare the pH profile of GCPIII with published data on GCPII and with the residual activity in GCPII knockout mouse brain, Tris-Cl buffer was used across the pH range. Recognizing that this buffer has very limited buffering capacity on the acidic side of the pH curve, we determined the pH in duplicate samples at the beginning and end of the incubation intervals and verified that each sample maintained the correct pH value.

Dipeptidyl peptidase IV activity was determined using 250 mm Gly-Pro-AMC as a substrate in 20 mm Tris-HCl, pH 7.5 and 100 µg membrane protein in a total volume of 0.5 mL. Fluorescence was measured with excitation at 360 nm and emission at 415 nm. Membranes from mouse cortical astrocytes were used as positive control.

Tissue distribution analysis

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

For northern blot analysis, Mouse Blot I was purchased from Ambion (Austin, TX, USA). The blot was hybridized with 30 bp, 32P end-labeled oligonucleotide probe (5′-CATGCATTCTAATGTTCCTGGAATATTCAC-3′) at 38°C in Ultrahyb-Oligo hybridization buffer (Ambion). The blot was washed twice in 2 × saline sodium citrate buffer/0.5% sodium dodecyl sulfate at 42°C and exposed to X-ray film.

Distribution of GCPIII message in mouse brain and primary neuronal and astrocytic cultures was determined using real-time RT-PCR method. Total RNA was extracted using guanidinium thiocyanate method (TRI Reagent, Sigma). RT reaction was conducted using M-MLV reverse transcriptase (Invitrogen), random hexamers as primers, and 0.25 µg of total RNA as a template. Primers for PCR amplification were: 5′-ATGATGCAGAGAGACTATT-3′ and 5′-TTTGTCTGGTGGAGCAGCT-3′. The product of amplification spans two exons. The primer's sequences, as well as probe for northern blot, were checked against GCPII sequence, and the ones with least possible homology were used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified in parallel reactions as an endogenous control. Real-time amplifications were performed using ABI Prism 7900HT Sequence Detection System (Applied Biosystems) and SYBR Green QPCR Core Reagent Kit (Stratagene). Dissociation curve analysis was performed as part of each real-time PCR run to ensure that primer dimers were not present. The products of real-time PCR were additionally verified on agarose gel. The relative quantification of target RNA was achieved by comparative threshold cycle (CT) method. The target threshold cycle number was normalized to an endogenous reference (GAPDH), and relative amount of message was calculated relative to the sample showing the least abundance (Applied Biosystems, User Bulletin #2).

Primary cell cultures

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Primary cultures of cerebellar granule cells were prepared from 8-day-old SW mice, according to the method of Gallo et al. (1982). The cells were grown on dishes (Nunc) coated with poly l-lysine (Sigma) in basal Eagle's medium (BME, Gibco) supplemented with 10% fetal calf serum (Gibco, Rockville, MD, USA), 25 mm KCl, 2 mm l-glutamine, and 10 µg/mL gentamycin. To prevent the proliferation of non-neuronal cells, cytosine arabinofuranoside (10 µm) was added 20–24 h after plating. Cells cultured for 7–14 days in vitro were used in all experiments. Immunocytochemical studies indicate that primary cultures of cerebellar granule cells contain about 97% neurons (Nicoletti et al. 1986). Cerebellar astrocytes were prepared according to the same method, but resuspended in MEM medium supplemented with l-glutamine, 10% fetal bovine serum (heat –inactivated) and 10 µg/mL gentamycin.

Primary cultures of mouse cortical neurons were prepared according to the method of Rose et al. (1993). In brief, cortical tissues from embryos (E-17, SW mice) were dissected and meninges were removed. Cells were dissociated with trypsin, mechanically dissociated by trituration and plated on poly l-lysine covered dishes (Nunc) in neurobasal medium supplemented with B-27, l-glutamine (2 mm) and antibiotics. Astrocytes were grown in MEM medium (Gibco) supplemented with 10% fetal bovine serum, l-glutamine and antibiotics.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

A similarity search of the GenBank mouse EST database using human GCPIII (termed NAALADase II in Pangalos et al. 1999) as a query sequence found several matching ESTs. However, none contained the entire coding sequence of the putative mouse peptidase. Moreover, part of the open reading frame sequence (bp 602–872, Fig. 1) was not found in any EST clone that we sequenced. Therefore, we amplified the region between bp 22 and 956 by RT-PCR using mouse brain RNA as a template and joined it with EST clone AU018710 (see Materials and methods). Sequencing of the resulting construct revealed a 2223-bp open reading frame (Fig. 1) that is 85% identical to human NAALADase II (Pangalos et al. 1999), and 70% identical to mouse glutamate carboxypeptidase II (Bacich et al. 2001). Blast search of the mouse genome with this sequence found 19 matching sequences in a supercontig mapped to chromosome 9. Alignment with these sequences revealed complete identity, except one silent mutation C/T at bp 999 (Fig. 1). The deduced 740-amino acid protein, which we termed mouse glutamate carboxypeptidase III (GCPIII), has a predicted non-glycosylated molecular mass of 82 801 Da. Analysis of this sequence using the SignalP v2.0 program (Nielsen and Krogh 1998) predicted that amino acids 9–31 serve as a transmembrane anchor of a type II integral membrane protein. The extracellular domain contains seven potential N-glycosylation sites, the putative zinc binding and active site domains that are present in GCPII (Fig. 1).

image

Figure 1. Nucleotide sequence and predicted amino acid sequence of mouse GCPIII. The putative transmembrane domain is underlined. Potential N -glycosylation sites are shaded in gray. Residues presumed to be involved in zinc coordination are shaded in black, and glutamate proposed to be general base in catalysis is boxed.

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Northern blot analysis of mRNA from various mouse tissues revealed the presence of transcripts of the appropriate size in ovary, testes, thymus, lung and kidney, but transcripts were not detected in heart or brain (Fig. 2). Transcripts were detected, however, in specific mouse brain regions by real time RT-PCR with the highest levels being present in the cerebellum and hippocampus with lowest levels in cortex and brainstem (Fig. 3). Real time RT-PCR analysis showed similar levels of GCPIII message in mouse testis and ovary, while about sixfold less message was present in mouse brain (data not shown), confirming results from northern blot.

image

Figure 2. Northern blot hybridization analysis of GCPIII expression. Mouse Blot I (Ambion) containing 2 µg of poly (A) + RNA per lane was hybridized with 32 P labeled oligonucleotide probe. Sizes of RNA molecular mass markers are indicated.

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image

Figure 3. Real-time RT-PCR analysis of GCPIII expression in mouse brain regions. Samples of total RNA (0.25 µg) were reverse transcribed with random oligohexamers, and then amplified with primers for GCPIII and GAPDH. Relative levels of gene expression were calculated as described in Materials and methods. Values are mean ± SEM of triplicate assays. CX, cortex; CB, cerebellum; HIP, hippocampus; STEM, brain stem; S.C., spinal cord; O.B., olfactory bulb. Negative controls (no mRNA added) failed to reach fluorescence threshold for detection.

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GCPII expression in the rat nervous system appears to be restricted to glia (Berger et al. 1999). However, mouse astrocytes and neurons from cortex and cerebellum grown separately in culture all expressed messages for GCPIII as determined by real time RT-PCR (Fig. 4). Cerebellar granule cells and cortical neurons appear to express more message than do astrocytes cultured from the same brain regions. This contrasts with GCPII, which appears to be exclusively glial (Berger et al. 1999)

image

Figure 4. Real-time RT-PCR analysis of GCPIII expression in mouse neurons and astrocytes in culture. Amplification was performed as described in Fig. 3 . Values are mean ± SEM of triplicate assays. CXA, cortical astrocytes; CXN, cortical neurons; CBA, cerebellar astrocytes; CBN, cerebellar neurons. t -test p -values are 0.05 for cortex and 0.1 for cerebellum.

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GCPIII message levels were compared in whole brain tissue obtained from wild-type and GCPII knockout mice and found to be nearly identical in real time PCR assays (relative values of 1.00 ± 0.08 in wild type and 1.00 ± 0.15 in GCPII knockout). These data argue against the up-regulation of GCPIII in the knockout mice. As would be expected, we found that the total NAAG peptidase activity/mg protein in cerebral cortical astrocytes grown from the knockout mice was less than half of that found in cultures of astrocytes from wild-type mice (14.7 ± 0.62 pmol/mg/min for wild type and 6.1 ± 0.65 pmol/mg/min for knockout). However, the peptidase activity in cerebellar astrocytes cultured from the knockout mice was no different from that found in wild-type cerebellar astrocytes (16.4 ± 1.12 pmol/mg/min for wild type and 18.3 ± 0.11 pmol/mg/min for knockout).

Michaelis–Menten and Eadie Hofstee analyses of the kinetic data obtained from assays of cloned rat GCPII and mouse GCPIII under the same conditions (Fig. 5) indicate that these two membrane bound enzymes possess very similar Vmax and Km values (1.4 µm and 54 pmol/min/mg; 3.5 µm and 71 pmol/min/mg, for GCPII and GCPIII, respectively). It should be noted though, that Vmax comparison is of a limited value, as levels of protein expression of both enzymes by the transfected cells are not known. EDTA eliminates the enzyme activity of both enzymes, and metal ions, including cobalt and zinc, stimulate the enzyme activities found in membranes that have been rinsed with and suspended in buffer to remove unbound metals (Fig. 6). Both enzymes have a pH optimum that extends from 6 to 8 (Fig. 7).

image

Figure 5. Kinetic characterization of NAAG hydrolyzing activities by GCPIII and GCP II. Membranes from CHO cells transfected with GCPIII (a) or GCP II (b) were incubated with increasing concentrations of NAAG at 37°C, and the activity determined as described in Materials and methods. Results are mean activities ± SEM from triplicate assays. Inserts: Eadie–Hofstee analysis of the kinetic data. The rate values shown were calculated by subtracting the baseline activity values of CHO cells transfected with vector alone.

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image

Figure 6. Effects of metal ions on NAAG peptidase activity in membranes from CHO cells transfected with GCPIII (a) or GCP II (b). Membranes were incubated at 37°C with 4 µ m NAAG in the presence or absence of metal ions (1 m m ) or EDTA. Activity was determined as described in Materials and methods. Results are the mean ± SEM from triplicate assays. The V -values shown were calculated by subtracting the baseline activity values of CHO cells transfected with vector alone.

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image

Figure 7. Effect of pH on NAAG peptidase activity in membranes from CHO cells transfected with GCPIII (a) or GCP II (b). Membranes were incubated in 50 m m Tris-HCl buffers of different pH. Activity was determined as described in Materials and Methods. Results are the mean ± SEM from triplicate assays. The V -values shown were calculated by subtracting the baseline activity values of CHO cells transfected with vector alone.

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Using cortical astrocyte cell membranes as a positive control, we detected high levels of dipeptidyl peptidase IV activity. However, membranes and whole cell extracts from cells expressing cloned rat GCPPII or mouse GCPIII failed to exhibit this activity. These data contrast a prior report (Pangalos et al. 1999) in which this activity was detected in whole cell preparations from COS cells transfected with the human homologues of these enzymes.

The residual NAAG peptidase activity found in the brains of mice in which GCPII has been deleted was found to be less sensitive (IC50≈ 90 nm) to the peptidase inhibitor, 2-PMPA, than wild-type mouse brain (Bacich et al. 2002). For this reason, the sensitivity of mouse GCPIII activity to this inhibitor was examined (Table 1). The cloned mouse GCPIII and rat GCPII were found to have a similarly high level of sensitivity to inhibition by 2-PMPA and another potent NAAG peptidase inhibitor, FN6.

Table 1.  Inhibition of GCPIII and GCP II activities by FN6 and 2-PMPA. Membranes from CHO cells transfected with GCPIII or GCP II were incubated with 4 µ m NAAG containing 19 pmol of [ 3 H]-NAAG, 15 µg of membrane protein and increasing concentrations (0.1 n m – 10 µ m ) of FN6 and 2-PMPA in a final volume of 50 µL
Peptidase inhibitorsIC50 (nm)
FN62-PMPA
GCPIII0.50 ± 0.270.94 ± 0.19
GCPII4.52 ± 0.476.74 ± 1.31

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

NAAG is a prevalent and widely distributed vertebrate neurotransmitter that is coexpressed with glutamate, GABA, acetylcholine and other small amine transmitters (reviewed in Neale et al. 2000). This peptide selectively activates the group II metabotropic receptor, mGluR3, with about 15-fold less efficacy at mGluR2 (Wroblewska et al. 1997; Cartmell et al. 1998; Schweitzer et al. 2000). NAAG inhibits voltage-dependent calcium current (Bischofberger and Schild 1996) and inhibits transmitter release via presynaptic mGluR3 receptors (Zhao et al. 2001; Garrido Sanabria et al. 2004). While some data have been taken to suggest that NAAG acts as an NMDA receptor antagonist (Puttfarcken et al. 1993; Greene 2001), we tested this hypothesis directly using highly purified NAAG on cerebellar granule cell NMDA receptors and found no physiologically relevant antagonist activity and minimal, if any, agonist activity (Losi et al. 2003).

A growing body of data supports the hypothesis that the synaptic release of NAAG influences the release of other transmitters, particularly glutamate, and that inhibition of NAAG peptidase activity may have considerable therapeutic potential. Critical to this hypothesis is a series of experiments in which novel and highly potent NAAG peptidase inhibitors have successfully mitigated the influence of glutamatergic transmission under pathological conditions. NAAG peptidase inhibitors reduced the excitoxic effects of anoxia and NMDA administration (Kozikowski et al. 2001; Slusher et al. 2001; Thomas et al. 2003), reduced pain perception in models of inflammatory pain and allodynia (Yamamoto et al. 2001a,b; Chen et al. 2002; Hirasawa et al. 2003; Kozikowski et al. 2004), and reduced motoneuron loss in a model of chronic neurodegenerative motoneuron disease (Ghadge et al. 2003).

As in the case of other peptide transmitters, the inactivation of NAAG is primarily by extracellular peptidase activity (Riveros and Orrego 1984; Robinson et al. 1987; Serval et al. 1990; Slusher et al. 1990; Williamson and Neale 1992; Cassidy and Neale 1993; Berger and Schwab 1996; Berger et al. 1999). The identification of NAAG peptidase activity as a property of prostate specific membrane antigen and the cloning of this gene from the rat and human nervous system (Carter et al. 1996; Bzdega et al. 1997; Luthi-Carter et al. 1998b,c) led to speculation that this enzyme, GCPII, was responsible for the inactivation of synaptically released NAAG. However, mice that are null mutants for GCPII appear normal on neurological examination and express low, but significant levels of NAAG peptidase activity (Bacich et al. 2002). While this result does not argue against a central role for GCPII in the inactivation of NAAG, it supports the hypothesis that this inactivation may be achieved by more than one enzyme.

A series of genes have been cloned that possess sequence homology with GCPII and thus have the potential to influence NAAG hydrolysis. One such enzyme with dipeptidyl peptidase IV activity was cloned from human ileum. It has 35% amino acid sequence identity with human GCPII (Shneider et al. 1997) but exhibits no apparent NAAG peptidase activity (Pangalos et al. 1999).

Gingras et al. (1999 ) cloned and expressed a plasma glutamate carboxypeptidase peptidase from human placenta that had 27% amino acid identity with human GCPII. This secreted human protein was reported to exhibit glutamate carboxypeptidase activity, including activity against 1 m m NAAG, and endopeptidase activity. A homologous rat sequence was submitted to GenBank ( AF009513 ) in 1998 as hematopoietic lineage switch 2 protein (HLS2). When we expressed this rat cDNA (graciously provided by David Talmage and Yachi Chen, Institute of Nutrition and Department of Pediatrics, Columbia University) in CHO cells, we failed to detect significant NAAG peptidase activity, although we found expression of this message in rat brain using semiquantitative RT-PCR and nested PCR (unpublished data).

Pangalos et al. (1999 ) identified a novel NAAG peptidase activity when expressing a human gene first identified as a lung carcinoma EST. This enzyme has 67% amino acid sequence identity with human GCPII. In the present paper, we describe cloning the homologous gene using mouse expressed sequence tags and RT-PCR. This enzyme has kinetic qualities and ion requirements that are very similar to those of cloned rat zinc metallopeptidase, GCPII, providing support for the hypothesis that cells in the nervous system express at least two distinct peptidases with high affinity for NAAG.

GCPII has pteroylpoly γ-glutamate hydrolase activity and the catalytic domain has been assigned to the m28 family of cocatalytic zinc metallopeptidases (Rawlings and Barrett 1997). Zinc binding residues and active site residues have been identified in GCPII. These domains are shared by GCPIII. Comparing the sequence homology between the two human enzymes (Pangalos et al. 1999) and the two mouse enzymes, we conclude that GCPIII also is a member of the m28 family of cocatalytic zinc metallopeptidases.

GCPII has been localized exclusively on the surface of glia in rats (Berger and Schwab 1996; Berger et al. 1999) raising a question as to the time course of its access to synaptically released NAAG. Using real time PCR methods, we found higher levels of mouse GCPIII message in primary cell cultures of neurons from cortex and cerebellum than from astrocytes grown from these same brain regions. The expression of GCPIII by neurons, depending upon its site of expression, would suggest that this enzyme might be more available for NAAG hydrolysis in the synaptic cleft. Beyond this, the higher level of expression of GCPII message and enzyme activity in brain may simply reflect the much greater quantity of astrocytes relative to neurons.

We found that astrocytes grown from GCPII knockout cerebellum had similar levels of NAAG peptidase activity as those grown from wild-type mice. This would suggest that GCPIII, rather than GCPII, is the primary peptidase expressed by cerebellar astrocytes. These data emphasize the importance of more rigorously defining the cellular localization and regulation of expression of this second NAAG peptidase. Perhaps more important from a preclinical perspective is the development of novel compounds that differentially inhibit the activity of these two enzymes. We have shown that two different classes of inhibitors, FN6 and 2-PMPA, are equally potent as inhibitors of these two enzymes.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Cloning of glutamate carboxypeptidase III
  5. Expression of GCPIII
  6. Enzymatic activity assays
  7. Tissue distribution analysis
  8. Primary cell cultures
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References
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