The Neuroactive Peptide N-Acetylaspartylglutamate (NAAG...

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The Neuroactive Peptide N-Acetylaspartylglutamate (NAAG)

is not an Agonist at the mGluR3 subtype of Metabotropic

Glutamate Receptor

Maninder Chopra, Yi. Yao, Timothy J. Blake, David. R. Hampson, Edwin. C.

Johnson

Dept of Neuroscience (MC, ECJ), and Dept of Chemistry (TJB), CNS/Pain Discovery,

AstraZeneca Pharmaceuticals, Wilmington, Delaware

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada

(YY, DRH)

JPET Fast Forward. Published on April 23, 2009 as DOI:10.1124/jpet.109.152553

Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 23, 2009 as DOI: 10.1124/jpet.109.152553

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Running Title: NAAG is not an agonist at mGluR3 Receptor

Corresponding Author: Edwin C. Johnson

Address:

AstraZeneca Pharmaceuticals

1800 Concord Pike, CNS/Pain Discovery

Wilmington, Delaware 19850

Phone: (302) 886-4970

Fax: (302) 886-4140

Email: [email protected]

Number of Text Pages: 16

Number of Tables: 1

Number of Figures: 6

Number of References: 27

Number of Words:

Abstract: 220

Introduction: 654

Discussion: 979

List of abbreviations used in the paper:

4C3HPG: 4-carboxy-3-hydroxyphenylglycine; DCG-IV: 2S, 1'R, 2'R, 3'R)-2-(2, 3-

dicarboxycyclopropyl)glycine; HPLC: High Performance Liquid Chromatography;

mGluR3: Metabotropic Glutamate Receptor Subtype 3; NAAG: N-

acetylaspartylglutamate; NAALADase: N-acetyl-α-linked acidic dipeptidase

Section: Neuropharmacology


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ABSTRACT

The peptide N-acetylaspartylglutamate (NAAG) is present in high concentrations in

the mammalian central nervous system. Various mechanisms have been proposed for its

action including selective activation of the metabotropic glutamate receptor, mGluR3, its

action at NMDA receptor, or to produce glutamate by its hydrolysis catalyzed by an

extracellular protease. To re-examine its agonist activity at mGluR3, we co-expressed

human or rat mGluR3 with G-protein inward rectifying channels in Xenopus oocytes.

HPLC analysis of commercial sources of NAAG showed 0.38%-0.48% glutamate

contamination. While both human and rat mGluR3 were highly sensitive to glutamate

with EC50 of 58 and 28 nM respectively, purified NAAG (100 μM) had little activity

(7.7% of full activation by glutamate). Only in the millimolar range did it show

significant activity, possibly due to residual traces of glutamate remaining in the purified

NAAG preparations. In contrast, the unpurified NAAG sample did produce a full agonist

response with mGluR3 co-expressed with Gα15 with an EC50 of 120 μM, as measured by

a calcium release assay. This response can be explained by the 0.38-0.48% glutamate

contamination. Our results suggest that NAAG may not have a direct agonist activity at

the mGluR3 receptor. Thus, a number of in vivo and in vitro published results that did

not address the issue of glutamate contamination of NAAG preparations may need to be

re-evaluated.


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INTRODUCTION

The neurochemistry of the modified dipeptide N-acetylaspartylglutamate (NAAG)

has been studied extensively over the past 25 years. It is present in millimolar

concentrations in many neuronal pathways throughout the central nervous systems of

mammals (Coyle, 1997). NAAG is contained in vesicles and released from neurons in a

calcium-dependent manner (Neale et al., 2000). In the brain, the major mode of

metabolism of NAAG is via glutamate carboxypeptidase II, also known as either

prostate-specific membrane antigen, or N-acetyl-α-linked acidic dipeptidase

(NAALADase). In the CNS, this enzyme is present on astrocytes where it cleaves the

dipeptide into N-acetylaspartate and L-glutamate (Berger et al., 1999). The development

of inhibitors of this peptidase has been an active area of study because they have been

shown to produce beneficial effects in a wide variety of in vitro and in vivo cellular and

animal models of neurological disorders including amyotrophic lateral sclerosis (Ghadge

et al., 2003), pain, particularly inflammatory (Yamamoto et al., 2007), ischemia (Slusher

et al., 1999), and diabetic retinopathy (Neale et al., 2005).

Based on neurochemical profiling, it has been assumed for some time that

neuroprotective properties of NAAG stem from its reported ability to act as (a) a partial

agonist or antagonist at the NMDA receptor, and (b) a highly selective agonist at the

mGluR3 subtype of metabotropic glutamate receptor (mGluR3) with the latter being the

most widely accepted mechanism of action (Wroblewska et al., 1997; Neale et al., 2000).

The interaction of NAAG at the NMDA receptor has been controversial and it is not


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completely clear how the peptide affects NMDA receptor activity. For example, NAAG

was reported to activate NMDA receptors with an EC50 of 0.7 mM (Westbrook et al.,

1986), while others have reported that it possess antagonist activity at this receptor

(Bergeron et al., 2005; 2007). Among G-protein-coupled receptors, NAAG has been

reported to be a highly selective agonist at mGluR3 with an EC50 of 65 μM at a chimeric

receptor consisting of the extracellular ligand-binding domain of mGluR3 and the

transmembrane domain and carboxy terminus of mGluR1a (Wroblewska et al., 1997).

As with glutamate stimulation of mGluR3, NAAG stimulation is assumed to inhibit

the release of glutamate from nerve terminals, thereby affording neuroprotection in

excitoxic testing paradigms. Another potential mGluR3-mediated neuroprotective

mechanism is via the release of growth factors. Activation of mGluR3, which is

expressed in both neurons and astrocytes, by the Group II agonists DCG-IV and

4C3HPG, has been shown to protect neurons from excitotoxic cell death by inducing the

release of transforming growth factor beta from astrocytes (Bruno et al., 1998). In fact,

experiments conducted on mGluR2 and mGluR3 knockout mice strongly indicate that

much of the anti-excitotoxic properties of Group II mGluR agonists are mediated by the

mGluR3 receptor subtype rather than the mGluR2 subtype (Corti et al., 2007). Thus

mGluR3 may play a heretofore under-appreciated role in both normal brain function, and

in disease states.

The two Group II mGluRs, mGluR2 and mGluR3, are highly homologous with each

other. The rat sequences are 65% identical in the extracellular ligand binding domain and

75% identical in the transmembrane domain region. This high degree of amino acid

sequence homology has stymied the development of highly receptor subtype-specific


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compounds (Conn and Pin, 1997; Brauner-Osborne et al., 2007). Nevertheless, NAAG

has been suggested to be a selective agonist for mGluR3 (Wroblewska et al., 1997).

However, the molecular basis for this specificity has not been reported. A molecular

docking analysis conducted on the mGluRs has shown that the glutamate binding site is

rather restricted in terms of the space available for larger ligands, such as peptides, to

bind (Wang and Hampson, 2006; Wang et al., 2006). In the present study, we re-

examined the interaction of NAAG at Group II mGluRs. We found that unpurified

NAAG but not purified NAAG activated mGluR3, and that commercial preparations of

NAAG are contaminated with L-glutamate; the later finding likely explains the

previously reported activity at this receptor.


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METHODS

Purification of NAAG

NAAG purchased from Sigma was dissolved in ultrapure water. Prepacked poly-prep

cation exchange columns (2ml) from Bio-Rad (AG 50W-X8 resin, 200-400 mesh, Cat no:

731-6214) were washed with water and then with 5 bed volumes of 10 mM HCl (pH 2).

One ml of 10 mM NAAG was loaded onto the column; the unretained fraction containing

NAAG was collected and the column was washed with 1 bed volume of water followed

by 5 bed volumes of 10 mM HCl. The bound glutamate can be eluted with 1M

ammonium.

HPLC analysis

Amino acid analyses of unpurified commercial samples and purified samples of

NAAG were carried out using PITC derivatized amino acids on a Waters Alliance

Separation Module equipped with a Hypersil ODS column and a Waters 2487 dual

wavelength absorbance detector (set at 254 nm). The injection volume was 20 μl, the

mobile phase was 60% acetonitrile, and the column temperature was 48ºC. The data

were analyzed using Waters Empower 2 chromatography software.

Mass Spectrometric analysis of NAAG samples

The NAAG samples were analyzed by electrospray LCMS by loop injection and

gradient elution on a Waters QTOF-1 and an Agilent 6210 TOF (for accurate mass

measurements) fronted with Agilent 1100 and 1200 HPLC systems respectively. A


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purified batch from Sigma was diluted (x10) with water:acetonitrile:formic acid (1:1:0.1

v/v) prior to analysis (1 μl aliquot injections). The compound was studied under both

positive and negative ionization conditions. For gradient elution, a Discovery HS C18

75x2.1mm, 3 μM column was used at a flow rate of 0.5 ml/min. The analysis was

performed using mobile phases of water/acetonitrile/formic acid at ratios of 98:2:0.1 v/v

(phase A) and 2:98:0.05 (phase B). The gradient was ramped linearly from 2% to 95% B

over 10 min.

Functional analysis of mGluRs in HEK293 cells

Human embryonic kidney 293 cells (American Type Culture Collection # CRL 1573)

were transiently transfected in 6 well plates using 2 μg of rat wild-type mGlu3 (or rat

mGluR2) cDNA plus 2 μg of Gα15 cDNA using the Fugene 6 reagent (Roche

Biochemicals) or Lipofectamine 2000 (InVitrogen Inc.) as instructed by the

manufacturers. Twenty-four hours after transfection, the cells were plated in

polyornithine pre-treated 96-well microtiter plates (Costar black microtiter plates) at

100,000 cells/100μl of DMEM medium. Forty hours post-transfection, cells were

washed once with calcium assay buffer (20 mM HEPES, pH 7.4, 146 mM NaCl, 5 mM

KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml bovine serum albumin and 1 mg/ml glucose)

and incubated at 37ºC in 100 μl of assay buffer. After a 2-hour incubation, the assay

buffer was changed and the cells were incubated for another 1 hour at 37ºC. The assay

buffer was removed and 30 μl of assay buffer containing 6 μM of Fluo-4 (Molecular

Probes Inc.) was added into each well; the cells were incubated for 1 hour at room

temperature in the dark. The cells were washed 3 times with assay buffer and then


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incubated in 150 μl of assay buffer for 30 min. at room temperature in the dark. Agonists

were dissolved in assay buffer and the responses were recorded on a FLEXstation

benchtop scanning fluorometer (Molecular Devices Corp., Sunnyvale CA) at room

temperature with settings of 485 nm for excitation and 525 nm for emission. Maximal

responses were observed with 1 mM L-glutamate. Thus, in each experiment the results

are expressed as a percentage of the response obtained with 1 mM L-glutamate, and

described as % activation. The Graphpad Prism software was used to plot fluorescence

intensities and calculate the EC50 values.

Preparation of Xenopus oocytes

Surgically removed ovarian lobes of Xenopus laevis frogs were obtained

commercially (Nasco), and teased out in OR2 solution (82 NaCl, 2.5 KCl, 5 HEPES, 1.5

NaH2PO4, 1 MgCl2, 0.1 EDTA, in mM, pH 7.4). The oocytes were defolliculated by

incubation in 25 mL OR2 containing 2 mg/mL collagenase 1A (Sigma) two times for

about 60 minutes on a platform vibrating at 1Hz and stored in 0.5 X Leibovitz’s L-15

medium containing 50mg/ml gentamycin, 10 units/ml penicillin, and 10mg/ml

streptomycin.

Preparation and injection of cRNA

Capped cRNAs from the linearized vectors containing human mGluR3, Human Kir

3.1 and Kir 3.4 genes were prepared using mMESSAGE mMACHINE T7 Ultra Kit

(Ambion, Cat#AM1345). For rat mGluR3 cRNA, N-cmyc-tagged mGluR3 or mGluR2

DNA was linearized by NotI and 1 μg of purified linearized DNA was mixed with

NTP/ARCA, T7 reaction buffer and T7 enzyme mix in total volume of 20 μl, and


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incubated at 370C for 2 hours. After adding 1 μl of DNase I, the mixture was incubated

at 370C for 15 min, then 20 μl of 5xE-PAP buffer, 10 μl of 25 mM MnCl2, 10 ul of ATP

solution, 4 μl of E-PAP and 36 μl of RNAse-free water were added. After 45 min

incubation at 37ºC, the mixture was placed on ice. cRNA was purified by

phenol:chloroform extraction and isopropanol precipitation. Freshly harvested oocytes

(0-1 day old) were injected with 25-50 nL of RNA cocktail with approximately 5-10 ng

of the mGluR3 and 1 ng each of Kir 3.1 and 3.4. The injected oocytes were incubated in

at room temperature for 3-7 days before recording.

Two-Electrode Voltage-Clamping Measurements

All measurements were done in a medium containing ND-96 (96 NaCl, 2 KCl, 1.8

CaCl2.2H2O, 1 MgCl2.6H2O, 5 HEPES, in mM, pH 7.5). Two-electrode voltage-clamp

recording was carried out using OpusXpress amplifier (Axon Instruments, Foster City,

CA), which allows simultaneous recording from 8 oocytes. The assays for GIRK-based

mGluR2 and mGluR3 activation in oocytes have been described (Saugstad et al, 1996,

Sharon et al., 1997). Oocytes were impaled with two electrodes of 0.5-2 MΩ tip

resistance when filled with 3M KCl. Membrane potential was clamped at -60mV.

Current amplitude was measured from baseline to peak using Clampfit (Axon Inst.,

Foster City, CA). Various other data shown here were fitted and plotted using GraphPad

Prism (GraphPad Software, Inc. San Diego, CA). The GIRK currents mediated through

Kir 3.1/3.4 are K+ channels were uncovered by perfusing the oocytes with High K buffer

(2 NaCl, 96 KCl, 1.8 CaCl2.2H2O, 1 MgCl2.6H2O, 5 HEPES, in mM, pH 7.5), typically

for 120-180 seconds. A pulse of 60-120 seconds of agonist(s) was applied during the


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High K+ pulse, to measure agonist activation of mGluR3 receptor and Gβγ production,

which in turn modulates the GIRK currents. For LY341495 antagonist experiments, the

agonist pulse was followed by a solution with added antagonist. Application of 3 mM

Ba2+ in the buffer blocked most or all of the currents, showing that the current being

measured is largely mediated through GIRK channels (data not shown).

Data analysis

Dose-response curves for agonists and antagonists were fitted by nonlinear regression

to the equations: I = Imax/[1 + (EC50/Ag)n] or I = Imax Imax/[1 + (IC50/An)n] wherein

Imax = maximal normalized current response (in the absence of antagonist for inhibitory

curves), Ag = agonist concentration, An = antagonist concentration, EC50 = agonist

concentration eliciting half maximal current, IC50 = antagonist concentration eliciting half

maximal current, and n = Hill coefficient. Antagonist curves were constrained to Imax=1

and Imin= 0. For agonist efficacy curves, Imin was constrained to 0, but Imax was not

constrained. For the antagonists, IC50 values were converted to Kb values using the Leff-

Dougall (Leff and Dougall, 1993) variant of the Cheng-Prusoff equation: Kb =

IC50/((2 + ([Ag]/[EC50])n)1/n 1), where Ag is the agonist, and n = Hill coefficient.


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RESULTS

Purification of NAAG

There have been previous speculations that the commercial NAAG may have

significant impurities, including glutamate (Losi et al., 2004). NAAG obtained from

Sigma was purified on a cation exchange column essentially as described earlier in Losi

et al., (2004). Figure 1 shows HPLC chromatogram of unpurified (upper panel) and

purified (lower panel). Quantification of the glutamate peak area showed that the

unpurified NAAG from various batches of Sigma were contaminated with glutamate in

the range of 0.38-0.48%, as seen in the Figure 1 peak marked Glu. The batches purified

in this study had significantly lower glutamate levels (0.01%), as shown by the

diminished glutamate peak in the chromatogram in Figure 1. Since NAAG does not

possess a free amino group to react with the derivitizing reagent, it is not present in the

chromatogram. Losi et al (2004) reported that all commercial NAAG sources contain

between 0.1-0.5% glutamate, and they had to purify NAAG from Tocris (to < 0.1% in

their studies). Our qualitative HPLC analysis supports this observation that NAAG from

Tocris also contained glutamate. A recent study further confirmed glutamate

contamination in NAAG from Tocris to be ~0.5% (Fricker et al. 2009). We focused all

our studies starting with commercial NAAG from Sigma.

We went on to establish unequivocally if the purified NAAG indeed was structurally

intact. For that purpose, purified NAAG was spectroscopically confirmed by electrospray

mass spectrometric analysis. The ESI(+) and ESI(-) mass spectra obtained from analysis

of the purified sample from Sigma are shown in Figure 2. The ESP(+) mass spectrum


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shows the protonated molecular ion at m/z 305 together with the protonated dimer at m/z

609. The corresponding ESP(-) negative ions were observed at m/z 303 and 607

respectively. The spectra confirm the molecular mass of the neutral molecule as 304 Da.

The elemental composition of the protonated molecular ions (m/z 305) from the two

NAAG samples was confirmed, by accurate mass measurement to within 1.04 ppm

respectively.

Analysis of Purified and Unpurified NAGG in a Calcium Release Assay.

The effect of commercial (Sigma) unpurified NAAG on mGluR3 co-expressed with

Gα15 was measured by calcium-based fluorescence on a FLEXstation. Unpurified NAAG

elicited robust activation with EC50 of 120 μM (Figure 3). In contrast, purified

preparations of NAAG elicited no measurable response in this assay up to 5 mM on

mGluR3 or mGluR2.

Effect of NAAG on mGluR3 expressed in Xenopus oocytes

To assess functional activity of unpurified and purified NAAG, human and rat

mGluR3 receptors were co expressed with GIRK1 and GIRK4. Glutamate elicited robust

responses to the GIRK current, which enabled us to characterize effect of NAAG as

compared to glutamate in a more quantitative fashion. Concentration response curves

showed that both human and rat mGluR3 are highly sensitive to glutamate, with 58 and

28 nM EC50 respectively (Figure 5). Application of commercial unpurified NAAG at 10

or 100 μM elicited strong current responses in both human (Figure 4b) and rat mGluR3

(data not shown). Responses to 100 μM NAAG were comparable to that with 1 mM


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glutamate applied to same at the end of experiment (Figure 4b). The glutamate response

was slightly smaller than the impure NAAG response, possibly due to desensitization or

run-down. Over multiple studies we did not observe a difference between maximal

stimulation by glutamate or unpurified NAAG. Purified NAAG, on the other hand, had

significantly reduced responses to both human (Figure 4a) and rat mGluR3 (Figure 4c).

The purified NAAG preparations still had a low level of residual glutamate, which could

be responsible for residual activity seen in Figure 4a and 4c. Employing the numbers

from the HPLC assay for the purified NAAG (0.01% glutamate), 100 μM purified

NAAG would still have 10 nM of glutamate, sufficient to elicit a small response. An

application of 10 nM glutamate elicited a response 5.5 + 1.1% of the full agonist response

on human mGluR3 (data from Figure 5), which fully explains residual current (~7.7 %)

by 100 μM purified NAAG in Figure 4a. Table 1 summarizes the effect of unpurified

and purified NAAG on human mGluR3, showing that purified NAAG had highly

diminished responses as compared to commercial unpurified NAAG.

If residual glutamate in the commercial NAAG preparations is responsible for its

activity on mGluR3, a competitive antagonist should inhibit the activation with similar

potency. Thus, if we compare the inhibition by mGluR3 directly activated by glutamate

(1 μM) to that activated by NAAG, where the concentration of the calculated

contaminating glutamate in the NAAG sample is also 1 μM, the inhibition curves should

be the same or very similar. The batch of NAAG used at 250 μM has approximately 1

μM glutamate, and is expected to fully activate human mGluR3 receptors (Figure 5).

Human mGluR3 receptors were stimulated with either 250 μM commercial NAAG

(Sigma) or 1 μM glutamate and dose-dependently inhibited with the mGluR2/3 selective


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orthosteric antagonist, LY341495 in the Xenopus oocyte assay. The inhibition curves

from this study were essentially identical (Figure 6a). LY341495 inhibited both NAAG-

and glutamate-activated mGluR3 with an IC50 of 57 and 71 nM respectively (Figure 6a).

This corresponds to a calculated Kb of 4.8 and 3.0 nM or log(Kb) of –8.3 or –8.5

respectively. (Note, the Kb used here is the Leff-Dougall variant of the Cheng-Prusoff

equation that determines antagonist affinity in functional tests correcting for the agonist

EC50, Hill number and agonist concentration (Leff and Dougall, 1993).

As a final demonstration of the presence of glutamate in commercial unpurified

NAAG, we tested NAAG on human mGluR2 co-expressed with GIRK channels in

Xenopus oocytes. Application of 1 mM commercial NAAG (Sigma) elicited a response

that was blocked by LY341495 in a dose-dependent manner with an IC50 of 58 nM

(Figure 6b). The response to 1 mM NAAG was 64% (63.4 + 0.087; n=7) as compared

to100 μM glutamate, which causes 100% activation. The glutamate contamination in 1

mM NAAG was calculated to be about 4-5 μM; the activation observed with commercial

1 mM NAAG on mGluR2 was very similar to that with equivalent glutamate (EC50 for

glutamate on human mGluR2 is 2.6 μM, data not shown).


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DISCUSSION

We explored the possibility that the activity of NAAG at mGluR3 is due to glutamate

contamination in commercial samples rather than direct activation of mGluR3 by NAAG,

in part, because we had variable results with commercial, “off the shelf” samples. Other

studies have questioned the purity of commercial NAAG (Losi et al., 2004) using

purified NAAG to reassess its at native NMDA receptors and concluded that NAAG was

not active at NMDA receptor in cerebellar granule cells. Given this, we set out to

examine the purity of NAAG and whether the purity of NAAG altered its agonist activity

at mGluR3.

When rat mGluR3 co-expressed with Gα15 was tested the in FLEXstation calcium

release assay, we observed that commercial NAAG preparations had low potency (EC50 =

120 μM) consistent with previous publications (Wroblewska et al., 1997). HPLC analysis

of these NAAG preparations showed that there was significant contamination with

glutamate. We estimated that this contamination is approximately 0.38-0.48%. NAAG.

Commercial NAAG preparation was then purified through an ion exchange column,

which substantially reduced the glutamate level to 0.01%. This purified NAAG

preparation was inactive at mGluR3 in the Flexstation assay. Mass spectrometric

analysis confirmed that intact NAAG was present in the purified samples.

To independently confirm this observation, we assessed the activity of glutamate,

commercial NAAG, and the glutamate-depleted NAAG in Xenopus oocytes expression

rat or human mGluR3 with GIRK. In this preparation, we confirmed the high potency of


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glutamate (EC50= 58 nM at human mGluR3 and 28 nM at rat mGluR3). Commercial

NAAG showed robust currents confirming the Flexstation observations. Additionally the

glutamate-depleted NAAG was nearly inactive; residual activity could be due to the trace

remaining glutamate. Thus, in the NAAG dose-response study in the Flexstation, at the

EC50 (120 μM) there would have been approximately 0.6 μM glutamate. In the oocytes

studies, the 10 and 100 μM NAAG would have approximately 0.5 and 5 μM glutamate

respectively. Given the high potency of glutamate at mGluR3, this level of glutamate

contamination is sufficient to explain the NAAG responses in both assays. The

observation that the inhibition curves of NAAG or glutamate by LY341495 overlap, and

their corresponding Kb values are similar, constitutes independent support for our

hypothesis that the activation of mGluR3 by NAAG is through contaminating glutamate.

Finally, the commercial NAAG sample was able to activate mGluR2 at high doses

predicted by the level of glutamate contamination. Thus, these data demonstrate that at

recombinant rat and human mGluR3, N-acetylaspartylglutamate, NAAG, is not an

agonist.

Demonstrating that NAAG is not a selective mGluR3 agonist has implications on

studies published previously and their resulting interpretations. Schweitzer et al. (2000)

demonstrated, using 3H-LY354740 that glutamate (from Tocris) had an affinity of 0.44

μM at mGlu3 and 1.2 μM at mGluR2 while NAAG showed affinities of 19 μM and 236

μM respectively. These NAAG affinity determinations can be explained by glutamate

contamination in the NAAG preparation. Similarly, a number of studies have used

NAAG to demonstrate an activity in native preparations and ascribe this to activation of

mGluR3.


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Our findings suggest that conclusions from earlier studies investigating the effects of

NAAG on mGluR3 will need to be re-evaluated. A number of studies have used NAAG

in native preparations and shown a lack of activity and interpreted the result as

demonstration that mGluR3 is not involved in the observed activity. For example, Neale

and Salt (2006) showed that the mGluR2/3 agonist, LY354740 modulated the IPSCs in

the superior colliculus while NAAG (up to 500 μM) was without activity. These results

were interpreted to demonstrate that the activity of LY354740 was mediated by mGluR2.

Another study (Poschel et al., 2005) suggested that “mGluR3 is critically required for

hippocampal LTP” based on the activity of commercial NAAG in an in vivo preparation.

In these studies, and others, the use of NAAG to show the role of mGluR3 needs to be re-

evaluated since almost all have not have addressed the issue of glutamate contamination.

Thus, the in vivo effects of NAAG previously described are likely due to other

mechanisms, such as NAAG as a source of glutamate released by the action of NAAG

peptidases. The interpretation of different studies is further confounded by the possibility

that mGluR3 receptor may have coupling to different G-proteins in different systems

(Wroblewska et al., 2006). A number of publications have linked mGluR3 to the

pathophysiology of schizophrenia (Coyle, 2006) through candidate gene association

studies (Addington et al., 2004). These hypotheses on the role of mGluR3 in

schizophrenia have been, at least in part, based on using NAAG to demonstrate a

selective mGluR3 action. A recent study further supported our findings that commercial

sources of NAAG are contaminated with glutamate. They showed that purified NAAG

had little effect on GIRK currents from HEK cells transfected with human mGluR2 or

mGluR3 and human GIRK1/2 (Fricker et al., 2009). They also showed that 1 μM


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LY341495 abolished glutamate evoked GIRK. They did not show if LY341495 inhibits

currents stimulated by commercial NAAG. We have explored this in quite more detail in

a Xenopus oocyte based assay. First we show that commercial NAAG induced currents

are inhibited by LY341495 in a dose-dependent manner in both human mGluR2 and

mGluR3. Detailed Concentration-response curves on both human mGluR3 and human

mGluR2 allowed us to calculate the affinity of the active moiety in commercial NAAG to

be identical to that of glutamate, suggesting that glutamate is indeed likely

pharmacological moiety responsible for this activity in impure NAAG. We further

confirmed purity of NAAG in our purified preparations by mass-spectroscopic

techniques.

In summary, we have shown that NAAG is not an agonist at mGluR3 receptor. We

speculate that it may have an alternative role, such as acting as a NAALAdase-regulated

reservoir of glutamate. The hypothesis that the high concentration of NAAG in the CNS

could NAAG may serve as an important source of physiological glutamate requires

further investigation.


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ACKNOWLEDGEMENTS

We wish to thank Geeta Ayer and Alan Robbins for generating the human mGluR3

and Kir constructs.


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FOOTNOTES

This work was supported in part by a grant (to DRH) from the Canadian Institutes for

Health Research [grant number MOP-81179].


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LEGENDS FOR FIGURES

Figure 1: Amino acid analysis of the commercial (unpurified sample from Sigma) and

purified NAAG samples. HPLC chromatograms showing unpurified (upper

panel) and purified (lower panel at twice the gain) NAAG. The glutamate peak

(arrow) is substantially reduced in the purified preparation. The peaks labeled

“RgntPK1 and RgntPK2” are a breakdown product of the derivatizing reagent.

The peak labeled with an asterisk is an unidentified substance, possibly

corresponding to another breakdown product of the derivatizing reagent.

Figure 2: ESP(+) (top) and ESP(-) (bottom) mass spectra from the gradient LC-MS

analysis of the purified Sigma NAAG sample. The compound eluted with a

retention time of 0.72 min. The protonated monomer and the dimer molecular

ions (m/z 305 and 609 respectively) and the equivalent deprotonated molecular

anions (m/z 303 and 607) confirmed the molecular mass of NAAG as 304 Da.

Figure 3: Concentration-response curve for the effect of unpurified NAAG on rat

mGluR3 co-expressed with Gα15 in HEK 293 cells. Various concentrations of

commercial NAAG (from Sigma) were added in assay buffer to mGluR3

expressing cells loaded with Fluo-4, and responses were measured in a

FLEXstation. Each point represents the mean + SEM of 5 determinations.

Commercial unpurified NAAG (from Sigma) elicited strong responses with an

EC50 of 120 μM. Purified NAAG at 5 mM elicited no responses in this assay

on rat mGluR3 (open circle, n = 3) or rat mGluR2 (open triangle, n = 2).


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Figure 4: Current traces from a representative Xenopus oocyte expressing human (a and

b) or rat (c) mGluR3 and Kir3.1/3.4 in response to high potassium buffer,

followed by addition of various concentrations of purified (from a Sigma batch

shown in panels a and c) or unpurified NAAG (from Sigma shown in panel b)

or glutamate, as indicated, 120 seconds after the start of high potassium pulse.

NAAG is indicated as P or U in parentheses for the purified and unpurified

samples respectively. The actual time is somewhat longer than 120 seconds

due to delay of pipet tip exchange by the robotics. Cells were perfused for 5

minutes with normal ND96 between the pulses. Currents were normalized to

the current at the end of high potassium buffer solution, just before agonist

application, to compensate for the variability of individual GIRK current

responses.

Figure 5: Concentration-response curve for the effect of glutamate on human and rat

mGluR3 co-expressed with Kir3.1/3.4 in Xenopus oocytes. The glutamate-

stimulated currents were expressed as a fraction of maximum fold potentiation

from basal GIRK current responses induced by high K+ alone. Glutamate

potently stimulated both human and rat mGluR3 with EC50 values of 58 and 28

nM respectively. Data for human mGluR3 is from 7 oocytes, while that for rat

mGluR3 is from 6 oocytes.

Figure 6: Concentration-response curves for the inhibition of human mGluR3 (Figure

6a) or human mGluR2 (Figure 6b) mediated GIRK-current by the mGluR

antagonist, LY341495. (a). Inhibition of commercial NAAG (Sigma, 250 μM)

or glutamate (1 μM) mediated activation of mGluR3 GIRK currents in Xenopus


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oocytes by the mGluR antagonist, LY341495. Relative Responses was

calculated as a decrease in the agonist-induced GIRK current with application

of the inhibitor relative to the stimulation seen with the agonist alone. The IC50

values for inhibition for both NAAG and glutamate mediated activation were

57 and 71 nM respectively. The numbers of oocytes tested were 2, 6, 10, 11,

11, 4 for concentrations 1, 10, 100, 300 nM, 1 μM and 10 μM respectively.

Error bars are standard error of means. (b), Inhibition of human mGluR2

current activated by commercial NAAG (Sigma, 1 mM) by LY341495 with a

IC50 of 58 nM. The numbers of oocytes tested were 5, 10, 10, 4, 6, 3 for

concentrations 1, 10, 100, 300 nM, 1 μM and 10 μM respectively. error bars are

standard error of means. Upper right corner shows a current trace from a

representative oocyte expressing human mGluR2 and Kir3.1/3.4. The upper

bar above the trace (1) show the application of high potassium buffer, middle

bar (2) shows 1 mM Sigma NAAG, and lower bar (3) shows 1 μM LY341495.

The vertical and horizontal scales are 1 μA and 50 seconds respectively.


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TABLES

Table 1:

Table 1: Effect of unpurified and purified NAAG (from Sigma) on human mGluR3. The

relative effect represent average of fraction of fold potentiation of GIRK currents relative

to that obtained with full agonist response with 1 mM glutamate in the each cell tested.

Even this small level of activation may represent residual glutamate in the purified prep,

with an estimated concentration from 0.1-100 nM.

Purified NAAG Unpurified NAAG

Conc Relative Effect (avg) SD n Relative

Effect (avg) SD n

1 μM 0.023 0.065 8

10 μM 0.048 0.051 11 0.455 0.239 4

100 μM 0.077 0.085 11 0.917 0.409 4

300 μM 0.081 0.060 7

1 mM 0.039 0.074 11


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Unpurified NAAG

Purified NAAG

Figure 1

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