The FASEB Journal • Research Communication
Glutamate carboxypeptidase II is not an amyloid peptide-degrading enzyme Jesse Alt,* Marigo Stathis,* Camilo Rojas,* and Barbara Slusher*,†,‡,1 *Brain Science Institute Neurotranslational Drug Discovery Program, and †Department of Neurology, and ‡ Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Glutamate carboxypeptidase II (GCPII) is an exopeptidase that catalyzes the hydrolysis of N-acetylated aspartate-glutamate (NAAG) to N-acetyl aspartate (NAA) and glutamate. Consequently, GCPII inhibition has been of interest for the treatment of central and peripheral nervous system diseases associated with excess glutamate. Recently, it was reported that GCPII can also serve as an endopeptidase cleaving amyloid  (A) peptides and that its inhibition could increase the risk of Alzheimer’s disease by increasing brain A levels. This study aimed to corroborate and extend these new findings. We incubated A peptides (20 M) with human recombinant GCPII (300 ng/ml) and monitored the appearance of degradation products by mass spectrometry. A peptides remained intact after 18 h incubation with GCPII. Under the same experimental conditions, A1– 40 (20 M) was incubated with neprilysin (300 ng/ml), an endopeptidase known to hydrolyze A1– 40 and the expected cleavage products were observed. GCPII was confirmed active by catalyzing the complete hydrolysis of NAAG (100 M). We also studied the hydrolysis of [3H]-NAAG (30 nM) catalyzed by GCPII (40 pM) in the presence of A peptides (picomolar to micromolar range). The addition of A peptides did not alter [3H]-NAAG hydrolysis. We conclude that GCPII is not an amyloid peptidedegrading enzyme.—Alt, J., Stathis, M., Rojas, C., Slusher. Glutamate carboxypeptidase II is not an amyloid peptidedegrading enzyme. FASEB J. 27, 2620 –2625 (2013). www.fasebj.org ABSTRACT
Key Words: Alzheimer’s disease 䡠 prostate-specific membrane antigen Glutamate carboxypeptidase II (GCPII; E.C. 3.4.17.21) is an enzyme that catalyzes the hydrolysis of N-acetylated aspartate-glutamate (NAAG) to N-acetyl aspartate (NAA) and glutamate (1–3). GCPII resides on the Abbreviations: 2-MPPA, 2-(3-mercaptopropyl) pentanedioic acid; A, amyloid ; AD, Alzheimer’s disease; AUC, area under curve; CNS, central nervous system; GCPII, glutamate carboxypeptidase II; NAA, N-acetyl aspartate; NAAG, N-acetylated aspartate-glutamate; NEP, neprilysin; PSMA, prostatespecific membrane antigen; MS, mass spectrometry; rhGCPII, recombinant human glutamate carboxypeptidase II; QTOF MS, quadrupole time-of-flight mass spectrometry 2620
membranes of astrocytes in the central nervous system (CNS) so that when NAAG is released from presynaptic terminals, it can be readily hydrolyzed by GCPII to release glutamate (4 – 6). While glutamate release plays a pivotal role in normal neurotransmission, accumulation of excess glutamate in the extracellular space as a consequence of neuronal damage due to CNS trauma, neurodegenerative disease, infection, or deregulation of glutamate clearance results in excitotoxicity (7, 8). Inhibition of GCPII would prevent glutamate release from NAAG while increasing NAAG levels. NAAG itself is known to prevent glutamate release through agonism at the mGluR3 receptor (5, 9). As a result, GCPII inhibition could help ameliorate excess glutamate in the extracellular space and has the potential of being used as treatment of diseases associated with glutamate excitotoxicity. This hypothesis has been substantiated by a multitude of reports where GCPII inhibitors have shown neuroprotective activity in ⬎20 animal models of disease (10), including inflammatory and neuropathic pain (11–13), brain ischemia (14), motor neuron disease (15), spinal cord and traumatic brain injury (16, 17), peripheral neuropathy (18, 19), epilepsy (20), and drug abuse (21, 22). Further, a GCPII inhibitor, 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA), was used in humans in an exploratory study to assess safety and tolerability of GCPII inhibition. 2-MPPA did not provoke any adverse CNS effects and was well tolerated at exposures that in animals were pharmacologically active (23). The enzyme also catalyzes the sequential hydrolysis of carboxy-terminal glutamate residues from folate polyglutamate in the membrane brush border of the small intestine. The folate hydrolase activity of GCPII is thought to expedite intestinal uptake of folate (24). A 1561 C¡T polymorphism in the GCPII gene was reported to be associated with impaired intestinal absorption of dietary folates, resulting in low blood folate levels and consequent hyperhomocysteinemia (25). GCPII has been shown to be identical to human prostate-specific membrane antigen (PSMA; ref. 26). PSMA is highly expressed in prostate cancer, especially in late-stage and metastatic disease, so it has been used 1 Correspondence: Brain Science Institute, Johns Hopkins University School of Medicine, 855 North Wolfe St., Baltimore, MD 21205, USA. E-mail:
[email protected] doi: 10.1096/fj.12-225102
0892-6638/13/0027-2620 © FASEB
as an imaging biomarker as well as a target for therapeutic intervention. However, the functional role of the enzyme in prostate cancer is not known (26). Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the accumulation of amyloid  (A) peptide in the brain. Processes that increase A synthesis or that impede A degradation are thought to contribute to A deposition, possibly leading to AD (27). A recent report indicated that GCPII could act as an amyloid peptide-degrading enzyme (28). This finding suggested that GCPII plays a role in A clearance and that inhibition of GCPII activity could result in increased A levels and a higher risk for AD. Given the potential of GCPII inhibitors for the treatment of disease on one hand, and the suggestion that GCPII inhibition could result in increased AD risk on the other, it was important to corroborate the finding that GCPII acts as A-degrading enzyme. In the studies reported here, we investigated A1– 40 and A1– 42 degradation after incubation with GCPII and found no evidence of GCPII acting as an A-degrading enzyme.
MATERIALS AND METHODS Reagents A1– 40 and A1– 42 were purchased from AnaSpec (Fremont, CA, USA) and Sigma (St. Louis, MO, USA). Recombinant human brain GCPII (EC 3.4.17.21) purified to homogeneity (29) was kindly provided by Dr. Jan Konvalinka (Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic). Sample preparation A1– 40 and A1– 42 monomers Lyophilized A1– 40 or A1– 42 peptide was dissolved according to manufacturer’s instructions in HPLC-grade water (A1– 40) or DMSO (A1– 42) at 6 mg/ml, then further diluted in Tris-HCl buffer (pH 7.4, 50 mM, calcium and phosphate free) to 200 M. After centrifugation at 10,000 g for 10 min to separate soluble from insoluble fractions, the supernatant was taken and used in the GCPII activity assay. A1– 40 and A1– 42 oligomers Peptides were solubilized as with monomers and then incubated at 37°C for 4 d. After centrifugation at 10,000 g for 10 min, the aggregated fraction (pellet) was separated from the soluble peptide, resolubilized in Tris-HCl buffer (pH 7.4, 50 mM, calcium and phosphate free), and used in the GCPII activity assay. Measurement of GCPII activity in vitro GCPII activity measurements were carried out following published procedures (2, 30). Briefly, the reaction mixture (total volume of 90 l) contained [3H]-NAAG (30 nM, 30 Ci/ mmol) and recombinant human GCPII (rhGCPII; 40 pM) in Tris-HCl (pH 7.4, 40 mM) containing 1 mM CoCl2. The reaction was carried out at 37°C for 20 min, and stopped with ice-cold sodium phosphate buffer (pH 7.4, 0.1 M, 90 l). GCPII DOES NOT HYDROLYZE A-PEPTIDES
Blanks were obtained by incubating the reaction mixture without GCPII. Duplicate aliquots of 90 l from each terminated reaction was transferred to a well in a 96-well spin column containing AG1X8 ion-exchange resin (Bio-Rad, Richmond, CA,USA); the plate was centrifuged at 1000 rpm for 5 min using a Beckman GS-6R centrifuge equipped with a PTS-2000 rotor (Beckman Coulter, Fullerton, CA, USA). [3H]-NAAG bound to the resin and [3H]-glutamate eluted in the flow through. Columns were then washed twice with formate (1 M, 90 l) to ensure complete elution of [3H]glutamate. The flow through and the washes were collected in a deep 96-well block; from each well with a total volume of 270 l, a 200-l aliquot was transferred to a glass scintillation vial, to which 10 ml of Ultima-Gold (Perkin Elmer, Wellesley, MA, USA) was added. The radioactivity in each vial corresponding to [3H]-glutamate was determined via a Beckman LS-6000IC scintillation counter. Measurement of A1– 40 and A1– 42 by quadrupole time-offlight mass spectrometry (QTOF MS) Peptides were dissolved at 0.2 mM in 0.3% ammonium hydroxide and frozen at ⫺80°C until use. The day of the experiment, A1– 40 and A1– 42 (20 M) were incubated with GCPII (300 ng/ml in 60 mM Tris, pH 7.7) for 18 h at 37°C in a total volume of 100 l. A concentrations and buffer composition were not mentioned in the earlier report (28), so we chose the conditions mentioned above, which would be amenable to enzyme activity and QTOF analysis. Hydrolysis of A1– 40 (20 M) in the presence of neprilysin (NEP; 300 ng/ml) and hydrolysis of NAAG (100 M) in the presence of GCPII were carried out as positive controls. At the end of the incubation, peptide samples were placed on ice until analyzed. NAAG and NAA samples were derivatized by 30 min incubation with 3N HCl n-butanol at 60°C. An aliquot (20 l) was injected and separated on an Agilent 1290 UPLC system with a Zorbax SB-aq column (Agilent Technologies, Santa Clara, CA, USA) using a gradient run of 15– 80% acetonitrile over 6 min and detected on an Agilent 6520 QTOF mass spectrometer. Total ion chromatograms spanning 50 –3200 m/z were analyzed to visualize the formation of peaks not present in control conditions resulting from the degradation of A peptide. In addition, the extracted ion chromatogram of the base peak for each A peptide, NAAG, and NAA was integrated and analyzed to quantify the loss or appearance of the analyte by incubation with enzyme.
RESULTS Neither A1– 40 nor A1– 42 degrades in the presence of GCPII Monomeric forms of A1– 40 and A1– 42 were incubated for 18 h at 37°C with GCPII, denatured GCPII, or vehicle. These incubations were carried out under similar conditions as previously reported (28). After incubation, the loss of parent peptides or the appearance of new peptide fragments was monitored by QTOF MS. A1– 40 with a deconvoluted peak of m/z 4329.15 (Fig. 1A) exhibited the same size chromatographic peak after treatment with GCPII as when denatured GCPII or nothing was added (Fig. 1B). Further, no new fragments were observed after any of these incubations. When A1– 42 with a deconvoluted peak of m/z 4513.28 (Fig. 1C) was used, the same 2621
Figure 1. GCPII-catalyzed peptide hydrolysis. A1– 40 (20 M), A1– 42 (20 M), and NAAG (100 M) were incubated with GCPII, denatured GCPII, or vehicle for 18 h at 37°C. Samples were then analyzed by QTOF MS. A) Deconvoluted mass spectrum of A1– 40 (m/z 4329.2). B) Chromatographic peaks corresponding to the base peak of A1– 40 after incubation with GCPII (black), denatured GCPII (red), and vehicle only (blue). C) Deconvoluted mass spectrum of A1– 42 (m/z 4513.3). D) Chromatographic peaks corresponding to base peak of A1– 42 after incubation with GCPII (black), denatured GCPII (red), and vehicle only (blue). E) Quantification of chromatographic peaks (AUC) shown in panels B and D. F) NAAG (200 M) was incubated for 18 h with GCPII, denatured GCPII, or vehicle only. Samples were analyzed for NAAG and NAA levels, and AUC of chromatographic peaks was determined. (Peaks corresponding to derivatized NAAG and NAA were m/z 473.3 and 288.2, respectively). G) Chromatogram of A1– 40 (20 M) after incubation with NEP (300 ng/ml) for 18 h. Peaks corresponding to peptide fragments resulting from expected cleavage sites are identified with an arrow and their amino acid sequence.
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results were obtained (Fig. 1D). The area under the curve (AUC) corresponding to both A1– 40 and A1– 42 was the same under the three different treatments, further corroborating the visual observation (Fig. 1E). The results showed no degradation of either A1– 40 or A1– 42 on incubation with GCPII. As positive control, NAAG was also incubated with GCPII under the same conditions. In this instance, product analysis by QTOF MS showed nearly complete NAAG (derivatized m/z of 473.2850) disappearance and the concomitant appearance of NAA (derivatized m/z of 288.1804) (Fig. 1F). When denatured or no GCPII was used, NAAG did not disappear, and NAA did not appear (Fig. 1F). In another positive control experiment, the monomeric form of A1– 40 was incubated with NEP, a neutral endopeptidase known to hydrolyze A1– 40 (31). After incubation, we observed the appearance of new peaks in the mass spectrum corresponding to the expected cleavage of A1– 40 by NEP (31) (Fig. 1G). The results of these experiments showed that A1– 40 and A1– 42 were not degraded by GCPII. Neither A1– 40 nor A1– 42 competes with NAAG as a substrate for GCPII We also examined whether monomeric or oligomeric A1– 40 or A1– 42 could act as a substrate for GCPII by performing competition experiments with NAAG, a known substrate for GCPII. GCPII was incubated with [3H]-NAAG in the presence of picomolar to micromolar concentrations of monomeric and oligomeric forms of both A1– 40 and A1– 42. If these peptides are substrates for GCPII acting at the known catalytic domain,
they would be expected to compete with [3H]-NAAG for GCPII, with a corresponding inhibition of [3H]NAAG hydrolysis. Neither monomeric nor oligomeric forms of A1– 40 or A1– 42 inhibited [3H]-NAAG hydrolysis (Fig. 2A–D). In contrast, the prototype GCPII inhibitors 2-PMPA and 2-MPPA inhibited the enzyme with IC50 values of 400 pM (Fig. 2E) and 60 nM (Fig. 2F), respectively, similar to IC50 values previously described (32).
DISCUSSION GCPII is a zinc-containing exopeptidase with strong preference for cleaving C-terminal glutamate/aspartate residues (33). GCPII is known to catalyze the hydrolysis of glutamate from the abundant neurodipeptide NAAG (2, 3). Acute and chronic administration of GCPII inhibitors have shown neuroprotective activity in ⬎20 CNS/PNS animal models of disease wherein excess glutamate is presumed pathogenic (10). The GCPII inhibitor, 2-MPPA, has been administered to humans, and it has been reported to be well tolerated, with no adverse CNS effects (23). The recent report characterizing GCPII as an endopeptidase capable of catalyzing the degradation A peptides was surprising (28). Further, the corresponding implication that the GCPII enzyme is involved in A clearance created a concern about the use of GCPII inhibitors in the clinic; inhibition of GCPII could disrupt normal degradation of A peptide and possibly increase the risk or expedite the onset of AD. Conse-
Figure 2. Inhibition of GCPII-catalyzed [3H]-NAAG hydrolysis. [3H]-NAAG was incubated with GCPII in the presence and absence of various concentrations of both monomeric and oligomeric A1– 40 and A 1– 42, as well as the GCPII prototype inhibitors 2-PMPA and 2-MPPA (Materials and Methods). Amount of [3H] glutamate produced was measured and compared to that in the absence of potential inhibitors to obtain percent inhibition. A) Monomer of A1– 40. B) Monomer of A1– 42. C) Oligomer of A1– 40. D) Oligomer of A1– 42. E) 2-PMPA IC50 ⫽ 400 pM. F) MPPA IC50 ⫽ 60 nM. GCPII DOES NOT HYDROLYZE A-PEPTIDES
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quently, it was important to determine whether the finding on GCPII acting as an A-degrading enzyme could be reproduced in an independent laboratory. In our experiments, we incubated A1– 40 and A1– 42 in the presence of GCPII and monitored degradation. In the presence of a high concentration of GCPII (300 ng/ml) after 18 h incubations, we found that neither A1– 40 nor A1– 42 exhibited any degradation, as measured by MS. The reason for the discrepancy between our results and those recently reported in the literature (28) is not clear to us. Detailed conditions for monomeric and oligomeric preparations were not provided in the earlier report, so the conditions we used were standard conditions provided by the peptide manufacturer and may have been different than those used previously (28). In a second set of experiments, we examined whether monomeric or oligomeric forms of A1– 40 and of A1– 42 could compete with NAAG as substrates for GCPII. We found that neither monomeric nor oligomeric forms of A1– 40 or A1– 42 in the picomolar to micromolar range could inhibit the ability of GCPII to hydrolyze NAAG. The results indicated that A1– 40 and A1– 42 are not substrates at the known active site for GCPII, corroborating the first set of experiments. The GCPII enzyme used in this study, as well as that by Kim et al. (28), was human brain GCPII purified to homogeneity, so the discrepancy in results cannot be explained on the basis of using different enzyme sources. The report on GCPII acting as an endopeptidase and catalyzing hydrolysis of H14-Q15, V18-F19, and M35V36 in A peptides (28) is hard to reconcile with the detailed GCPII X-ray structure studies (33–35). These studies have documented an active site that is finely tuned to catalyze the hydrolysis of glutamate at the carboxylic end of NAAG and folate polyglutamate. GCPII can also hydrolyze other acidic dipeptides, namely Asp-Glu, Glu-Glu, and ␥Glu-Glu (36), consistent with the ability to catalyze the hydrolysis of dipeptides with glutamate at the carboxylic end. Studies on GCPII substrate specificity have identified additional acetylated dipeptides containing methionine at the carboxylic end; in these limited cases, however, even when an acetylated dipeptide is still used, GCPII catalytic efficiency compared to NAAG is rapidly lost from 250-fold (Ac-Glu-Met) to 30,000-fold (Ac-Ala-Met) (29). The report on GCPII-catalyzed degradation of A1– 40 and A1– 42 involves hydrolysis of two nonterminal peptide bonds with amino acids containing hydrophobic residues (V18-F19 and M35-V36) and another nonterminal peptide bond with amino acids containing basic and carboxamide side chains (H14-Q15) (28); this level of catalytic diversity and nonspecificity would necessitate significant rearrangements of GCPII architecture for which no experimental evidence exists. Given these findings, the results described by Kim et al. (28) demonstrating alterations in A levels in GCPII-transfected cells or in mice treated with the GCPII inhibitor 2-PMPA cannot be explained on the basis of GCPII’s amyloid peptide-degrading catalytic 2624
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activity. One possible explanation of the results in the mouse study is that 2-PMPA could be inhibiting other closely related enzymes belonging to the GCPII family. However, GCPIII, the only other member of this protein family exhibiting similar catalytic activity as GCPII, has been shown to be in very low abundance in mouse brain (1). In summary, we showed that both A peptides remained intact after 18 h incubations at 37°C with GCPII, as measured by QTOF MS, and that NAAG hydrolysis catalyzed by GCPII was not inhibited by either A1– 40 or A1– 42. The results indicate that GCPII is not an amyloid peptide-degrading enzyme.
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