Glutamate carboxypeptidase II (GCPII) (also known as NAAG Peptidase) is a zinc metalloenzyme that resides in membranes. Most of the enzyme resides in the extracellular space. GCPII is a class II membrane glycoprotein. It is also known as NAAG peptidase and NAALADase. It catalyzes the hydrolysis of N-Acetylaspartylglutamate (NAAG) to Glutamate and NAA according to the following reaction scheme (Mesters et al., 2005; Rojas et al, 2002). Human GCPII contains 750 amino acids and weighs approximately 84 kD (Barinka et al, 2004).
GCPII is also known as folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1). Neuroscientist primarily use the term NAALADASE in their studies, those studying folate metabolism use folate hydrolase, and those studying prostate cancer or oncology, PSMA. All of which refer to the same protein designation as glutamate carboxypeptidase II.
GCPII is expressed in many tissues, including the prostate, kidney, the small intestine, and the central and peripheral nervous system (Barinka et al, 2004).
Indeed the initial cloning of the CDNA encoding the gene expressing PSMA was accomplished with RNA from a prostate tumor cell line, LNCaP (Israeli et al 1993). PSMA shares homology with the transferrin receptor and undergoes endcytosis but the ligand for inducing internalization has not been identified (Goodman et al 2007). It was found that PSMA was the same as the membrane protein in the small intestine responsible for removal of gamma-linked glutamates from polygammaglutamate folate. This enables the freeing of folic acid which then can be transported into the body for use as a vitamin. This resulted in the cloned genomic designation of PSMA as FOLH1 for folate hydrolase (Pinto et al Clin Cancer Res 1996).
PSMA(FOLH1)+ folate polygammaglutamate(n 1-7)---> PSMA (FOLH1) + folate(poly)gammaglutamate(n-1) + glutamate continuing until releasing folate.
Role in prostate cancer
It was found that there were multiple potential start sites for PSMA as well as multiple alternative splice forms that vary in the type of membrane protein formed or having a cytosolic location and each form probably varies regarding caboxypeptidase activity given the restriction for enzymatic activity for PSMA (O'Keefe Prostate Cancer: Biology Genetics and the New Therapeutics 2003 and Barinka 2004). PSMA is strongly expressed in the human prostate being a hundred fold greater than the expression in most other tissues. In cancer it is upregulated in expression and has been called the second most up-regulated gene in prostate cancer being increased 8- 12 fold over the non cancerous prostate (O'Keefe Prostate 2004). Because of this high expression it is being developed as a target for therapy and imaging (Wang X, J Cell Biochem 2007). In human prostate cancer the higher expressing tumors are associated with quicker time to progression and a greater percentage of patients suffering relapse (Ross et al Clin Cancer res 2003 and Perner et al Hum Pathol 2007). PSMA is the target of an approved imaging agent for prostate cancer, capromabpentide, PROSTASCINT. Second generation antibodies and low molecular weight ligands for imaging and therapy are being developed (Wang X, J Cell Biochem 2007, Foss et al Clin Cancer Res 2005, and Milowsky J Clin Oncol 2007). In addition to the expression in the human prostate and prostate cancer, PSMA is also found to be highly expressed in tumor neovasculature but not normal vasculature of all types of solid tumors as kidney, breast, colon etc (Chang S Cancer Res 1999). In terms of imaging no non- tumor site such as normal kidney, small intestine, CNS has been imaged using second generation antibody imaging agents while sites even in bone are being detected with better sensitivity than than with technetium scans and the tumors expressing PSMA in their neovasculature are also being imaged (Milowsky et al 2007). Low molecular weight ligands exhibit different binding with imaging seen in the kidney of the mouse, however mouse has much higher levels of PSMA in kidney and brain than the human and in the mouse it was only the normal kidney and prostate tumors that were imaged and not other tissues, not even CNS suggesting the imprortance of the blood brain barrier (Foss et al Clin Cancer Res 2005). In kidney it is a subset of tubules that contain PSMA. Thus imaging studies will have to be performed in humans with the low molecular weight ligands to define their potential for imaging and targeting. Still, in terms of potential toxicities, knockout animals were normal on most tests which reduces somewhat concerns about toxicity in targeting PSMA(Bacich J Neurochem 2005). In the CNS, PSMA is present only in a sub-set of glial cells, again suggesting that toxin trageting would likely have minimal toxicity to the host even if the blood brain barrier were not intact (Sacha 2006).
For those studying neural based diseases, NAAG is one of the three most prevalent neurotransmitters found in the central nervous system (Reviewed in Zhou et al 2005) and when it catylizes the reaction to produce glutamate it is also producing another neurotransmitter (Mesters et al 2005). Glutamate is a common and abundant excitatory neurotransmitter in the central nervous system; although if there is too much glutamate transmission this can kill or at least damage neurons and has been implicated in many neurological diseases and disorders (Reviewed in Zhou et al 2005) therefore the balance that NAAG peptidase contributes to is quite important.
The three domains
of the extracellular portion of GCPII –the protease, apical and C-terminal domains- collaborate in substrate recognition (Mesters et al, 2005). The protease domain is a central seven-stranded mixed β-sheet. The β-sheet is flanked by 10 α-helices. The apical domain is located between the first and second strands of the central β-sheet of the protease domain. The apical domain creates a pocket that facilitates substrate binding. The C-terminal domain is an Up-Down-Up-Down four-helix bundle.
The central pocket is approximately 2 nanometres in depth and opens from the extracellular space to the active site (Mesters et al, 2005). This active site contains two zinc ions. During inhibition, each acts as a ligand to an oxygen in 2-PMPA or phosphate.
There is also one calcium ion coordinated in GCPII, far from the active site. It has been proposed that calcium holds together the protease and apical domains (Mesters et al, 2005).
In addition, ten glycosylation sites have been identified in human GCPII (Barinka et al, 2004). Glycosylation far from the catalytic domain still affects the ability of GCPII to hydrolyze NAAG (Barinka et al, 2004).
The hydrolysis of NAAG by GCPII obeys Michaelis-Menten kinetics (Mesters et al, 2005). Mesters et al. (2005) calculated the binding constant (Km) for NAAG as approximately 130 nM and the turnover constant (kcat) as approxately 4 s-1. The apparent second-order rate constant is approximately 3 x 107 (M·s)-1.
GCPII inhibition and its possible therapeutic uses
What happens in the presence of NAAG peptidase in the brain?
GCPII has been shown to both indirectly and directly increase the concentration of glutamate in the extra cellular space (Reviewed in Zhou et al., 2005). Directly, GCPII cleaves NAAG into NAA and glutamate (Mesters et al., 2005; Rojas et al, 2002). Indirectly, NAAG has been shown, in high concentration, to inhibit the release of neutrotransmitters, such as GABA and glutamate. It does this through interaction with, and activation of presynaptic group II mGluRs (Reviewed in Zhou et al., 2005). Thus, in the presence of NAAG peptidase, the concentration of NAAG is kept in check and glutamate and GABA, among other neurotransmitters, are not inhibited.
Researchers have been able to show that effective and selective GCPII inhibitors are able to decrease the brains levels of glutamate and even provide protection from apoptosis or degradation of brain neurons in many animal models of stroke, amyotrophic lateral sclerosis, and neuropathic pain (Mesters et al 2005). This inhibition of these NAAG peptidases sometimes referred to as NPs are thought to provide this protection from apoptosis or degradation of brain neurons by elevating the concentrations of NAAG within the synapse of neurons (Reviewed in Zhou et al 2005). NAAG then reduces the release of glutamate while stimulating the release of some trophic factors from the glia cells in the central nervous system and this results with the protection from apoptosis or degradation of brain neurons (Reviewed in Zhou et al 2005). The important thing however is that these NP inhibitors do not seem to have any effect on normal glutamate function (Reviewed in Zhou et al 2005). The NP inhibition is able to improve the naturally occurring regulation instead of activating or inhibiting receptors that would disrupt this process (Reviewed in Zhou et al 2005). Research has also shown that small-molecule-based NP inhibitors are beneficial in animal models that are relevant to neurodegenerative diseases (Reviewed in Zhou et al 2005). Some specific applications of this research include neuropathic and inflammatory pain, traumatic brain injury, ischemic stroke, schizophrenia, diabetic neuropathy, amyotrophic lateral sclerosis as well as drug addiction (Reviewed in Zhou et al 2005). Previous research has found that drugs that are able to reduce glutamate transmission can relieve the neuropathic pain, although the side effects of this have limited a great deal of their clinical applications (Zhang et al 2006). Therefore it appears that since GCPII is exclusively recruited for the purpose of providing a glutamate source in hyperglutamatergic and excitotoxic conditions this could be an alternative to get rid of these side effects (Zhang et al 2006). More research findings have shown that the hydrolysis of NAAG is disrupted in schizophrenia and they have shown that specific anatomical regions of the brain may even show discrete abnormalities in the GCP II synthesis so NPs may also be therapeutic for patients suffering with schizophrenia (Ghose et al 2004). One major hurdle with using many of the potent GCPII inhibitors that have been prepared to date are typically highly polar compounds which causes problems because they do not then penetrate the blood-brain barrier easily (Kozikowski et al 2001).
How could NAAG peptidase inhibition have therapeutic value?
Glutamate is the “primary excitatory neurotransmitter in the human nervous system” (Reviewed in Zhou et al., 2005), participating in a multitude of brain functions. Over stimulation and activation of glutamate receptors as well as “disturbances in the cellular mechanisms that protect against the adverse consequences of physiological glutamate receptor activation” (Kowzikowski et al., 2001) have been known to cause neuron damage and death which has been associated with multiple neurological diseases (Reviewed in Zhou et al., 2005).
Due to the pure range of glutamate function and presence, it has been difficult to create glutamatergic drugs that do not negatively affect other necessary functions and cause unwanted side effects (Nagel et al., 2006). NAAG peptidase inhibition has offered the possibility for specific drug targeting.
What can inhibit GCPII?
Since its promise for possible neurological disease therapy and specific drug targeting, NAAG peptidase inhibitors have been widely created and studied. A few small molecule examples are those that follow (Reviewed in Zhou et al., 2005):
- 2-PMPA and analogues
- Thiol and indole thiol derivatives
- Hydroxamate derivatives
- Conformationally constricted dipeptide mimetics
- PBDA- and urea based inhibitors.
What diseases and conditions are being examined for NAAG inhibition therapy?
Neuropathic and inflammatory pain
Pain cause by injury to CNS or PNS has been associated with increase glutamate concentration. NAAG inhibition reduced glutamate presence and could thus diminish pain (Reviewed in Zhou et al., 2005). Nagel et al. (2006) used the inhibitor 2-PMPA to show the analgesic effect of NAAG peptidase inhibitions. This study followed one by Chen et al. (2002) which showed similar results (Nagel et al., 2006)
Severe head injury (SHI) and traumatic brain injury (TBI) are widespread and have a tremendous impact. “They are the leading cause of death in children and young adults (<25 years) and account for a quarter of all deaths in the five to 15 years age group” (Tolias, 2002). Following initial impact, glutamate levels rise and cause excitotoxic damage in a process that has been well characterized (Reviewed in Zhou et al., 2005). With its ability to reduced glutamate levels, NAAG inhibition has shown promise in prevent neurological damage associated with SHI and TBI.
According to the National Stroke Association, stroke
is the third leading cause of death and the leading cause of adult disability. It is thought that glutamate levels cause underlying ischemic damage during a stroke and thus NAAG inhibition might be able to diminish this damage (Reviewed in Zhou et al., 2005).
is a mental disorder which affects 1% of people throughout the world (Schizophrenia). It can be modeled by PCP in laboratory animals and it has been shown that mGluR agonists have reduced the effects of the drug. NAAG is such an mGluR agonist. Thus, through inhibition of the enzyme which reduces NAAG concentration, NAAG peptidase, could provide a practical treatment for reduction of schizophrenic symptoms. (Reviewed in Zhou et al., 2005).
can lead to damaged nerves causing loss of sensation, pain or, if autonomic nerves are associated, damage to the circulatory, reproductive, or digestive systems, among others. Over 60% of diabetic patients are said to have some form of neuropathy (Reviewed in Zhou et al., 2005), however the severity ranges dramatically. Neuropathy not only directly causes harm and damage, but also can indirectly lead to such problems as diabetic ulcerations, which in turn can lead to amputations. In fact, over half of all lower limb amputations in the United States are of patients with diabetes ("Diabetic Neuropathies").
In a study performed by Berent-Spillson et al. (2004), it was shown that through the use of the NAAG peptidase inhibitor, 2-PMPA, NAAG cleavage was inhibited and with it, programmed DRG neuronal cell death in the presence of high glucose levels. The researchers have proposed the cause of this being NAAG’s agonistic activity at mGluR3. Additionally, NAAG also “prevented glucose-induced inhibition of neurite growth” (Berent- Spillson, et al. 2004). Overall, this makes GCPIII inhibition a clear model target for combating diabetic neuropathy.
Schizophrenia, as previously described, is normally modeled in the laboratory through a PCP animal model. As GCPIII inhibition was shown to possibly limit schizophrenic behavior in this model (Reviewed in Zhou et al., 2005), this suggests that GCPIII inhibition thus reduces the effect of PCP. Additionally, the reward action of many drugs (cocaine, PCP, alcohol, nicotine, etc.) have been shown with increasing evidence to be related to glutamate levels, which NAAG and GCPIII can have some regulatory effect (Reviewed in Zhou et al., 2005).
In their review, Zhou et al. (2005) summarized the findings of multiple drug studies to conclude that:
- NAAG/NP system might be involved in neuronal mechanisms regulating cue-induced cocaine craving, the development of cocaine seizure kindling, and management of opioid addiction and alcohol consumptive behaviour. NP inhibitors could therefore provide a novel therapy for such conditions.
Other diseases and disorders
NAAG inhibition has also been studied as a treatment against prostate cancer, ALS, and other neurodegenerative diseases such as Parkinson’s disease and Huntington’s disease (Reviewed in Zhou et al., 2005).
Bacich DJ, Wozniak KM, Lu X-CM et al, Mice lacking glutamate carboxypeptidase II are protected from peripheral neuropathy and ischemic brain injury. J Neurochem 95: 314-323, 2005.
- Barinka C, Sácha P, Sklenár J, et al (2004). "Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity". Protein Sci. 13 (6): 1627–35.
- Berent-Spillson A, Robinson AM, Golovoy D, Slusher B, Rojas C, Russell JW (2004). "Protection against glucose-induced neuronal death by NAAG and GCP II inhibition is regulated by mGluR3". J. Neurochem. 89 (1): 90–9.
Chang SS, Reuter VE, Heston WD et al, Five different anti-prostate-specific- membraen antigen antibodies confirm PSMA expression in tumor assoicated neovasculature. Cancer Res. 59: 3192-3198, 1999.
- Chen SR, Wozniak KM, Slusher BS, Pan HL (2002). "Effect of 2-(phosphono-methyl)-pentanedioic acid on allodynia and afferent ectopic discharges in a rat model of neuropathic pain". J. Pharmacol. Exp. Ther. 300 (2): 662–7.
- “Diabetic Neuropathies: The Nerve Damage of Diabetes.” (2002). National Institute of Diabetes and Digestive and Kidney Diseases. http://diabetes.niddk.nih.gov/dm/pubs/neuropathies/ Accessed 22 April 2007.
Foss CA, Mease RC, Fan H et al: Radiolabeled small molecules ligands for prostate specific membrane antigen: In vivo imaging in experimental models of prostate cancer. Clin Cancer Res. 11: 4022-4028, 2005.
- Ghose S, Weickert CS, Colvin SM, et al (2004). "Glutamate carboxypeptidase II gene expression in the human frontal and temporal lobe in schizophrenia". Neuropsychopharmacology 29 (1): 117–25.
Goodman OB Jr, Barwe SP, Ritter B et al, Interaction of prostate specific membrane antigen with clathrin and the adapter protein complex-2. Int J Oncol. 31 (5) 1199-1203, 2007.
Israeli RS, Powell CT, Fair WR, Heston WDW: Molecular cloning of a complimentary DNA encoding a prostate specific membrane antigen. Cancer Res 53: 227-230, 1993.
- Kozikowski AP, Nan F, Conti P, et al (2001). "Design of remarkably simple, yet potent urea-based inhibitors of glutamate carboxypeptidase II (NAALADase)". J. Med. Chem. 44 (3): 298–301.
- Mesters JR, Barinka C, Li W, et al (2006). "Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer". EMBO J. 25 (6): 1375–84.
Milowsky MI, Nanus DM Kostakoglu L et al, Vascular targeted therapy with anti-prostate-specific- membrane antigenmonoclonal antibody J591 in advanced solid tumors. J Clin Oncol 25: (5) 540-547 2007.
- Nagel J, Belozertseva I, Greco S, et al (2006). "Effects of NAAG peptidase inhibitor 2-PMPA in model chronic pain - relation to brain concentration". Neuropharmacology 51 (7-8): 1163–71.
O'Keefe DS, Bacich DJ, Heston WDW. Comparitive analysis of prostate specific membrane andtigen (PSMA) versus a prostate specific membrane antigen like gene. Prostate 58 (2), 200-210, 2004.
O'Keefe DS, Bacich DJ, Heston WDW Prostate Specific Membraen Antigen in Prostate Cancer: Biology, Genetics, and the new Therapeutics (Eds Chung, Isaacs, Simons) Humana press Totawa NJ chapter 18 pp 307-326, 2003.
Perner S, Hofer MD, Kim R et al: Prostate specific membrane antigen expression as a predictor of prostate cancer progression. Hum Pathol 38: 696-701, 2007.
Pinto JT Suffoletto BP, Berzin TM et al, Prostate Specific Membraen antigen, a novel folate hydrolase in human prostatic carcinoma cells. Clin Cancer Res 2 (9) 1445-1451, 1996.
- Rojas C, Frazier ST, Flanary J, Slusher BS (2002). "Kinetics and inhibition of glutamate carboxypeptidase II using a microplate assay". Anal. Biochem. 310 (1): 50–4.
Ross JS, Sheehan CE, Fisher HA et al: Correlation of primary tumor prostate specific membraen antigen expression with disease recurrence in prostate cancer.
Clin Cancer Res. 9: 6357-6362, 2003.
Sacha P, Zamecnik J, Barinka C; Expression of glutamate carboxypeptidase II in human brain. Neurosci. 144: 1361-1372, 2006.
- “Schizophrenia.” (2007) National Institute of Mental Health. http://www.nimh.nih.gov/healthinformation/schizophreniamenu.cfm. Accessed 22 April 2007.
- Segel, I. (1975). Enzyme Kinetics. Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley: New York.
- Tolias, Christos and Jonathan Wasserberg (2002). “Critical decision making in sever head injury management.” Trauma 4: 211.
- “What is Stroke.” National Stroke Association. http://www.stroke.org/site/PageServer?pagename=STROKE. Accessed 22 April 2007.
Wang X, Yin L, Rao P et al: Targeted treatment of prostate cancer. J Cell Biochem 102: 571-579, 2007.
- Zhang W, Murakawa Y, Wozniak KM, Slusher B, Sima AA (2006). "The preventive and therapeutic effects of GCPII (NAALADase) inhibition on painful and sensory diabetic neuropathy". J. Neurol. Sci. 247 (2): 217–23.
- Zhou J, Neale JH, Pomper MG, Kozikowski AP (2005). "NAAG peptidase inhibitors and their potential for diagnosis and therapy". Nature reviews. Drug discovery 4 (12): 1015–26.