Biochemical characterization of human glutamate carboxypeptidase III

Abstract

Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N-acetyl-L-aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostatespecific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N-glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N-acetyl-L-aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes. Keywords: folate hydrolase, metallopeptidase, molecular modeling, N-acetylated-alpha-linked-acidic dipeptidase II, neurodegeneration, prostate-specific membrane antigen. Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21), also known as N-acetylated-alpha-linked-acidic dipeptidase (NAALADase), is a type-II transmembrane metallopeptidase found in a variety of human tissues, primarily in the central nervous system, small intestine, and prostate (Israeli et al. 1994;Troyer et al. 1995;Silver et al. 1997;Chang et al. 1999;Renneberg et al. 1999;Sokoloff et al. 2000). In the brain, it hydrolyzes the peptide neurotransmitter N-acetyl-Laspartyl-glutamate (NAAG), thus liberating free glutamate (Robinson et al. 1987). Inhibition of GCPII has been shown to be neuroprotective in animal models of stroke, neuropathic pain and other neurodegenerative states (Slusher et al. 1999;Harada et al. 2000;Zhang et al. 2002;Ghadge et al. 2003), and thus it is being considered and tested as a potential therapeutic target (Subasinghe et al. 1990;Jackson et al. 1996;Nan et al. 2000;Whelan 2000;Neale et al. 2005;Tsukamoto et al. 2005;Zhou et al. 2005). In the small intestine, GCPII has been shown to facilitate the absorption Abbreviations used: b-NAAG, N-acetyl-L-aspartyl-b-linked L-glutamate; 2-PMPA, 2-phosphonomethyl-pentanedioic acid; AccQ, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; DPP IV, dipeptidyl peptidase IV; HPLC, high-performance liquid chromatography; KO, knock-out; MD, molecular dynamics; MOPS,propanesulfonic acid; NAAG, N-acetyl-L-aspartyl-L-glutamate; NAALADase, N-acetylated-alpha-li...

Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3

“…and GCP3-Ser509-As previously noted by others, the major difference between the catalytic sites of GCP2 and GCP3 is the replacement of a conserved asparagine (Asn-519) in GCP2 by a conserved serine (Ser-505) in GCP3 (18). Structural modeling of ␤-citrylglutamate in the catalytic site of GCP2 and GCP3 (see "Discussion") suggested that the bulkier asparagine could impede proper binding of ␤-citrylglutamate to the GCP2 binding site, explaining that the latter enzyme was not able to hydrolyze the citrate derivative.…”

“…Inhibitors of GCP2 have shown neuroprotective effects in animal models of cerebral ischemia, as well as analgesic activity (16). Noteworthy, NAAG can also be hydrolyzed by glutamate carboxypeptidase 3 (GCP3), also designated as N-acetylated ␣-linked acidic dipeptidase 2 (NAALAD2), which shares about 67% sequence identity with GCP2, but with a 10-fold lower catalytic efficiency (17,18). Like GCP2, GCP3 is a membrane-bound, glycosylated ectoenzyme, and its mRNA is abundant in testes and ovaries and detectable in placenta, spleen, prostate, and brain, whereas the mRNA of GCP2 is mainly expressed in kidneys, prostate, liver, and brain (17).…”

PMPA for Nephroprotection in PSMA-Targeted Radionuclide Therapy of Prostate Cancer

“…In 1997, immunostaining had already revealed a physiologic expression of PSMA in the kidney tubules (1). Glutamate carboxypeptidase III (GCP III), a GCP II homolog with 67% amino acid identity, was cloned and evaluated on RNA level in 2007, but because of the lack of a GCP III-specific antibody, the effect of different amounts of GCP II and III on the cell surface of different tissues could not be assessed on protein level until now (19). A third enzyme highly similar to GCP II is NAALADase L (20).…”

Structural Basis of Interactions between Human Glutamate Carboxypeptidase II and Its Substrate Analogs

“…20 In contrast, little is understood about the interaction of the penultimate residue of GCPII substrates with the S1 site of the enzyme, despite the important role of this residue in substrate recognition. [21][22][23] To address this issue, we utilized three phosphapeptide analogs of GCPII substrates in which the planar scissile peptide bond is substituted by a phosphinate moiety. These compounds mimic unstable tetrahedral transition state formed during the course of substrate hydrolysis 24 and potently inhibit GCPII by interacting with both S1 and S1′ sites of the enzyme (Fig.…”