Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3

Abstract

␤-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre-and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify ␤-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca 2؉ , the purified enzyme specifically hydrolyzed ␤-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn 2؉ . This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that ␤-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of ␤-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to ␤-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed ␤-citrylglutamate in the presence of Ca 2؉ , and acted on both N-acetyl-aspartylglutamate and ␤-citrylglutamate in the presence of Mn 2؉ , whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze ␤-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its ␤-citrylglutamate hydrolase activity. ␤-Citrylglutamate (BCG)3 is a pseudodipeptide first identified in newborn rat brain at a concentration of 0.5 to 1 mol/g. BCG is also detected in kidneys, heart, and to a much lower extent in intestine, spinal cord, and lungs of young rats. The content of BCG in all organs decreases rapidly after birth to the noticeable exception of testes, where its concentration increases during sexual maturation and remains constant during adulthood (1-3). Although the exact physiological function of BCG is presently unknown, different observations suggest that it may play an important role during brain development and spermatogenesis (4, 5). Recently, BCG has been proposed to be an iron and copper chelator (6, 7).BCG is structurally close to N-acetyl-aspartylglutamate (NAAG), the most abundant dipeptide in the adult brain. NAAG is secreted by neurons upon calcium-dependent depolarization and has long been thought to bind to the glutamate metabotropic receptor mGluR3, possibly attenuating the glutamate-induced excitotoxicity (8). However, this neurotransmitter function of NAAG is still debated (9 -11). NAAG is hydrolyzed into N-acetyl-aspartate (NAA) and glutamate by glutamate carboxypeptidase 2 (GCP2), a membrane-bound, glycosylat...

ATP-binding Cassette Subfamily C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs

“…Analogous to NAAG, BCG is formed by RIMKLB and hydrolyzed back to citrate and glutamate by GCP3 (58), which is ubiquitously expressed in mouse brain (59). We now show that RIMKLA can also ligate a second glutamate to BCG, forming the previously unknown BCG 2 .…”

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

“…N-Acetyl-aspartyl-glutamate is degraded extracellularly by the membrane-bound dipeptidyl peptidase encoded by NAALAD2 (38). NAALAD2 mRNA levels were induced ϳ3-fold 2 h after irradiation in WT cells but Ͼ10-fold in CD47(Ϫ) cells (p Ͻ 0.05; Fig.…”

GCPII Variants, Paralogs and Orthologs

“…When Mn 2+ is present, GCPIII cleaves both NAAG and BCG while in the presence of Ca 2+ it is specific for BCG only. Remarkably, GCPII did not show any BCG hydrolyzing activity [34].…”

“…Interestingly, a very recent study identified GCPIII as acitrylglutamate (BCG) hydrolase in mouse testis [34]. BCG is a pseudodipeptide found in adult rat testis and rat pre-and perinatal CNS.…”