N-Acetylaspartylglutamate Synthetase II Synthesizes N-Acetylaspartylglutamylglutamate

Abstract

N-Acetylaspartylglutamate (NAAG) is found at high concentrations in the vertebrate nervous system. NAAG is an agonist at group II metabotropic glutamate receptors. In addition to its role as a neuropeptide, a number of functions have been proposed for NAAG, including a role as a non-excitotoxic transport form of glutamate and a molecular water pump. We recently identified a NAAG synthetase (now renamed NAAG synthetase I, NAAGS-I), encoded by the ribosomal modification protein rimK-like family member B (Rimklb) gene, as a member of the ATP-grasp protein family. We show here that a structurally related protein, encoded by the ribosomal modification protein rimK-like family member A (Rimkla) gene, is another NAAG synthetase (NAAGS-II), which in addition, synthesizes the N-acetylated tripeptide N-acetylaspartylglutamylglutamate (NAAG 2 ). In contrast, NAAG 2 synthetase activity was undetectable in cells expressing NAAGS-I. Furthermore, we demonstrate by mass spectrometry the presence of NAAG 2 in murine brain tissue and sciatic nerves. The highest concentrations of both, NAAG 2 and NAAG, were found in sciatic nerves, spinal cord, and the brain stem, in accordance with the expression level of NAAGS-II. To our knowledge the presence of NAAG 2 in the vertebrate nervous system has not been described before. The physiological role of NAAG 2 , e.g. whether it acts as a neurotransmitter, remains to be determined. N-Acetylaspartylglutamate (NAAG)3 is an abundant peptide in the vertebrate nervous system, found at high micromolar to low millimolar concentrations (1-3). A number of studies demonstrated that NAAG acts as a specific agonist at the group II metabotropic mGluR3 glutamate receptors (4 -6). Agonistic and antagonistic effects of NAAG at N-methyl-D-asparatate receptors have been described (5, 7, 8), but could not be confirmed in later studies (9). Several reports indicate a neuroprotective role of NAAG (10 -12), and in line with this, inhibitors of the NAAG hydrolyzing glutamate carboxypeptidase (GCP)-II have a significant neuroprotective effect in different model systems (13). Increasing NAAG concentrations by GCP-II inhibition appear to reduce glutamate release through activation of presynaptic mGluR3 receptors (for review, see Ref. 13).NAAG may also be involved in neuron-glia signaling (14), although its specific role is not fully understood. Theoretically, synthesis of NAAG could also be an efficient way to transfer large amounts of glutamate from neurons to the extracellular fluid, avoiding the excitotoxic effect of free glutamate (15). A possible role of NAAG as a molecular water pump has also been suggested (16).NAAG is synthesized independently of ribosome from N-acetylaspartate (NAA) and glutamate by NAAG synthetases. Although neurons are the major source of NAAG, it is also present in cultured oligodendrocytes and activated microglia (17). In the mammalian nervous system, the highest NAAG levels have been found in the brain stem, spinal cord, and peripheral nerves (1, 18 -21). NAAG is released from synaptic ...

Growth Hormone–Releasing Hormone Effects on Brain γ-Aminobutyric Acid Levels in Mild Cognitive Impairment and Healthy Aging

“…37 A secondary form of NAAG, NAAG2, was recently identified, and its function and distribution are still undetermined. 39 In the absence of clear evidence to indicate that NAAG has effects other than inhibiting neurotransmission, perhaps the most parsimonious conclusion from our data at this point is that 20 weeks of GHRH treatment enhances inhibitory features of brain network activity (GABA and NAAG).…”

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

“…5). The concentration of the newly discovered metabolite BCG 2 was also strongly increased in RIMKLA-overexpressing cells, indicating that RIMKLA ligates a second glutamate not only to NAAG (39), but also to BCG.…”

“…NAAG is formed from NAA and glutamate by RIMKLA, which is also known as NAAG synthase 2. It was recently shown that the same enzyme can also ligate a second glutamate to NAAG, forming NAAG 2 (39). NAAG 2 also accumulated in Abcc5 Ϫ/Ϫ brain, but its function, if any, is currently not known.…”

Advances in understanding the peptide neurotransmitter NAAG and appearance of a new member of the NAAG neuropeptide family

“…More stunning was the very recent report that NAAG synthetase II also mediates the synthesis of the tripeptide N ‐acetylaspartylglutamylglutamate (NAAG 2 ) and that this peptide is found in brain at 30‐ to 50‐fold lower concentrations than NAAG (Lodder‐Gadaczek et al. 2011).…”

“…A central question arising from the discovery of NAAG 2 is its role relative to that of NAAG in nervous system function. Extracellular GCPII hydrolyzes NAAG 2 to NAAG and glutamate and hydrolyzes NAAG to N ‐acetylaspartate, releasing a second glutamate (Lodder‐Gadaczek et al. 2011).…”