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 ...

N‐acetylaspartylglutamate (NAAG) is an abundant neuropeptide in the mammalian nervous system, synthesized by two related NAAG synthetases I and II (NAAGS‐I and ‐II) encoded by the genes Rimklb and Rimkla, respectively. NAAG plays a role in cognition and memory, according to studies using inhibitors of the NAAG hydrolase glutamate carboxypeptidase II that increase NAAG concentration. To examine consequences of reduced NAAG concentration, Rimkla‐deficient (Rimkla−/−) mice were generated. These mice exhibit normal NAAG level at birth, likely because of the intact Rimklb gene, but have significantly reduced NAAG levels in all brain regions in adulthood. In wild type mice NAAGS‐II was most abundant in brainstem and spinal cord, as demonstrated using a new NAAGS‐II antiserum. In the hippocampus, NAAGS‐II was only detectable in neurons expressing parvalbumin, a marker of GABAergic interneurons. Apart from reduced open field activity, general behavior of adult (6 months old) Rimkla−/− mice examined in different tests (dark‐light transition, optokinetic behavior, rotarod, and alternating T‐maze) was not significantly altered. However, Rimkla−/− mice were impaired in a short‐term novel object recognition test. This was also the case for mice lacking NAA synthase Nat8l, which are devoid of NAAG. Together with results from previous studies showing that inhibition of the NAAG degrading enzyme glutamate carboxypeptidase II is associated with a significant improvement in object recognition, these results suggest a direct involvement of NAAG synthesized by NAAGS‐II in the memory consolidation underlying the novel object recognition task.

NAAG (N-acetylaspartylglutamate) is an abundant neuropeptide in the vertebrate nervous system. It is released from synaptic terminals in a calcium-dependent manner and has been shown to act as an agonist at the type II metabotropic glutamate receptor mGluR3. It has been proposed that NAAG may also be released from axons. So far, however, it has remained unclear how NAAG is transported into synaptic or other vesicles before it is secreted. In the present study, we demonstrate that uptake of NAAG and the related peptide NAAG2 (N-acetylaspartylglutamylglutamate) into vesicles depends on the sialic acid transporter sialin (SLC17A5). This was demonstrated using cell lines expressing a cell surface variant of sialin and by functional reconstitution of sialin in liposomes. NAAG uptake into sialin-containing proteoliposomes was detectable in the presence of an active H+-ATPase or valinomycin, indicating that transport is driven by membrane potential rather than H+ gradient. We also show that sialin is most probably the major and possibly only vesicular transporter for NAAG and NAAG2, because ATP-dependent transport of both peptides was not detectable in vesicles isolated from sialin-deficient mice.