Synthesis of N-Acetylaspartylglutamate (NAAG) and N-Acetylaspartate (NAA) in Axons and Glia of the Crayfish Medial Giant Nerve Fiber

Lieberman, Edward M.; Achreja, Mohit; Urazaev, A. Kh.

doi:10.1007/0-387-30172-0_22

Cited by 7 publications

(11 citation statements)

References 34 publications

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“…Previous reports indicated that NAAG is synthesized from N-acetylaspartate and glutamate in the presence of ATP (12,19,20). This suggests that the synthesis of NAAG is catalyzed by a ligase using ATP.…”

Section: Resultsmentioning

confidence: 94%

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Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

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Stroobant

Lamosa

et al. 2010

Journal of Biological Chemistry

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The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and ␤-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and L-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of ␤-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and ␤-citrylglutamate. N-Acetylaspartylglutamate (NAAG),3 the most abundant dipeptide in the CNS, is present in brain and in the spinal cord, most particularly in neurons of the anterior horn (1). NAAG can be released from neurons upon calcium depolarization (reviewed in Ref. 1) and is a substrate for two glial peptidases, glutamate carboxypeptidase-II (2) and, to a lower extent, glutamate carboxypeptidase-III (3), which are anchored to the plasma membrane with their catalytic site oriented toward the outside of the cell. NAAG long has been thought to be a neurotransmitter able to bind to the metabotropic glutamate receptors mGluR3 (4). Two recent reports suggest, however, that the effects of NAAG as a neurotransmitter are due to a 0.5% glutamate contamination present in commercial NAAG (5, 6). The function of NAAG is, therefore, presently unknown.␤-Citrylglutamate (BCG), which is structurally close to NAAG, is less well characterized. It was first identified in newborn rat brain, where its concentration reaches 0.5-1 mol/g at birth and then decreases with age (7). BCG also was detected in kidneys and heart and to a much lower extent in intestine, spinal cord, and lung of newborn rats. The content of BCG in all organs decreased with age to the noticeable exception of testes, where its concentration increases during sexual maturation and remains constant in adulthood (8,9). BCG is present not only in the testes of mammals but also in those of amphibians and fishes. There is evidence in germinal cells for a role in spermatogenesis (9). BCG rec...

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Section: Resultsmentioning

confidence: 94%

“…NAA and NAAG have been described in the crayfish giant axon (20), and it is therefore likely that these molecules are synthesized in invertebrates by homologues of mammalian NAT8L and RIMKLA/B. Unfortunately, no nuclear genomic sequence is available for the crayfish or any other crustacean.…”

Section: Agreement With Previous Results Suggesting That Naag Ismentioning

confidence: 99%

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

Collard

Stroobant

Lamosa

et al. 2010

Journal of Biological Chemistry

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“…NAAG metabolism in the WM differs from that in the GM. Axons and glia have shown the ability to produce NAAG using Glu 69,70 . According to Lieberman et al, 71 Asp and NAA are also able to be used as substrates for NAAG formation; however, Glu is preferable.…”

Section: Discussionmentioning

confidence: 99%

“…Metabotropic GluRs are responsible for regulating glial membrane potential. 70 It has been shown that modification of the membrane potential activates the phosphoinositide second-messenger system. 72 Thus, alterations in NAAG binding with the receptor in the context of mTBI could modify the membrane potential of astrocytes and impact [mI] through the phosphoinositide second-messenger system, because mI is a member of the phosphoinositide pathway.…”

Section: F I G U R Ementioning

confidence: 99%

Separate N‐acetyl aspartyl glutamate, N‐acetyl aspartate, aspartate, and glutamate quantification after pediatric mild traumatic brain injury in the acute phase

Menshchikov

Ivantsova

Манжурцев

et al. 2020

Magnetic Resonance in Med

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Purpose To separately measure N‐acetyl aspartul glutamate (NAAG), N‐acetyl aspartate (NAA), aspartate (Asp), and glutamate (Glu) concentrations in white matter (WM) using J‐editing techniques in patients with mild traumatic brain injury (mTBI) in the acute phase. Methods Twenty‐four patients with closed concussive head injury and 29 healthy volunteers were enrolled in the current study. For extended 1H MRS examination, patients and controls were equally divided into two subgroups. In subgroup 1 (12 patients/15 controls), NAAG and NAA concentrations were measured in WM separately with MEGA‐PRESS (echo time/repetition time [TE/TR] = 140/2000 ms; δONNAA/ δOFFNAA = 4.84/4.38 ppm, δONNAAG/ δOFFNAAG = 4.61/4.15 ppm). In subgroup 2 (12 patients/14 controls), Asp and Glu concentrations were acquired with MEGA‐PRESS (TE/TR = 90/2000 ms; δONAsp/ δOFFAsp = 3.89/5.21 ppm) and TE‐averaged PRESS (TE from 35 ms to 185 ms with 2.5‐ms increments; TR = 2000 ms) pulse sequences, respectively. Results tNAA and NAAG concentrations were found to be reduced, while NAA concentrations were unchanged, after mild mTBI. Reduced Asp and elevated myo‐inositol (mI) concentrations were also found. Conclusion The main finding of the study is that the tNAA signal reduction in WM after mTBI is associated with a decrease in the NAAG concentration rather than a decrease in the NAA concentration, as was thought previously. This finding highlights the importance of separating these signals, at least for WM studies, to avoid misinterpretation of the results. NAAG plays an important role in selectively activating mGluR3 receptors, thus providing neuroprotective and neuroreparative functions immediately after mTBI. NAAG shows potential for the development of new therapeutic strategies for patients with injuries of varying severity.

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“…As expected for Asp-NAT, close homologues of NAT8L were found in vertebrates, whose brain contains NAA, but not in insects and Caenorhabditis elegans, where NAA has never been reported. Unfortunately, the genome of the crayfish, in which NAA has been described [35], has not yet been sequenced.…”

Section: Molecular Identification Of Aspartate N-acetyltransferasementioning

confidence: 99%

Molecular identification of aspartate N-acetyltransferase and its mutation in hypoacetylaspartia

Wiame

Tyteca

Pierrot

et al. 2009

Biochemical Journal

147

122

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The brain-specific compound NAA (N-acetylaspartate) occurs almost exclusively in neurons, where its concentration reaches approx. 20 mM. Its abundance is determined in patients by MRS (magnetic resonance spectroscopy) to assess neuronal density and health. The molecular identity of the NAT (N-acetyltransferase) that catalyses NAA synthesis has remained unknown, because the enzyme is membrane-bound and difficult to purify. Database searches indicated that among putative NATs (i.e. proteins homologous with known NATs, but with uncharacterized catalytic activity) encoded by the human and mouse genomes two were almost exclusively expressed in brain, NAT8L and NAT14. Transfection studies in HEK-293T [human embryonic kidney-293 cells expressing the large T-antigen of SV40 (simian virus 40)] indicated that NAT8L, but not NAT14, catalysed the synthesis of NAA from L-aspartate and acetyl-CoA. The specificity of NAT8L, its Km for aspartate and its sensitivity to detergents are similar to those described for brain Asp-NAT. Confocal microscopy analysis of CHO (Chinese-hamster ovary) cells and neurons expressing recombinant NAT8L indicates that it is associated with the ER (endoplasmic reticulum), but not with mitochondria. A mutation search in the NAT8L gene of the only patient known to be deficient in NAA disclosed the presence of a homozygous 19 bp deletion, resulting in a change in reading frame and the absence of production of a functional protein. We conclude that NAT8L, a neuron-specific protein, is responsible for NAA synthesis and is mutated in primary NAA deficiency (hypoacetylaspartia). The molecular identification of this enzyme will lead to new perspectives in the clarification of the function of this most abundant amino acid derivative in neurons and for the diagnosis of hypoacetylaspartia in other patients.

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Synthesis of N-Acetylaspartylglutamate (NAAG) and N-Acetylaspartate (NAA) in Axons and Glia of the Crayfish Medial Giant Nerve Fiber

Cited by 7 publications

References 34 publications

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

Separate N‐acetyl aspartyl glutamate, N‐acetyl aspartate, aspartate, and glutamate quantification after pediatric mild traumatic brain injury in the acute phase

Molecular identification of aspartate N-acetyltransferase and its mutation in hypoacetylaspartia

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