.BETA.-Citryl-L-glutamate Is an Endogenous Iron Chelator That Occurs Naturally in the Developing Brain

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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“…There is evidence in germinal cells for a role in spermatogenesis (9). BCG recently has been proposed to be an iron chelator (10).…”

mentioning

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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“…In addition, our previous findings suggested that b-CG is an endogenous low molecular weight Fe chelator. 6) It has been reported that citrate interacts with the fourth iron atom (Fe a ) of the [4Fe-4S] 2ϩ cubane cluster of aconitase in vitro 7) and prevents cluster disassembly, while it is also required for enzyme reactivation in rat cardiac mitochondria treated with H 2 O 2 . Recently, the mitochondrial matrix protein frataxin was also shown to play a role in the storage of iron within mitochondria and promote Fe(II) availability.…”

Section: Discussionmentioning

confidence: 99%

“…Using the same protocol as noted above, 2ϫ10 6 cells were cultured in 24-well plates for 3 d, then solubilized in 1 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 100 mM NaCl, 1% SDS). The solution was transferred into tubes and incubated with proteinase K (6 mg/ml) at 50°C for 2 h, then cooled on ice and incubated with RNase A (10 mg/ml) at 37°C for 30 min.…”

mentioning

confidence: 99%

.BETA.-Citryl-L-glutamate Acts as an Iron Carrier to Activate Aconitase Activity

Hamada-Kanazawa

Narahara

Takano

et al. 2011

Biological & Pharmaceutical Bulletin

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We previously isolated a unique compound containing glutamate and citrate residues from the brains of newborn rats, and identified it as b-citryl-L-glutamate (b-CG).1) This compound appears in high concentrations (300-600 mM) during the period characterized by growth and differentiation of neurons in developing rats, and guinea pigs, and then decreases with maturation. [2][3][4] In another study, we found developmental changes in the concentrations of b-CG in the optic lobes of developing chick brains as well as in primary cultured neuronal cells from chick embryo optic lobes.5) A sharp peak of b-CG was shown, with a maximal concentration at 13 d of incubation in the optic lobe, while similar developmental changes were observed in primary cultured neuronal cells. In additions, the synthetic activity of b-CG was significantly increased on day 3 of the culture, whereas the hydrolyzing enzyme activity was low until day 3, and then it increased significantly until day 12.5) Together, these findings suggested that b-CG plays roles in regulating the differentiation or growth of neuronal cells. However, its functional roles in the developing brain remain unclear.The tetra-carboxyl nature of b-CG, in which a b-carboxylresidue of citrate and amino-residue of glutamate are linked with an amide bond, indicates that it may coordinate with metal ions. To evaluate that coordination, we previously performed pH titration experiments.6) The stability constant was calculated from pH titration data, which showed that b-CG forms relatively strong complexes with Fe(III), Cu(II), and Fe(II). Therefore, we examined the effects of b-CG on Fe-dependent reactive oxygen species (ROS)-generating systems, as well as the potential ROS-scavenging activities of b-CG and metal ion-(b-CG) complexes. b-CG inhibited the Fe-dependent degradation of deoxyribose, as well as Fe-dependent damage to DNA or plasmid DNA in a dose-dependent manner, whereas it had no effect on Cu-mediated DNA damage. In addition, thermodynamic data showed that b-CG in a physiological pH solution is an Fe(II) chelator rather than an Fe(III) chelator. Taken together, these findings suggest that b-CG is an endogenous low molecular weight Fe chelator.Aconitase, a critical citric acid-cycle enzyme that converts citrate to isocitrate, belongs to the family of iron-sulfur-containing dehydratases, whose activities depend on an intact 8) The active center of purified mitochondrial aconitase has been reported to be protected completely from oxidative damage by citrate, an enzyme substrate.9,10) Indeed, citrate interacts with the fourth iron atom (Fe a ) of the [4Fe-4S] 2ϩ cubane cluster, 11) prevents cluster disassembly, while it is also required for enzyme reactivation in rat cardiac mitochondria treated with H 2 O 2 .In the present study, we investigated whether b-CG could insert Fe into [3Fe-4S] 0 of aconitase. Our results showed that the [Fe(II)(b-CG)] complex plays a role as an Fe-carrier for mitochondrial aconitase, and then activates it, whereas no Fe complex with citrate, glu...

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“…As shown in Fig. 4C Recently, we found that β-citrylglutamate (β-CG), isolated from newborn rat brains, was an endogenous low molecular weight Fe chelator, 36) while the [Fe(II)(β-CG)] complex plays a role as an Fe-carrier for mitochondrial aconitase, and then activates it. 16) Therefore, we used the[Fe(II)(β-CG)] complex as an Fe-complex containing moderately bound Fe in our experiments.…”

Section: Comparison Of Effects Of Nitroprusside With Those Of Other Imentioning

confidence: 98%

“…46) Therefore, since the [Fe(II)(β-CG)] complex have a higher affinity to aconitase in mitochondria and can insert its Fe into the [3Fe-4S] cluster of aconitase, 16,36) it is considered that NO reacts with the Fe moiety of the [Fe(II)(β-CG)] complex, then forms an [(NO) Fe(II)(β-CG)] complex and reaches to mitochondria, resulting in activation of aconitase. β-CG seems to function as a tag addressed to aconitase in mitochondria, though further investigation is required to fully explain this interesting possibility.…”

Section: No]mentioning

confidence: 99%

Nitric Oxide Promotes Survival of Cerebral Cortex Neurons with Simultaneous Addition of [Fe(II)(β-Citryl-L-glutamate)] Complex in Primary Culture

Hamada-Kanazawa

Narahara

Takano

et al. 2013

Biological & Pharmaceutical Bulletin

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It has been reported that the activity of mitochondrial aconitase (m-aconitase) is rapidly inhibited in a variety of cells when exposed to nitric oxide (NO).NO is a free radical produced by a wide variety of cell types, that reacts predominantly with Fe(III) or Fe(II) ions of heme proteins and Fe-S centers, as well as molecular oxygen, and superoxide anion. 4,5) Moreover, NO easily reaches mitochondria from cytosolic or extracellular sources, due to its low molecular radius and hydrophobic nature.6) It has also been reported that induction of NO synthesis or exposure of different cell types to NO-donors resulted in early loss of mitochondrial aconitase (m-aconitase) activity. 1)NO-mediated inactivation of m-aconitase has been reported in a variety of cells including macrophages, 7) fibroblasts, 8) tumor cells, 9,10) and Escherichia (E.) coli, 11) though results from in vitro studies are somewhat contradictory. Indeed, in vitro studies that used porcine heart m-aconitase found that low concentrations of NO did not inactivate aconitase, whereas high concentrations led to moderate inhibition.12) Resistance of purified E. coli aconitase and human recombinant cytosolic aconitase (c-aconitase) to NO-dependent inactivation has also been reported. 13) On the contrary, inactivation by either NO or a NO-donor was reported for both m-aconitase and c-aconitase in the presence and absence of a substrate. 14)In agreement with those findings, it was shown that the active form [4Fe-4S] of m-aconitase is rapidly and directly oxidized by ONOO − , to [3Fe-4S], causing a loss of catalytic activity. 12)Another report 11) also noted that E. coli aconitase is highly sensitive to NO-mediated inactivation apparently independent of ONOO − formation. Recently, it was also reported that NO binds to Fe a in the [4Fe-4S] cluster of recombinant porcine maconitase and slowly promotes complete cluster disassembly. 6)The present findings demonstrate that NO-donors promote Fe-dependent activation of m-aconitase ([3Fe-4S] form) in vitro. In intact mitochondria, the NO-donors also promoted Fe-dependent reactivation of the enzyme disassembled by ammonium peroxodisulfate (APS). In mixed neuronal and

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.BETA.-Citryl-L-glutamate Is an Endogenous Iron Chelator That Occurs Naturally in the Developing Brain

Cited by 18 publications

References 47 publications

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

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

.BETA.-Citryl-L-glutamate Acts as an Iron Carrier to Activate Aconitase Activity

Nitric Oxide Promotes Survival of Cerebral Cortex Neurons with Simultaneous Addition of [Fe(II)(β-Citryl-L-glutamate)] Complex in Primary Culture

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