Are Astrocytes the Missing Link Between Lack of Brain Aspartoacylase Activity and the Spongiform Leukodystrophy in Canavan Disease?

Canavan disease (CD) is a genetic degenerative brain disorder associated with mutations of the gene encoding aspartoacylase (ASPA). In humans, the CD syndrome is marked by early onset, hydrocephalus, macroencephaly, psychomotor retardation, and spongiform myelin sheath vacuolization with progressive leukodystrophy. Metabolic hallmarks of the disease include elevated N-acetylaspartate (NAA) levels in brain, plasma and CSF, along with daily excretion of large amounts of NAA and its anabolic metabolite, N-acetylaspartylglutamate (NAAG). Of the observed neuropathies, the most important appears to be the extensive demyelination that interferes with normal neuronal signaling. However, finding the links between the lacks of ASPA activity in oligodendrocytes, the buildup of NAA in white matter (WM) and the mechanisms underlying the edematous spongiform leukodystrophy have remained elusive. In this analytical review we consider what those links might be and propose that in CD, the pathological buildup of NAA in limited WM extracellular fluid (ECF) is responsible for increased ECF osmotic–hydrostatic pressure and initiation of the demyelination process. We also hypothesize that NAA is not directly liberated by neurons in WM as it is in gray matter, and that its source in WM ECF is solely as a product of the catabolism of axon-released NAAG at nodes of Ranvier by astrocyte NAAG peptidase after it has docked with the astrocyte surface metabotropic glutamate receptor 3. This hypothesis ascribes for the first time a possible key role played by astrocytes in CD, linking the lack of ASPA activity in myelinating oligodendrocytes, the pathological buildup of NAA in WM ECF, and the spongiform demyelination process. It also offers new perspectives on the cause of the leukodystrophy in CD, and on possible treatment strategies for this inherited metabolic disease. CD, a rare genetic disorder that compromises a physiologically important tri-cellular brain metabolic system.     

N-acetylaspartate in the vertebrate brain: metabolism and function

N-Acetyl-l-aspartate (NAA) is an amino acid that is present in the vertebrate brain. Its concentration is one of the highest of all free amino acids and, although NAA is synthesized and stored primarily in neurons, it cannot be hydrolyzed in these cells. Furthermore, neuronal NAA is dynamic and turns over more than once each day by virtue of its continuous efflux, in a regulated intercompartmental cycling via extracellular fluids, between neurons and a second compartment in oligodendrocytes. The metabolism of NAA, between its anabolic compartment in neurons and its catabolic compartment in oligodendrocytes, and its possible physiological role in the brain has been the subject of much speculation. There are two human inborn errors in metabolism of NAA. One is Canavan disease (CD), in which there is a buildup of NAA (hyperacetylaspartia) and associated spongiform leukodystrophy, caused by a lack of aspartoacylase activity. The other is a singular human case of lack of NAA (hypoacetylaspartia), where the enzyme that synthesizes NAA is apparently absent. There are two animal models currently available for studies of CD. One is a rat with a natural deletion of the catabolic enzyme, and the other a gene knockout mouse. In addition to the presence of NAA in neurons, its prominence in ¹H nuclear magnetic resonance spectroscopic studies has led to its wide use in diagnostic human medicine as both an indicator of brain pathology and of disease progression in a variety of CNS diseases. In this review, various hypotheses regarding the metabolism of NAA and its possible role in the CNS are evaluated. Based on this analysis, it is concluded that although NAA may have several functions in the CNS, an important role of the NAA intercompartmental system is osmoregulatory, and in this role it may be the primary mechanism for the removal of intracellular water, against a water gradient, from myelinated neurons.     

N-Acetylaspartate (NAA) and N-Acetylaspartylglutamate (NAAG) Promote Growth and Inhibit Differentiation of Glioma Stem-like Cells

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.     

Evidence that the tri-cellular metabolism of N-acetylaspartate functions as the brain’s “operating system”: how NAA metabolism supports meaningful intercellular frequency-encoded communications

N-acetylaspartate (NAA), an acetylated derivative of l-aspartate (Asp), and N-acetylaspartylglutamate (NAAG), a derivative of NAA and l-glutamate (Glu), are synthesized by neurons in brain. However, neurons cannot catabolize either of these substances, and so their metabolism requires the participation of two other cell types. Neurons release both NAA and NAAG to extra-cellular fluid (ECF) upon stimulation, where astrocytes, the target cells for NAAG, hydrolyze it releasing NAA back into ECF, and oligodendrocytes, the target cells for NAA, hydrolyze it releasing Asp to ECF for recycling to neurons. This sequence is unique as it is the only known amino acid metabolic cycle in brain that requires three cell types for its completion. The results of this cycling are two-fold. First, neuronal metabolic water is transported to ECF for its removal from brain. Second, the rate of neuronal activity is coupled with focal hyperemia, providing stimulated neurons with the energy required for transmission of meaningful frequency-encoded messages. In this paper, it is proposed that the tri-cellular metabolism of NAA functions as the “operating system” of the brain, and is essential for normal cognitive and motor activities. Evidence in support of this hypothesis is provided by the outcomes of two human inborn errors in NAA metabolism.     

Functions of N-acetyl-L-aspartate and N-acetyl-L-aspartylglutamate in the vertebrate brain: role in glial cell-specific signaling

N-Acetyl-L-aspartate (NAA) and its derivative N-acetylaspartylglutamate (NAAG) are major osmolytes present in the vertebrate brain. Although they are synthesized primarily in neurons, their function in these cells is unclear. In the brain, these substances undergo intercompartmental cycles in which they are released by neurons in a regulated fashion and are then rapidly hydrolyzed by catabolic enzymes associated with glial cells. Recently, the catabolic enzyme for NAA hydrolysis has been found to be expressed only in oligodendrocytes, and the catabolic enzyme for NAAG expressed only in astrocytes. These results indicate an unusual tricellular metabolic sequence for the synthesis and hydrolysis of NAAG wherein it is synthesized in neurons from NAA and L-glutamate, hydrolyzed to NAA and L-glutamate by astrocytes, and further hydrolyzed to L-aspartate and acetate by oligodendrocytes. Since the discovery that the NAA and NAAG anabolic products of neurons are specifically targeted to oligodendrocytes and astrocytes, respectively, this unique metabolic compartmentalization also suggests that these substances may play an important role in cell-specific glial signaling. In this review, it is hypothesized that a key function of NAA and NAAG in the vertebrate brain is in cell signaling and that these substances are important in the regulation of interactions of brain cells and in the establishment and maintenance of the nervous system.     

Molecular Characterization of N-Acetylaspartylglutamate Synthetase

The dipeptide N-acetylaspartyl-glutamate (NAAG) is an abundant neuropeptide in the mammalian brain. Despite this fact, its physiological role is poorly understood. NAAG is synthesized by a NAAG synthetase catalyzing the ATP-dependent condensation of N-acetylaspartate and glutamate. In vitro NAAG synthetase activity has not been described, and the enzyme has not been purified. Using a bioinformatics approach we identified a putative dipeptide synthetase specifically expressed in the nervous system. Expression of the gene, which we named NAAGS (for NAAG synthetase) was sufficient to induce NAAG synthesis in primary astrocytes or CHO-K1 and HEK-293T cells when they coexpressed the NAA transporter NaDC3. Furthermore, coexpression of NAAGS and the recently identified N-acetylaspartate (NAA) synthase, Nat8l, in CHO-K1 or HEK-293T cells was sufficient to enable these cells to synthesize NAAG. Identity of the reaction product of NAAGS was confirmed by HPLC and electrospray ionization tandem mass spectrometry (ESI-MS). High expression levels of NAAGS were restricted to the brain, spinal cord, and testis. Taken together our results strongly suggest that the identified gene encodes a NAAG synthetase. Its identification will enable further studies to examine the role of this abundant neuropeptide in the vertebrate nervous system.     

A radiometric assay for aspartoacylase activity in cultured oligodendrocytes

Recent studies have shown that aspartoacylase (ASPA), the defective enzyme in Canavan disease, is detectable in the brain only in the oligodendrocytes. Studying the regulation of ASPA is central to the understanding the pathogenesis of Canavan disease and to the development of therapeutic strategies. Toward this goal, we have developed a sensitive method for the assay of ASPA in cultured oligodendrocytes. The method involves: (a) chemical synthesis of [14C]N-acetylaspartate (NAA) from L-[14C]Asp; (b) use of [14C]NAA as substrate in the assay; and (c) separation and quantitation of the product L-[14C]Asp using a TLC system. This method can detect as low as 10pmol of product and has been optimized for cultured oligodendrocytes. Thus, this method promises to be a valuable tool for understanding the biochemical mechanisms involved in the cell-specific expression and regulation of ASPA in oligodendrocytes.     

N-acetylaspartylglutamate synthetase II synthesizes N-acetylaspartylglutamylglutamate

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.     

Loss of Central Auditory Processing in a Mouse Model of Canavan Disease

Canavan Disease (CD) is a leukodystrophy caused by homozygous null mutations in the gene encoding aspartoacylase (ASPA). ASPA-deficiency is characterized by severe psychomotor retardation, and excessive levels of the ASPA substrate N-acetylaspartate (NAA). ASPA is an oligodendrocyte marker and it is believed that CD has a central etiology. However, ASPA is also expressed by Schwann cells and ASPA-deficiency in the periphery might therefore contribute to the complex CD pathology. In this study, we assessed peripheral and central auditory function in the Aspa^lacZ/lacZ rodent model of CD using auditory brainstem response (ABR). Increased ABR thresholds and the virtual loss of waveform peaks 4 and 5 from Aspa^lacZ/lacZ mice, indicated altered central auditory processing in mutant mice compared with Aspa^wt/wt controls and altered central auditory processing. Analysis of ABR latencies recorded from Aspa^lacZ/lacZ mice revealed that the speed of nerve conduction was unchanged in the peripheral part of the auditory pathway, and impaired in the CNS. Histological analyses confirmed that ASPA was expressed in oligodendrocytes and Schwann cells of the auditory system. In keeping with our physiological results, the cellular organization of the cochlea, including the organ of Corti, was preserved and the spiral ganglion nerve fibres were normal in ASPA-deficient mice. In contrast, we detected substantial hypomyelination in the central auditory system of Aspa^lacZ/lacZ mice. In summary, our data suggest that the lack of ASPA in the CNS is responsible for the observed hearing deficits, while ASPA-deficiency in the cochlear nerve fibres is tolerated both morphologically and functionally.     

N-Glycosylation pattern of recombinant human CD82 (KAI1), a tumor-associated membrane protein

The membrane glycoprotein CD82 (KAI1) has attracted increasing attention as a suppressor of cell migration, related tumor invasion, as well as metastasis. The glycosylation of CD82 has been shown to be involved in a correlative cell adhesion and motility. However, the N-glycosylation pattern of CD82 has not been described yet. In the current study, a detailed characterization of the recombinant human CD82 N-linked glycosylation pattern was conducted by employing an integrative proteomic and glycomic approach, including glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots. The results reveal three N-glycosylation sites, and further demonstrate a putative glycosylation site at Asn₁₅₇ for the first time. A highly heterogeneous pattern of N-linked glycans is described, which express distinct carbohydrate epitopes, such as bisecting N-acetylglucosamine, (α-2,6) N-acetylneuraminic acid, and core fucose. These epitopes are highly associated with various biological functions, including cell adhesion and cancer metastasis, and can possibly influence the anti-cancer inhibition ability of CD82.     

Myelin Lipid Abnormalities in the Aspartoacylase-Deficient Tremor Rat

The high concentration of N-acetylaspartate (NAA) in neurons of the central nervous system and its growing clinical use as an indicator of neuronal viability has intensified interest in the biological function of this amino acid derivative. The biomedical relevance of such inquiries is highlighted by the myelin-associated pathology of Canavan disease, an inherited childhood disorder resulting from mutation of aspartoacylase (ASPA), the NAA-hydrolyzing enzyme. This enzyme is known to be localized in oligodendrocytes with bimodal distribution in cytosol and the myelin sheath, and to produce acetyl groups utilized in myelin lipid synthesis. Loss of this acetyl source in Canavan disease and rodent models such as the tremor rat are thought to account for the observed myelin deficit. This study was undertaken to further define and quantify the specific lipid abnormalities that occur as a result of ASPA deficit in the tremor rat. Employing mass spectrometry together with high performance thin-layer chromatography, we found that myelin from 28-day-old animals showed major reduction in cerebrosides (CB) and sulfatides (Sulf) with unsubstituted fatty acids, and equal if not greater changes in myelin from 7-month-old tremors. Cerebrosides with 2-hydroxyfatty acids showed little if any change at either age; Sulf with 2-hydroxyfatty acids showed no significant change at 28 days, but surprisingly a major increase at 7 months. Two species of phosphatidylcholine, 32:0 and 34:1, also showed significant increase, but only at 28 days. One form of phosphatidylethanolamine, PE36:1, was reduced a modest amount at both ages, whereas the plasmalogen form did not change. The dysmyelination that results from inactivation of ASPA is thus characterized by selective decreases as well as some increases in specific lipids. Special issue article in honor of Dr. George DeVries. Fatty acid designations (e.g. 18:1) indicate carbon number and number of double bonds.     

N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Effect of N-acetylaspartic acid on the diffusion coefficient of water: a proton magnetic resonance phantom method for measurement of osmolyte-obligated water

N-acetyl-L-aspartic acid (NAA) is an amino acid present in the vertebrate brain that is synthesized and stored primarily in neurons, although it cannot be hydrolyzed in these cells. Nonetheless, neuronal NAA is dynamic and turns over more than once each day by cycling, via extracellular fluids (ECF), between neurons and catabolic compartments in oligodendrocytes. One important role of the NAA intercompartmental cycle appears to be osmoregulatory, and in this role it may be the primary mechanism for the removal of metabolic water, against a water gradient, from myelinated neurons. However, the number of water molecules that might be cotransported to ECF per NAA molecule released is as yet unclear. In this investigation, using a proton nuclear magnetic resonance method and diffusion measurements at two magnetic field strengths on water and NAA phantoms in vitro, the effect of NAA on the diffusion coefficient of water has been measured, and a ratio (K) of obligated water molecules per molecule of NAA has been determined. For NAA measured at 100mM and 3 Tesla K=24 and at 7 Tesla K=14. Based on these results, apparent K(NAA) varies inversely with field strength, and with a computed field strength factor of 2.55mmol water/unit Tesla, K(NAA) in the absence of any applied magnetic field strength would be 32.     

Aspartoacylase-lacZ knockin mice: an engineered model of Canavan disease

Canavan Disease (CD) is a recessive leukodystrophy caused by loss of function mutations in the gene encoding aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme that hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. The neurological phenotypes of different rodent models of CD vary considerably. Here we report on a novel targeted aspa mouse mutant expressing the bacterial β-Galactosidase (lacZ) gene under the control of the aspa regulatory elements. X-Gal staining in known ASPA expression domains confirms the integrity of the modified locus in heterozygous aspa lacZ-knockin (aspa(lacZ/+)) mice. In addition, abundant ASPA expression was detected in Schwann cells. Homozygous (aspa(lacZ/lacZ)) mutants are ASPA-deficient, show CD-like histopathology and moderate neurological impairment with behavioural deficits that are more pronounced in aspa(lacZ/lacZ) males than females. Non-invasive ultrahigh field proton magnetic resonance spectroscopy revealed increased levels of NAA, myo-inositol and taurine in the aspa(lacZ/lacZ) brain. Spongy degeneration was prominent in hippocampus, thalamus, brain stem, and cerebellum, whereas white matter of optic nerve and corpus callosum was spared. Intracellular vacuolisation in astrocytes coincides with axonal swellings in cerebellum and brain stem of aspa(lacZ/lacZ) mutants indicating that astroglia may act as an osmolyte buffer in the aspa-deficient CNS. In summary, the aspa(lacZ) mouse is an accurate model of CD and an important tool to identify novel aspects of its complex pathology.     

Synthesis and characterization of chitosan-homocysteine thiolactone as a mucoadhesive polymer

Two mucoadhesive thiolated polymers were synthesized by the covalent attachment of homocysteine thiolactone (HT) to chitosan and N,N,N-trimethyl-chitosan (TM-chitosan) at various chitosan:HT ratios. The amount of thiol and disulphide groups immobilized on the chitosan influenced the polymer's mucoadhesion positively and negatively, respectively, with the optimal chitosan:HT (w/w) ratio being found to be 1:0.1. The interaction between mucin and chitosan and its three derivatives was highest for the thiolated chitosan derivatives but was pH dependent. HT-chitosan and TM-HT-chitosan, with the thiol groups of 64.15 and 32.48 μmol/g, respectively, displayed a 3.67- and 6.33-fold stronger mucoadhesive property compared to that of the unmodified chitosan at pH 1.2, but these differences were only ∼1.7-fold at pH 6.4. The swelling properties of TM-HT-chitosan and HT-chitosan were higher than that of chitosan and TM-chitosan, attaining a swelling ratio of up to 240% and 140%, respectively, at pH 1.2 within 2 h.     

The ESR2 AluI 1730G>A (rs4986938) gene polymorphism is associated with fibrinogen plasma levels in postmenopausal women

Canavan disease (CD) is a neurodegenerative disorder usually presenting in the first six months of life. CD patients can be identified via elevated levels of N-acetyl-l-aspartate in the pattern of urinary organic acids assessed by gas chromatography-mass spectrometry. They are characterized by deficiency of aspartoacylase (aminoacylase 2; ASPA) due to mutations in the ASPA gene. Information on the molecular basis of CD is rather sparse. A lack of expression studies of ASPA mutant proteins in appropriate expression systems has prompted this investigation. Studies with overexpressed ASPA mutant proteins were carried out in the HEK293 cell line, which provides the authentic human machinery for posttranslational modifications. All ASPA mutants tested (ASPA Arg168His, ASPA Pro181Thr, ASPA Tyr288Cys, ASPA Phe295Ser, and ASPA Ala305Glu) showed loss of ASPA activity, which can be explained by the intramolecular effects of the mutations in the enzyme. The mutation p.Phe295Ser even leads to absent ASPA mRNA expression, as revealed by quantitative real-time PCR. Using this approach, ASPA gene expression analysis yielded high levels of human ASPA gene expression not only in brain and kidney, but also in lung and liver. More information of ASPA localization in human organs and detailed characterization of mutations leading to a deficiency of ASPA can contribute to a better understanding of this inborn error of metabolism.     

N-Acetyl-Aspartyl-Glutamate: Regional Levels in Rat Brain and the Effects of Brain Lesions as Determined by a New HPLC Method

Abstract: An isocratic HPLC method to measure endogenous N -acetyl-aspartyl-glutamate (NAAG) and N -acetyl-aspartate (NAA) is described. After removal of primary amines by passage of tissue extracts over AG-50 resin, the eluate was subject to HPLC anion-exchange analysis and eluted with phosphate buffer with absorbance monitored at 214 nm. The retention time for NAA was 5.6 min and for NAAG 11.4 min with a limit sensitivity of 0.1 nmol. The levels of NAA and NAAG were measured in 16 regions of rat brain and in heart and liver. NAAG was undetectable in heart and liver and exhibited 10-fold variation in concentration among brain regions; the highest levels were found in spinal cord. In contrast, low concentrations of NAA were detectable in heart and liver, and the regional distribution of NAA in brain varied only twofold. The regional distribution of NAA and NAAG correlated poorly. To assess the neuronal localization of these two compounds, the effects of selective brain lesions on their levels were examined. Decortication caused a 28% decrease in NAAG levels in the ipsi-lateral striatum while NAA decreased 38%. Kainate lesion of the striatum resulted in a 31% decrease in NAAG in the ipsilateral striatum, whereas NAA fell by 58%. Kainate lesion of the hippocampus resulted in significant decrements in NAAG and NAA in the hippocampus and septum. Transection of the spinal cord at midthorax resulted in a 51% decrease in NAAG levels immediately caudal and a 40% decrease immediately rostral to the lesion; however, NAA decreased only 30% in these areas. These results are consistent with a neuronal localization of NAAG in brain. Combined with the fact that NAAG interacts with a subpopulation of glutamate receptors, these results suggest that NAAG may serve as an excitatory neurotransmitter.     

N-acylation of phosphatidylethanolamine and its biological functions in mammals

N-acylphosphatidylethanolamine (NAPE) and N-acylplasmenylethanolamine (pNAPE) are widely found phospholipids, and they are precursors for N-acylethanolamines, a group of compounds that has a variety of biological effects and encompasses the endocannabinoid anandamide. NAPE and pNAPE are synthesized by the transfer of an acyl chain from a donor phospholipid, to the amine in phosphatidylethanolamine or plasmenylethanolamine. NAPE has been reported to stabilize model membranes during brain ischemia, and to modulate food intake in rodents, thus having bioactive effects besides its precursor role. This paper reviews the metabolism, occurrence and assay of NAPE and pNAPE, and discusses the putative biological functions in mammals of these phospholipids. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

The molecular basis of aminoacylase 1 deficiency

Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.     

Effect of pH and temperature on nitrosamide-induced mutation in Escherichia coli

N-Nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) induced reversions in four mutant auxotropic strains of E. coli. Among other nitroso compounds tested only N-methyl-N′-nitro-N-nitrosoguanidine (MNG) was an active mutagen in the system used.