Masumi Katane

d-Aspartate—An important bioactive substance in mammals: A review from an analytical and biological point of view☆

It was long believed that D-amino acids were either unnatural isomers or laboratorial artifacts and that the important functions of amino acids were exerted only by l-amino acids. However, recent investigations have shown that a variety of D-amino acids are present in mammals and that they play important roles in physiological functions in the body. Among the free d-amino acids that have been identified in mammals, D-aspartate (D-Asp) has been shown to play a crucial role in the neuroendocrine and endocrine systems as well as in the central nervous system. Here, we present an overview of recent studies of free D-Asp, focusing on the analytical methods in real biological matrices, expression and localization in tissues and cells, biological and physiological activities, biosynthesis, degradation, cellular transport, and possible relevance to disease. In addition to frequently used techniques for the enantiomeric determination of amino acids, including high-performance liquid chromatography and enzymatic methods, the recent development of analytical methods is also described.

Cloning and functional characterization of Arabidopsis thaliana d‐amino acid aminotransferase – d‐aspartate behavior during germination

The understanding of d‐amino acid metabolism in higher plants lags far behind that in mammals, for which the biological functions of these unique amino acids have already been elucidated. In this article, we report on the biochemical behavior of d‐amino acids (particularly d‐Asp) and relevant metabolic enzymes in Arabidopsis thaliana. During germination and growth of the plant, a transient increase in d‐Asp levels was observed, suggesting that d‐Asp is synthesized in the plant. Administration of d‐Asp suppressed growth, although the inhibitory mechanism responsible for this remains to be clarified. Exogenous d‐Asp was efficiently incorporated and metabolized, and was converted to other d‐amino acids (d‐Glu and d‐Ala). We then studied the related metabolic enzymes, and consequently cloned and characterized A. thalianad‐amino acid aminotransferase, which is presumably involved in the metabolism of d‐Asp in the plant by catalyzing transamination between d‐amino acids. This is the first report of cDNA cloning and functional characterization of a d‐amino acid aminotransferase in eukaryotes. The results presented here provide important information for understanding the significance of d‐amino acids in the metabolism of higher plants.

Suppression of protein l-isoaspartyl (d-aspartyl) methyltransferase results in hyperactivation of EGF-stimulated MEK-ERK signaling in cultured mammalian cells.

l-Aspartyl (l-Asp) and l-asparaginyl residues in proteins isomerize or racemize to d,l-isoaspartyl (d,l-isoAsp) or d-aspartyl (d-Asp) residues during protein aging. These atypical aspartyl residues can interfere with the biological function of the protein and lead to cellular dysfunction. Protein l-isoaspartyl (d-aspartyl) methyltransferase (PIMT) is a repair enzyme that facilitates conversion of l-isoAsp and d-Asp to l-Asp. PIMT deficient mice exhibit accumulation of l-isoAsp in several tissues and die, on average, 12 days after birth from progressive epileptic seizures with grand mal and myoclonus features. However, little is known about the molecular mechanisms by which accumulation of the aberrant residues leads to cellular abnormalities. In this study, we established PIMT-knockdown cells using a short interfering RNA expression system and characterized the resultant molecular abnormalities in intracellular signaling pathways. PIMT-knockdown cells showed significant accumulation of proteins with isomerized residues, compared to control cells. In the PIMT-knockdown cells, Raf-1, MEK, and ERK, members of the MAPK cascade, were hyperphosphorylated after EGF stimulation compared to control cells. These results suggest that PIMT repair of abnormal proteins is necessary to maintain normal MAPK signaling.

The role of protein L-isoaspartyl/D-aspartyl O-methyltransferase (PIMT) in intracellular signal transduction.

Under physiological conditions, L‐aspartyl (L‐Asp) and L‐asparaginyl residues in proteins are spontaneously isomerized or racemized to D‐aspartyl (D‐Asp) or D,L‐isoaspartyl (D,L‐isoAsp) residue. These atypical Asp residues can interfere with protein activity and lead to disruption of cellular function. Protein L‐isoaspartyl/D‐aspartyl O‐methyltransferase (PIMT) is a repair enzyme that initiates the conversion of L‐isoAsp (or D‐Asp) residues to L‐Asp residues. PIMT‐Deficient mice exhibit accumulation of L‐isoAsp in several tissues and die from progressive epileptic seizures at a mean age of 42 days. However, the biological roles of PIMT are still largely unknown. To further our understanding of the function of this protein, we developed an assay to measure PIMT activity in cell lysates. Additionally, we generated PIMT‐knockdown cells by stable transfection of HEK293 cells with PIMT small interfering (si) RNA. Northern blotting and immunoblot analysis revealed that PIMT mRNA and protein levels were significantly decreased in the knockdown cells. In addition, significant levels of proteins that contained isoAsp residues accumulated in these cells, and immunoblot analysis revealed that Raf‐1, MEK, and ERK were hyperphosphorylated upon EGF stimulation compared to control cells. These results indicate that the ability to repair atypical Asp residues is important for normal MAP kinase signaling.

Identification of Novel d-Amino Acid Oxidase Inhibitors by in Silico Screening and Their Functional Characterization in Vitro

D-amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for D-amino acids, including D-serine and D-alanine, which are potential coagonists of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders such as schizophrenia. Hence, a DAO inhibitor that augments the brain levels of D-serine and/or D-alanine and thereby activates NMDA receptor function is expected to be an antipsychotic drug, for instance, in the treatment of schizophrenia. In the search for potent DAO inhibitor(s), a large number of compounds were screened in silico, and several compounds were estimated as candidates. These compounds were then characterized and evaluated as novel DAO inhibitors in vitro. The results reported in this study indicate that some of these compounds are possible lead compounds for the development of a clinically useful DAO inhibitor and have the potential to serve as active site probes to elucidate the structure-function relationships of DAO.

Comparative characterization of three D-aspartate oxidases and one D-amino acid oxidase from Caenorhabditis elegans.

Previously, we cloned cDNAs for four Caenorhabditis elegans genes (F20 Hp, C47Ap, F18Ep, and Y69Ap genes) that were annotated in the database as encoding D‐amino acid oxidase (DAO) or D‐aspartate oxidase (DDO) proteins. These genes were expressed in Escherichia coli, and the recombinant C47Ap and F18Ep were shown to have functional DDO activities, while Y69Ap had functional DAO activity. In this study, we improved the E. coli culture conditions for the production of recombinant F20 Hp and, following purification of the protein, revealed that it has functional DDO activity. The kinetic properties of recombinant C47Ap (DDO‐1), F18Ep (DDO‐2), F20 Hp (DDO‐3), and Y69Ap (DAO) were also determined and compared with recombinant human DDO and DAO. In contrast to the low catalytic efficiency of human DDO for D‐Glu, all three C. elegans DDOs showed higher catalytic efficiencies for D‐Glu than D‐Asp or N‐methyl‐D‐Asp. The catalytic efficiency of C. elegans DAO for D‐Ser was substantially lower than that of human DAO, while the C. elegans DAO was more efficient at deamination of basic D‐amino acids (D‐Arg and D‐His) than human DAO. Collectively, our results indicate that C. elegans contains at least three genes that encode functional DDOs, and one gene encoding a functional DAO, and that these enzymes have different and distinctive properties.

Caenorhabditis elegans has two genes encoding functional d‐aspartate oxidases

Four cDNA clones that were annotated in the database as encoding d‐amino acid oxidase (DAAO) or d‐aspartate oxidase (DASPO) were isolated by RT‐PCR from Caenorhabditis elegans RNA. The proteins (Y69Ap, C47Ap, F18Ep, and F20Hp) encoded by the cloned cDNAs were expressed in Escherichia coli as recombinant proteins with an N‐terminal His‐tag. All proteins except F20Hp were recovered in the soluble fractions. The recombinant Y69Ap has functional DAAO activity, as it can deaminate neutral and basic d‐amino acids, whereas the recombinants C47Ap and F18Ep have functional DASPO activities, as they can deaminate acidic d‐amino acids. Additional experiments using purified recombinant proteins revealed that Y69Ap deaminates d‐Arg more efficiently than d‐Ala and d‐Met, and that C47Ap and F18Ep show distinct kinetic properties against d‐Asp, d‐Glu, and N‐methyl‐d‐Asp. This is the first time that cDNA cloning of invertebrate DAAO and DASPO genes has been reported. In addition, our study reveals for the first time that C. elegans has at least two genes encoding functional DASPOs and one gene encoding DAAO, although it had previously been thought that organisms only bear one copy each of these genes. The two C. elegans DASPOs differ in their substrate specificities and possibly also in their subcellular localization.

Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis

Summary.The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT–PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.

Biosynthesis of D-aspartate in mammals: the rat and human homologs of mouse aspartate racemase are not responsible for the biosynthesis of D-aspartate.

Abstractd-Aspartate (d-Asp) has important physiological functions, and recent studies have shown that substantial amounts of free d-Asp are present in a wide variety of mammalian tissues and cells. Biosynthesis of d-Asp has been observed in several cultured rat cell lines, and a murine gene (glutamate-oxaloacetate transaminase 1-like 1, Got1l1) that encodes Asp racemase, a synthetic enzyme that produces d-Asp from l-Asp, was proposed recently. The product of this gene is homologous to mammalian glutamate-oxaloacetate transaminase (GOT). Here, we tested the hypothesis that rat and human homologs of mouse GOT1L1 are involved in Asp synthesis. The following two approaches were applied, since the numbers of attempts were unsuccessful to prepare soluble GOT1L1 recombinant proteins. First, the relationship between the d-Asp content and the expression levels of the mRNAs encoding GOT1L1 and d-Asp oxidase, a primary degradative enzyme of d-Asp, was examined in several rat and human cell lines. Second, the effect of knockdown of the Got1l1 gene on d-Asp biosynthesis during culture of the cells was determined. The results presented here suggest that the rat and human homologs of mouse GOT1L1 are not involved in d-Asp biosynthesis. Therefore, d-Asp biosynthetic pathway in mammals is still an urgent issue to be resolved.

Spatiotemporal Localization of d-Amino Acid Oxidase and d-Aspartate Oxidases during Development in Caenorhabditis elegans

ABSTRACT Recent investigations have shown that a variety of d-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. d-Amino acid oxidase (DAO) and d-aspartate oxidase (DDO) are degradative enzymes stereospecific for d-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of d-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several d- and l-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and d-amino acids in multicellular organisms.