Distinctive Roles of D-Amino Acids in the Homochiral World: Chirality of Amino Acids Modulates Mammalian Physiology and Pathology.

Living organisms enantioselectively employ L-amino acids as the molecular architecture of protein synthesized in the ribosome. Although L-amino acids are dominantly utilized in most biological processes, accumulating evidence points to the distinctive roles of D-amino acids in non-ribosomal physiology. Among the three domains of life, bacteria have the greatest capacity to produce a wide variety of D-amino acids. In contrast, archaea and eukaryotes are thought generally to synthesize only two kinds of D-amino acids: D-serine and D-aspartate. In mammals, D-serine is critical for neurotransmission as an endogenous coagonist of N-methyl D-aspartate receptors. Additionally, D-aspartate is associated with neurogenesis and endocrine systems. Furthermore, recognition of D-amino acids originating in bacteria is linked to systemic and mucosal innate immunity. Among the roles played by D-amino acids in human pathology, the dysfunction of neurotransmission mediated by D-serine is implicated in psychiatric and neurological disorders. Non-enzymatic conversion of L-aspartate or L-serine residues to their D-configurations is involved in age-associated protein degeneration. Moreover, the measurement of plasma or urinary D-/L-serine or D-/L-aspartate levels may have diagnostic or prognostic value in the treatment of kidney diseases. This review aims to summarize current understanding of D-amino-acid-associated biology with a major focus on mammalian physiology and pathology.

Serine racemase is involved in d-aspartate biosynthesis.

d-Aspartate is found in the nervous and reproductive system and participates in various physiological roles. While several lines of evidence suggest that this amino acid has an endogenous origin, the enzyme responsible for mammalian d-Asp biosynthesis has not yet been identified. We show that mammalian serine racemase (SRR), the primary enzyme responsible for brain d-Ser production, catalyses Asp racemization via a two-base mechanism. We observed that overexpression of SRR in rat pheochromocytoma PC12 cells resulted in an increase in intracellular d-Asp compared with control cells, demonstrating that SRR functions as an Asp racemase in the cells. To investigate the impact of endogenous SRR on endogenous d-Asp levels in the cells, we generated SRR-knockout (SRR-KO) PC12 cells. The SRR-KO cells exhibited decreased intracellular d-Ser levels, but production levels of d-Asp were unaffected. In contrast, SRR-KO mice showed significantly decreased d-Asp levels in their frontal cortices and hippocampi, where SRR is normally highly expressed, while d-Asp levels in the cerebellum and testes remained unchanged. Our results indicate that SRR indeed acts as a d-Asp biosynthetic enzyme in some organs and/or tissues, and also provide evidences that there should be some additional enzyme for d-Asp synthesis in mammals.

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

D-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.

D-Aspartate acts as a signaling molecule in nervous and neuroendocrine systems.

D-Aspartate (D-Asp) is an endogenous amino acid in the central nervous and reproductive systems of vertebrates and invertebrates. High concentrations of D-Asp are found in distinct anatomical locations, suggesting that it has specific physiological roles in animals. Many of the characteristics of D-Asp have been documented, including its tissue and cellular distribution, formation and degradation, as well as the responses elicited by D-Asp application. D-Asp performs important roles related to nervous system development and hormone regulation; in addition, it appears to act as a cell-to-cell signaling molecule. Recent studies have shown that D-Asp fulfills many, if not all, of the definitions of a classical neurotransmitter-that the molecule's biosynthesis, degradation, uptake, and release take place within the presynaptic neuron, and that it triggers a response in the postsynaptic neuron after its release. Accumulating evidence suggests that these criteria are met by a heterogeneous distribution of enzymes for D-Asp's biosynthesis and degradation, an appropriate uptake mechanism, localization within synaptic vesicles, and a postsynaptic response via an ionotropic receptor. Although D-Asp receptors remain to be characterized, the postsynaptic response of D-Asp has been studied and several L-glutamate receptors are known to respond to D-Asp. In this review, we discuss the current status of research on D-Asp in neuronal and neuroendocrine systems, and highlight results that support D-Asp's role as a signaling molecule.

Aspartate racemase, generating neuronal D-aspartate, regulates adult neurogenesis.

D-aspartic acid is abundant in the developing brain. We have identified and cloned mammalian aspartate racemase (DR), which converts L-aspartate to D-aspartate and colocalizes with D-aspartate in the brain and neuroendocrine tissues. Depletion of DR by retrovirus-mediated expression of short-hairpin RNA in newborn neurons of the adult hippocampus elicits profound defects in the dendritic development and survival of newborn neurons and survival. Because D-aspartate is a potential endogenous ligand for NMDA receptors, the loss of which elicits a phenotype resembling DR depletion, D-aspartate may function as a modulator of adult neurogenesis.

Abundance and activity of Chloroflexi-type SAR202 bacterioplankton in the meso- and bathypelagic waters of the (sub)tropical Atlantic.

The contribution of Chloroflexi-type SAR202 cells to total picoplankton and bacterial abundance and uptake of D- and L-aspartic acids (Asp) was determined in the different meso- and bathypelagic water masses of the (sub)tropical Atlantic (from 35 degrees N to 5 degrees S). Fluorescence in situ hybridization (FISH) revealed that the overall abundance of SAR202 was < or = 1 x 10(3) cells ml(-1) in subsurface waters (100 m layer), increasing in the mesopelagic zone to 3 x 10(3) cells ml(-1) and remaining fairly constant down to 4000 m depth. Overall, the percentage of total picoplankton identified as SAR202 increased from < 1% in subsurface waters to 10-20% in the bathypelagic waters. On average, members of the SAR202 cluster accounted for about 30% of the Bacteria in the bathypelagic waters, whereas in the mesopelagic and subsurface waters, SAR202 cells contributed < 5% to total bacterial abundance. The ratio of D-Asp : L-Asp uptake by the bulk picoplankton community increased from the subsurface layer (D-Asp : L-Asp uptake ratio approximately 0.03) to the deeper layers reaching a ratio of approximately 1 at 4000 m depth. Combining FISH with microautoradiography to determine the proportion of SAR202 cells taking up D-Asp versus L-Asp, we found that approximately 30% of the SAR202 cells were taking up L-Asp throughout the water column while D-Asp was essentially not taken up by SAR202. This D-Asp : L-Asp uptake pattern of SAR202 cells is in contrast to that of the bulk bacterial and crenarchaeal community in the bathypelagic ocean, both sustaining a higher fraction of D-Asp-positive cells than L-Asp-positive cells. Thus, although the Chloroflexi-type SAR202 constitutes a major bathypelagic bacterial cluster, it does not contribute to the large fraction of d-Asp utilizing prokaryotic community in the meso- and bathypelagic waters of the North Atlantic, but rather utilizes preferentially L-amino acids.

Latitudinal trends of Crenarchaeota and Bacteria in the meso- and bathypelagic water masses of the Eastern North Atlantic.

The distribution and activity of the bulk picoplankton community and, using microautoradiography combined with catalysed reported deposition fluorescence in situ hybridization (MICRO-CARD-FISH), of the major prokaryotic groups (Bacteria, marine Crenarchaeota Group I and marine Euryarchaeota Group II) were determined in the water masses of the subtropical North Atlantic. The bacterial contribution to total picoplankton abundance was fairly constant, comprising approximately 50% of DAPI-stainable cells. Marine Euryarchaeota Group II accounted always for < 5% of DAPI-stainable cells. The percentage of total picoplankton identified as marine Crenarchaeota Group I was approximately 5% in subsurface waters (100 m depth) and between 10% and 20% in the oxygen minimum layer (250-500 m) and deep waters [North East Atlantic Deep Water (NEADW) and Lower Deep Water (LDW), 2750-4800 m depth]. Single-cell activity, determined via a quantitative MICRO-CARD-FISH approach and taking only substrate-positive cells into account, ranged from 0.05 to 0.5 amol D-aspartic acid (Asp) cell(-1) day(-1) and 0.1-2 amol L-Asp cell(-1) day(-1), slightly decreasing with depth. In contrast, the D-Asp:L-Asp cell-specific uptake ratio increased with depth. By combining data reported previously using the same method as applied here and data reported here, we found a decreasing relative abundance of marine Crenarchaeota Group I throughout the meso- and bathypelagic water column from 65 degrees N to 5 degrees N in the eastern basin of the North Atlantic. Thus, the relative contribution of marine Crenarchaeota Group I to deep-water prokaryotic communities might be more variable than previous studies have suggested. This apparent variability in the contribution of marine Crenarchaeota Group I to total picoplankton abundance might be related to successions and ageing of deep-water masses in the large-scale meridional ocean circulation and possibly, the appearance of crenarchaeotal clusters other than the marine Crenarchaeota Group I in the (sub)tropical North Atlantic.

D-aspartate modulates transcriptional activity in Harderian gland of frog, Rana esculenta: Morphological and molecular evidence.

In the green frog, Rana esculenta, a substantial amount of D-aspartate (D-Asp) is found endogenously within the Harderian gland (HG) following its synthesis from L-aspartate (L-Asp) by an aspartate racemase. The frog HG is an orbital seromucoid gland that displays seasonal changes in secretory activity. Our in vivo experiments, consisting of i.p. injection of 2.0 mumol/g b.w. D-Asp in frogs collected during two periods of differing glandular activity (high or medium-low secretory activity), revealed that HG can to take up and accumulate D-Asp and that this amino acid may modulate the exocrine secretion through a kinase pathway. At a time when the gland shows relatively low secretory activity, i.p. administration of D-Asp rapidly induced activation of ERK1 and an increase in cells active in RNA synthesis. This increase in transcriptional activity was followed by a significant increase in mucous secretion. By contrast, administration of exogenous D-Asp when HG was showing high activity rapidly induced inhibition of both ERK1 and transcriptional activity. Since D-Asp is known to be recognized by receptors for N-methyl-D-aspartic acid (NMDA), it is possible that in the HG, D-Asp mediated NMDA activation may enhance the kinase pathway. The above activation of opposing stimulatory and inhibitory processes could reflect different levels of NMDA-receptor activity, which could vary as a function of the level of gland activity. This study provides the first evidence of a role for this excitatory amino acid in exocrine secretion. The effects of D-Asp in HG appear to be specific since they were not seen in frogs treated with other D- or L-amino acids with known excitatory effects on neurosecretion.

Endocrine roles of D-aspartic acid in the testis of lizard Podarcis s. sicula.

In the lizard Podarcis s. sicula, a substantial amount of D-aspartate (D-Asp) is endogenous to the testis and shows cyclic changes of activity connected with sex hormone profiles during the annual reproductive phases. Testicular D-Asp content shows a direct correlation with testosterone titres and a reverse correlation with 17beta-estradiol titres. In vivo experiments, consisting of i.p. injections of 2.0 micromol/g body weight of D-Asp or other amino acids, in lizards collected during the three main phases of the reproductive cycle (pre-reproductive, reproductive and post-reproductive period), revealed that the testis can specifically take up and accumulate D-Asp alone. Moreover, this amino acid influences the synthesis of testosterone and 17beta-estradiol in all phases of the cycle. This phenomenon is particularly evident during the pre- and post-reproductive period, when endogenous testosterone levels observed in both testis and plasma were the lowest and 17beta-estradiol concentrations were the highest. D-Asp rapidly induces a fall in 17beta-estradiol and a rise in testosterone at 3 h post-injection in the testis and at 6 h post-injection in the blood. In vitro experiments show that testicular tissue converted L-Asp into D-Asp through an aspartate racemase. D-Asp synthesis was measured in all phases of the cycle, but was significantly higher during the reproductive period with a peak at pH 6.0. The exogenous D-Asp also induces a significant increase in the mitotic activity of the testis at 3 h (P < 0.05) and at 6 h (P < 0.01). Induction of spermatogenesis by D-Asp is recognized by an intense immunoreactivity of the germinal epithelium (spermatogonia and spermatids) for proliferation cell nuclear antigen (PCNA). The effects of D-Asp on the testis appear to be specific since they were not seen in lizards injected with other D- or L-forms of amino acids with known excitatory effects on neurosecretion. Our results suggest a regulatory role for D-Asp in the steroido-genesis and spermatogenesis of the testis of the lizard Podarcis s. sicula.