D-amino Acid Oxidase Activity Research Articles

Insights into the role of d-amino acid oxidase mutations in amyotrophic lateral sclerosis.

Missense mutations in the coding region of d-amino acid oxidase (DAO) have been found in patients suffering from amyotrophic lateral sclerosis (ALS). Mutations primarily impair the enzymatic activity of DAO and cause neurodegeneration due to an abnormal accumulation of d-serine in the spinal cord. However, the structural and dynamic changes that lead to impaired enzymatic activity are not fully understood. We present here extensive molecular dynamics simulations of wild-type, and all reported ALS-associated DAO mutants to elucidate the plausible mechanisms of impaired enzymatic activity, a critical function needed for neuroprotection. Simulation results show that DAO mutations disrupt several key interactions with the active site residues and decrease the conformational flexibility of active site loop comprising 216 to 228 residues, necessary for substrate binding and product release. This conformational restriction of the active site loop in the mutants is mainly due to the distortion of critical salt bridge and hydrogen bond interactions compared with wild-type. Furthermore, binding free energy calculations show that DAO mutants have a lower binding affinity toward cofactor flavin adenine dinucleotide and substrate imino-serine than the wild-type. A closer look at the cofactor and substrate interaction profiles further show that DAO mutants have lost several critical interactions with the neighboring residues as compared with wild-type. Taken together, this study provides first-hand explanation of crucial structural features that lead to the loss of enzymatic function in DAO mutants and highlights the need of further genomic scans of patients withALS to map the association of novel DAO variants in ALS pathophysiology.

Mitochondrial H2O2 Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death.

Among reactive oxygen species (ROS), H2O2 alone acts as a signaling molecule that promotes diverse phenotypes depending on the intracellular concentration. Mitochondria have been suggested as both sources and sinks of cellular H2O2, and mitochondrial dysfunction has been implicated in diseases such as cancer. A genetically encoded H2O2 generator, d-amino acid oxidase (DAAO), was targeted to the mitochondria of human cells, and its utility in investigating cellular response to a range of H2O2 doses over time was assessed. Organelle-specific peroxiredoxin dimerization and protein S-glutathionylation were measured as indicators of increased H2O2 flux due to the activity of DAAO. Cell death was observed in a concentration- and time-dependent manner, and protein oxidation shifted in localization as the dose increased. This work presents the first systematic study of H2O2-specific perturbation of mitochondria in human cells, and it reveals a marked sensitivity of this organelle to increases in H2O2 in comparison with prior studies that targeted the cytosol.

Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access.

D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.

Enzyme assay for d-amino acid oxidase using optically gated capillary electrophoresis-laser induced fluorescence detection

Because d-amino acids (AAs) play an essential role in the regulation of many processes in living cells, detection of D-AAs and assay of d-amino acid oxidase (DAAO) activity are of vital importance in bioanalytical science. However, the reliability and accuracy of DAAO assays could be interfered due to the facts that DAAO presents broad substrate activity towards different D-AAs and there could be abundant L-AA enantiomers in biological samples. In this study we presented the first application of optically gated capillary electrophoresis with LIF detection (OGCE-LIF) for efficient assay of DAAO activity. High repeatability of the OGCE-LIF assay of amino acids (AAs) was achieved with relative standard deviation (RSD) (n = 15) less than 1.5% and 2.7% for migration time and peak height, respectively. Under the optimal OGCE-LIF conditions, five pairs of D/L-AA enantiomers were efficiently separated in less than 1 min with low limit of detection of 1.3 μM. Enzymatic assays of DAAO were successfully achieved by detection the substrate consumption with OGCE-LIF, for either single or mixed AA substrates. Kinetic analysis of the parallel oxidation reactions of two different substrates was performed, which was in good agreement with the experimental results. Our study indicates OGCE-LIF can perform rapid and efficient separation of mixed pairs of AA enantiomers and is a promising method for quantitatively assaying DAAO catalyzed reaction with the presence of L-AA enantiomers in the sample. Our study would pave the way for accurate determination of D-AAs and DAAO enzymes in complicated biological samples.

Variations of L- and D-amino acid levels in the brain of wild-type and mutant mice lacking D-amino acid oxidase activity.

D-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some D-amino acids are metabolized by D-amino acid oxidase (DAO), while D-Asp and D-Glu are metabolized by D-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO+/+), mutant mice lacking DAO activity (ddY/DAO-/-), and the heterozygous mice (ddY/DAO+/-) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for L-amino acid levels among the three strains except for L-Trp which was markedly elevated in the DAO+/- and DAO-/- mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of L-amino acids were L-Glu, L-Asp, and L-Gln in all the three strains. The lowest L-amino acid level was L-Cys in ddY/DAO+/- and ddY/DAO-/- mice, while L-Trp showed the lowest level in ddY/DAO+/+mice. The highest concentration of D-amino acid was found to be D-Ser, which also had the highest % D value (~ 25%). D-Glu had the lowest % D value (~ 0.01%) in all the three strains. Significant differences of D-Leu, D-Ala, D-Ser, D-Arg, and D-Ile were observed in ddY/DAO+/- and ddY/DAO-/- mice compared to ddY/DAO+/+ mice. This work provides the most complete baseline analysis of L- and D-amino acids in the brains of ddY/DAO+/+, ddY/DAO+/-, and ddY/DAO-/- mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.

Assays of D-Amino Acid Oxidase Activity.

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

Selenoprotein W as a molecular target of d-amino acid oxidase is regulated by d-amino acid in chicken neurons.

Selenoprotein W (SelW) is an important member of the avian selenoprotein family. It is well known for its important role in protecting neurons from oxidative stress during neuronal development. d-Amino acid (d-serine), as a neurotransmitter in the central nervous system (CNS), can mediate neurotoxicity. d-Amino acid oxidase (DAAO) is responsible for regulating the d-serine levels in cells. However, the correlation between SelW and DAAO is not clear yet. To investigate the regulations between SelW and DAAO, chicken embryo monolayer neurons were treated with d-serine and/or Se. In this study, we predicted molecular binding between SelW and DAAO. These results showed that the 9-16, 18, 41-47 and 66 residues of SelW could combine with the DAAO, which suggested that chicken SelW might be the target of DAAO. We determined the DAAO activity and the mRNA expression of SelW in in vitro cultured chicken embryo primitive neuron cells. d-Serine influenced the activity of DAAO and, moreover, a significant increase in the mRNA expression of SelW was found in neurons treated with Se. Notably, we also observed changes in the expression of SelW and DAAO when neurons were treated with various concentrations of d-serine and Se. In conclusion, these data suggest that d-serine could regulate the mRNA expression of SelW by interfering with the activity of DAAO in chicken embryo neurons.

Optimization of D-Amino Acid Oxidase Production by Trigonopsis variabilis Using Glucose Syrup from Cassava as Carbon Source

Rusli Z, Suryadi H, Wibisana A. 2018. Optimization of D-amino acid oxidase production by Trigonopsis variabilis using glucose syrup from cassava as carbon source. Bioteknologi 15: xxxx. Glucose syrup from cassava (GSC) contains glucose as the main sugar suitable for an economical and efficient production of D-amino acid oxidase (DAAO) by Trigonopsis variabilis. The aim of this study is to optimize the DAAO production by T. variabilis using GSC as the carbon source via response surface methodology (RSM) with Face-centered Central Composite Design (FCCD). In the first step, the effects of six factors (concentration of GSC and DL-alanine, pH, temperature, incubation time, and inoculum concentration) were screened to obtain the significant factors on DAAO production by Plackett-Burman design. As a result, the incubation time, the concentration of GSC and DL-alanine showed to be significant factors for DAAO production (P<0.05). These factors were then optimized using RSM with FCCD. The optimized RSM results demonstrated that a quadratic polynomial model was found suitable to define the relation between the incubation times, concentration of GSC and DL-alanine parameters. Moreover, the observed high R2 value (0.9782) confirms a strong evaluation of the experimental data. The optimum condition for DAAO production is as follows: GSC and DL-alanine concentration was 12.3% and 0.3%, respectively, and the culture incubation time was 56.1 hours. Using this condition, we got a DAAO activity of 195.38 U/g dry weight of yeast.

Human d-amino acid oxidase: The inactive G183R variant

In the brain, the enzyme d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-serine, a main positive modulator of the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR). Dysregulation in d-serine signaling is implicated in the NMDAR dysfunctions observed in various brain diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia. A strain of ddY mice lacking DAAO activity due to the G181R substitution (DAAOG181R mice) and exhibiting increased d-serine concentration as compared to wild-type mice shows altered pain response, improved adaptative learning and cognitive functions, and larger hippocampal long-term potentiation. In past years, this mice line has been used to shed light on physiological and pathological brain functions related to NMDAR. Here, we decided to introduce the corresponding substitution in human DAAO (hDAAO). The recombinant G183R hDAAO is produced as an inactive apoprotein: the substitution alters the protein conformation that negatively affects the ability to bind the flavin cofactor in the orientation required for hydride-transfer during catalysis. At the cellular level, the overexpressed G183R hDAAO is not fully targeted to peroxisomes, forms protein aggregates showing a strong colocalization with ubiquitin, and significantly (7-fold) increases both the d-serine cellular concentration and the D/(D+L)-serine ratio. Taken together, our investigation warrants caution in using DAAOG181R mice: the abolition of enzymatic activity is coupled to DAAO aggregation, a central process in different pathological conditions. The effect due to G181R substitution in DAAO could be misleading: the effects due to impairment of d-serine degradation overlap with those related to aggregates accumulation.

Prediction Analysis for Transition to Schizophrenia in Individuals at Clinical High Risk for Psychosis: The Relationship of DAO, DAOA, and NRG1 Variants with Negative Symptoms and Cognitive Deficits.

Schizophrenia is characterized by positive and negative symptoms and cognitive dysfunction. The glutamate hypothesis of schizophrenia has been hypothesized to explain the negative symptoms and cognitive deficits better than the dopamine hypothesis alone. Therefore, we aimed to evaluate whether glutamatergic variants such as d-amino acid oxidase (DAO), DAO activator (DAOA)/G72, and neuregulin 1 (NRG1) single-nucleotide polymorphisms (SNPs) and their mRNA levels predicted (i) transition to schizophrenia spectrum disorders and (ii) research domain criteria (RDoC) domains, mainly negative valence and cognitive systems. In a 3-year prospective study cohort of 185 individuals (age: 13–35 years) at high risk and ultra-high risk (UHR) for psychosis, we assessed DAO (rs3918347, rs4623951), DAOA (rs778293, rs3916971, rs746187), and NRG1 (rs10503929) SNPs and their mRNA expression. Furthermore, we investigated their association with RDoC domains, mainly negative valence (e.g., anxiety, hopelessness) and cognitive (e.g., perception disturbances, disorganized symptoms) systems. NRG1 rs10503929 CC + CT versus TT genotype carriers experienced significantly more disorganized symptoms. DAOA rs746187 CC versus CT + TT genotype, DAOA rs3916971 TT versus TC + CC genotype, and DAO rs3918347 GA + AA versus GG genotype carriers experienced nominally more hopelessness, visual perception disturbances, and auditory perception disturbances, respectively. The schizophrenia risk G-allele of DAO rs3918347 nominally increased risk for those UHR individuals with attenuated positive symptoms syndrome. No association between DAO, DAOA, NRG1 SNPs, and conversion to schizophrenia spectrum disorders was observed. Our findings suggest that DAO, DAOA, and NRG1 polymorphisms might influence both RDoC negative valence and cognitive systems, but not transition to schizophrenia spectrum disorders.

Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates.

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties.

The interaction of NOS1AP, DISC1, DAOA, and GSK3B confers susceptibility of early-onset schizophrenia in Chinese Han population

Although many major breakthrough had identificated potential susceptibility genes for schizophrenia, the aetiology of schizophrenia is still unknown. In the present study, we focused on the N-methyl-Daspartate receptors related genes nitric oxide synthase 1 adaptor gene (NOS1AP), disrupted in schizophrenia 1 gene (DISC1), d-amino acid oxidase activator gene (DAOA), and glycogen synthase kinase 3-beta gene (GSK3B). A family-based genetic association study (459 Han Chinese subjects in 153 nuclear families) using 3 single nucleotide polymorphisms in NOS1AP, 2 in DISC1, 1 in DAOA and 1 in GSK3B was conducted. We found rs12742393 have just positive trend with schizophrenia (SCZ) (p=0.07) after FDR correction. NOS1AP mRNA and serum levels were significantly elevated in SCZ patients (p<0.001; p<0.001) compared with healthy control. However, expression Quantitative Trait Loci (eQTL) analysis have demonstrated that rs12742393 genotype were not significantly associated with the NOS1AP mRNA expression. GMDR identified a significant seven-locus interaction model involving (NOS1AP-rs348624, rs12742393, rs1415263, DISC1-rs821633, rs1000731, DAOA-rs2391191and GSK3B- rs6438552) with a good testing accuracy (0.72). Our finding suggested statistically significant role of interaction of NOS1AP, DISC1, DAOA, and GSK3B polymorphisms (NOS1AP-rs348624, rs12742393, rs1415263, DISC1-rs821633, rs1000731, DAOA-rs2391191and GSK3B-rs6438552) in EOS susceptibility.

Controversial Effects of D-Amino Acid Oxidase Activator (DAOA)/G72 on D-Amino Acid Oxidase (DAO) Activity in Human Neuronal, Astrocyte and Kidney Cell Lines: The N-methyl D-aspartate (NMDA) Receptor Hypofunction Point of View.

Dysfunction of D-amino acid oxidase (DAO) and DAO activator (DAOA)/G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA) receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y), astrocyte-like (1321N1) and kidney-like (HEK293) human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO) showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD)] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD), which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.

Haplotype-based association analysis of general cognitive ability in Generation Scotland, the English Longitudinal Study of Ageing, and UK Biobank

Background: Cognitive ability is a heritable trait with a polygenic architecture, for which several associated variants have been identified using genotype-based and candidate gene approaches. Haplotype-based analyses are a complementary technique that take phased genotype data into account, and potentially provide greater statistical power to detect lower frequency variants. Methods: In the present analysis, three cohort studies (n total = 48,002) were utilised: Generation Scotland: Scottish Family Health Study (GS:SFHS), the English Longitudinal Study of Ageing (ELSA), and the UK Biobank. A genome-wide haplotype-based meta-analysis of cognitive ability was performed, as well as a targeted meta-analysis of several gene coding regions. Results: None of the assessed haplotypes provided evidence of a statistically significant association with cognitive ability in either the individual cohorts or the meta-analysis. Within the meta-analysis, the haplotype with the lowest observed P-value overlapped with the D-amino acid oxidase activator ( DAOA) gene coding region. This coding region has previously been associated with bipolar disorder, schizophrenia and Alzheimer’s disease, which have all been shown to impact upon cognitive ability. Another potentially interesting region highlighted within the current genome-wide association analysis (GS:SFHS: P = 4.09 x 10 -7), was the butyrylcholinesterase ( BCHE) gene coding region. The protein encoded by BCHE has been shown to influence the progression of Alzheimer’s disease and its role in cognitive ability merits further investigation. Conclusions: Although no evidence was found for any haplotypes with a statistically significant association with cognitive ability, our results did provide further evidence that the genetic variants contributing to the variance of cognitive ability are likely to be of small effect.

A systematic meta-analysis of the association of Neuregulin 1 (NRG1), D-amino acid oxidase (DAO), and DAO activator (DAOA)/G72 polymorphisms with schizophrenia.

The glutamate hypothesis of schizophrenia is related to the proposed dysregulation of D-amino acid oxidase (DAO), DAO activator (DAOA)/G72, and Neuregulin 1 (NRG1) genes. Genetic studies have shown significant associations between DAO, DAOA, NRG1 single-nucleotide polymorphisms (SNPs), and schizophrenia. The systematic literature search yielded 6, 5, and 18 new studies for DAO, DAOA, and NRG1 published after 2011 and not included in the previous SchizophreniaGene (SZGene) meta-analysis. We conducted meta-analyses of 20, 23, and 48 case-control studies, respectively, to comprehensively evaluate the association of 8 DAO, 12 DAOA, and 14 NRG1 SNPs with schizophrenia. The updated meta-analyses resulted in the following findings: the C-allele of DAO rs4623951 was associated with schizophrenia across all pooled studies [Odds ratio (OR)=0.88, 95% confidence interval (CI)=0.79-0.98, p=0.02, N=3143]; however, no new reports could be included. The G-allele of DAOA rs778293 was associated with schizophrenia in Asian patients (OR=1.17, 95% CI=1.08-1.27, p=0.00008, N=6117), and the T-allele of DAOA rs3916971 was associated with schizophrenia across all studies (OR=0.84, 95% CI=0.73-0.96, p=0.01, N=1765). Again, for both SNPs, no new eligible studies were available. After adding new reports, the T-allele of NRG1 SNP8NRG241930 (rs62510682) across all studies (OR=0.95, 95% CI=0.91-0.997, p=0.04, N=22,898) and in Caucasian samples (OR=0.95, 95% CI=0.90-0.99, p=0.03, N=16,014), and the C-allele of NRG1 rs10503929 across all studies (OR=0.89, 95% CI=0.81-0.97, p=0.01, N=6844) and in Caucasian samples (OR=0.89, 95% CI=0.81-0.98, p=0.01, N=6414) were protective against schizophrenia. Our systematic meta-analysis is the most updated one for the association of DAO, DAOA, and NRG1 SNPs with schizophrenia.

Haplotype-based association analysis of general cognitive ability in Generation Scotland, the English Longitudinal Study of Ageing, and UK Biobank.

Cognitive ability is a heritable trait with a polygenic architecture, for which several associated variants have been identified using genotype-based and candidate gene approaches. Haplotype-based analyses are a complementary technique that take phased genotype data into account, and potentially provide greater statistical power to detect lower frequency variants. In the present analysis, three cohort studies (n total = 48,002) were utilised: Generation Scotland: Scottish Family Health Study (GS:SFHS), the English Longitudinal Study of Ageing (ELSA), and the UK Biobank. A genome-wide haplotype-based meta-analysis of cognitive ability was performed, as well as a targeted meta-analysis of several gene coding regions. None of the assessed haplotypes provided evidence of a statistically significant association with cognitive ability in either the individual cohorts or the meta-analysis. Within the meta-analysis, the haplotype with the lowest observed P-value overlapped with the D-amino acid oxidase activator ( DAOA) gene coding region. This coding region has previously been associated with bipolar disorder, schizophrenia and Alzheimer's disease, which have all been shown to impact upon cognitive ability. Another potentially interesting region highlighted within the current genome-wide association analysis (GS:SFHS: P = 4.09 x 10 -7), was the butyrylcholinesterase ( BCHE) gene coding region. The protein encoded by BCHE has been shown to influence the progression of Alzheimer's disease and its role in cognitive ability merits further investigation. Although no evidence was found for any haplotypes with a statistically significant association with cognitive ability, our results did provide further evidence that the genetic variants contributing to the variance of cognitive ability are likely to be of small effect.

Markers of enteral adaptation in pediatric short bowel syndrome.

The aim of this study was to ascertain if prospective determination of specific gut hormones and growth factors could predict bowel adaptation in children with short bowel syndrome (SBS). We studied independence from parenteral nutrition (PN) as the short-term result and discontinuation of enteral nutrition (EN) as the long-term result from a retrospective chart review of seven patients with SBS, who were managed in the absence of growth retardation. The correlation between increased number of enteral feeds or enteral nutrients and fasting serum gastrin, glucagon-like peptide 2 (GLP-2), citrulline, and D-amino acid oxidase (DAO) activity was analyzed. Five patients were weaned from PN, and two from EN. Fasting serum gastrin was significantly higher and serum GLP-2 lower in the PN-dependent patients than in the patients weaned from EN. The upper limit of fasting serum gastrin for PN independence and for EN independence was 300 and 200 pg/mL, respectively. The lower limit of fasting serum citrulline for PN independence was 15 μmol/L. The relationship between serum citrulline and DAO and the course of bowel adaptation, however, was poor. Serum citrulline is a predictor of PN independence in children with SBS. Fasting serum gastrin and GLP-2 are indicators for adaptation of the residual intestine, but this was a small study and further larger prospective trials are required to confirm these results.

Efficient production of d-amino acid oxidase in Escherichia coli by a trade-off between its expression and biomass using N-terminal modification

Native d-amino acid oxidase (DAAO) that is expressed mostly as inclusion body and its toxicity for E. coli hamper efficient heterologous expression. In this study, the soluble expression of DAAO from Rhodosporidium toruloides (RtDAAO) was improved in E. coli through N-terminal modification, but the cell biomass was decreased. Then a trade-off between DAAO expression and biomass was achieved to obtain the highest volumetric activity of DAAO through regulated the number of N-terminus histidine residues. When variant 2H3G was fused with three N-terminus histidine residues, the volumetric activity was increased by 3.1 times and the biomass was not significant change compared with the wild type. Finally, the N-terminus disordered region of RtDAAO (HSQK) was replaced with HHHG and the variant enzyme activity reached 80.7U/mL (with a 40 percent of inactive DAAO reduced) in a 7.5L fermenter in 24h.

Expression of D-Amino Acid Oxidase (DAO/DAAO) and D-Amino Acid Oxidase Activator (DAOA/G72) during Development and Aging in the Human Post-mortem Brain.

In the brain, D-amino acid oxidase (DAO/DAAO) mainly oxidizes D-serine, a co-agonist of the N-methyl-D-aspartate (NMDA) receptors. Thus, DAO can regulate the function of NMDA receptors via D-serine breakdown. Furthermore, DAO activator (DAOA)/G72 has been reported as both DAOA and repressor. The co-expression of DAO and DAOA genes and proteins in the human brain is not yet elucidated. The aim of this study was to understand the regional and age span distribution of DAO and DAOA (mRNA and protein) in a concomitant manner. We determined DAO and DAOA mRNA and protein expression across six brain regions in normal human post-mortem brain samples (16 weeks of gestation to 91 years) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found higher expression of DAO mRNA in the cerebellum, whereas lower expression of DAO protein in the cerebellum compared to the other brain regions studied, which suggests post-transcriptional regulation. We detected DAOA protein but not DAOA mRNA in all brain regions studied, suggesting a tightly regulated expression. To understand this regulation at the transcriptional level, we analyzed DNA methylation levels at DAO and DAOA CpG sites in the cerebellum and frontal cortex of control human post-mortem brain obtained from Gene Expression Omnibus datasets. Indeed, DAO and DAOA CpG sites in the cerebellum were significantly more methylated than those in the frontal cortex. While investigating lifespan effects, we found that DAO mRNA levels were positively correlated with age <2 years in the cerebellum and amygdala. We also detected a significant positive correlation (controlled for age) between DAO and DAOA protein in all of the brain regions studied except for the frontal cortex. In summary, DAO and DAOA expression in the human brain are both age and brain region dependent.

The DAOA gene is associated with schizophrenia in the Taiwanese population

The gene D-amino acid oxidase activator (DAOA), which has a former name of G72, and the D-amino acid oxidase (DAO) gene have been suggested as candidate genes of schizophrenia. However, association studies have so far yielded equivocal results. We analyzed one single nucleotide polymorphism (SNP) for DAO (rs3741775) and seven SNPs for G72 (rs3916956, rs2391191, rs9558562, rs947267, rs778292, rs3918342, and rs1421292) in this study enrolling 248 schizophrenia cases and 267 controls in the Taiwanese samples. In SNP-based single locus association analyses, the rs778292 in the DAOA gene showed significant association with schizophrenia. The rs3741775 in the DAO gene could not withstand statistically significant after multiple corrections. Additionally, a three-SNP haplotype (rs778292-rs3918342-rs1421292) in the DAOA gene were observed to be significantly associated with schizophrenia. Among them, the TCT haplotype presented higher prevalence in controls than in cases whereas the TTT and CTT haplotype were significantly more frequent in cases than in controls. The study also provides significant evidence for epistatic interactions among DAOA and DAO gene in the development of schizophrenia. These results provide additional evidence and indicate that the DAOA gene and DAOA-DAO gene-gene interactions might play a role for schizophrenia in a Taiwanese sample.