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PubMed1996

Serine isotopmer analysis by 13C-NMR defines glycine-serine interconversion in situ in the renal proximal tubule.

[2-(13)C]glycine metabolism was studied in freshly isolated rat renal proximal tubules. Mitochondrial coupling of the glycine cleavage complex (GC) and serine hydroxymethyltransferase (SHMT) was confirmed by the formation of three serine isotopomers, [2-(13)C]-, [3-(13)C]- and [2,3-(13)C]serine, detected by 13C-NMR. Incubation with different fractions of 13C-labelled glycine altered the labelling pattern of the serine isotopomers predictably and allowed calculation of the 13C-labelled fractions of total glycine and methylene in N5,N10-methylenetetrahydrofolate (m-THF) available for serine metabolism. Within 20 min there was a fall in labelled glycine (to 42 +/- 3, 68 +/- 3 and 93 +/- 2%, (n = 4, mean +/- S.D.) from 50%, 75% and 100% 13C-labelled added glycine respectively), followed by a slow rate of endogenous glycine formation for up to 80 min incubation. The C2 of glycine was the source of more than 90% of the methylene group of m-THF formed. Gas chromatography-mass spectroscopy (GC-MS) showed that greater than 50% of serine formed was unlabelled. GC and SHMT proceeded in the direction of serine formation. Serine isotopomer analysis by NMR and GC-MS allowed the actions of GC and SHMT and de novo contributions to glycine, serine and m-THF to be monitored in situ in fresh renal proximal tubules.

Animals↗
PubMed2004

Effect of systemic administration of D-serine on the levels of D- and L-serine in several brain areas and periphery of rat.

To obtain further insight into the distribution and metabolism of exogenous D-serine, we have investigated the effect of the intraperitoneal administration of D-serine (10 mmol/kg) on the concentrations of D- and L-serine in several brain areas and periphery of infant and adult rats. The administration produced a significant augmentation of the D-serine levels not only in the cortex but also in the hippocampus, striatum, cerebellum and periphery. The rapid decline in the enhanced D-serine levels was observed in the periphery and cerebellum, whereas the injection caused a prolonged elevation of the D-serine levels in the cortex and hippocampus. The application caused a slight increase in the L-serine levels in several brain areas and periphery 3 or 6 h after the injection, whereas a significant decrease in the L-serine concentration was observed in the periphery, diencephalon and cerebellum 3 or 7 days after the injection. Because a structural abnormality and N-methyl-D-aspartate (NMDA) receptor hypofunction has been demonstrated in the cortex and hippocampus of schizophrenic subjects, D-serine treatment may offer a new therapeutic approach to diseases related to the hypofunction of NMDA receptors such as schizophrenia.

Adrenal Glands↗
PubMed2005

Reduced D-serine to total serine ratio in the cerebrospinal fluid of drug naive schizophrenic patients.

Several lines of evidence suggest that D-serine, an endogenous agonist of the glycine site on the NMDA receptors, might play a role in the pathophysiology of schizophrenia. The purpose of this study was to determine whether levels of D- and L-serine or D-serine ratio (D-serine/total serine) in cerebrospinal fluid (CSF) were altered in first episode and drug-naive schizophrenic patients. The CSF levels of D- and L-serine in 25 male first episode and drug-naive schizophrenic patients and 17 age-matched male healthy subjects were measured using a column-switching high performance liquid chromatography system. The percentage of D-serine in the total serine of patients was significantly (z = -2.01, p = 0.044) lower than that of controls. This study suggests that synthetic or metabolic pathways of D-serine may be abnormal in the brain of drug-naive schizophrenic patients, supporting the NMDA receptor dysfunction hypothesis of schizophrenia.

Adolescent↗
PubMed2004

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.

Aldehydes↗
PubMed1998

Occurrence of an unusual phospholipid, phosphatidyl-L-threonine, in cultured hippocampal neurons. Exogenous L-serine is required for the synthesis of neuronal phosphatidyl-L-serine and sphingolipids.

We have recently reported that L-serine released from astroglial cells supports the survival and neuritogenesis of hippocampal neurons under a serum- and glia-free culture condition (Mitoma, J., Furuya, S., and Hirabayashi, Y. (1998) Neurosci. Res. 30, 195-199). In this study, we show that exogenous L-serine is required for the synthesis of phosphatidyl-L-serine (PS) and sphingolipids in hippocampal neurons. When hippocampal neurons were maintained under an astroglial cell-free condition, the levels of sphingolipids and phosphatidyl-L-serine in the neurons were greatly reduced in the absence of external L-serine or glycine. Instead, a novel phospholipid appeared just ahead of PS on TLC. This novel lipid was determined to be phosphatidyl-L-threonine by TLC blotting/negative secondary ion mass spectrometry and amino acid analysis. Biochemical studies on rat brain microsomes have indicated that phosphatidyl-L-threonine is synthesized by the base exchange enzyme that is involved in PS synthesis with much lower affinity, that is, approximately (1)/(150) of L-serine. Addition of L-serine or glycine to the culture medium restored the synthesis of PS and sphingolipids in the neurons. These observations show that hippocampal neurons require exogenous L-serine for the synthesis of PS and sphingolipids in the absence of astroglial cells and suggested that astroglial cells contribute to neuronal lipid synthesis through the supply of L-serine.

Animals↗
PubMed1974

Evidence for the involvement of serine transhydroxymethylase in serine and glycine interconversions in Salmonella typhimurium.

Salmonella typhimurium can normally use glycine as a serine source to support the growth of serine auxotrophs. This reaction was presumed to occur by the reversible activity of the enzyme, serine transhydroxymethylase (E. C. 2. 1. 2. 1; L-serine: tetrahydrofolic-5, 10 transhydroxymethylase), which is responsible for glycine biosynthesis. However, this enzyme had not been demonstrated to be solely capable of synthesizing serine from glycine in vivo. The isolation and characterization of a mutant able to convert serine to glycine but unable to convert glycine to serine supports the conclusion that a single enzyme is involved in this reversible interconversion of serine and glycine. The mutation conferring this phenotype was mapped with other mutations affecting serine transhydroxymethylase (glyA) and assays demonstrated reduced activities of this enzyme in the mutant.

Chromosome Mapping↗
PubMed1997

Isolation and characterization of an Escherichia coli mutant lacking the major serine transporter, and cloning of a serine transporter gene.

L-Serine as well as L-valine inhibits the growth of Escherichia coli cells, and L-isoleucine releases this growth inhibition. We isolated an E. coli mutant (designated as WAT9) that was able to grow on lactate (or glucose) as a carbon source even in the presence of L-serine, the parent not being able to. Cells of WAT9 were not able to grow on L-serine as a carbon source even if L-isoleucine was present in the culture medium, while the parental cells grew. This mutant was shown to lack the principal L-serine transporter in E. coli, the Na+/ serine symporter. This mutant is useful for analysis of the role(s) of the Na+/serine symporter in cell physiology and as a host for the cloning of L-serine transporter gene(s). In fact, we cloned a gene encoding a serine transporter from chromosomal DNA of E. coli using WAT9 as the host. The gene enabled the mutant cells to grow on L-serine. Transport activity for L-serine was restored in the mutant cells harboring a plasmid carrying the gene. We partially sequenced the gene and found that it was the tdcC gene. We showed that TdcC is an H+/serine symporter.

Amino Acid Transport Systems↗
PubMed1991

Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine.

Factor VII is a multidomain, vitamin K-dependent plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. Earlier studies demonstrated a novel disaccharide (Xyl-Glc) or trisaccharide (Xyl2-Glc) O-glycosidically linked to serine 52 in human plasma factor VII (Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Shimonishi, Y., and Iwanaga, S. (1989) J. Biol. Chem. 264, 20320-20325). In the present study, human plasma and recombinant factor VII were isolated and subjected to enzymatic fragmentation. Peptides comprising residues 48-62 of the first epidermal growth factor-like domain of each factor VII preparation were isolated for comparative analysis. Using a combined strategy of amino acid sequencing, carbohydrate and amino acid composition analysis, and mass spectrometry, three different glycan structures consisting of either glucose, glucose-xylose, or glucose-(xylose)2 were detected O-glycosidically linked to serine 52 in plasma and recombinant factor VII. Approximately equal amounts of the three glycan structures were observed in plasma factor VII, whereas in recombinant factor VII the glucose and the glucose-(xylose)2 structures predominated. In addition to the O-linked glycan structures observed at serine 52, a single fucose was found to be covalently linked at serine 60 in both human plasma and recombinant factor VII. Carbohydrate and mass spectrometry analyses indicated that the fucosylation of serine 60 was virtually quantitative. Metabolic labeling studies using [14C]fucose confirmed the presence of O-linked fucose at serine 60. In order to assess whether the carbohydrate moiety at serine 52 contributes to the biological activity of factor VII, we have constructed a site-specific mutant of recombinant factor VII in which serine 52 has been replaced with an alanine residue. Mutant factor VIIa exhibited approximately 60% of the coagulant activity of wild-type factor VIIa in a clotting assay. The amidolytic activity of mutant factor VIIa was indistinguishable from that observed for recombinant wild-type factor VIIa. In addition, the ability of mutant factor VIIa in complex with either purified relipidated tissue factor apoprotein or tissue factor on the surface of a human bladder carcinoma cell line (J82) to activate either factor X or factor IX was virtually identical to that observed for wild-type factor VIIa. These results indicate that the carbohydrate moiety O-glycosidically linked to serine 52 does not appear to be involved either in the interaction of factor VIIa with tissue factor, or the expression of its proteolytic activity toward factor X or factor IX following complex formation with tissue factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2005

Regulation of serine racemase activity by D-serine and nitric oxide in human glioblastoma cells.

In the human glioblastoma cell line U87, the activity of serine racemase (SR), catalyzing the isomerisation of serine, was inversely regulated by D-serine and nitric oxide (NO), a neuromodulator and a neurotransmitter, respectively. SR activity was dose-dependently enhanced up to five times in cells treated with 10 mM D-serine, whereas it was inhibited by NO. Furthermore, D-serine was found to induce the denitrosylation of SR purified from mouse brain. These results suggest that serine racemase activity in astrocyte is regulated inversely by d-serine and NO. SR should be inhibited through nitrosylation by NO and activated through denitrosylation elicited by D-serine.

Analysis of Variance↗
PubMed2003

Mouse brain serine racemase catalyzes specific elimination of L-serine to pyruvate.

D-Serine was previously identified in mammalian brain and was shown to be a co-agonist at the 'glycine' site of the N-methyl-D-aspartate (NMDA)-type receptors. Racemization of serine is catalyzed by serine racemase, a pyridoxal 5'-phosphate-dependent enzyme expressed mainly in brain and liver. NMDA receptor overactivation has been implicated in a number of pathological conditions and inhibitors of serine racemase are thus potentially interesting targets for therapy. We expressed recombinant mouse serine racemase in insect cells and purified it to near homogeneity. The enzyme is a non-covalent homodimer in solution and requires divalent cations Mg(2+), Ca(2+) or Mn(2+) for activity but not for dimerization. In addition to the racemization it also catalyzes specific elimination of L-Ser to pyruvate. D-Serine is eliminated much less efficiently. Both L-serine racemization and elimination activities of serine racemase are of comparable magnitude, display alkaline pH optimum and are negligible below pH 6.5.

Animals↗
PubMed1999

Serine racemase: a glial enzyme synthesizing D-serine to regulate glutamate-N-methyl-D-aspartate neurotransmission.

Although D amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of D-serine have been found in mammalian brain where it activates glutamate/N-methyl-D-aspartate receptors by interacting with the "glycine site" of the receptor. Because amino acid racemases are thought to be restricted to bacteria and insects, the origin of D-serine in mammals has been puzzling. We now report cloning and expression of serine racemase, an enzyme catalyzing the formation of D-serine from L-serine. Serine racemase is a protein representing an additional family of pyridoxal-5' phosphate-dependent enzymes in eukaryotes. The enzyme is enriched in rat brain where it occurs in glial cells that possess high levels of D-serine in vivo. Occurrence of serine racemase in the brain demonstrates the conservation of D-amino acid metabolism in mammals with implications for the regulation of N-methyl-D-aspartate neurotransmission through glia-neuronal interactions.

Amino Acid Sequence↗
PubMed2002

In vivo phosphorylation of insulin receptor substrate 1 at serine 789 by a novel serine kinase in insulin-resistant rodents.

Insulin resistance is a key pathophysiologic feature of obesity and type 2 diabetes and is associated with other human diseases, including atherosclerosis, hypertension, hyperlipidemia, and polycystic ovarian disease. Yet, the specific cellular defects that cause insulin resistance are not precisely known. Insulin receptor substrate (IRS) proteins are important signaling molecules that mediate insulin action in insulin-sensitive cells. Recently, serine phosphorylation of IRS proteins has been implicated in attenuating insulin signaling and is thought to be a potential mechanism for insulin resistance. However, in vivo increased serine phosphorylation of IRS proteins in insulin-resistant animal models has not been reported before. In the present study, we have confirmed previous findings in both JCR:LA-cp and Zucker fatty rats, two genetically unrelated insulin-resistant rodent models, that an enhanced serine kinase activity in liver is associated with insulin resistance. The enhanced serine kinase specifically phosphorylates the conserved Ser(789) residue in IRS-1, which is in a sequence motif separate from the ones for MAPK, c-Jun N-terminal kinase, glycogen-synthase kinase 3 (GSK-3), Akt, phosphatidylinositol 3'-kinase, or casein kinase. It is similar to the phosphorylation motif for AMP-activated protein kinase, but the serine kinase in the insulin-resistant animals was shown not to be an AMP-activated protein kinase, suggesting a potential novel serine kinase. Using a specific antibody against Ser(P)(789) peptide of IRS-1, we then demonstrated for the first time a striking increase of Ser(789)-phosphorylated IRS-1 in livers of insulin-resistant rodent models, indicating enhanced serine kinase activity in vivo. Taken together, these data strongly suggest that unknown serine kinase activity and Ser(789) phosphorylation of IRS-1 may play an important role in attenuating insulin signaling in insulin-resistant animal models.

Amino Acid Sequence↗
PubMed1997

STAT3 is a serine kinase target in T lymphocytes. Interleukin 2 and T cell antigen receptor signals converge upon serine 727.

Interleukin 2 (IL-2) induces tyrosine phosphorylation of STATs 3 and 5 (signal transducer and activator of transcription). We now show that IL-2 regulation of STAT3 proteins in T cells is a complex response involving activation of two forms of STAT3: 90-kDa STAT3alpha and an 83-kDa carboxyl-terminal truncated STAT3beta. The phosphorylation of STAT proteins on serine residues is also required for competent STAT transcription. A critical serine phosphorylation site in STAT3alpha is at position 727. In this study we have produced an antisera specific for STAT3alpha proteins phosphorylated on serine 727 and used this to monitor the phosphorylation of this residue during T lymphocyte activation. Our results show that phosphorylation of STAT3alpha on serine 727 is not constitutive in quiescent T cells but can be induced by the cytokine IL-2. Interestingly, triggering of the T cell antigen receptor complex or activation of protein kinase C with phorbol esters also induces phosphorylation of serine 727 but without simultaneously inducing STAT3 tyrosine phosphorylation or DNA binding. Hence, the present results show that STAT3 serine phosphorylation can be regulated independently of the tyrosine phosphorylation of this molecule. IL-2 and T cell antigen receptor complex induction of STAT3alpha serine 727 phosphorylation is dependent on the activity of the MEK/ERK pathway. Previous studies have identified H-7-sensitive kinase pathways that regulate STAT3 DNA binding. We show that H-7-sensitive pathways regulate STAT3 DNA binding in T cells. Nevertheless, we show that H-7-sensitive kinases do not regulate STAT3 tyrosine phosphorylation or phosphorylation of serine 727. These results thus show that STAT3 proteins are targets for multiple kinase pathways in T cells and can integrate signals from both cytokine receptors and antigen receptors.

Amino Acid Sequence↗
PubMed1997

Mapping of Stat3 serine phosphorylation to a single residue (727) and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3.

During their polypeptide ligand-induced activation Stats (signaltransducers andactivators oftranscription) 1 and 3 acquire, in addition to an obligatory tyrosine phosphorylation, phosphorylation on serine which boosts their transactivating potential [Wen, Z., Zhong, Z. and Darnell, J. E. Jr. (1995) Cell 82, 241-250]. By examining phosphopeptide maps of wild-type and mutant protein we show here that the Stat3 serine phosphorylation, like the Stat1 serine phosphorylation, occurs on a single residue, serine 727. Neither the DNA binding of Stat1 nor Stat3 is demonstrably affected by the presence or absence of the serine phosphorylation. Thus the earlier demonstration that transcription is enhanced by the presence of the serine 727 residue likely occurs after DNA binding. These findings do not agree with earlier claims of excess serine to tyrosine phosphorylation in activated Stats 1 and 3 or to claims of more stable DNA binding of serine phosphorylated Stat dimers.

Animals↗
PubMed1990

Purification and characterization of serine-glyoxylate aminotransferase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.

Serine--glyoxylate aminotransferase was purified to complete homogeneity from a serine-producing methylotrophic bacterium, Hyphomicrobium methylovorum GM2, which possesses the serine pathway. This is the first microbial serine--glyoxylate aminotransferase to be purified. The enzyme has a molecular mass of about 140 kDa and consists of four subunits of identical mass, i.e. 40 kDa. The holoenzyme exhibited absorption maxima at 282 nm and 408 nm, and a shoulder at about 315-345 nm in potassium phosphate pH 7.0; it contained 4 mol pyridoxal 5'-phosphate/mol enzyme. Isoelectric focusing showed that the enzyme had a pI value of 6.9. The Km values for glyoxylate and L-serine were 0.23 mM and 4.98 mM, respectively, and the enzyme showed high specificity for these substrates. The transamination between glyoxylate and L-serine seemed to be nearly irreversible. These data indicated that this serine--glyoxylate aminotransferase plays an essential role in methanol assimilation through the serine pathway in H. methylovorum GM2.

Bacteria↗
PubMed2003

The temporal and spatial pattern of histone H3 phosphorylation at serine 28 and serine 10 is similar in plants but differs between mono- and polycentric chromosomes.

Immunolabeling using site-specific antibodies against phosphorylated histone H3 at serine 10 or serine 28 revealed in plants an almost similar temporal and spatial pattern of both post-translational modification sites at mitosis and meiosis. During the first meiotic division the entire chromosomes are highly H3 phosphorylated. In the second meiotic division, like in mitosis, the chromosomes contain high phosphorylation levels in the pericentromeric region and very little H3 phosphorylation along the arms of monocentric species. In the polycentric plant Luzula luzuloides phosphorylation at both serine positions occurs along the whole chromosomes, whereas in monocentric species, only the pericentromeric regions showed strong signals from mitotic prophase to telophase. No phosphorylated serine 10 or serine 28 was detectable on single chromatids at anaphase II resulting from equational segregation of rye B chromosome univalents during the preceding anaphase I. In addition, we found a high level of serine 28 as well as of serine 10 phosphorylation along the entire mitotic monocentric chromosomes after treatment of mitotic cells using the phosphatase inhibitor cantharidin. These observations suggest that histone H3 phosphorylation at serine 10 and 28 is an evolutionarily conserved event and both sites are likely to be involved in the same process, such as sister chromatid cohesion.

Cantharidin↗