Journal of Biochemistry - recent issues

A Simple and Immediate Method for Simultaneously Evaluating Expression Level and Plasmid Maintenance in Yeast

2009-06-01

To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.

Saccharomyces cerevisiae Na+/H+ Antiporter Nha1p Associates with Lipid Rafts and Requires Sphingolipid for Stable Localization to the Plasma Membrane

Mitsui, K., Hatakeyama, K., Matsushita, M., Kanazawa, H. — 2009-06-01

The plasma membrane-type Na⁺/H⁺ antiporter Nha1p from budding yeast plays an important role in intracellular Na⁺ and pH homeostasis by mediating the exchange of Na⁺ for H⁺ across the plasma membrane. However, the mechanism of intracellular targeting of Nha1p to the plasma membrane remains unknown. Here, we found that Nha1p exists predominantly in detergent-resistant membrane fractions (DRMs) following density gradient centrifugation. When ergosterol was extracted from membranes, Nha1p was transferred to a detergent-soluble fraction, suggesting that Nha1p associates with ergosterol-containing DRMs, also known as lipid rafts. Density gradient centrifugation of cell extracts of yeast mutants that were defective in different stages of the secretory pathway revealed that, unlike previously identified raft proteins, the association of Nha1p with DRMs occurs mainly at the plasma membrane. In lcb1-100 cells, which are temperature-sensitive for sphingolipid synthesis, newly synthesized Nha1p failed to localize to the plasma membrane at the non-permissive temperature. Rather, Nha1p was distributed in an intracellular punctate pattern. The addition of phytosphingosine or the inhibition of endocytosis in lcb1-100 cells restored the targeting of Nha1p to the plasma membrane. The results of the current study suggest that sphingolipids are required for the stable localization of Nha1p to the plasma membrane.

Guanidine Hydrochloride- and Urea-Induced Unfolding of Toxoplasma gondii Ferredoxin-NADP+ Reductase: Stabilization of a Functionally Inactive Holo-Intermediate

Singh, K., Bhakuni, V. — 2009-06-01

Usually during the folding/unfolding of flavoproteins, an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii ferredoxin-NADP⁺ reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations, were found to interact selectively with the NADP⁺-binding domain of TgFNR and to induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding.

Post-Transcriptional Regulation of the Expression of Ferrochelatase by Its Variant mRNA

Sakaino, M., Kataoka, T., Taketani, S. — 2009-06-01

Ferrochelatase (FECH) catalyses the insertion of ferrous ions into protoporphyrin IX to produce haem at the haem-biosynthetic pathway. The present study characterized a variant mRNA of mouse FECH, which was generated by skipping exon II (FECH-v). FECH-v mRNA was expressed in various tissues, including the liver and kidney, of mice. The mRNA was also expressed in mouse and human non-erythroid and erythroid cells to a different extent but could not be translated into functional FECH. The ratio of FECH-v/FECH increased in hemin-treated Balb/3T3 cells, while it decreased after treatment with succinylacetone, an inhibitor of haem biosynthesis, strongly suggesting that FECH expression was decreased by increasing the level of intracellular haem. These results demonstrated the haem-dependent negative feedback regulation of the expression of FECH at a post-transcriptional level.

Evaluation of Conditions for Release of Mucin-Type Oligosaccharides from Glycoproteins by Hydrazine Gas Treatment

Goso, Y., Tsubokawa, D., Ishihara, K. — 2009-06-01

By using commercially available anhydrous hydrazine in the gas-phase, mucin-type oligosaccharides were released from porcine gastric mucin (PGM) and bovine fetuin. The data indicated that a certain amount of the oligosaccharides from PGM were further degraded. Despite this, the HPLC elution profile of the anthranilic acid (AA)-derivatized oligosaccharides obtained by the treatment with hydrazine at 65°C for 6 h resembled those obtained from the alkaline-borohydride treatment, except for the additional disaccharide fractions derived from the core 1 side of the oligosaccharides by further degradation. The other degraded products derived from the core 2 side could not be derivatized by AA, therefore, not visible by fluorescence detection. Liberation of the oligosaccharides was incomplete by the hydrazine treatment for 6 h. Although almost complete liberation was achieved by extending the treatment to 18 h, the degraded products also increased. In this case, the addition of barium oxide to the reaction vessel decreased the degree of further degradation. Results similar to PGM were obtained from bovine fetuin, but with less degradation. Application of this method for the analysis of rat gastric mucin (RGM) obtained from the corpus and antral region revealed that RGM has a large oligosaccharide portion on the core 1 side.

Sequence Perturbation Analysis: Addressing Amino Acid Indices to Elucidate the C-Terminal Role of Escherichia Coli Dihydrofolate Reductase

Takahashi, H., Yokota, A., Takenawa, T., Iwakura, M. — 2009-06-01

Because amino acid residues intrinsically possess many factors participating in protein structures and functions, to determine main (or unique) factors at a specific site in a protein sequence should be of great help for understanding how a protein obtains its structure and function. In this study, we proposed a means of sequence perturbation analysis to address the above concerns involving comprehensive AA indices. We constructed all 19 possible single mutant proteins as to the three sites in the C-terminal of Escherichia coli dihydrofolate reductase (DHFR), and measured the activity and thermal stability of each of all the single mutant proteins. The significantly perturbed properties with each systematic single mutation at each mutational site were examined in terms of the linear correlation with each AA index. As a result, at each of Arg158 and Arg159 of DHFR, the AA index for the isoelectric points of amino acids showed strong correlation with the transition temperature of thermal denatuation, suggesting that the electrostatic interaction is the main factor influencing the C-terminal role of the DHFR. The feasibility and general versatility of our sequence perturbation analysis were also examined by application to other sites of DHFR.

Probing the haem d-binding site in cytochrome bd quinol oxidase by site-directed mutagenesis

Mogi, T. — 2009-06-01

Cytochrome bd is a cyanide-resistant terminal quinol oxidase under micro-aerophilic growth conditions and generates a proton motive force via scalar protolytic reactions. Protons used for dioxygen reduction are taken up from the cytoplasm and delivered to haem d through a proton channel. Electrons are transferred from quinols to haem d through haem b₅₅₈ and haem b₅₉₅. All three haems are bound to subunit I but only the axial ligand of haem d remains to be determined. Haems b₅₉₅ and d form a haem–haem binuclear centre and substitutions of either His19 in helix I (haem b₅₉₅ ligand) and Glu99 in helix III eliminated or severely reduced both haems. To probe the location of the haem d ligand, we introduced mutations around His19 and Glu99 and examined the cyanide-resistance of the oxidase activity and spectroscopic properties. In contrast to mutations around His19, I98F and L101T reduced the IC₅₀ for cyanide to 0.18 and 0.41 mM, respectively, from 1.4 mM of the wild-type. Blue shifts in the peak of I98F suggest that Ile98 is in the vicinity of the haem d-binding site. Our data are consistent with the proposal that Glu99 serves as a haem d ligand of cytochrome bd.

Simvastatin Suppresses Leptin Expression in 3T3-L1 Adipocytes via Activation of the Cyclic AMP-PKA Pathway Induced by Inhibition of Protein Prenylation

Maeda, T., Horiuchi, N. — 2009-06-01

Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyses conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at 1 µM markedly inhibited adipocyte differentiation measured by Oil Red O staining in preadipocyte cells (3T3-L1), while expression of leptin, a marker of adipocyte differentiation, was suppressed by 1 µM simvastatin for up to 12 days of culture. Next, to elucidate mechanisms underlying the reduction of leptin expression induced by simvastatin, differentiated 3T3-L1 adipocytes were treated with various inhibitors with mevalonate or its metabolite in the presence or absence of simvastatin. Simvastatin time- and dose-dependently suppressed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by 10 µM simvastatin, while stability of the mRNA was not changed by treatment with simvastatin in transcription-arrested 3T3-L1 cells. Simvastatin inhibition of leptin gene transcription was not abrogated by pre-treatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate (GGPP), a mevalonate metabolite, abolished simvastatin-induced inhibition of leptin expression in 3T3-L1 cells. Suppression of expression was observed upon addition of GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor. Expression was suppressed by treatment with hydroxyfasudil, a protein prenylation inhibitor. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells. Simvastatin dose-dependently increased intra-cellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells, with maximal stimulation obtained at 10 µM. Addition of GGPP abolished simvastatin-induced stimulation of cAMP accumulation and protein kinase A (PKA) activity. H89, an inhibitor of PKA, completely abolished simvastatin-induced suppression of leptin expression. These results suggested that simvastatin reduced geranylgeranylprotein prenylation followed by deactivation of PI3K, leading to cAMP accumulation and subsequent activation of PKA in differentiated 3T3-L1 adipocytes. Finally, PKA inhibited leptin gene transcription without new protein synthesis.

Role of the Stem Domain of Matriptase in the Interaction with its Physiological Inhibitor, Hepatocyte Growth Factor Activator Inhibitor Type I

Kojima, K., Tsuzuki, S., Fushiki, T., Inouye, K. — 2009-06-01

Matriptase is a type II transmembrane serine protease containing the non-catalytic domains (stem domain) and catalytic domain in the extra-cellular region. Our aim is to address the role of the stem domain in the interaction between matriptase and its physiological inhibitor, hepatocyte growth factor activator inhibitor type I (HAI-1). We prepared secreted variants of recombinant matriptase containing the entire extra-cellular domain (HL-matriptase) or only the catalytic domain (L-matriptase), and compared the inhibition activities of a cell membrane-anchored form of recombinant HAI-1 (maHAI-1) against the matriptase variants in the hydrolysis of peptidyl–4-methyl-coumaryl-7-amide (MCA) substrates. HL-matriptase and L-matriptase were inhibited by purified maHAI-1 with a similar extent when t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA (1) and acetyl-Lys-Thr-Lys-Gln-Leu-Arg-MCA (2) were used as substrates. However, HL-matriptase was inhibited more strongly than L-matriptase by maHAI-1 in the hydrolysis of Boc-[(2S)-2-amino-3-(benzyloxycarbonyl)propionyl]-Pro-Arg-MCA (3). These results show that the stem domain of matriptase facilitates the inhibitory interaction of this protease with maHAI-1 in the hydrolysis of substrate 3, although it has no effect in the hydrolysis of substrates 1 and 2. To our knowledge, this is the first evidence that the stem domain of matriptase can affect the interaction between this protease and HAI-1.

Comparison of Total Protein Concentration in Skeletal Muscle as Measured by the Bradford and Lowry Assays

Seevaratnam, R., Patel, B. P., Hamadeh, M. J. — 2009-06-01

The Lowry and Bradford assays are the most commonly used methods of total protein quantification, yet vary in several aspects. To date, no comparisons have been made in skeletal muscle. We compared total protein concentrations of mouse red and white gastrocnemius, reagent stability, protein stability and range of linearity using both assays. The Lowry averaged protein concentrations 15% higher than the Bradford with a moderate correlation (r = 0.36, P = 0.01). However, Bland–Altman analysis revealed considerable bias (15.8 ± 29.7%). Both Lowry reagents and its protein–reagent interactions were less stable over time than the Bradford. The linear range of concentration was smaller for the Lowry (0.05–0.50 mg/ml) than the Bradford (0–2.0 mg/ml). We conclude that the Bradford and Lowry measures of total protein concentration in skeletal muscle are not interchangeable. The Bradford and Lowry assays have various strengths and weaknesses in terms of substance interference and protein size. However, the Bradford provides greater reagent stability, protein–reagent stability and range of linearity, and requires less time to analyse compared to the Lowry assay.

Effective Induction of Cell Death on Adult T-Cell Leukaemia Cells by HLA-DR{beta}-Specific Small Antibody Fragment Isolated from Human Antibody Phage Library

2009-06-01

By a biopanning method using cell sorter, we quickly isolated an antibody phage clone (S1T-A3) specific to human T-lymphotropic virus type 1-carrying T-cell line S1T from a human single chain Fv (scFv) antibody phage library. This scFv antibody bound to HTLV-1-carrying T-cell lines including MT-2, MT-4 and M8166 other than S1T, but not to non-HTLV-1-carrying T-cell lymphomas such as Jurkat and MOLT4 cells. Interestingly, this antibody induced the cell death on S1T cells very quickly (< 30 min). We tried to identify the target molecules by western blotting and mass spectrometric analysis, revealing that the target antigen was HLA class II DR. The cell death was induced only in dimmer form of scFv (diabody) and at 15-fold lower concentration than that of a fusion protein of scFv and human IgG Fc [(scFv)₂-Fc] or anti HLA-DR mouse whole antibody L243. Thus, S1T-A3 diabody is a small antibody fragment with agonistic activity to induce cell death through HLA-DR. This is the first report elucidating that diabody specific to HLA-DR is effective to induce the cell death in T-cell malignancy especially adult T-cell leukaemic cell line.

Isolation of ON Bipolar Cell Genes via hrGFP-coupled Cell Enrichment Using the mGluR6 Promoter

Nakajima, Y., Moriyama, M., Hattori, M., Minato, N., Nakanishi, S. — 2009-06-01

mGluR6 expression is a characteristic property of retinal ON bipolar cells. mGluR6 is also the causal gene for a form of congenital night blindness. To elucidate physiological and pathological functions of ON bipolar cells and mGluR6, we thought it important to identify genes specifically expressed in them. We thus made transgenic mouse lines expressing humanized Renilla reniformis green fluorescent protein (hrGFP), under the control of the mGluR6 promoter. From their retina, we isolated hrGFP-positive cells by cell sorting, and analysed the gene-expression profile of these cells by using DNA microarray. Further analysis revealed that about half of the initially selected ON bipolar cell genes were expressed in the expected retinal layer. We confirmed previously ambiguous retinal localization of regulator of G-protein signalling 11 (RGS11) and transient receptor potential cation channel, subfamily M, member 1 (TRPM1). In addition, we showed the expression of calcium channel, voltage-dependent, alpha2/delta subunit 3 (Cacna2d3) in ON bipolar cells for the first time. Although we could not completely exclude the possibility that a small population of hrGFP-positive cells might not be ON bipolar cells, these mice as well as our strategy would be highly valuable for the further analysis of ON bipolar cells.

Two Crystal Structures of Lysyl-tRNA Synthetase from Bacillus stearothermophilus in Complex with Lysyladenylate-Like Compounds: Insights into the Irreversible Formation of the Enzyme-Bound Adenylate of L-Lysine Hydroxamate

Sakurama, H., Takita, T., Mikami, B., Itoh, T., Yasukawa, K., Inouye, K. — 2009-04-30

Aminoacyl-tRNA synthetase forms an enzyme-bound intermediate, aminoacyladenylate in the amino-acid activation reaction. This reaction is monitored by measuring the ATP-PPi exchange reason in which [³²P]PPi is incorporated into ATP. We previously reported that l-lysine hydroxamate completely inhibited the l-lysine-dependent ATP-PPi exchange reaction catalysed by lysyl-tRNA synthetase from Bacillus stearothermophilus (BsLysRS). Several experiments suggested that BsLysRS can adenylate l-lysine hydroxamate, but the enzyme-bound lysyladenylate-like compound does not undergo the nucleophilic attack of PPi. This contrasts with the two reports for seryl-tRNA synthetase (SerRS): (i) l-serine hydroxamate was utilized by yeast SerRS as a substrate in the ATP-PPi exchange; and (ii) a seryladenylate-like compound was formed from l-serine hydroxamate in the crystal structure of Thermus thermophilus SerRS. To gain clues about the mechanistic difference, we have determined the crystal structures of two complexes of BsLysRS with the adenylate of l-lysine hydroxamate and with 5'-O-[N-(l-Lysyl)sulphamoyl] adenosine. The comparisons of the two BsLysRS structures and the above SerRS structure revealed the specific side-chain shift of Glu411 of BsLysRS in the complex with the adenylate of l-lysine hydroxamate. In support of other structural comparisons, the result suggested that Glu411 plays a key role in the arrangement of PPi for the nucleophilic attack.

Impaired chemotaxis and cell adhesion due to decrease in several cell-surface receptors in cathepsin E-deficient macrophages

2009-04-30

Cathepsin E is an endo-lysosomal aspartic proteinase exclusively present in immune system cells. Previous studies have shown that cathepsin E-deficient (CatE^–/–) mice display aberrant immune responses such as atopic dermatitis and higher susceptibility to bacterial infection. However, the mechanisms underlying abnormal immune responses induced by cathepsin E deficiency are still unclear. In this study, we found that the cell-surface levels of chemotactic receptors, including chemokine receptor (CCR)-2 and N-formyl peptide receptors (FPRs), were clearly diminished in CatE^–/–macrophages compared with those in wild-type cells. Consistently, chemotaxis of CatE^–/–macrophages to MCP-1 and N-formyl-methionyl-leucyl-phenylalanine was also decreased. Similar to the chemotactic receptors, the surface expressions of the adhesion receptors CD18 (integrin β₂) and CD 29 (integrin β₁) in CatE^–/– macrophages were significantly decreased, thereby reducing cell attachment of CatE^–/– macrophages. These results indicate that the defects in chemotaxis and cell adhesion are likely to be involved in the imperfect function of CatE^–/–macrophages.

The Influence of Domain Structures on the Signal Transduction of Chimeric Receptors Derived from the Erythropoietin Receptor

Liu, W., Kawahara, M., Ueda, H., Nagamune, T. — 2009-04-30

Although cytokine receptors regulate many cellular functions, contribution of receptor's domains and their conformation to signal transduction remains unclear. In this study, we designed a series of chimeric erythropoietin receptor (EpoR) variants encoding a haemagglutinin epitope-tagged anti-fluorescein single-chain Fv and different combinations of extracellular D1/D2 domain(s) of EpoR as the extracellular domain to allow the receptor to be activated by multiple ligands. Furthermore, one to four Ala residues were inserted at the intracellular juxtamembrane region of each chimeric receptor to modulate the conformation of the intracellular domain. When the chimeric receptors were expressed in Ba/F3 cells, cell-surface expression levels of chimeric receptors without D2 domain were markedly lowered, suggesting a role of D2 domain for stabilizing the receptor. Furthermore, the ligand-dependent cell proliferation was strongly affected by extracellular domain structures and the number of inserted Ala residues. Moreover, the conformational change of chimeric receptors was induced by various ligands to detect the phosphorylation of JAK2, STAT5 and ERK2, whose activations are characteristics of EpoR signalling. Consequently, the phosphorylation pattern of these signal transducers was significantly influenced by ligands and receptor variants. These results indicate that signal transduction of EpoR is strongly affected by conformation of both extracellular and intracellular domains.

Inspection of the Activator Binding Site for 4-{alpha}-Glucanotransferase in Porcine Liver Glycogen Debranching Enzyme with Fluorogenic Dextrins

Yamamoto, E., Watanabe, Y., Makino, Y., Omichi, K. — 2009-04-30

Recently, we found that -, β- and -cyclodextrins accelerated the 4--glucanotransferase action of porcine liver glycogen debranching enzyme (GDE) on Glc1-4Glc1-4Glc1-4(Glc1-4Glc1-4Glc1-4Glc1-6)Glc1-4Glc1-4Glc1-4Glc1-4GlcPA (B5/84), and proposed the presence of an activator binding site in the GDE molecule. In liver cells, the structures of -glucans proximal to the site GDE acts are not cyclodextrins, but glycogen and its degradation products. To estimate the structural characteristics of intrinsic activators and to inspect the features of the activator binding site, we examined the effects of four fluorogenic dextrins, (Glc1-6)_mGlc1-4(Glc1-4)_nGlcPA (B5/51, m = 1, n = 3; B6/61, m = 1, n = 4; B7/71, m = 1, n = 5; G6PA, m = 0, n = 4), on the debranching of B5/84 by porcine liver GDE. The GDE 4--glucanotransferase removed the maltotriosyl residue from the maltotetraosyl branch of B5/84, producing Glc1-4Glc1-4Glc1-4(Glc1-6)Glc1-4Glc1-4Glc1-4Glc1-4GlcPA (B5/81). In the presence of G6PA, the removed maltotriosyl residue was transferred to G6PA to give Glc1-4Glc1-4Glc1-4Glc1-4Glc1-4Glc1-4Glc1-4Glc1-4GlcPA (G9PA). In the absence of G6PA, the removed maltotriosyl residue was transferred to water. B7/71, B6/61 and B5/51 did not undergo any changes by the GDE, but they accelerated the action of the 4--glucanotransferase in removing the maltotriosyl residue. Of the four fluorogenic dextrins examined, B6/61 most strongly accelerated the 4--glucanotransferase action. The activator binding site is likely to be a space that accommodates the structure of Glc1-6Glc1-4Glc1-4Glc1-4Glc1-4Glc.

Maturation of the Extracellular Matrix and Cell Adhesion Molecules in Layered Co-cultures of HepG2 and Endothelial Cells

Ohno, M., Motojima, K., Okano, T., Taniguchi, A. — 2009-04-30

We previously reported that using thermo-responsive culture surfaces, a layered co-culture was achieved by placing an endothelial cell sheet onto a layer of human hepatoma cell line HepG2 in order to develop a culture model that mimics hepatic lobules. In the layered co-culture cells, the expression levels of liver-specific genes gradually increased. A cross-sectional view of the layered co-culture cells showed that the thickness of the layer slowly increased after layering, as did extracellular matrix (ECM) deposition around HepG2 cells. In this study, we report that the molecular compositions of ECM and cell-adhesion molecules changed in the layered co-culture cells. Gene expression of integrin 4 and decorin gradually increased after layering, and the time-course pattern of these genes was correlated with that of liver-specific genes. Moreover, the layered co-culture system has the ability to assemble a branching network of fibronectin fibrils. These results suggest that a vastly different extracellular environment in layered co-culture cells may induce an increase in liver-specific functions.

Effects of Replacement of Low-Spin Haem b by Haem O on Escherichia coli Cytochromes bo and bd Quinol Oxidases

Mogi, T. — 2009-04-30

Cytochromes bo and bd are terminal ubiquinol oxidases in the aerobic respiratory chain of Escherichia coli and generate proton motive force across the membrane. To probe roles of haem species in the oxidation of quinols, intramolecular electron transfer and the dioxygen reduction, we replaced b-haems with haem O by using the haem O synthase-overproducing system, which can accumulate haem O in cytoplasmic membranes. Characterizations of spectroscopic properties of cytochromes bo and bd isolated from BL21 (DE3)/pLysS and BL21 (DE3)/pLysS/pTTQ18-cyoE after 4 h of the aerobic induction of haem O synthase (CyoE) showed the specific incorporation of haem O into the low-spin haem-binding site in both oxidases. We found that the resultant haem oo- and obd-type oxidase severely reduced the ubiquinol-1 oxidase activity due to the perturbations of the quinol oxidation site. Our observations suggest that haem B is required at the low-spin haem site for the oxidation of quinols by cytochromes bo and bd.

Gender Disparity of Hepatic Lipid Homoeostasis Regulated by the Circadian Clock

Yang, X., Zhang, Y.-K. J., Esterly, N., Klaassen, C. D., Wan, Y.-J. Y. — 2009-04-30

The mammalian clock regulates major aspects of energy metabolism, including glucose and lipid homoeostasis as well as mitochondrial oxidative metabolism. This study is to identify specific patterns of circadian rhythms for lipid homoeostasis in both female and male mouse livers, and to clarify gender disparity in coupling the peripheral circadian clock to lipid metabolic outputs by nuclear receptors. To achieve this, profiling the diurnal hepatic expression of genes encoding circadian clocks, nuclear receptors and lipid metabolic enzymes was performed. Hepatic lipid levels including cholesterol, triglyceride and non-esterified fatty acids (NEFAs) were monitored over a 24-h period. The cosinor analysis revealed that several genes encoding nuclear receptors and enzymes involved in the lipid metabolic pathway were rhythmically expressed in liver in phase with the peripheral clocks, which were correlated with the diurnal changes of hepatic lipid levels. Gender disparity was observed for circadian characteristics including mesor and amplitude values, accompanied with advances in acrophases in female mouse livers. Accordingly, gender differences were also observed in diurnal lipid homoeostasis. The identification of cycling patterns for lipid metabolic pathways in both female and male mouse livers may shed light on the development of gender-based treatment for human diseases related to the coordination of the cellular clock and control of lipid homoeostasis.

Probing Structure of Heme A Synthase from Bacillus subtilis by Site-Directed Mutagenesis

Mogi, T. — 2009-04-30

Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme A synthase in Bacillus subtilis (CtaA) has eight transmembrane helices and oxidizes a methyl group on pyrrole ring D of heme O to an aldehyde. In this study, to explore structure of heme binding site(s) in heme A synthase, we overproduced the B. subtilis His₆-CtaA in Escherichia coli and characterized spectroscopic properties of the purified CtaA. On the contrary to a previous report (Svensson, B., Andersson, K.K., and Hederstedt, L. (1996) Low-spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis. Eur. J. Biochem. 238, 287–295), we found that two molecules of heme B were bound to CtaA. Further, we demonstrated that substitutions of His60 and His126 did not affect heme binding while His216 and His278 in the carboxy-halves are essential in heme binding. And we found that Ala substitutions of Cys191 and Cys197 in loop 5/6 reduced heme content to a half of the wild-type level. On the basis of our findings, we proposed a helical-wheel-projection model of CtaA.

Molecular Recognition of Tryptophan tRNA by Tryptophanyl-tRNA Synthetase from Aeropyrum pernix K1

Tsuchiya, W., Hasegawa, T. — 2009-04-30

The identity elements of transfer RNA are the molecular basis for recognition by each cognate aminoacyl-tRNA synthetase. In the archaea system, the tryptophan tRNA identity has not been determined in detail. To investigate the molecular recognition mechanism of tryptophan tRNA by tryptophanyl-tRNA synthetase (TrpRS) from the hyperthermophilic and aerobic archaeon, Aeropyrum pernix K1, various mutant transcripts of tryptophan tRNA prepared by an in vitro transcription system were examined by overexpression of A. pernix TrpRS. Substitution of the discriminator base, A73, impaired tryptophan incorporation activity. Changing the G1–C72 base pair to other base pairs also decreased the aminoacylation activity. Substitutions of anticodon CCA revealed that the C34 and C35 mutants dramatically reduced aminoacylation with tryptophan, but the A36 mutants had the same activity as the wild type. The results indicate that the anticodon nucleotides C34, C35, discriminator base A73 and G1–C72 base pair are major recognition sites for A. pernix TrpRS.

Requirement of Fut8 for the expression of vascular endothelial growth factor receptor-2: a new mechanism for the emphysema-like changes observed in Fut8-deficient mice

2009-04-30

1,6-Fucosylation plays key roles in many biological functions, as evidenced by the study of 1,6-fucosyltransferase (Fut8) knockout (Fut8^–/–) mice. Phenotypically, Fut8^–/– mice exhibit emphysema-like changes in the lung, and severe growth retardation. Fut8^–/– cells also show marked dysregulation of the TGF-β1 receptor, EGF receptor, integrin activation and intracellular signalling, all of which can be rescued by reintroduction of Fut8. The results of the present study demonstrated that vascular endothelial growth factor receptor-2 (VEGFR-2) expression was significantly suppressed in Fut8^–/– mice, suggesting that Fut8 was required for VEGFR-2 expression. The expression of VEGFR-2 mRNA and protein was consistently down-regulated by knockdown of the Fut8 gene with small interference RNA in A549 cells, as well as in TGP49 cells, suggesting that suppression occurs at the level of transcription. In contrast, the expression level of ceramide, an inducer of cell apoptosis, was increased in the lungs of Fut8^–/– mice. The terminal transferase dUTP nick end-labelling (TUNEL) assay was used to identify apoptotic cells. The number of TUNEL-positive septal epithelia and endothelia cells was significantly increased in the alveolar septa of lungs from Fut8^–/– mice when in comparison with lungs from wild-type mice. It is well known that, in emphysema, ceramide expression can be greatly enhanced by blockade of the VEGFR-2. Thus, suppression of VEGFR-2 expression may provide a novel explanation for the emphysema-like changes in Fut8^–/– mice.

Induction and Expression of Anti-Angiogenic Vasohibins in the Hematopoietic Stem/Progenitor Cell Population

Naito, H., Kidoya, H., Sato, Y., Takakura, N. — 2009-04-30

Haematopoiesis and blood vessel formation are closely associated, with several molecules employed by both systems. Recently, vasohibin-1 (VASH1), an endothelium-derived negative feedback regulator of angiogenesis, has been isolated and characterized. VASH1 is induced by VEGF or bFGF in endothelial cells (ECs) and inhibits their proliferation and migration. However, there are no data on the induction and expression of VASH1 in haematopoietic cells (HCs). Here, we show that the haematopoietic stem cell (HSC) population, but not haematopoietic progenitors (HPs) or mature HCs from adult bone marrow (BM) constitutively express VASH1. However, HPs, but not HSCs, can be induced to express VASH1 after BM suppression by 5-FU. Knock-down of the VASH1 gene in VASH1⁺ leukaemia cells induced cell proliferation. These results suggest a role for VASH1 in negative feedback regulation of HP proliferation during recovery following BM ablation.

Gene Cloning and Expression of Pyridoxal 5'-Phosphate-Dependent L-threo-3-Hydroxyaspartate Dehydratase from Pseudomonas sp. T62, and Characterization of the Recombinant Enzyme

Murakami, T., Maeda, T., Yokota, A., Wada, M. — 2009-04-30

L-threo-3-Hydroxyaspartate dehydratase (L-THA DH, EC 4.3.1.16), which catalyses the cleavage of L-threo-3-hydroxyaspartate (L-THA) to oxalacetate and ammonia, has been purified from the soil bacterium Pseudomonas sp. T62. In this report, the gene encoding L-THA DH was cloned and expressed in Escherichia coli, and the gene product was purified and characterized in detail. A 957-bp nucleotide fragment was confirmed to be the gene encoding L-THA DH, based on the agreement of internal amino acid sequences. The deduced amino acid sequence, which belongs to the serine/threonine dehydratase family, shows similarity to YKL218c from Saccharomyces cerevisiae (64%), serine racemase from Schizosaccharomyces pombe (64%) and Mus musculus (36%), and biodegradative threonine dehydratase from E. coli (38%). Site-directed mutagenesis experiments revealed that lysine at position 53 is an important residue for enzymatic activity. This enzyme exhibited dehydratase activity specific only to L-THA [K_m = 0.54 mM, V_max = 39.0 µmol min^–1 (mg protein)^–1], but not to other 3-hydroxyaspartate isomers, and exhibited no detectable serine/aspartate racemase activity. This is the first report of an amino acid sequence of the bacterial enzyme that acts on L-THA.

Over-Expression and Characterization of Bacillus subtilis Heme O Synthase

Mogi, T. — 2009-04-30

Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme O synthase in Bacillus subtilis (CtaB) has eight transmembrane helices and catalyses the transfer of a farnesyl group from farnesyl diphosphate to the 2-vinyl group on pyrrole ring A of ferrous heme B. In this study, we constructed the overproduction system for the B. subtilis CtaB in Escherichia coli. We isolated His₇-CtaB by affinity chromatography and demonstrated the presence of the heme-binding site in heme O synthase. His₇-CtaB binds substoichiometric amounts of heme B and O, substrate and unreleased product, respectively. Mutagenesis studies suggest that strictly conserved His199 present at the extra-cellular side of helix 5 would serve as the heme-binding site. We are hoping that the overproducing system for heme O synthase would help understanding of detailed mechanism on heme O biosynthesis and X-ray crystallographic studies.

Characterization of Type I Collagen Fibril Formation Using Thioflavin T Fluorescent Dye

Morimoto, K., Kawabata, K., Kunii, S., Hamano, K., Saito, T., Tonomura, B. — 2009-04-30

Collagen is composed of fibrils that are formed by self-assembly of smaller units, monomers which are triple-helical polypeptide. However, the mechanism of fibril formation at the level of individual molecules has remained to be clarified. We found that the fluorescence of thioflavin T, which has been widely used as a specific dye for amyloid fibrils, also increased by binding with fibrils of atelocollagen prepared from yellowfin tuna skin. There was a linear correlation between the fluorescence increase and the amount of atelocollagen within a collagen concentration range of 0–0.15 mg/ml at pH 6.5 with 50 µM thioflavin T. In contrast, neither actinidain-processed collagen that keeps monomeric nature nor heat-denatured collagen could cause the fluorescence increase of thioflavin T at all. The relationship between the fluorescence increase and thioflavin T concentration was fit to a theoretical binary binding curve. An apparent dissociation constant, K_d, and a maximal fluorescence increase, F_max, were calculated at various pHs. The values of K_d and F_max were dependent on pH (K_d was 9.4 µM at pH 6.5). The present finding demonstrates that thioflavin T specifically binds to collagen fibrils and may be used as a sensitive tool for the study of collagen structure.

Intramolecular Electron Transfer Processes in CuB-deficient Cytochrome bo Studied by Pulse Radiolysis

Kobayashi, K., Tagawa, S., Mogi, T. — 2009-04-30

The Escherichia coli cytochrome bo is a heme-copper terminal ubiquinol oxidase, and functions as a redox-driven proton pump. We applied pulse radiolysis technique for studying the one-electron reduction processes in the Cu_B-deficient mutant, His333Ala. We found that the Cu_B deficiency suppressed the heme b-to-heme o electron transfer two order of the magnitude (4.0x10² s^–1), as found for the wild-type enzyme in the presence of 1 mM KCN (3.0x10² s^–1). Potentiometric analysis of the His333Ala mutant revealed the 40 mV decrease in the E_m value for low-spin heme b and the 160 mV increase in the E_m value of high-spin heme o. Our results indicate that Cu_B not only serves as one-electron donor to the bound dioxygen upon the O-O bond cleavage, but also facilitates dioxygen reduction at the heme-copper binuclear centre by modulating the E_m value of heme o through magnetic interactions. And the absence of a putative OH^– bound to Cu_B seems not to affect the uptake of the first chemical proton via K-channel in the His333Ala mutant.

Epitope Mapping Using Ribosome Display in a Reconstituted Cell-Free Protein Synthesis System

Osada, E., Shimizu, Y., Akbar, B. K., Kanamori, T., Ueda, T. — 2009-04-30

Ribosome display is a powerful technology for selecting ligand-binding peptides or proteins. We demonstrate here that the ribosome display using the reconstituted cell-free protein synthesis system can be applied for the epitope mapping of monoclonal antibodies (mAbs). Using this technology, we selected peptides that specifically bind to three mAbs from random peptide library. When selection was performed against the anti-FLAG M2 antibody, selected peptides contained previously characterized consensus epitope, indicating that the methodology can be applied for the epitope mapping. When the selection was carried out against two anti-β-Catenin (anti-β-Cat) mAbs, selected peptides had a homology for the partial peptide sequences of β-Cat. Western blot analysis showed that these putative epitopes had affinity for the corresponding mAbs and β-Cat mutants that lack these regions did not bind to the antibodies, indicating we correctly mapped the epitope for these mAbs. The study shown here provides a way for the quick identification of the epitope of mAbs.

Molecular Phenotyping of Mannosyltransferases-Deficient Candida albicans Cells by High-Resolution Magic Angle Spinning NMR

Maes, E., Mille, C., Trivelli, X., Janbon, G., Poulain, D., Guerardel, Y. — 2009-04-03

The yeast Candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is directly mediated by cell wall components of the yeast, including a wide range of abundantly expressed glycoconjugates. Of particular interest in C. albicans are the β-mannosylated epitopes that show a complex expression pattern on N-glycan moiety of phosphopeptidomannans and are absent in the non-pathogenic species Saccharomyces cerevisiae. Being known as potent antigens for the adaptive immune response and elicitors of specific infection-protective antibodies, the exact delineation of β-mannosides regulation and expression pathways has lately become a major milestone toward the comprehension of host-pathogen interplay. Using the newly developed HR-MAS NMR methodology, we demonstrate the possibility of assessing the general profiles of cell-surface-exposed glycoconjugates from intact living yeast cells without any prior purification step. This technique permitted to directly observe structural modifications of surface expressed phosphodiester-linked β-mannosides on a series of deletion strains in β-mannosyltransferases and phospho-mannosyltransferases compared with their parental strains

Serine Racemase with Catalytically Active Lysinoalanyl Residue

2009-04-03

Serine racemase synthesizes d-serine, a physiological agonist of the NMDA receptor in mammalian brains. Schizosaccharomyces pombe produces serine racemase (spSR) that is highly similar to the brain enzyme. Our mass-spectrometric and X-ray studies revealed that spSR is modified with its natural substrate serine. spSR remains partially active even though its essential Lys57 inherently forming a Schiff base with the coenzyme pyridoxal 5'-phosphate is converted to N(6)-(R-2-amino-2-carboxyethyl)-l-lysyl (lysino-d-alanyl) residue. This indicates that the -amino group of the d-alanyl moiety of the lysino-d-alanyl residue serves as a catalytic base in the same manner as the -amino group of Lys57 of the original spSR.

Peroxiredoxin III-deficiency Sensitizes Macrophages to Oxidative Stress

Li, L., Kaifu, T., Obinata, M., Takai, T. — 2009-04-03

As a mitochondrial scavenger of reactive oxygen species (ROS), peroxiredoxin III (PrxIII) plays an important role in regulating intracellular ROS level. We previously found that PrxIII knockout (PrxIII^–/–) mice were more sensitive than wild-type (PrxIII^+/+) controls to intratracheal inoculation of lipopolysaccharide (LPS), but the precise mechanism remained to be obscure. In the present study, we detected the levels of ROS and tumour necrosis factor alpha (TNF-) in mouse bone-marrow-derived macrophages. LPS stimulation induced transient increase of ROS production and augmentation of TNF- accumulation in PrxIII^–/– macrophages. In addition, we observed reduced viability and increased apoptosis in PrxIII^–/– macrophages exposed to LPS. Our results provide direct evidence that PrxIII is necessary for macrophages to protect against LPS-induced oxidative stress.

Concerted Effects of Two Activator Modules on the Group I Ribozyme Reaction

Ikawa, Y., Shiohara, T., Ohuchi, S., Inoue, T. — 2009-04-03

Group I intron ribozymes have a modular architecture and structural elements essential for catalysis. The elements are located in the conserved modular domain P3–P7 that is stabilized by another conserved module, P4–P6. It has been reported that artificial modules can complement the function of the native P4–P6. To exploit the modular architecture of group I ribozyme, we have constructed a hybrid ribozyme by attaching an artificial activator module to the wild-type T4 td ribozyme. Kinetic analysis of the hybrid ribozyme revealed that the artificial module and P4–P6 have unusual positive and negative concerted effects in activating the ribozyme.

Identification and Functional Characterization of Rat Riboflavin Transporter 2

2009-04-03

We have newly identified rat riboflavin transporter 2 (rRFT2) and its human orthologue (hRFT2), and carried out detailed functional characterization of rRFT2. The mRNA of rRFT2 was highly expressed in jejunum and ileum. When transiently expressed in human embryonic kidney (HEK) 293 cells, rRFT2 could transport riboflavin efficiently. Riboflavin transport mediated by rRFT2 was Na⁺-independent but moderately pH-sensitive, being more efficient in acidic conditions than in neutral and basic conditions. Kinetic analysis indicated that rRFT2-mediated riboflavin transport was saturable with a Michaelis constant (K_m) of 0.21 µM. Furthermore, it was specifically and strongly inhibited by lumiflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), and to a lesser extent by amiloride. Such ability to transport riboflavin in a specific manner, together with its high expression in the small intestine, indicates that RFT2 may play a role in the intestinal absorption of riboflavin.

Functional Expression of Miraculin, a Taste-Modifying Protein in Escherichia Coli

Matsuyama, T., Satoh, M., Nakata, R., Aoyama, T., Inoue, H. — 2009-04-03

Miraculin isolated from red berries of Richadella dulcifica, a native shrub of West Africa, has the unusual property of modifying a sour taste into a sweet one. This homodimer protein consists of two glycosylated polypeptides that are cross-linked by a disulfide bond. Recently, functional expression of miraculin was reported in host cells with the ability to glycosylate proteins, such as lettuce, tomato and the microbe Aspergillus oryzae, but not Escherichia coli. Thus, a question remains as to whether glycosylation of miraculin is essential for its taste-modifying properties. Here we show that recombinant miraculin expressed in E. coli has taste-modifying properties as a homodimer, not as a monomer, indicating that glycosylation is not essential for the taste-modifying property.

Highly Active Low Magnesium Hammerhead Ribozyme

2009-04-03

Hammerhead (HH) ribozymes can be used for highly specific inhibition of gene expression through the degradation of target mRNA. In vitro experiments with minimal HH domains demonstrated that the efficiency of catalysis is highly dependent on concentration of magnesium ions. Optimal ion requirements for HH-catalysed RNA cleavage are far from these found in the cell. Recently, it has been proposed that the efficiency of HH ribozymes can be increased at low magnesium concentration through stabilization of a catalytically active conformation by tertiary interactions between helices I and II. We designed a ribozyme stabilized by GAAA tetraloop and its receptor motifs and demonstrated that it can efficiently catalyse target RNA hydrolysis at submillimolar Mg²⁺ concentrations in vitro as well as in cultured cells. Both unmodified and locked nucleic acid-modified extended ribozymes proved superior to the minimal core ribozyme and DNAzyme against the same target sequence.

Colicin E5 Ribonuclease Domain Cleaves Saccharomyces cerevisiae tRNAs Leading to Impairment of the Cell Growth

Ogawa, T., Hidaka, M., Kohno, K., Masaki, H. — 2009-04-03

Colicin E5 is a ribonuclease that specifically cleaves tRNA^Tyr, tRNA^His, tRNA^Asn and tRNA^Asp of sensitive Escherichia coli cells by recognizing their anticodon sequences. Since all organisms possess universal anticodons of these tRNAs, colicin E5 was expected to potentially cleave eukaryotic tRNAs. Here, we expressed the active domain of colicin E5 (E5-CRD) in Saccharomyces cerevisiae and investigated its effects on growth. E5-CRD impaired growth of host cells by cleaving tRNA^Tyr, tRNA^His, tRNA^Asn and tRNA^Asp in S. cerevisiae, which is the same repertoire as that in E. coli. This activity of E5-CRD was inhibited by the co-expression of its cognate inhibitor (ImmE5). Notably, the growth impairment by E5-CRD was reversible; cells restored the colony-forming activity after suppression of the E5-CRD expression. This seems different from the sharp killing effect of E5-CRD on E. coli. These results may provide insights into the role and behaviour of cytosolic tRNAs on cell growth and proliferation.

Closed Complex of the D-3-Hydroxybutyrate Dehydrogenase Induced by an Enantiomeric Competitive Inhibitor

Nakashima, K., Ito, K., Nakajima, Y., Yamazawa, R., Miyakawa, S., Yoshimoto, T. — 2009-04-03

d-3-Hydroxybutyrate dehydrogenase (HBDH) from Pseudomonas fragi showed a strict stereospecificity to the d-enantiomer of 3-hydroxybutyrate (d-3-HB) as a substrate. The l-enantiomer acts as a competitive inhibitor, with a K_i value comparable to the K_m value for d-3-HB. We have determined the crystal structures of the ternary complex of HBDH–NAD⁺–l-3-HB and the binary complex of HBDH–NAD⁺. The former structure showed a so-called closed-form conformation, which is considered an active form for catalysis, while the latter stayed mostly in a open-form conformation. The determined structures along with the site-directed mutagenesis confirmed the substrate recognition mechanism that we proposed previously. The hydrogen bonding interaction between Gln196, located in the moving helix, and the carboxyl group of the substrate/inhibitor is important for the stable ternary complex formation. Finally, the crystal structures of the Thr190 mutants, T190S and T190A, indicate that the Thr190 is a key residue for the open-closed conformational change. T190S retained 37% of the activity. In T190A, however, the activity decreased to 0.1% that of the wild-type enzyme. Fixing the position of the hydroxyl group of Thr190 to form hydrogen bonds to the pyrophosphate moiety and the carboxamide of NAD⁺ seems to be a significant factor for the open-closed conformational change.

In Vitro and In Vivo Interactions of Ferredoxin-NADP+ Reductases in Pseudomonas putida

Yeom, J., Jeon, C. O., Madsen, E. L., Park, W. — 2009-04-03

Ferredoxin-NADP⁺ reductase (Fpr) is known to control NADP⁺/NADPH pool in proteobacteria. There is only one fpr gene present in most proteobacteria, but Pseudomonas putida has two Fprs (FprA and FprB). We elucidated the functional relationships between the two types of Fpr and their electron transport partners [ferredoxin (Fd) and flavodoxin (Fld)] by cloning, expressing and preparing these proteins in various combinations and assessing their properties in vitro and in vivo using biochemical assays, the Far-western analysis, the yeast two-hybrid assay and structural molecular modelling. Both of the Fprs have a lower K_m value for NADPH than for NADH in the diaphorase assays. With NADH as electron donor, FprB also has a high specific constant (k_cat/K_m) in the diaphorase assay. The catalytic efficiency of FprA is higher when Fld is present as its redox partner, compared to the kinetics observed with other electron transport partners in a NADPH-dependent cytochrome c reduction assay. The highest specific constant (k_cat/K_m) of FprB was observed in the presence of FdA. FprB's K_m value and catalytic activity (k_cat) with NADH were significant in cytochrome c reduction assays. Strong kinetic interactions of Fprs with their redox partners were also demonstrated by homology modelling, the Far-western analysis and the in vivo yeast two-hybrid system. This study demonstrates for the first time that Fprs in P. putida function as diaphorase, Fd/Fld reductases and determines their preferred redox partner in vivo and in vitro.

Gene Identification and Characterization of 5-Formyl-3-Hydroxy-2-Methylpyridine 4-Carboxylic Acid 5-Dehydrogenase, an NAD+-Dependent Dismutase

Yokochi, N., Yoshikane, Y., Matsumoto, S., Fujisawa, M., Ohnishi, K., Yagi, T. — 2009-04-03

A chromosomal gene, mlr6793, in Mesorhizobium loti was identified as the gene encoding 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) dehydrogenase (dismutase) involved in the degradation pathway for pyridoxine (vitamin B₆). The homogenously purified recombinant enzyme has a molecular mass of 59.1 kDa and is a homodimeric protein. FHMPC dehydrogenase catalyses practically irreversible oxidation (k_cat = 204 s^–1) of FHMPC (K_m = 48.2 µM) by NAD⁺ (K_m = 34.3 µM) to 3-hydroxy-2-methyl-pyridine 4, 5-dicarboxylic acid (HMPDC), and practically irreversible reduction (k_cat = 217 s^–1) of FHMPC (K_m = 24.9 µM) by NADH (K_m = 12.4 µM) to 4-pyridoxic acid. When the enzyme reaction was started with the combination of FHMPC and NAD⁺ or that of FHMPC and NADH, HMPDC and 4-pyridoxic acid were produced in an almost equimolar ratio throughout the reaction. FHMPC dehydrogenase belongs to the 3-hydroxyacyl-CoA dehydrogenase family with 31% identity with the human enzyme: it has probable catalytic diad residues, i.e. His137 and Glu149. The H137L mutant enzyme showed no measurable activity. The E149Q one was stable in contrast to the corresponding human 3-hydroxyacyl-CoA dehydrogenase mutant, and showed unique pH optima depending on the co-substrates used for the reaction.

Cooperative Binding of L-Trp to Human Tryptophan 2,3-Dioxygenase: Resonance Raman Spectroscopic Analysis

Fukumura, E., Sugimoto, H., Misumi, Y., Ogura, T., Shiro, Y. — 2009-04-03

Tryptophan 2,3-dioxygenase (TDO) is a tetrameric enzyme that catalyses the oxidative cleavage of l-tryptophan (l-Trp) to N-formylkynurenine by the addition of O₂ across the 2,3-bond of the indole ring. This reaction is the first and rate-limiting step in the kynurenine pathway in mammals. In the present study, we measured the conformational changes in the haem pocket of recombinant human TDO (rhTDO) in ferric form that are induced by l-Trp binding using both resonance Raman and optical absorption spectroscopies. The deconvolution analysis of the haem Raman bands at various concentrations of l-Trp revealed that the wild-type enzyme exhibits homotropic cooperativity in l-Trp binding, which was confirmed by a change in the optical absorption spectra. Mutation analysis showed that the Y42F mutant abolished the cooperative binding, and that the H76A mutant considerably reduced the catalytic activity. These data and the inter-subunit contacts reported in the bacterial TDO structure suggest that the Y42 of rhTDO is responsible for the cooperative binding of l-Trp by participating in the active site of the adjacent subunit.

Protein Oxidation During Long Storage: Identification of the Oxidation Sites in Dihydrofolate Reductase from Escherichia coli through LC-MS and Fragment Studies

Takenawa, T., Yokota, A., Oda, M., Takahashi, H., Iwakura, M. — 2009-04-03

An LC–MS study revealed some heterogeneity in terms of molecular mass of a cysteine-free mutant of dihydrofolate reductase (DHFR) after long storage of the highly purified protein as an ammonium sulfate precipitate, but not in the case of a cysteine- and methioneine-free mutant of DHFR. One-third of the cysteine-free DHFR sample stored for a long time, around 18 months, comprised molecular species with molecular masses increased by 16, 32 and 48 Da. A peptide mapping study revealed that at least one of the methionine residues at positions 1, 16 and 20 was oxidatively modified to a methione-sulfoxide residue, while those at positions 42 and 92 were essentially unaffected. Each of the oxidized species of the DHFR exhibiting different degrees or sites of oxidation was further purified to essentially homogeneity in terms of molecular mass from the stored sample, and its enzyme activity was determined. One oxidized DHFR showed higher activity than that of the non-oxidized enzyme, while the other four oxidized DHFRs showed less activity. This agrees with the observation that the enzyme activity of the stored sample, a mixture in terms of oxidation, was apparently the same as that of the non-oxidized enzyme. This suggests that the activity itself is not a proper measure for quality control of proteins.

Cloning and Expression of the MutM Gene from Obligate Anaerobic Bacterium Desulfovibrio vulgaris (Miyazaki F)

Sanada, H., Nakanishi, T., Inoue, H., Kitamura, M. — 2009-04-03

The gene encoding a MutM from Desulfovibrio vulgaris (Miyazaki F) was cloned and expressed in Escherichia coli. A 5.9-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by XhoI and PvuII, contained a MutM gene and other open reading frames. The nucleotide sequence of the MutM gene indicated that the protein was composed of 336 amino acids. The amino-acid sequence deduced from the MutM gene was highly homologous with the MutM of other bacteria; however an additional insert consisted of 64 amino acids. An expression system for the MutM gene under the control of the T7 promoter was constructed in E. coli. From the kinetic analysis results, the purified His-tagged MutM showed 8-oxoguanine-DNA glycosylase activity comparable with that of MutM from E. coli. In this study, the amounts of mRNA and protein for MutM were scant in the D. vulgaris (Miyazaki F). MutM activity may be induced by oxidative stress. However, its induction may not be frequently generated because sulfate-reducing bacteria generally grow in anaerobic conditions. MutM might play a role in the protection against the mutagenicity of oxygen when oxygen stress exceeded the capacity of the defense systems against oxygen toxicity.

Construction of the Plasmid, Expression by Chinese Hamster Ovary Cell, Purification and Characterization of the First Three Short Consensus Repeat Modules of Human Complement Receptor Type 1

Yamaguchi, A., Takagawa, H., Iwakaji, H., Miyagawa, S., Wang, P.-C., Ishii, N. — 2009-04-03

Short consensus repeat (SCR1-3), the first three SCR modules from N-terminus of type 1 complement receptor (CR1), is expected to accelerate dissociation of complement components and suppress complement activity by binding the main component of complement C4b. In order to clarify the three-dimensional structure, which triggers the activity of SCR1-3 on complement, we constructed an over-expression system in CHO DG44 cells which facilitated mass production of SCR1-3. The mass production was achieved by a two-stage culture system and optimum culture conditions using ASF104N medium and MTX-, NaBu-containing -MEM/10% FBS medium, respectively. The constructed gene of SCR1-3 was confirmed by restriction enzyme digestion and DNA sequence analysis, and the expressed protein by CHO DG44 cells was confirmed by western blotting. The expressed SCR1-3 was proved containing N-linked sugar chain, an important factor to the proper expression of protein, by the cleavage with glycosidase of N-linked oligosaccharide (PNGase F). The suppression effect of the yield protein on complement-mediated inflammation was investigated by haemolytic assay and necrosis assay of stromal cells. Both assays showed that SCR1-3 possessed complement control activity. However, residing sugar chain on SCR1-3 did not show significant difference in the complement control activity.

Interactions Between Histidine and Tryptophan Residues in the BM2 Proton Channel from Influenza B Virus

Otomo, K., Toyama, A., Miura, T., Takeuchi, H. — 2009-04-03

The BM2 protein of influenza B virus forms a transmembrane proton channel essential for the virus infection. We investigated the structure and mechanism of the BM2 proton channel by using a 31-mer peptide (BM2-TMP) representing the putative transmembrane domain of BM2, with special focus on His19, Trp23 and His27. Like the full-length protein, BM2-TMP formed a transmembrane proton channel activated at acidic pH with a midpoint of transition at pH 6.4 ± 0.1. Mutation of His19 to Ala almost abolished the channel activity, whereas the His27-to-Ala mutant retained partial activity. The proton selectivity of the channel was lost upon substitution of Phe for Trp23. Comparison of CD, fluorescence and Raman spectra measured for wild-type and mutated BM2-TMP at varied pH showed the pK_a of the imidazole ring to be ~6.5 for His19 and ~7.6 for His27. Analysis of the pH-dependent fluorescence and Raman intensities suggested the occurrence of cation– interaction between the protonated imidazole ring of His and the indole ring of Trp. The His19–Trp23 cation– interaction below pH 6.5 is likely to trigger the opening of the proton channel, whereas His27 is not essential but enhances the channel activity through interaction with Trp23, which constitutes the proton-selective gate.