Insight into the transition between the open and closed conformations of Thermus thermophilus carboxypeptidase.

Carboxypeptidase cleaves the C-terminal amino acid residue from proteins and peptides. Here, we report the functional and structural characterizations of carboxypeptidase belonging to the M32 family from the thermophilic bacterium Thermus thermophilus HB8 (TthCP). TthCP exhibits a relatively broad specificity for both hydrophilic (neutral and basic) and hydrophobic (aliphatic and aromatic) residues at the C-terminus and shows optimal activity in the temperature range of 75-80 °C and in the pH range of 6.8-7.2. Enzyme activity was significantly enhanced by cobalt or cadmium and was moderately inhibited by Tris at 25 °C. We also determined the crystal structure of TthCP at 2.6 Å resolution. Two dimer types of TthCP are present in the crystal. One type consists of two subunits in different states, open and closed, with a Cα RMSD value of 2.2 Å; the other type consists of two subunits in the same open state. This structure enables us to compare the open and closed states of an M32 carboxypeptidase. The TthCP subunit can be divided into two domains, L and S, which are separated by a substrate-binding groove. The L and S domains in the open state are almost identical to those in the closed state, with Cα RMSD values of 0.84 and 0.53 Å, respectively, suggesting that the transition between the open and closed states proceeds with a large hinge-bending motion. The superimposition between the closed states of TthCP and BsuCP, another M32 family member, revealed that most putative substrate-binding residues in the grooves are oriented in the same direction.

Determination of the active transport of fucoidan derived from Okinawa Mozuku across the human intestinal Caco-2 cells as assessed by size-exclusion chromatography.

INTRODUCTION: In order to clarify the mechanism of fucoidan transport, we developed the chromatographic determination method. METHOD: A size-exclusion chromatography (SEC) method for the determination of Okinawa-fucoidan using Develosil 300 Diol-5 (60×8.0mm I.D., 30nm pore-diameter) with the eluent containing 1% non-ionic detergent is developed. Determination range (UV at 210nm) is from 0 to 100ng of fucoidan with the linear calibration line inserting to zero. RESULTS: A transport activity of fucoidan is demonstrated by using Caco-2 cells (model of gut transport system); i.e., the initial transport velocity 12nmol/h/mg of protein (25-fold slower rate as compared to a bacterial l-alanine active-transport activity 300nmol/h/mg of protein) is found to occur. Since this fucoidan transport is inhibited by 10mM sodium azide (respiration inhibitor) and 0.05mM FCCP (uncoupler), this transport by Caco-2 cells is found to be an active one requiring energy-source. On the other hand, colchicine (inhibitor of phagocytosis/pinocytosis) and mannitol (putative competitive-inhibitor of tight-junction transport) cannot inhibit the fucoidan transport at all. CONCLUSION: We firstly report that the active transport occurs for such a high molecular-weight sulphated-polyfucose of fucoidan in vitro using Caco-2 cells.

Identification of functionally important residues of the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate neuromedin U receptor.

The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBAN(R2K)) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca(2+) mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca(2+) mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide.

Fucoidan-dependent increased membrane components in HepG2 cells: effect of fucoidan is not due to gene expression.

BACKGROUND: The precise mechanism of the therapeutic effects of fucoidan (sulphated polyfucose) on cultured hepatocarcinoma HepG2 cells is as yet unclear. MATERIALS AND METHODS: Protein components between fucoidan-treated and non-treated HepG2 cells were compared through a quantitative micro-sequencing method. RESULTS: A dramatic and immediate increment of the membrane compartment and a decrement of RNA virus by fucoidan, as an effect of the Ishi-Mozuku (an edible brown seaweed Mozuku of Japan), are demonstrated. The ratio of membrane glycoproteins to total cellular proteins increases from 28.9% to 43.2% (1.5-fold), and the positive-sense single-stranded RNA viral proteins among the total cellular proteins decrease from 5.3% to 0.29% (18-fold), respectively, in response to 0.102 mg/ml fucoidan in HepG2 cells over three days' period. CONCLUSION: Fucoidan seems to retard the growth of HepG2 cells through membrane glycoprotein metabolism. Therefore, fucoidan could be expected to have a therapeutic effect on hepatocellular carcinoma.

Biotin deficiency in a glycogen storage disease type 1b girl fed only with glycogen storage disease-related formula.

Glycogen storage disease type I is an autosomal recessive disorder caused by the defect in the glucose-6-phosphate enzyme system. Frequent intake of glucose-containing glycogen storage disease formula, uncooked cornstarch, or both, are usually needed to maintain normal blood glucose level. We report a glycogen storage disease type 1b girl with biotin deficiency caused by an exclusive glucose-containing glycogen storage disease formula for years, presenting with the appearance of severe skin lesions, and diagnosed by urinary organic acid analysis by gas chromato-spectrometry, and blood acylcarnitine analysis by tandem mass-spectrometry.

The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission.

Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 A crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants.

Effect of fucoidan on the biotinidase kinetics in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is difficult to treat with anticancer drugs. Therefore, development of new drugs for HCC is required. MATERIALS AND METHODS: The livers of 14 hepatoma patients accompanied by hepatitis B (2 cases) and hepatitis C (12 cases) were used. The biotinidase kinetics of HCC tissues were compared to those of the adjacent liver tissues of 13 liver cirrhosis (LC) and 1 chronic active hepatitis (CAH). RESULTS: The Kip (the inhibition constant by biotin) of HCC tissues were consistently higher than those of LC (plus CAH) tissues: the Kip was 450+/-231 micromol/l in HCC tissues and 240+/-111 micromol/l in LC (plus CAH) tissues, p<0.001. This increase of Kip is considered to be due to an increase of biotin repulsion by biotinidase in the HCC tissues. Fucoidan, a sulfated poly-fucose, was found to decrease the Kip of biotinidase in HCC tissues, and conversely to increase it in LC tissues. Fucoidan was also found to decrease the Kip of the hepatoma HuH-6 cells. CONCLUSION: These findings suggest that fucoidan has a potential therapeutic effect on HCC.

Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12.

The aim of this study is to assess whether fucoidan modulates the expression of chemokine ligand 12 (CXCL12)/chemokine receptor 4 (CXCR4) and exerts antitumor activity toward Huh7 hepatoma cells. According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, fucoidan inhibited the growth of Huh7 cells and HepG2 cells in a dose-dependent manner, with a 50% inhibition of cell growth (IC50) of 2.0 and 4.0 mg/ml, respectively. alpha-fetoprotein levels in medium collected from fucoidan-treated cells were significantly decreased in Huh7 cells but not in HepG2 cells. Western blotting revealed that the amount of alpha-fetoprotein was decreased by 1.0 mg/ml of fucoidan in Huh7 cells, whereas it was unchanged in HepG2 cells. In Huh7 cells, CXCL12 mRNA expression was significantly downregulated by 1.0 mg/ml of fucoidan, whereas CXCR4 mRNA expression was unchanged by fucoidan. CXCL12 and CXCR4 mRNA were barely expressed in HepG2 cells. In addition, 1.0 mg/ml of fucoidan mildly arrested the cell cycle and induced apoptosis in Huh7 cells. The findings suggest that fucoidan exhibits antitumor activity toward Huh7 cells through the downregulation of CXCL12 expression.

Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis.

Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 A resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 A, alpha = 90.96, beta = 103.41, gamma = 101.79 degrees. The Matthews coefficient (V(M) = 2.10 A(3) Da(-1)) indicated that the crystal contained two mAG molecules per asymmetric unit.

Wide Range of Biotin (Vitamin H) Content in Foodstuffs and Powdered Milks as Assessed by High-performance Affinity Chromatography.

The biotin (vitamin H) contents of various foodstuffs were determined by using a newly developed high-performance affinity chromatography with a trypsin-treated avidin-bound column. Biotin was derivatized with 9-anthryldiazomethane (ADAM) to fluorescent biotin-ADAM ester. A wide range of biotin contents were found in various foodstuffs depending upon the species (strain), season, organ (of plants and animals), geography, freshness, preparation method and storage method. Among the foodstuffs and fermented foods tested, it was found that wide distributions of biotin content were observed in powdered milk, natto, sake (rice wine), beer, edible oil and sea weed. Since powdered milk is important for child health and development, 14 kinds of powdered and special milks for use in children's diseases were intensively measured. We found that several special milk powders for children with allergies contained low levels of free biotin. Use of these powdered milks caused skin diseases and alopecia in some patients possessing thermolabile serum biotinidase, and administration of free biotin improved their symptoms dramatically. Therefore, it is essential to estimate the total and free biotin contents on each foodstuff in order to improve effective biotin intake and support better health and quality of life for people.

Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column.

A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1h at 120 degrees C and the recovery for biocytin was 65.7+/-2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, "natto", rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.

Determination of specific activities and kinetic constants of biotinidase and lipoamidase in LEW rat and Lactobacillus casei (Shirota).

Enzyme kinetic parameters, such as K(m), V(max) (or V), k(cat)/K(m), and K(i) (by biotin or lipoic acid) for biotinidase and lipoamidase were determined in Lewis (LEW) rat and Lactobacillus casei (Shirota) using fluorimetric high-performance liquid chromatography (HPLC). It was found that the final protein concentration below 0.1mg/ml is sufficient to obtain linear hydrolytic reaction and to determine the Michaelis-Menten type kinetic parameters (K(m), V, K(i)). We applied this HPLC enzyme assay method onto the rat and some bacteria. The highest specific activities (Vs) for biotinidase were found in Lactobacillus casei (Shirota) and rat kidney. It was also found that the largest K(i) by product for biotinidase and lipoamidase were present in the Lactobacillus casei (Shirota). There has been found specie (between rat and mouse) differences and tissue (organ) differences, together with tissue region differences and sex differences in some tissues. Summary of the distributions of both enzymes in LEW rat was also presented. Therefore, this HPLC determination method for the enzyme kinetic parameters in tissues is expected to be an indispensable tool for the investigation of the various diseases in humans.

Inhibition of allogeneic T-cell responses by dendritic cells expressing transduced indoleamine 2,3-dioxygenase.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an enzyme involved in the catabolism of tryptophan and has been shown to prevent rejection of the fetus during pregnancy by inhibiting alloreactive T cells. METHODS: In this study we investigated dendritic cells (DCs) that are transfected with IDO cDNA in the inhibition of T-cell proliferation after antigen-specific interaction. XS106 DCs, derived from A/J mice (H-2k), were transduced with IDO with a gene-delivery system using a recombinant adenoviral vector. RESULTS: Western blotting and immune staining revealed IDO expression in XS106 DCs transduced with IDO (XS106-IDO DCs), and its catabolic effect was confirmed by an increase in kynurenine concentration. Fluorescence-activated cell sorting revealed that XS106-IDO DCs were not changeable for Ia, CD80, and CD86 expression. After XS106-IDO DCs were co-cultured with C57BL/6 allogeneic splenic T cells, the proliferation of the T cell was significantly inhibited. The co-cultured T cells with XS106-IDO DCs exhibited cell-cycle arrest. Furthermore, injection of XS160-IDO DCs into the footpads of C57BL/6 (H-2b) mice demonstrated a reduced T-cell response against allo-antigen. CONCLUSIONS: These results suggest that overexpression of IDO in the DCs effectively inhibited T-cell proliferation, and may expand a new immunomodulatory strategy for the prevention of allo-rejection of organ transplantation.

Size-exclusion chromatography of biological samples which contain extremely alkaline proteins.

An improved size-exclusion chromatography (SEC) was developed to isolate extremely basic (alkaline) proteins, such as trypsin (pI=10.5), lysozyme (pI=11), and histone (pI=10.8). Develosil 300 Diol-5 (300 x 8 mm I.D., 30 nm average pore diameter) column was used with an eluent of 0.1 M sodium phosphate, 1.5 M sodium chloride, glycerol (40%, v/v), 2-propanol (10%, v/v), and Brij-58 (1%, v/v). Under these conditions, the final apparent pH becomes to 4.0, and pH adjustment is not necessary. Column temperature and flow rate were 15 degrees C and 0.2 ml/min, respectively. This elution system is stable and reliable, and applications onto human pancreatic juice, human bile, and tissue homogenates were successfully achieved. Since this system is convenient for protein analysis, it is expected to be generally applicable to clinical and biochemical research for identifying protein components in combination with microsequencing.