Journal of Biochemistry - current issue

Matrix Metalloproteinase-9 Associated with Heparan Sulphate Chains of GPI-Anchored Cell Surface Proteoglycans Mediates Motility of Murine Colon Adenocarcinoma Cells

2008-05-06

Using murine colon adenocarcinoma-derived clones with different metastatic potentials, the cellular localization of matrix metalloporteinase-9 (MMP-9) and its role in the cell motility were examined. Highly metastatic LuM1 clone aggressively invaded into adjacent tissue in vivo, but low metastatic NM11 clone did not. As compared with the NM11 clone, the LuM1 clone expressed and secreted a remarkably large amount of MMP-9, and exhibited higher abilities of cell migration and invasion in vitro, which were suppressed by MMP-2/MMP-9 inhibitor IV. MMP-9, exhibiting high affinity to heparin, was demonstrated to be condensed on tips of cellular podia. Treatment of the cells with heparitinase-I or heparin resulted in release of MMP-9 from the cell surface, which caused concomitant suppression of their motility to a similar level to that with the MMP inhibitor. Immunoprecipitation of a LuM1 cell lysate with an anti-MMP-9 antibody resulted in co-precipitation of phosphatidylinositol-specific phospholipase C-susceptible heparan sulphate proteoglycans having 66 and 64 kDa core proteins. Taken together, the present results demonstrate that secreted MMP-9 associates with glypican-like proteoglycans through their heparan sulphate chains, and plays a crucial role in cell motility of LuM1 cells.

Ig L-chain Shuffling for Affinity Maturation of Phage Library-derived Human Anti-human MCP-1 Antibody Blocking its Chemotactic Activity

2008-05-06

Monocyte chemotactic protein-1 (MCP-1, CC-chemokine ligand 2; CCL2) is involved in the development of various forms of chronic inflammations. Employing the naive human single-chain Fv displaying phage library, we established seven MCP-1-specific scFvs. The MC8 and MC32 clones exhibited blocking activity for the MCP-1-induced chemotaxis of THP-1 cells, in spite of their monovalency. The analysis of V gene usage showed that all clones bore the identical Vh1 gene, IGHV1-24*01, with variable DJ joining sequences, while their Vl usage was relatively varied, suggesting the preferential contribution of the Vh gene. Based on these findings, to minimize the deteriorative influences on the MCP-1 specificity of MC32, we aimed to achieve the affinity maturation of MC32 using MC32 L-chain shuffling library and select MC32 variants. Most MC32 variants increased their affinity by reducing the k_off value with no influence of the antigen specificity. MC32 variants #22 or #56 showed ~15-fold higher affinity than MC32, indicating that the L-chain shuffling library is useful if the Vh is dominantly involved in the determination of the antigen specificity.

Gene Identification and Characterization of the Pyridoxine Degradative Enzyme 4-Pyridoxic Acid Dehydrogenase from the Nitrogen-fixing Symbiotic Bacterium Mesorhizobium loti MAFF303099

Ge, F., Yokochi, N., Yoshikane, Y., Ohnishi, K., Yagi, T. — 2008-05-06

The gene encoding 4-pyridoxic acid dehydrogenase was identified as mlr6792 in a chromosome of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099. The enzyme is the fourth enzyme in the vitamin B₆ (pyridoxine)-degradation pathway I. The recombinant enzyme with a his-tag over-expressed in Escherichia coli cells was a membrane-bound protein, and purified to homogeneity. The enzyme was a monomeric protein with a molecular weight of 59,000, and a flavoprotein containing one mole of FAD per mole of subunit. The optimum pH and temperature, and K_m for 4-pyridoxic acid were pH 8.5 and 30°C, and 29 µM, respectively. The enzyme was a glucose-methanol-choline (GMC) family protein with two signature patterns, FAD-binding residues, a putative active site histidine residue and a probable transmembrane segment.

Reactivity of Astrocytes to Fibroblast Growth Factor-1 for Biogenesis of Apolipoprotein E-High Density Lipoprotein is Down-regulated by Long-time Secondary Culture

Nagayasu, Y., Ito, J.-i., Nishida, T., Yokoyama, S. — 2008-05-06

We previously showed that astrocytes produce and release fibroblast growth fator-1 (FGF-1) upon 1-month primary and 1-week secondary culture (M/W cells) and stimulate themselves by an autocrine manner to produce apoE-high-density lipoproteins (HDL), closely associated with their generation of apoE-HDL in brain injury. Astrocytes prepared by 1-week primary and 1-month secondary culture (W/M cells), however, expressed FGF-1 as much as M/W cells but produce apoE-HDL much less. The W/M cells conditioned medium in fact contained FGF-1 activity to stimulate astrocytes prepared by 1-week primary and 1-week-secondary culture (W/W cells). FGF-1 did not stimulate W/M cells for apoE-HDL biogenesis while it stimulated W/W cells. Phosphorylation of Akt, ERK and MEK were induced by FGF-1 in W/W cells but not in W/M cells. Finally, fibroblast growth factor receptor-1 in the membrane decreased in W/M cells in comparison to W/W cells. Interestingly, the reactivity of astrocytes to FGF-1 was recovered when W/M cells were transferred to the tertiary culture of 1 week. We concluded that astrocytes decrease their reactivity to FGF-1 for apoE-HDL biogenesis in certain conditions. The findings indicate astrocyte FGF-1 enhances biogenesis of apoE-HDL also by a paracrine mechanism.

Characterization of Dihydrodipicolinate Reductase from Thermotoga maritima Reveals Evolution of Substrate Binding Kinetics

Pearce, F. G., Sprissler, C., Gerrard, J. A. — 2008-05-06

In lysine biosynthesis, dihydrodipicolinate reductase (DHDPR) catalyses the formation of tetrahydrodipicolinate. Unlike DHDPR enzymes from Escherichia coli and Mycobacterium tuberculosis, which have dual specificity for both NADH and NADPH as co-factors, the enzyme from Thermotoga maritima has a significantly greater affinity for NADPH. Despite low sequence identity with the E. coli and M. tuberculosis DHDPR enzymes, DHDPR from T. maritima has a similar catalytic site, with many conserved residues involved in interactions with substrates. This suggests that as the enzyme evolved, the co-factor specificity was relaxed. Kinetic studies show that the T. maritima DHDPR enzyme is inhibited by high concentrations of its substrate, DHDP, and that at high concentrations NADH also acts as an inhibitor of the enzyme, suggesting a novel method of regulation for the lysine biosynthetic pathway. Increased thermal stability of the T. maritima DHDPR enzyme may be associated with the lack of C-terminal and N-terminal loops that are present in the E. coli DHDPR enzyme.

Role of Disulphide Bonds in a Thermophilic Serine Protease Aqualysin I from Thermus aquaticus YT-1

2008-05-06

A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulphide bonds, which are also found in a psychrophilic serine protease from Vibrio sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulphide bonds in aqualysin I, we prepared mutants C99S, C194S and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194 and both of these disulphide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in Escherichia coli. The C99S mutant was 68% as active as the wild-type enzyme at 40°C in terms of k_cat value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulphide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90°C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7°C and 86.9°C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulphide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.

Characterization of the Molecular Interaction between Tropoelastin and DANCE/Fibulin-5

2008-05-06

Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca²⁺, Ba²⁺ and Mg²⁺ significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2–15 and exons 16–36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.

Identification of Amino Acid Positions Involved in HLA-E Expression

Matsunami, K., Okura, E., Uchida, H., Otsuka, H., Fukuzawa, M., Miyagawa, S. — 2008-05-06

The cell surface expression of HLA-E molecules by transfection is faint in xenogeneic cells. Therefore, this study was done for the aim of better expression of HLA-E molecules on the surface of pig cells in order to overcome xenograft rejection mediated by human natural killer (NK) cells. The importance of the loading peptide sequence for HLA-E expression has been studied extensively, but much less information is available concerning the HLA-E heavy chain sequence. In our previous study, we developed the S147C substitution of HLA-E as a useful gene tool for xenotransplantation. In this study, a more extensive substitution analysis throughout the entire region led to the identification of nine amino acid positions, positions-9, 11, 25, 40, 66, 67, 74, 99 and 174, that are significantly involved in the cell surface expression of HLA-E molecules. In view of xenotransplantation usage, double and triple point substitutions, HLA-Ev(11,147) and HLA-Ev(11,66,147), were constructed. These constructs led to a high expression on the xenogeneic cell surface and possessed inhibitory functions against human NK cell-mediated cytolysis in an in vitro pig to human xenotransplantation model system.

Contribution of Peroxisome-specific Isoform of Lon Protease in Sorting PTS1 Proteins to Peroxisomes

Omi, S., Nakata, R., Okamura-Ikeda, K., Konishi, H., Taniguchi, H. — 2008-05-06

Using an organelle proteomics approach, we previously studied the rat peroxisome in order to characterize the proteins participating in its biogenesis. A peroxisome-specific isoform of Lon (pLon) protein was accordingly identified. However, the precise role of pLon in peroxisomes remains to be elucidated. Here, we demonstrate that pLon plays a role in processing and activating a specific regulatory protein belonging to the peroxisome targeting signal (PTS) 1-containing proteins. Proteomic analysis of proteins co-immunoprecipitated with Lon suggested that Lon interacts with PMP70 and several enzymes involved in β-oxidation, including acyl-CoA oxidase (AOX). The processing of AOX for its activation in peroxisomes was strongly inhibited in cells expressing a dominant negative form of pLon. Furthermore, a catalase possessing a modified PTS1 sequence was misdistributed in this cell line. pLon exhibits little, if any, in vitro AOX processing activity, and does not process PTS2-containing 3-ketoacyl-coenzyme A thiolase (PTL). Therefore, pLon may specifically control, sort and process PTS1 proteins. Based on the relationship between pLon and the β-oxidation enzymes that regulate peroxisomal morphology, the observation of enlarged peroxisomes in cells expressing recombinant pLon suggests that pLon is a critical factor determining peroxisome morphology.

Vertebrate Genomes Code Excess Proteins with Charge Periodicity of 28 Residues

Ke, R., Sakiyama, N., Sawada, R., Sonoyama, M., Mitaku, S. — 2008-05-06

All amino acid sequences derived from 248 prokaryotic genomes, 10 invertebrate genomes (plants and fungi) and 10 vertebrate genomes were analysed by the autocorrelation function of charge sequences. The analysis of the total amino acid sequences derived from the 268 biological genomes showed that a significant periodicity of 28 residues is observable for the vertebrate genomes, but not for the other genomes. When proteins with a charge periodicity of 28 residues (PCP28) were selected from the total proteomes, we found that PCP28 in fact exists in all proteomes, but the number of PCP28 is much larger for the vertebrate proteomes than for the other proteomes. Although excess PCP28 in the vertebrate proteomes are only poorly characterized, a detailed inspection of the databases suggests that most excess PCP28 are nuclear proteins.

Yeast-Based Fluorescence Reporter Assay of G Protein-coupled Receptor Signalling for Flow Cytometric Screening: FAR1-Disruption Recovers Loss of Episomal Plasmid Caused by Signalling in Yeast

2008-05-06

Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.

Transcriptional Regulation of the Human PNRC Promoter by NFY in HepG2 Cells

Zhang, Y., Chen, B., Li, Y., Chen, J., Lou, G., Chen, M., Zhou, D. — 2008-05-06

PNRC (proline-rich nuclear receptor co-activator) was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC has been demonstrated to be a novel co-activator for multiple nuclear receptors. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, potential transcriptional start site was determined by 5' RACE analysis. Functional analysis of the 5' flanking region of the human PNRC gene by deletion mutagenesis, transient transfection and luciferase assays revealed that the –123/+27 region is the minimal promoter of the human PNRC gene. Within this promoter region, there is one putative binding site for the transcription factor NFY (nuclear factor Y). EMSA and ChIP analyses demonstrated the specific binding of NFY protein to the human PNRC promoter. Transient transfection and luciferase assays further revealed that over-expression of NFY represses promoter activity of PNRC gene in a dose-dependent manner. These results indicate that the transcription factor NFY specifically binds to promoter region of PNRC and negatively regulates the transcription of the human PNRC gene.

Interaction of Glutathione and Sodium Selenite In vitro Investigated by Electrospray Ionization Tandem Mass Spectrometry

Cui, S.-Y., Jin, H., Kim, S.-J., Kumar, A. P., Lee, Y.-I. — 2008-05-06

Selenite has been found to be an active catalyst for the oxidation of sulphhydryl compounds, such as glutathione (GSH). Considering the biological importance of GSH oxidation and the implication of sulphhydryl compounds in selenium poisoning and other biological activities, more information on selenite oxidation of GSH in enzyme-free conditions is desirable. Herein, we describe glutathione and sodium selenite simply mixed in aqueous solutions. The interaction products and transient intermediate are identified and characterized using electrospray ionization (ESI) tandem mass spectrometry. In the first step, GSH directly reacts to form diglutathione (GSSG) and unstable selenodiglutathione (GS-Se-SG). Then selenodiglutathione further reacted with remaining GSH to form diglutathione and elemental selenium, Se⁰. As the amount of GSSG significantly increased or acidity of the solution increased, the redox potential of glutathione [E^0'(GSSG/2GSH) –250 mV (NHE)] significantly shifted to the positive direction. This makes the GSSG react with elemental selenium formed in the solution, which can be demonstrated by another unstable intermediate ion identified at m/z 418 by mass spectrometry with the elemental composition of [GSS-Se]^–. The reaction mechanism between GSH and sodium selenite has been proposed according to the ESI-MS, NMR and UV-vis spectrometric measurements.

Physical and Functional Interactions between STAT5 and Runx Transcription Factors

Ogawa, S., Satake, M., Ikuta, K. — 2008-05-06

The signal transducers and activators of transcription (STAT) and the Runt-related (Runx) are two of major transcription factor families that play essential roles in lymphocyte development. Although the interaction of Runx2 with STAT1 and STAT3 has been reported before, the interaction between STAT5 and Runx family proteins has not been characterized. In this study, we first showed that STAT5 physically interacts with Runx1, Runx2 and Runx3 by co-immunoprecipitation experiments. The Runt domain of Runx proteins and the DNA-binding domain and -helix loop structure of STAT5 are responsible for the interaction. When expressed in CHO cells, STAT5 inhibits the nuclear localization of Runx proteins and retains them in the cytoplasm. In addition, we showed by reporter assay that the interaction between STAT5 and Runx proteins mutually inhibits their transcriptional activity. Furthermore, Runx proteins inhibit the DNA-binding activity of STAT5. Finally, we found that Runx proteins suppress the transcription of an endogenous STAT5 target gene, cytokine-inducible SH2 protein-1, in an interleukin-3-dependent pro-B cell line, Ba/F3. These results collectively suggested that STAT5 and Runx proteins physically and functionally interact to mutually inhibit their transcriptional activity. Thus, this study implies a potential role of the STAT5–Runx interaction in lymphocyte development.

Cloning, Expression, Purification and Characterization of an Isotype of Clytin, a Calcium-Binding Photoprotein from the Luminous Hydromedusa Clytia gregarium

Inouye, S. — 2008-05-06

The cDNA for an isotype of clytin, a calcium-binding photoprotein from the luminous jellyfish Clytia gregarium, was identified and named clytin-II. The histidine-tagged apoprotein of clytin-II expressed into the periplasmic space of Escherichia coli cells was isolated by nickel chelate affinity chromatography. Recombinant clytin-II regenerated from apoprotein by incubation with coelenterazine was purified. The yield of purified clytin-II was 13 mg from 2 l of cultured cells with purity >95%. The luminescence properties of clytin-II were characterized by comparison with clytin-I and aequorin, and semi-synthetic clytin-II was also prepared. The initial luminescence intensity of clytin-II triggered by Ca²⁺ was 4.5 times higher than that of clytin-I and aequorin, but the luminescence capacity was close to clytin-I and aequorin. Thus, clytin-II is a useful protein, showing high sensitivity in the signal-to-noise ratio of luminescence intensity.