72 results on '"Wada, Y."'
Search Results
2. “Module” substitution in hemoglobin subunits. Preparation and characterization of a “chimera beta alpha-subunit”.
- Author
-
Wakasugi, K., primary, Ishimori, K., additional, Imai, K., additional, Wada, Y., additional, and Morishima, I., additional
- Published
- 1994
- Full Text
- View/download PDF
3. Charged collagen structure mediates the recognition of negatively charged macromolecules by macrophage scavenger receptors.
- Author
-
Doi, T., primary, Higashino, K., additional, Kurihara, Y., additional, Wada, Y., additional, Miyazaki, T., additional, Nakamura, H., additional, Uesugi, S., additional, Imanishi, T., additional, Kawabe, Y., additional, and Itakura, H., additional
- Published
- 1993
- Full Text
- View/download PDF
4. Structure, organization, and chromosomal mapping of the human macrophage scavenger receptor gene.
- Author
-
Emi, M., primary, Asaoka, H., additional, Matsumoto, A., additional, Itakura, H., additional, Kurihara, Y., additional, Wada, Y., additional, Kanamori, H., additional, Yazaki, Y., additional, Takahashi, E., additional, and Lepert, M., additional
- Published
- 1993
- Full Text
- View/download PDF
5. Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. I. Isolation and characterization of two classes of vam mutants.
- Author
-
Wada, Y, primary, Ohsumi, Y, additional, and Anraku, Y, additional
- Published
- 1992
- Full Text
- View/download PDF
6. Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. II. VAM7, a gene for regulating morphogenic assembly of the vacuoles.
- Author
-
Wada, Y, primary and Anraku, Y, additional
- Published
- 1992
- Full Text
- View/download PDF
7. Three subunit a isoforms of mouse vacuolar H(+)-ATPase. Preferential expression of the a3 isoform during osteoclast differentiation.
- Author
-
Toyomura, T, Oka, T, Yamaguchi, C, Wada, Y, and Futai, M
- Abstract
Vacuolar H(+)-ATPase (V-ATPase) is a multi-subunit enzyme with a membrane peripheral catalytic (V(1)) and an intrinsic (V(o)) sector. We have identified three cDNA clones coding for isoforms of mouse V(o) subunit a (a1, a2, and a3). They exhibit 48-52% identity with each other and high similarity to subunit a of other species. The a1 isoform was mainly expressed in brain and liver. The a2 isoform was observed in heart and kidney in addition to brain and liver. Transcripts for the a3 isoform were strongly expressed in heart and liver. The a3 isoform was induced during osteoclast differentiation, and localized in the plasma membrane and cytoplasmic filamentous structures. In contrast to a3, the a1 isoform was constitutively expressed and localized in the cytoplasmic endomembrane compartments of the same cells. These findings suggest that the a3 isoform is a component of the plasma membrane V-ATPase essential for bone resorption.
- Published
- 2000
8. Syntaxin 7 mediates endocytic trafficking to late endosomes.
- Author
-
Nakamura, N, Yamamoto, A, Wada, Y, and Futai, M
- Abstract
The lysosome functions are ensured by accurate membrane trafficking in the cell. We found that mouse syntaxin 7 could complement yeast vam3 and pep12 mutants defective in docking/fusion to vacuolar and prevacuolar membranes, respectively. Immunohistochemical studies showed that syntaxin 7 is localized to late endosomes, but not to early endosomes. Induced expression of mutant syntaxin 7 blocked endocytic transport from early to late endosomes but did not block the transport of cathepsin D and lamp-2 from the trans-Golgi network to lysosomes. Thus, syntaxin 7 mediates the endocytic trafficking from early endosomes to late endosomes and lysosomes. These results also suggest that the biosynthetic pathway utilizes a different machinery from that of the endocytic pathway in the docking/fusion to late endosomes.
- Published
- 2000
9. Regulation and reversibility of vacuolar H(+)-ATPase.
- Author
-
Hirata, T, Nakamura, N, Omote, H, Wada, Y, and Futai, M
- Abstract
Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.
- Published
- 2000
10. Vam2/Vps41p and Vam6/Vps39p are components of a protein complex on the vacuolar membranes and involved in the vacuolar assembly in the yeast Saccharomyces cerevisiae.
- Author
-
Nakamura, N, Hirata, A, Ohsumi, Y, and Wada, Y
- Abstract
The VAM2/VPS41 and VAM6/VPS39 were shown to encode hydrophilic proteins of 113 and 123 kDa, respectively. Deletion of the VAM2 and VAM6 functions resulted in accumulation of numerous vacuole-related structures of 200-400 nm in diameter that were much smaller than the normal vacuoles. Loss of functions of Vam2p and Vam6p resulted in inefficient processings of a set of vacuolar proteins, including proteinase A, proteinase B, and carboxypeptidase Y (CPY), and in severely defective maturation of another vacuolar protein, alkaline phosphatase. A part of newly synthesized CPY was missorted to the cell surface in the mutants. Epitope-tagged versions of Vam2p and Vam6p retained their functions, and they were found mostly in sedimentable fractions. The epitope-tagged Vam2p and Vam6p remained in the sedimentable fractions in the presence of Triton X-100, but they were extracted by urea or NaCl. Vam2p and Vam6p were cross-linked by the treatment of a chemical cross-linker. These observations indicated that Vam2p and Vam6p physically interact with each other and exist as components of a large protein complex. Vam6p fused with a green fluorescent protein were highly accumulated in a few specific regions of the vacuolar membranes. Large portions of Vam2p and Vam6p were fractionated into a vacuolar enriched fraction, indicating that they were localized mainly in the vacuolar membranes. These results showed that Vam2p and Vam6p execute their function in the vacuolar assembly as the components of a protein complex reside on the vacuolar membranes.
- Published
- 1997
11. Effects of glucose and insulin on rat apolipoprotein A-I gene expression.
- Author
-
Murao, K, Wada, Y, Nakamura, T, Taylor, A H, Mooradian, A D, and Wong, N C
- Abstract
We have examined the regulation of apolipoprotein A-I (apoA-I) gene expression in response to glucose and insulin. In Hep G2 cells, endogenous apoA-I mRNA was suppressed by one-half or induced 2-fold following 48 h of exposure to high concentrations of glucose (22.4 mM) or insulin (100 microunits/ml), respectively, compared with control. Transcriptional activity of the rat apoA-I promoter (-474 to -7) in Hep G2 cells paralleled endogenous mRNA expression, and this activity was dependent on the dose of glucose or insulin. Deletional analysis showed that a 50-base pair fragment spanning -425 to -376 of the promoter mediated the effects of both insulin and glucose. Within this DNA fragment there is a motif (-411 to -404) that is homologous to a previously identified insulin response core element (IRCE). Mutation of this motif abolished not only the induction of the promoter by insulin but also abrogated its suppression by glucose. Electrophoretic mobility shift assay analysis of nuclear extracts from Hep G2 cells revealed IRCE binding activity that formed a duplex with radiolabeled probe. The IRCE binding activity correlated with insulin induction of apoA-I expression. In summary, our data show that glucose decreases and insulin increases apoA-I promoter activity. This effect appears to be mediated by a single cis-acting element.
- Published
- 1998
12. Human SPA-1 gene product selectively expressed in lymphoid tissues is a specific GTPase-activating protein for Rap1 and Rap2. Segregate expression profiles from a rap1GAP gene product.
- Author
-
Kurachi, H, Wada, Y, Tsukamoto, N, Maeda, M, Kubota, H, Hattori, M, Iwai, K, and Minato, N
- Abstract
Mouse Spa-1 gene with a region homologous to the human rap1GAP gene is transcriptionally induced in the lymphocytes by mitogenic stimulation. Herein we have cloned a cDNA for its human counterpart. SPA-1 cDNA encodes a 130-kDa protein (p130(SPA-1)) consisting of proline-rich regions and rap1GAP-related domain followed by a coiled-coil stretch. Baculovirally expressed p130(SPA-1) exhibited GTPase-activating protein (GAP) activity for Rap1 and Rap2, but not for Ras, Rho, Cdc42, Rac, and Ran, with comparable specific activity to the rap1GAP gene product (p85/95(rap1GAP)). In the cells, p130(SPA-1) was mostly localized at the perinuclear membranous region co-localizing with Rap1 and Rap2. Expression of SPA-1 and rap1GAP genes tended to be segregate in various tissues, lymphoid tissues expressing abundant SPA-1 transcript without rap1GAP, while those such as brain, kidney, and pancreas exhibiting rap1GAP mRNA with little SPA-1. Promyelocytic HL-60 cells, which expressed p130(SPA-1) with little p85/95(rap1GAP) in uninduced state, showed progressive decline in p130(SPA-1) and conversely drastic increase in p85/95(rap1GAP) as they ceased from proliferation and differentiated into macrophages by 12-O-tetradecanoylphorbol-13-acetate. These results suggested that products of SPA-1 and rap1GAP genes, albeit comparable GAP activity for Rap1 and Rap2, functioned in the distinct contexts depending on cell types and/or states.
- Published
- 1997
13. In vitro differential expression of human globin genes.
- Author
-
Wada, Y and Noguchi, C T
- Abstract
K562 human erythroleukemia cells can be induced by hemin and other stimuli to produce hemoglobin and actively express epsilon- and gamma-globin genes but not the adult-like beta-globin gene. The control of globin gene expression is regulated by both cis-acting DNA sequences, such as the 5′-promoter region and 3′- and 5′-enhancers, and trans-acting factors. The activation of 5′-promoter DNA sequences mediated by trans-acting factors can be examined in a cell-free system by in vitro transcription. We report here the differential transcriptional activation of globin genes by nuclear extracts from K562 cells without the need of supplemental HeLa whole cell extracts. Transcription of the epsilon-globin gene which is more active in K562 cells than the gamma-globin gene was greater at low nuclear extract concentrations (protein concentration less than 1.5 mg/ml) than the gamma-globin gene. However, neither beta-globin nor insulin promoters (which are not active in intact K562 cells) were active in the nuclear extract in vitro transcription system. The accurate initiation and specific transcription of globin genes by K562 nuclear extracts suggest that trans-acting factors interacting with the 5′-promoter gene sequence regulate in part the extent and specificity of gene expression and that the activity of these factors can be preserved during their isolation.
- Published
- 1988
- Full Text
- View/download PDF
14. The AtVAM3 encodes a syntaxin-related molecule implicated in the vacuolar assembly in Arabidopsis thaliana.
- Author
-
Sato, M H, Nakamura, N, Ohsumi, Y, Kouchi, H, Kondo, M, Hara-Nishimura, I, Nishimura, M, and Wada, Y
- Abstract
The vacuole constitutes a large compartment in plant and fungal cells. The VAM3 gene of Saccharomyces cerevisiae encodes a syntaxin-related protein required for vacuolar assembly. An Arabidopsis thaliana cDNA library, designed for expression in S. cerevisiae, was screened for cDNAs able to complement defective vacuolar assembly of the Deltavam3 mutation. One cDNA, encoding a 33-kDa protein with structural similarities to the other syntaxins, was identified. The product of AtVAM3 (AtVam3p) was expressed in various tissues including roots, leaves, inflorescence stems, flower buds, and young siliques. The AtVAM3 transcripts were abundant in undifferentiated cells in the meristematic region. AtVam3p fractionated predominantly to an 8,000 x g pellet fraction where a vacuolar membrane protein H+-translocating inorganic pyrophosphatase (H+-PPase) also fractionated. Immunoelectron microscopy showed that AtVam3p was localized to restricted regions on the vacuolar membranes. We propose that AtVam3p provides the t-SNARE function in the vacuolar assembly in A. thaliana.
- Published
- 1997
15. Molecular cloning and expression of cDNA encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase.
- Author
-
Honke, K, Tsuda, M, Hirahara, Y, Ishii, A, Makita, A, and Wada, Y
- Abstract
We have isolated a cDNA clone encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11). Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from human renal cancer cell cDNA by the polymerase chain reaction method. The amplified cDNA fragment was then used as probe to screen a human renal cancer cell cDNA library. The isolated cDNA clone contained an open reading frame encoding 423 amino acids including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. There is no significant homology between this sequence and either the sulfotransferases cloned to date or other known proteins. Northern blot analysis demonstrated that a 1.9-kilobase mRNA was unique to renal cancer cells. When the cDNA was inserted into the expression vector pSVK3 and transfected into COS-1 cells, galactosylceramide sulfotransferase activity in the transfected cells increased from 8- to 16-fold over that of controls, and the enzyme product, sulfatide, was expressed on the transformed cells.
- Published
- 1997
16. Molecular cloning, sequencing, and expression of the genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca.
- Author
-
Tobimatsu, T, Hara, T, Sakaguchi, M, Kishimoto, Y, Wada, Y, Isoda, M, Sakai, T, and Toraya, T
- Abstract
The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.
- Published
- 1995
17. Vacuolar ion channel of the yeast, Saccharomyces cerevisiae.
- Author
-
Wada, Y, Ohsumi, Y, Tanifuji, M, Kasai, M, and Anraku, Y
- Abstract
Ionic flux is most likely to regulate the chemiosmotic potential differences across vacuolysosomal membranes in animal, plant, and fungal cells. We found a membrane potential-dependent cation channel in yeast vacuolar membrane and characterized its several features by an electrophysiological method using artificial planar bilayer membranes incorporated with isolated yeast vacuolar membrane vesicles. This ion channel conducts K+ (single channel conductance, 435 pS in 0.3 M KCl) and several other monovalent cations (Cs+, Na+, and Li+) with broad selectivity, but does not conduct Cl-. The opening of this channel is regulated by the membrane potential and the presence of calcium ion on the cytoplasmic face. These characteristics suggested that the vacuolar cation channel functions as one of essential components for formation and regulation of the chemical and electrical potential differences across the vacuolar membrane.
- Published
- 1987
- Full Text
- View/download PDF
18. Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages.
- Author
-
Noda K, Lu SL, Chen S, Tokuda K, Li Y, Hao F, Wada Y, Sun-Wada GH, Murakami S, Fukuda M, Itoh T, and Noda T
- Abstract
Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
19. Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle.
- Author
-
Torii S, Orba Y, Sasaki M, Tabata K, Wada Y, Carr M, Hobson-Peters J, Hall RA, Takada A, Fukuhara T, Matsuura Y, Hall WW, and Sawa H
- Subjects
- Chikungunya virus genetics, HEK293 Cells, Humans, Chikungunya Fever metabolism, Chikungunya Fever virology, Chikungunya virus growth & development, Chikungunya virus metabolism, Endosomal Sorting Complexes Required for Transport metabolism, Virus Replication genetics
- Abstract
Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Torii et al.)
- Published
- 2020
- Full Text
- View/download PDF
20. Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells.
- Author
-
McGuire CM, Collins MP, Sun-Wada G, Wada Y, and Forgac M
- Subjects
- Breast Neoplasms genetics, Breast Neoplasms pathology, CRISPR-Cas Systems, Cell Line, Tumor, Cell Membrane enzymology, Enzyme Inhibitors pharmacology, Female, Humans, Isoenzymes antagonists & inhibitors, Isoenzymes genetics, Neoplasm Metastasis, RNA, Messenger genetics, Vacuolar Proton-Translocating ATPases antagonists & inhibitors, Vacuolar Proton-Translocating ATPases genetics, Breast Neoplasms enzymology, Isoenzymes metabolism, Neoplasm Invasiveness, Vacuolar Proton-Translocating ATPases metabolism
- Abstract
The vacuolar H
+ -ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype., (© 2019 McGuire et al.)- Published
- 2019
- Full Text
- View/download PDF
21. The Muscular Dystrophy Gene TMEM5 Encodes a Ribitol β1,4-Xylosyltransferase Required for the Functional Glycosylation of Dystroglycan.
- Author
-
Manya H, Yamaguchi Y, Kanagawa M, Kobayashi K, Tajiri M, Akasaka-Manya K, Kawakami H, Mizuno M, Wada Y, Toda T, and Endo T
- Subjects
- Cell Line, Dystroglycans chemistry, Dystroglycans genetics, Glycosylation, Humans, Membrane Proteins chemistry, Membrane Proteins genetics, Muscular Dystrophies genetics, Mutation, Missense, Nuclear Magnetic Resonance, Biomolecular, Pentosyltransferases, Transferases (Other Substituted Phosphate Groups) chemistry, Transferases (Other Substituted Phosphate Groups) genetics, Dystroglycans metabolism, Membrane Proteins metabolism, Muscular Dystrophies metabolism, Transferases (Other Substituted Phosphate Groups) metabolism
- Abstract
A defect in O-mannosyl glycan is the cause of α-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant α-dystroglycan (α-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAβ1-3Xylα1]
n -3GlcAβ1-4Xyl-Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4 (phospho-6)Manα1-, which is required for the binding of α-DG to extracellular matrix ligands, has been proposed. However, the linkage of the first Xyl residue to ribitol 5-phosphate (Rbo5P) is not clear. TMEM5 is a gene product responsible for α-dystroglycanopathy and was reported as a potential enzyme involved in this linkage formation, although the experimental evidence is still incomplete. Here, we report that TMEM5 is a xylosyltransferase that forms the Xylβ1-4Rbo5P linkage on O-mannosyl glycan. The anomeric configuration and linkage position of the product (β1,4 linkage) was determined by NMR analysis. The introduction of two missense mutations in TMEM5 found in α-dystroglycanopathy patients impaired xylosyltransferase activity. Furthermore, the disruption of the TMEM5 gene by CRISPR/Cas9 abrogated the elongation of the (-3GlcAβ1-3Xylα1-) unit on O-mannosyl glycan. Based on these results, we concluded that TMEM5 acts as a UDP-d-xylose:ribitol-5-phosphate β1,4-xylosyltransferase in the biosynthetic pathway of O-mannosyl glycan., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2016
- Full Text
- View/download PDF
22. The absence of core fucose up-regulates GnT-III and Wnt target genes: a possible mechanism for an adaptive response in terms of glycan function.
- Author
-
Kurimoto A, Kitazume S, Kizuka Y, Nakajima K, Oka R, Fujinawa R, Korekane H, Yamaguchi Y, Wada Y, and Taniguchi N
- Subjects
- Animals, Cells, Cultured, Mice, Mice, Inbred C57BL, Mice, Knockout, Oligonucleotide Array Sequence Analysis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Fucose metabolism, N-Acetylglucosaminyltransferases genetics, Polysaccharides metabolism, Up-Regulation, Wnt Proteins metabolism
- Abstract
Glycans play key roles in a variety of protein functions under normal and pathological conditions, but several glycosyltransferase-deficient mice exhibit no or only mild phenotypes due to redundancy or compensation of glycan functions. However, we have only a limited understanding of the underlying mechanism for these observations. Our previous studies indicated that 70% of Fut8-deficient (Fut8(-/-)) mice that lack core fucose structure die within 3 days after birth, but the remainder survive for up to several weeks although they show growth retardation as well as emphysema. In this study, we show that, in mouse embryonic fibroblasts (MEFs) from Fut8(-/-) mice, another N-glycan branching structure, bisecting GlcNAc, is specifically up-regulated by enhanced gene expression of the responsible enzyme N-acetylglucosaminyltransferase III (GnT-III). As candidate target glycoproteins for bisecting GlcNAc modification, we confirmed that level of bisecting GlcNAc on β1-integrin and N-cadherin was increased in Fut8(-/-) MEFs. Moreover using mass spectrometry, glycan analysis of IgG1 in Fut8(-/-) mouse serum demonstrated that bisecting GlcNAc contents were also increased by Fut8 deficiency in vivo. As an underlying mechanism, we found that in Fut8(-/-) MEFs Wnt/β-catenin signaling is up-regulated, and an inhibitor against Wnt signaling was found to abrogate GnT-III expression, indicating that Wnt/β-catenin is involved in GnT-III up-regulation. Furthermore, various oxidative stress-related genes were also increased in Fut8(-/-) MEFs. These data suggest that Fut8(-/-) mice adapted to oxidative stress, both ex vivo and in vivo, by inducing various genes including GnT-III, which may compensate for the loss of core fucose functions.
- Published
- 2014
- Full Text
- View/download PDF
23. Transglutaminase 2-dependent deamidation of glyceraldehyde-3-phosphate dehydrogenase promotes trophoblastic cell fusion.
- Author
-
Iwai K, Shibukawa Y, Yamazaki N, and Wada Y
- Subjects
- Amino Acid Sequence, Cell Fusion, Cell Line, Tumor, Enzyme Inhibitors pharmacology, GTP-Binding Proteins antagonists & inhibitors, Gene Knockdown Techniques, Giant Cells cytology, Giant Cells drug effects, Giant Cells metabolism, Glutamic Acid metabolism, Glutamine metabolism, Glyceraldehyde-3-Phosphate Dehydrogenases chemistry, Glyceraldehyde-3-Phosphate Dehydrogenases isolation & purification, Humans, Molecular Sequence Data, Mutant Proteins metabolism, Protein Glutamine gamma Glutamyltransferase 2, Protein Processing, Post-Translational drug effects, Transglutaminases antagonists & inhibitors, Trophoblasts drug effects, Amides metabolism, GTP-Binding Proteins metabolism, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Transglutaminases metabolism, Trophoblasts cytology, Trophoblasts enzymology
- Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.
- Published
- 2014
- Full Text
- View/download PDF
24. Chondroitin sulfate proteoglycan tenascin-R regulates glutamate uptake by adult brain astrocytes.
- Author
-
Okuda H, Tatsumi K, Morita S, Shibukawa Y, Korekane H, Horii-Hayashi N, Wada Y, Taniguchi N, and Wanaka A
- Subjects
- Age Factors, Animals, Animals, Newborn, Astrocytes ultrastructure, Cells, Cultured, Cerebral Cortex metabolism, Chondroitin Sulfates metabolism, Excitatory Amino Acid Transporter 1 metabolism, Glycoproteins metabolism, Male, Mice, Mice, Inbred C57BL, RNA, Messenger metabolism, RNA, Small Interfering genetics, Tenascin genetics, Astrocytes metabolism, Cerebral Cortex cytology, Glutamic Acid metabolism, Homeostasis physiology, Tenascin metabolism
- Abstract
In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.
- Published
- 2014
- Full Text
- View/download PDF
25. Suppression of heregulin β signaling by the single N-glycan deletion mutant of soluble ErbB3 protein.
- Author
-
Takahashi M, Hasegawa Y, Ikeda Y, Wada Y, Tajiri M, Ariki S, Takamiya R, Nishitani C, Araki M, Yamaguchi Y, Taniguchi N, and Kuroki Y
- Subjects
- Amino Acid Substitution, Antineoplastic Agents pharmacology, Cell Line, Tumor, ErbB Receptors genetics, ErbB Receptors metabolism, Humans, Lapatinib, Neuregulin-1 genetics, Protein Structure, Tertiary, Quinazolines pharmacology, Receptor, ErbB-2 genetics, Receptor, ErbB-2 metabolism, Receptor, ErbB-3 genetics, Receptor, ErbB-4, MAP Kinase Signaling System, Mutation, Missense, Neuregulin-1 metabolism, Protein Multimerization, Receptor, ErbB-3 metabolism
- Abstract
Heregulin signaling is involved in various tumor proliferations and invasions; thus, receptors of heregulin are targets for the cancer therapy. In this study we examined the suppressing effects of extracellular domains of ErbB2, ErbB3, and ErbB4 (soluble ErbB (sErbB)) on heregulin β signaling in human breast cancer cell line MCF7. It was found that sErbB3 suppresses ligand-induced activation of ErbB receptors, PI3K/Akt and Ras/Erk pathways most effectively; sErbB2 scarcely suppresses ligand-induced signaling, and sErbB4 suppresses receptor activation at ∼10% efficiency of sErbB3. It was revealed that sErbB3 does not decrease the effective ligands but decreases the effective receptors. By using small interfering RNA (siRNA) for ErbB receptors, we determined that sErbB3 suppresses the heregulin β signaling by interfering ErbB3-containing heterodimers including ErbB2/ErbB3. By introducing the mutation of N418Q to sErbB3, the signaling-inhibitory effects were increased by 2-3-fold. Moreover, the sErbB3 N418Q mutant enhanced anticancer effects of lapatinib more effectively than the wild type. We also determined the structures of N-glycan on Asn-418. Results suggested that the N-glycan-deleted mutant of sErbB3 suppresses heregulin signaling via ErbB3-containing heterodimers more effectively than the wild type. Thus, we demonstrated that the sErbB3 N418Q mutant is a potent inhibitor for heregulin β signaling.
- Published
- 2013
- Full Text
- View/download PDF
26. Mechanism for p38α-mediated experimental autoimmune encephalomyelitis.
- Author
-
Namiki K, Matsunaga H, Yoshioka K, Tanaka K, Murata K, Ishida J, Sakairi A, Kim J, Tokuhara N, Shibakawa N, Shimizu M, Wada Y, Tokunaga Y, Shigetomi M, Hagihara M, Kimura S, Sudo T, Fukamizu A, and Kasuya Y
- Subjects
- Animals, Electrophoretic Mobility Shift Assay, Encephalomyelitis, Autoimmune, Experimental drug therapy, Encephalomyelitis, Autoimmune, Experimental genetics, Enzyme Inhibitors therapeutic use, Female, Interleukin-17 genetics, Interleukin-17 metabolism, Male, Mice, Mitogen-Activated Protein Kinase 14 antagonists & inhibitors, Mitogen-Activated Protein Kinase 14 genetics, Promoter Regions, Genetic, RNA Stability genetics, Encephalomyelitis, Autoimmune, Experimental metabolism, Mitogen-Activated Protein Kinase 14 metabolism
- Abstract
One of the mitogen-activated protein kinases, p38, has been found to play a crucial role in various inflammatory responses. In this study, we analyzed the roles of p38α in multiple sclerosis, using an animal model, experimental autoimmune encephalomyelitis (EAE). p38α(+/-) mice (p38α(-/-) showed embryonic lethality) showed less severe neurological signs than WT mice. Adoptive transfer of lymph node cells (LNC) from sensitized WT mice with MOG(35-55) to naive WT-induced EAE was much more severe compared with the case using LNC from sensitized p38α(+/-) mice. Comprehensive analysis of cytokines from MOG(35-55)-challenged LNC by Western blot array revealed that production of IL-17 was significantly reduced by a single copy disruption of the p38α gene or a p38 inhibitor. Likewise, by a luciferase reporter assay, an electrophoresis mobility shift assay, and characterization of the relationship between p38 activity and IL-17 mRNA expression, we confirmed that p38 positively regulates transcription of the Il17 gene. Furthermore, oral administration of a highly specific p38α inhibitor (UR-5269) to WT mice at the onset of EAE markedly suppressed the progression of EAE compared with a vehicle group. These results suggest that p38α participates in the pathogenesis of EAE through IL-17 induction.
- Published
- 2012
- Full Text
- View/download PDF
27. Absence of post-phosphoryl modification in dystroglycanopathy mouse models and wild-type tissues expressing non-laminin binding form of α-dystroglycan.
- Author
-
Kuga A, Kanagawa M, Sudo A, Chan YM, Tajiri M, Manya H, Kikkawa Y, Nomizu M, Kobayashi K, Endo T, Lu QL, Wada Y, and Toda T
- Subjects
- Animals, Disease Models, Animal, Dystroglycans genetics, Female, Humans, Laminin genetics, Lung metabolism, Male, Mice, Mice, Transgenic, Muscular Dystrophies genetics, Pentosyltransferases, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Proteins genetics, Proteins metabolism, Testis metabolism, Transferases, Dystroglycans metabolism, Laminin metabolism, Muscular Dystrophies metabolism
- Abstract
α-Dystroglycan (α-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of α-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of α-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, FKRP (fukutin-related protein) is also involved in the post-phosphoryl modification of O-mannose on α-DG. Furthermore, we have found that the glycosylation status of α-DG in lung and testis is minimally affected by defects in fukutin, LARGE, or FKRP. α-DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α-DG functional expression as a laminin receptor in normal tissues and cells.
- Published
- 2012
- Full Text
- View/download PDF
28. Brain endothelial cells produce amyloid {beta} from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form.
- Author
-
Kitazume S, Tachida Y, Kato M, Yamaguchi Y, Honda T, Hashimoto Y, Wada Y, Saito T, Iwata N, Saido T, and Taniguchi N
- Subjects
- Alternative Splicing genetics, Alzheimer Disease genetics, Alzheimer Disease pathology, Amyloid Precursor Protein Secretases genetics, Amyloid Precursor Protein Secretases metabolism, Amyloid beta-Protein Precursor genetics, Animals, Biomarkers metabolism, Brain pathology, Cell Line, Female, Humans, Male, Mice, Mice, Transgenic, Middle Aged, Neurons pathology, Alzheimer Disease metabolism, Amyloid beta-Protein Precursor metabolism, Brain metabolism, Neurons metabolism, Protein Processing, Post-Translational
- Abstract
Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.
- Published
- 2010
- Full Text
- View/download PDF
29. An N-glycosylation site on the beta-propeller domain of the integrin alpha5 subunit plays key roles in both its function and site-specific modification by beta1,4-N-acetylglucosaminyltransferase III.
- Author
-
Sato Y, Isaji T, Tajiri M, Yoshida-Yamamoto S, Yoshinaka T, Somehara T, Fukuda T, Wada Y, and Gu J
- Subjects
- Animals, CHO Cells, Cell Adhesion physiology, Cricetinae, Cricetulus, Dimerization, Fibronectins, Glycosylation, HeLa Cells, Humans, Integrin alpha5 genetics, Integrin alpha5beta1 genetics, Integrin alpha5beta1 metabolism, Mutation, N-Acetylglucosaminyltransferases genetics, Cell Movement physiology, Integrin alpha5 metabolism, N-Acetylglucosaminyltransferases metabolism
- Abstract
Recently we reported that N-glycans on the beta-propeller domain of the integrin alpha5 subunit (S-3,4,5) are essential for alpha5beta1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type alpha5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the alpha5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type alpha5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the alpha5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.
- Published
- 2009
- Full Text
- View/download PDF
30. N-Glycosylation of laminin-332 regulates its biological functions. A novel function of the bisecting GlcNAc.
- Author
-
Kariya Y, Kato R, Itoh S, Fukuda T, Shibukawa Y, Sanzen N, Sekiguchi K, Wada Y, Kawasaki N, and Gu J
- Subjects
- Basement Membrane enzymology, Basement Membrane pathology, Cell Adhesion genetics, Cell Adhesion Molecules genetics, Cell Line, Tumor, Glycosylation, Humans, Integrin alpha3beta1 genetics, Integrin alpha3beta1 metabolism, N-Acetylglucosaminyltransferases genetics, Neoplasm Invasiveness genetics, Neoplasm Invasiveness pathology, Neoplasm Proteins genetics, Polysaccharides genetics, Polysaccharides metabolism, Protein Subunits genetics, Protein Subunits metabolism, Skin enzymology, Skin pathology, Skin Neoplasms genetics, Skin Neoplasms pathology, Kalinin, Cell Adhesion Molecules metabolism, Cell Movement genetics, N-Acetylglucosaminyltransferases metabolism, Neoplasm Proteins metabolism, Skin Neoplasms enzymology
- Abstract
Laminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of beta1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and beta1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnT-V-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired alpha3beta1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer.
- Published
- 2008
- Full Text
- View/download PDF
31. Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence.
- Author
-
Fukami M, Wada Y, Okada M, Kato F, Katsumata N, Baba T, Morohashi K, Laporte J, Kitagawa M, and Ogata T
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors genetics, Cell Line, Tumor, Cell Nucleus genetics, Cell Nucleus pathology, Codon, Nonsense, DNA-Binding Proteins genetics, Humans, Hypospadias genetics, Hypospadias metabolism, Hypospadias pathology, Leydig Cell Tumor genetics, Leydig Cell Tumor metabolism, Leydig Cell Tumor pathology, Male, Mice, Nerve Tissue Proteins genetics, Nuclear Proteins genetics, Open Reading Frames genetics, RNA Stability genetics, RNA, Small Interfering genetics, Repressor Proteins, Steroidogenic Factor 1 genetics, Steroidogenic Factor 1 metabolism, Trans-Activators, Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors metabolism, Cell Nucleus metabolism, DNA-Binding Proteins metabolism, Nerve Tissue Proteins metabolism, Nuclear Proteins metabolism, Promoter Regions, Genetic genetics, Testosterone biosynthesis, Transcription Factors metabolism, Transcriptional Activation genetics
- Abstract
Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wild-type CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure.
- Published
- 2008
- Full Text
- View/download PDF
32. Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the beta subunit.
- Author
-
Nakanishi-Matsui M, Kashiwagi S, Ubukata T, Iwamoto-Kihara A, Wada Y, and Futai M
- Subjects
- Adenosine Triphosphate metabolism, Amino Acid Substitution, Bacterial Proton-Translocating ATPases genetics, Bacterial Proton-Translocating ATPases metabolism, Catalytic Domain genetics, Escherichia coli genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Hydrolysis, Mutation, Missense, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Adenosine Triphosphate chemistry, Bacterial Proton-Translocating ATPases chemistry, Escherichia coli enzymology, Escherichia coli Proteins chemistry, Models, Molecular
- Abstract
A complex of gamma, epsilon, and c subunits rotates in ATP synthase (FoF(1)) coupled with proton transport. A gold bead connected to the gamma subunit of the Escherichia coli F(1) sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the beta subunit key region; consistent with its bulk phase ATPase activities, F(1) with the Ser-174 to Phe substitution (betaS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120 degrees positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/product release" dwell, since the ATP concentration used for the assay was approximately 30-fold higher than the K(m) value for ATP. A betaGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of betaS174F. The revertant (betaG149A/betaS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant betaG149A/betaS174L. These results indicate that the domain between beta-sheet 4 (betaSer-174) and P-loop (betaGly-149) is important to drive rotation.
- Published
- 2007
- Full Text
- View/download PDF
33. N-glycosylation of the beta-propeller domain of the integrin alpha5 subunit is essential for alpha5beta1 heterodimerization, expression on the cell surface, and its biological function.
- Author
-
Isaji T, Sato Y, Zhao Y, Miyoshi E, Wada Y, Taniguchi N, and Gu J
- Subjects
- Animals, CHO Cells, Calnexin metabolism, Cell Adhesion, Cell Movement, Cricetinae, Dimerization, Fibronectins metabolism, Glycosylation, Humans, Integrin alpha5 genetics, Mutation genetics, Protein Binding, Protein Processing, Post-Translational, Protein Subunits genetics, Protein Subunits metabolism, Cell Membrane metabolism, Integrin alpha5 metabolism, Integrin beta1 metabolism
- Abstract
The N-glycosylation of integrin alpha5beta1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the alpha5 integrin. Removal of the putative N-glycosylation sites on the beta-propeller, Thigh, Calf-1, or Calf-2 domains of the alpha5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1-5) on the beta-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the alpha5 subunit carrying only three potential N-glycosylation sites (3-5 sites) on the beta-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Delta3-5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the beta-propeller domain of the alpha5 subunit is essential for heterodimerization and biological functions of alpha5beta1 integrin and might also be useful for studies of the molecular structure.
- Published
- 2006
- Full Text
- View/download PDF
34. Stochastic high-speed rotation of Escherichia coli ATP synthase F1 sector: the epsilon subunit-sensitive rotation.
- Author
-
Nakanishi-Matsui M, Kashiwagi S, Hosokawa H, Cipriano DJ, Dunn SD, Wada Y, and Futai M
- Subjects
- Adenosine Triphosphate metabolism, Fluorescence Resonance Energy Transfer, Rotation, ATPase Inhibitory Protein, Proteins physiology, Proton-Translocating ATPases chemistry
- Abstract
The gamma subunit of the ATP synthase F(1) sector rotates at the center of the alpha(3)beta(3) hexamer during ATP hydrolysis. A gold bead (40-200 nm diameter) was attached to the gamma subunit of Escherichia coli F(1), and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40- and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is approximately 13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F(1) sector rotates much faster than expected from the bulk of ATPase activity, and that approximately 10% of the F(1) molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The epsilon subunit inhibited rotation and ATPase, whereas epsilon fused through its carboxyl terminus to cytochrome b(562) showed no effect. The epsilon subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.
- Published
- 2006
- Full Text
- View/download PDF
35. ATP-dependent rotation of mutant ATP synthases defective in proton transport.
- Author
-
Hosokawa H, Nakanishi-Matsui M, Kashiwagi S, Fujii-Taira I, Hayashi K, Iwamoto-Kihara A, Wada Y, and Futai M
- Subjects
- Blotting, Western, Electrophoresis, Polyacrylamide Gel, Hydrolysis, Mutagenesis, Proton-Translocating ATPases genetics, Protons, Adenosine Triphosphate metabolism, Proton-Translocating ATPases metabolism
- Abstract
During ATP hydrolysis, the gammaepsilon c10 complex (gamma and epsilon subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the alpha3beta3delta ab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.
- Published
- 2005
- Full Text
- View/download PDF
36. The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta1-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
- Author
-
Sirikantaramas S, Morimoto S, Shoyama Y, Ishikawa Y, Wada Y, Shoyama Y, and Taura F
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cannabis chemistry, Cannabis enzymology, Cannabis metabolism, Cells, Cultured, Cloning, Molecular, DNA, Complementary, Dronabinol chemistry, Insecta, Molecular Sequence Data, Oxidation-Reduction, Plant Roots physiology, Nicotiana, Transfection, Cannabis genetics, Dronabinol biosynthesis, Intramolecular Oxidoreductases genetics, Intramolecular Oxidoreductases metabolism
- Abstract
Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.
- Published
- 2004
- Full Text
- View/download PDF
37. The N-terminal region of NTAK/neuregulin-2 isoforms has an inhibitory activity on angiogenesis.
- Author
-
Nakano N, Higashiyama S, Ohmoto H, Ishiguro H, Taniguchi N, and Wada Y
- Subjects
- Amino Acid Sequence, Cell Line, Tumor, Cells, Cultured, Endothelium, Vascular metabolism, Humans, Molecular Sequence Data, Nerve Growth Factors chemistry, Nerve Growth Factors genetics, Phosphorylation, Protein Isoforms chemistry, Protein Isoforms genetics, Retinoblastoma Protein metabolism, Sequence Homology, Amino Acid, Transfection, Endothelium, Vascular cytology, Neovascularization, Physiologic physiology, Nerve Growth Factors physiology, Protein Isoforms physiology
- Abstract
NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2, is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Because ErbB signaling has been implicated in various angiogenic mechanisms, the effect of NTAK (which has at least nine isoforms due to alternative splicing) in angiogenesis is explored. One isoform, NTAKgamma, inhibited cell growth in terms of DNA synthesis and cell numbers in vascular endothelial cells specifically, whereas NTAKalpha and beta had no activity. On the other hand, NTAKgamma secreted by transfected MDA-MB-231 cells inhibited endothelial cell growth, and NTAKgamma expressed in endothelial cells by adenovirus infection suppressed cell growth in a dose-dependent manner. The EGF-like domain of NTAKgamma did not have this activity. The NTAKdelta isoform, which had the Ig-like domain but not the EGF-like domain, inhibited proliferation of endothelial cells. NTAKdelta prevented hyper-phosphorylation of the retinoblastoma tumor suppressor protein and caused G(1) arrest in endothelial cells. Both NTAKgamma and delta isoforms displayed anti-angiogenic activity in the chick embryo chorioallantoic membrane in vivo. These results suggest that the active site of NTAK is localized outside of the EGF-like domain but within the N-terminal region, including the Ig-like domain, of NTAK.
- Published
- 2004
- Full Text
- View/download PDF
38. Molecular dissection of the alpha-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10.
- Author
-
Ido H, Harada K, Futaki S, Hayashi Y, Nishiuchi R, Natsuka Y, Li S, Wada Y, Combs AC, Ervasti JM, and Sekiguchi K
- Subjects
- Amino Acid Sequence, Base Sequence, Binding Sites, DNA Primers, Dystroglycans, Heparin metabolism, Humans, Laminin chemistry, Laminin genetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Deletion, Cytoskeletal Proteins metabolism, Integrin alpha3beta1 metabolism, Integrin alpha6beta1 metabolism, Laminin metabolism, Membrane Glycoproteins metabolism
- Abstract
The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.
- Published
- 2004
- Full Text
- View/download PDF
39. Mouse proton pump ATPase C subunit isoforms (C2-a and C2-b) specifically expressed in kidney and lung.
- Author
-
Sun-Wada GH, Murata Y, Namba M, Yamamoto A, Wada Y, and Futai M
- Subjects
- Acid-Base Equilibrium, Alternative Splicing, Amino Acid Sequence, Blotting, Northern, Cell Membrane enzymology, Conserved Sequence, Humans, Immunohistochemistry, Immunosorbent Techniques, In Situ Hybridization, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes physiology, Kidney physiology, Kinetics, Microscopy, Immunoelectron, Molecular Sequence Data, Organ Specificity, Proton Pumps chemistry, Proton Pumps physiology, Pulmonary Alveoli enzymology, Reverse Transcriptase Polymerase Chain Reaction, Sequence Alignment, Vacuolar Proton-Translocating ATPases chemistry, Vacuolar Proton-Translocating ATPases physiology, Gene Expression, Kidney enzymology, Lung enzymology, Proton Pumps genetics, Vacuolar Proton-Translocating ATPases genetics
- Abstract
The vacuolar-type H+-ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes. We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lung-specific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal alpha and beta intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.
- Published
- 2003
- Full Text
- View/download PDF
40. Preferential de novo methylation of cytosine residues in non-CpG sequences by a domains rearranged DNA methyltransferase from tobacco plants.
- Author
-
Wada Y, Ohya H, Yamaguchi Y, Koizumi N, and Sano H
- Subjects
- Base Sequence, Kinetics, Molecular Sequence Data, Nuclear Proteins metabolism, Nuclear Proteins physiology, Plant Proteins metabolism, Plant Proteins physiology, Plant Structures enzymology, Substrate Specificity, Tissue Distribution, Cytosine metabolism, DNA (Cytosine-5-)-Methyltransferases metabolism, DNA (Cytosine-5-)-Methyltransferases physiology, DNA Methylation, Nicotiana enzymology
- Abstract
In plant DNA, cytosines in symmetric CpG and CpNpG (N is A, T, or C) are thought to be methylated by DNA methyltransferases, MET1 and CMT3, respectively. Cytosines in asymmetric CpNpN are also methylated, and genetic analysis has suggested the responsible enzyme to be domains rearranged methyltransferase (DRM). We cloned a tobacco cDNA, encoding a novel protein consisting of 608 amino acids, that resembled DRMs found in maize and Arabidopsis and designated this as NtDRM1. The protein could be shown to be localized exclusively in the nucleus and exhibit methylation activity toward unmethylated synthetic as well as native DNA samples upon expression in Sf9 insect cells. It also methylated hemimethylated DNA, but the activity was lower than that for unmethylated substrates. Methylation mapping of a 962-bp DNA, treated with NtDRM1 in vitro, directly demonstrated methylation of approximately 70% of the cytosines in methylatable CpNpN and CpNpG sequences but only 10% in CpG. Further analyses indicated that the enzyme apparently non-selectively methylates any cytosines except in CpG, regardless of the adjacent nucleotide at both 5' and 3' ends. Transcripts of NtDRM1 ubiquitously accumulated in all tissues and during the cell cycle in tobacco cultured BY2 cells. These results indicate that NtDRM1 is a de novo cytosine methyltransferase, which actively excludes CpG substrate.
- Published
- 2003
- Full Text
- View/download PDF
41. Involvement of syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA.
- Author
-
Suzuki J, Ohnishi H, Wada A, Hirayama T, Ohno H, Ueda N, Yasuda H, Iiri T, Wada Y, Futai M, and Mashima H
- Subjects
- Blotting, Northern, Blotting, Western, Coloring Agents pharmacology, DNA metabolism, Genes, Dominant, Green Fluorescent Proteins, Humans, Immunohistochemistry, Luminescent Proteins metabolism, Microscopy, Fluorescence, Mutation, Neutral Red pharmacology, Plasmids metabolism, Protein Structure, Tertiary, Qa-SNARE Proteins, RNA, Messenger metabolism, Stomach Neoplasms microbiology, Syntaxin 1, Time Factors, Transfection, Tumor Cells, Cultured, Up-Regulation, Vacuoles metabolism, Bacterial Proteins chemistry, Epithelial Cells metabolism, Helicobacter pylori metabolism, Membrane Proteins metabolism, Stomach cytology
- Abstract
The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.
- Published
- 2003
- Full Text
- View/download PDF
42. Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits.
- Author
-
Hirata T, Iwamoto-Kihara A, Sun-Wada GH, Okajima T, Wada Y, and Futai M
- Subjects
- Actin Cytoskeleton chemistry, Biotin, Histidine, Protein Engineering, Protein Subunits chemistry, Proton-Translocating ATPases, Rotation, Saccharomyces cerevisiae Proteins chemistry, Sequence Homology, Molecular Motor Proteins chemistry, Vacuolar Proton-Translocating ATPases chemistry
- Abstract
Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.
- Published
- 2003
- Full Text
- View/download PDF
43. From lysosomes to the plasma membrane: localization of vacuolar-type H+ -ATPase with the a3 isoform during osteoclast differentiation.
- Author
-
Toyomura T, Murata Y, Yamamoto A, Oka T, Sun-Wada GH, Wada Y, and Futai M
- Subjects
- 3T3 Cells, Animals, Antigens, CD metabolism, Carrier Proteins metabolism, Cell Differentiation, Cell Line, Cells, Cultured, Cytoskeleton metabolism, Endosomes metabolism, Immunohistochemistry, Lysosomal-Associated Membrane Protein 2, Lysosomal Membrane Proteins, Membrane Glycoproteins metabolism, Membrane Proteins metabolism, Mice, Microscopy, Electron, Microscopy, Fluorescence, Microtubules metabolism, Nocodazole pharmacology, Osteoclasts cytology, Protein Isoforms, Protein Structure, Tertiary, Qa-SNARE Proteins, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, Time Factors, Vacuolar Proton-Translocating ATPases metabolism, Cell Membrane metabolism, Lysosomes metabolism, Osteoclasts metabolism
- Abstract
Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification is carried out by vacuolar type H+-ATPase (V-ATPase) localized in the plasma membrane. We have shown that a3, one of the four subunit a isoforms (a1, a2, a3, and a4), is a component of the plasma membrane V-ATPase (Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760-8765). To establish the unique localization of V-ATPase, we have used a murine macrophage cell line, RAW 264.7, that can differentiate into multinuclear osteoclast-like cells on stimulation with RANKL (receptor activator of nuclear factor kappaB ligand). The V-ATPase with the a3 isoform was localized to late endosomes and lysosomes, whereas those with the a1 and a2 isoforms were localized to organelles other than lysosomes. After stimulation, the V-ATPase with the a3 isoform was immunochemically colocalized with lysosome marker lamp2 and was detected in acidic organelles. These organelles were also colocalized with microtubules, and the signals of lamp2 and a3 were dispersed by nocodazole, a microtubule depolymerizer. In RAW-derived osteoclasts cultured on mouse skull pieces, the a3 isoform was transported to the plasma membrane facing the bone and accumulated inside podosome rings. These findings indicate that V-ATPases with the a3 isoform localized in late endosomes/lysosomes are transported to the cell periphery during differentiation and finally assembled into the plasma membrane of mature osteoclasts.
- Published
- 2003
- Full Text
- View/download PDF
44. Differential localization of the vacuolar H+ pump with G subunit isoforms (G1 and G2) in mouse neurons.
- Author
-
Murata Y, Sun-Wada GH, Yoshimizu T, Yamamoto A, Wada Y, and Futai M
- Subjects
- Adenosine Triphosphatases metabolism, Amino Acid Sequence, Animals, Blotting, Northern, Blotting, Western, Cell Membrane metabolism, Cerebellum cytology, Cloning, Molecular, DNA, Complementary metabolism, Gene Deletion, Genetic Complementation Test, Hippocampus cytology, Immunohistochemistry, Kinetics, Liver metabolism, Lysosomes metabolism, Mice, Mice, Inbred ICR, Microscopy, Fluorescence, Molecular Sequence Data, Neurons cytology, Precipitin Tests, Protein Isoforms, Protons, RNA, Messenger metabolism, Sequence Homology, Amino Acid, Subcellular Fractions, Synaptophysin metabolism, Time Factors, Tissue Distribution, Transcription, Genetic, Vacuolar Proton-Translocating ATPases metabolism, Neurons metabolism, Vacuolar Proton-Translocating ATPases biosynthesis, Vacuolar Proton-Translocating ATPases chemistry
- Abstract
Vacuolar H(+)-ATPases (V-ATPases), a family of multimeric proton pumps, are involved in a wide variety of physiological processes. We have identified two mouse genes, Atp6g1 and Atp6g2, encoding the G1 and G2 isoforms of the V-ATPase G subunit, respectively. G1 was distributed ubiquitously in the tissues examined, whereas G2 was specifically distributed in central nervous system neurons. G1 was expressed at an early embryonic stage, whereas G2 transcription was significantly induced at 10.5 days postcoitus (embryonic day 10.5, i.e. 2 days before axon outgrowth). Both G1 and G2 were strongly expressed in cortical and hippocampal neurons, cerebellar granule cells, and Purkinje cells. Immunohistochemistry with isoform-specific antibodies revealed that G2 was localized in cell bodies, dendrites, and axons. In addition, electron microscopy and subcellular fractionation indicated that G2 was localized in synaptic vesicles, whereas G1 was not detectable. G1 and G2 exhibit 62% identity, and both isoforms were immunoprecipitated with the c and A subunits of V-ATPase. G2 could complement the yeast deletion mutant Deltavma10, which lacks the G subunit. The V-ATPases containing the G1 and G2 isoforms, respectively, showed similar K(m)((ATP)) values and maximal velocity. These results indicate that G1 and G2 are bona fide subunits of V-ATPases and that the enzyme with the G2 isoform is involved in synaptic vesicle acidification.
- Published
- 2002
- Full Text
- View/download PDF
45. A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification.
- Author
-
Sun-Wada GH, Imai-Senga Y, Yamamoto A, Murata Y, Hirata T, Wada Y, and Futai M
- Subjects
- Amino Acid Sequence, Animals, Blotting, Northern, Fertility, Hydrogen-Ion Concentration, In Situ Hybridization, Kinetics, Male, Mice, Molecular Sequence Data, Protein Conformation, Seminiferous Tubules enzymology, Sequence Alignment, Vacuolar Proton-Translocating ATPases chemistry, Vacuolar Proton-Translocating ATPases metabolism, Yeasts, Acrosome enzymology, Testis enzymology, Vacuolar Proton-Translocating ATPases genetics
- Abstract
The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase. Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.
- Published
- 2002
- Full Text
- View/download PDF
46. ATP synthase F(1) sector rotation. Defective torque generation in the beta subunit Ser-174 to Phe mutant and its suppression by second mutations.
- Author
-
Iko Y, Sambongi Y, Tanabe M, Iwamoto-Kihara A, Saito K, Ueda I, Wada Y, and Futai M
- Subjects
- Actins chemistry, Adenosine Triphosphate metabolism, Alanine chemistry, Escherichia coli chemistry, Escherichia coli enzymology, Glycine chemistry, Hydrolysis, Isoleucine chemistry, Models, Molecular, Mutation, Phenylalanine chemistry, Protein Conformation, Protein Structure, Tertiary, Time Factors, Proton-Translocating ATPases metabolism, Serine chemistry
- Abstract
Subunit gamma of the ATP synthase F(1) sector is located at the center of the alpha(3)beta(3) hexamer and rotates unidirectionally during ATP hydrolysis, generating the rotational torque of approximately 45 pN.nm. A mutant F(1) with the betaSer-174 to Phe substitution (betaS174F) in the beta subunit generated lower torque ( approximately 17 pN.nm), indicating that betaS174F is mechanically defective, the first such mutant reported. The defective rotation of betaS174F was suppressed by a second-site mutation, betaGly-149 to Ala, betaIle-163 to Ala, or betaIle-166 to Ala in the same subunit, but not by betaLeu-238 to Ala. These results suggest that the region between betaGly-149 and betaSer-174 plays an important role in the coupling between ATP hydrolysis and mechanical work.
- Published
- 2001
- Full Text
- View/download PDF
47. a4, a unique kidney-specific isoform of mouse vacuolar H+-ATPase subunit a.
- Author
-
Oka T, Murata Y, Namba M, Yoshimizu T, Toyomura T, Yamamoto A, Sun-Wada GH, Hamasaki N, Wada Y, and Futai M
- Subjects
- Acid-Base Equilibrium, Amino Acid Sequence, Animals, Cell Membrane enzymology, Cell Polarity, Gene Library, Kidney Cortex cytology, Kidney Cortex enzymology, Kidney Tubules, Collecting cytology, Kidney Tubules, Collecting enzymology, Mice, Molecular Sequence Data, Protein Isoforms genetics, Protein Isoforms isolation & purification, Protein Subunits, Sequence Homology, Amino Acid, Vacuolar Proton-Translocating ATPases genetics, Kidney enzymology, Vacuolar Proton-Translocating ATPases isolation & purification
- Abstract
The vacuolar-type H+ -ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit a of V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, and a3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show that a4 is localized in the apical and basolateral plasma membranes of cortical alpha- and beta-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis.
- Published
- 2001
- Full Text
- View/download PDF
48. Four subunit a isoforms of Caenorhabditis elegans vacuolar H+-ATPase. Cell-specific expression during development.
- Author
-
Oka T, Toyomura T, Honjo K, Wada Y, and Futai M
- Subjects
- Amino Acid Sequence, Animals, Animals, Genetically Modified, Caenorhabditis elegans embryology, Caenorhabditis elegans Proteins, Disorders of Sex Development, Embryo, Nonmammalian, Female, Genes, Reporter, Green Fluorescent Proteins, Intestines cytology, Intestines enzymology, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Luminescent Proteins genetics, Male, Molecular Sequence Data, Nervous System cytology, Nervous System enzymology, Organ Specificity, Protein Subunits, Proton Pumps chemistry, Proton Pumps genetics, Proton Pumps metabolism, Proton-Translocating ATPases chemistry, Proton-Translocating ATPases metabolism, RNA, Double-Stranded genetics, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins chemistry, Sequence Alignment, Sequence Homology, Amino Acid, Skin cytology, Skin enzymology, Uterus cytology, Uterus enzymology, Caenorhabditis elegans enzymology, Caenorhabditis elegans genetics, Gene Expression Regulation, Developmental, Gene Expression Regulation, Enzymologic, Proton-Translocating ATPases genetics, Vacuolar Proton-Translocating ATPases
- Abstract
We have identified four genes (vha-5, vha-6, vha-7, and unc-32) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit a in Caenorhabditis elegans, the first example of four distinct isoforms in eukaryotes. Their products had nine putative transmembrane regions, exhibited 43-60% identity and 62-84% similarity with the bovine subunit a1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activity (Leng, X. H., Manolson, M. F., and Forgac, M. (1998) J. Biol. Chem. 273, 6717-6723). The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase. Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells; vha-5 was strongly expressed in an H-shaped excretory cell, vha-6 was strongly expressed in intestine, vha-7 was strongly expressed in hypodermis, and unc-32 was strongly expressed in nerve cells. Furthermore, the vha-7 and unc-32 genes were also expressed in the uteri of hermaphrodites. RNA interference analysis showed that the double-stranded RNA for unc-32 caused embryonic lethality similar to that seen with other subunit genes (vha-1, vha-4, and vha-11) (Oka, T., and Futai, M. (2000) J. Biol. Chem. 275, 29556-29561). The progenies of worms injected with the vha-5 or vha-6 double-stranded RNA became died at a specific larval stage, whereas the vha-7 double-stranded RNA showed no effect on development. These results suggest that V-ATPases with these isoforms generate acidic compartments essential for worm development in a cell-specific manner.
- Published
- 2001
- Full Text
- View/download PDF
49. A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila.
- Author
-
Christensen ST, Guerra C, Wada Y, Valentin T, Angeletti RH, Satir P, and Hamasaki T
- Subjects
- Animals, Calcium metabolism, Cyclic AMP pharmacology, Electrophoresis, Polyacrylamide Gel, Phosphorylation, Tetrahymena thermophila drug effects, Cilia metabolism, Dyneins metabolism, Tetrahymena thermophila metabolism
- Abstract
Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.
- Published
- 2001
- Full Text
- View/download PDF
50. Rotation of a complex of the gamma subunit and c ring of Escherichia coli ATP synthase. The rotor and stator are interchangeable.
- Author
-
Tanabe M, Nishio K, Iko Y, Sambongi Y, Iwamoto-Kihara A, Wada Y, and Futai M
- Subjects
- Actins metabolism, Adenosine Triphosphate metabolism, Dicyclohexylcarbodiimide pharmacology, Enzyme Inhibitors pharmacology, Hydrolysis, Plasmids, Proton-Translocating ATPases antagonists & inhibitors, Proton-Translocating ATPases chemistry, Venturicidins pharmacology, Escherichia coli enzymology, Proton-Translocating ATPases metabolism
- Abstract
ATP synthase (F0F1) transforms an electrochemical proton gradient into chemical energy (ATP) through the rotation of a subunit assembly. It has been suggested that a complex of the gamma subunit and c ring (c(10-14)) of F0F1 could rotate together during ATP hydrolysis and synthesis (Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722-1724). We observed that the rotation of the c ring with the cI28T mutation (c subunit cIle-28 replaced by Thr) was less sensitive to venturicidin than that of the wild type, consistent with the antibiotic effect on the cI28T mutant and wild-type ATPase activities (Fillingame, R. H., Oldenburg, M., and Fraga, D. (1991) J. Biol. Chem. 266, 20934-20939). Furthermore, we engineered F0F1 to see the alpha(3)beta(3) hexamer rotation; a biotin tag was introduced into the alpha or beta subunit, and a His tag was introduced into the c subunit. The engineered enzymes could be purified by metal affinity chromatography and density gradient centrifugation. They were immobilized on a glass surface through the c subunit, and an actin filament was connected to the alpha or beta subunit. The filament rotated upon the addition of ATP and generated essentially the same frictional torque as one connected to the c ring. These results indicate that the gammaepsilonc(10-14) complex is a mechanical unit of the enzyme and that it can be used as a rotor or a stator experimentally, depending on the subunit immobilized.
- Published
- 2001
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.