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Taroh Kinoshita - One of the best experts on this subject based on the ideXlab platform.
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Glycosylphosphatidylinositol mannosyltransferase II is the rate-limiting enzyme in glycosylphosphatidylinositol biosynthesis under limited Dolichol-Phosphate mannose availability
Journal of Biochemistry, 2013Co-Authors: Tetsuya Hirata, Noriyuki Kanzawa, Yusuke Maeda, Morihisa Fujita, Yoshiko Murakami, Taroh KinoshitaAbstract:Although the genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI) are well characterized, the regulation of GPI biosynthesis remains unclear. We isolated and characterized a mutant cell line showing decreased surface expression of CD59 and the accumulation of GPI intermediates. The mutant cell line was partially defective in MPDU1, which encodes a protein required for the utilization of Dolichol-Phosphate mannose. Overexpression of PIGV, which encodes GPI mannosyltransferase II, restored the surface expression of CD59 and normalized the accumulation of GPI intermediates in the mutant cells. Among all known genes involved in GPI biosynthetic pathway, only PIGV had such suppressive activity. PIGV, however, did not restore the abnormality of N-glycosylation caused by MPDU1 mutation. Our results suggest that GPI mannosyltransferase II is the rate-limiting enzyme in GPI biosynthesis under limited Dolichol-Phosphate mannose availability.
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Dolichol-Phosphate mannose synthase: Structure, function and regulation
Biochimica et biophysica acta, 2008Co-Authors: Yusuke Maeda, Taroh KinoshitaAbstract:Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and Dolichol-Phosphate-sugars, in which the sugar moiety is mannose or glucose. The sugar transfer from Dolichol-Phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of Dolichol-Phosphate-mannose, and particularly on the mammalian enzyme complex involved in the reaction.
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DPM1, the Catalytic Subunit of Dolichol-Phosphate Mannose Synthase, Is Tethered to and Stabilized on the Endoplasmic Reticulum Membrane by DPM3
The Journal of biological chemistry, 2005Co-Authors: Hisashi Ashida, Yusuke Maeda, Taroh KinoshitaAbstract:Abstract Dolichol-Phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.
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mammalian pig x and yeast pbn1p are the essential components of glycosylphosphatidylinositol mannosyltransferase i
Molecular Biology of the Cell, 2005Co-Authors: Hisashi Ashida, Yusuke Maeda, Yoshiko Murakami, Yeongjin Hong, Nobue Shishioh, Nakaba Sugimoto, Taroh KinoshitaAbstract:Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from Dolichol-Phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using Dolichol-Phosphate-monosaccharide.
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PIG-B, GPI-Man Transferase III, Man- (EthanolaminePhosphate)Man-GlcN- (Acy1)PI Mannosyltransferase
Handbook of Glycosyltransferases and Related Genes, 2002Co-Authors: Taroh Kinoshita, Norimitsu InoueAbstract:The core backbone of the glycosylphosphatidylinositol (GPI) anchors contains three mannoses, each linked in different bonds. All three mannoses are donated by Dolichol- Phosphate-mannose. Three Dolichol-Phosphate-mannose-dependent mannosyltrans- ferases are therefore involved in biosynthesis of the GPI-anchors. PIG-B is necessary for transfer of the third mannose and is most likely α1,2-mannosyltransferase itself. PIG-B cDNA was expression-cloned using a mouse mutant cell line that is defective in transfer of the third mannose to GPI. PIG-B encodes a 554-amino-acid protein expressed in the endoplasmic reticulum (ER), where GPI is assembled. It has multiple hydrophobic regions, some of which may be transmembrane domains.
Everardo López-romero - One of the best experts on this subject based on the ideXlab platform.
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Protein glycosylation in Candida
Future microbiology, 2009Co-Authors: Héctor M. Mora-montes, Arturo Flores-carreón, Julio C. Villagómez-castro, Patricia Ponce-noyola, Neil A. R. Gow, Everardo López-romeroAbstract:Candidiasis is a significant cause of invasive human mycosis with associated mortality rates that are equivalent to, or worse than, those cited for most cases of bacterial septicemia. As a result, considerable efforts are being made to understand how the fungus invades host cells and to identify new targets for fungal chemotherapy. This has led to an increasing interest in Candida glycobiology, with an emphasis on the identification of enzymes essential for glycoprotein and adhesion metabolism, and the role of N- and O-linked glycans in host recognition and virulence. Here, we refer to studies dealing with the identification and characterization of enzymes such as Dolichol Phosphate mannose synthase, Dolichol Phosphate glucose synthase and processing glycosidases and synthesis, structure and recognition of mannans and discuss recent findings in the context of Candida albicans pathogenesis.
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Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases
Antonie van Leeuwenhoek, 2005Co-Authors: Estela Ruiz-baca, Arturo Flores-carreón, Julio C. Villagómez-castro, Carlos A. Leal-morales, Myrna Sabanero-lópez, Everardo López-romeroAbstract:A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into Dolichol Phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by Dolichol Phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous Dolichol monoPhosphate, membranes failed to use exogenous Dolichol Phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via Dolichol Phosphate mannose and the reaction was stimulated several fold by Mg^2+ and Mn^2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A–Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26–112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O -glycosylation.
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Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans : Activation of Dolichol Phosphate mannose synthase by cAMP-mediated protein phosphorylation
FEMS immunology and medical microbiology, 2005Co-Authors: Blanca L. Arroyo-flores, Carlos Calvo-méndez, Arturo Flores-carreón, Everardo López-romeroAbstract:Following incubation with ATP and a cAMP-dependent protein kinase under optimal conditions of lipid acceptor, phospholipid and metal ion requirements, the transfer activity of partially purified Dolichol Phosphate mannose synthase (DPMS) increased about 60% and this activation correlated with a 50% increase in Vmax with no alteration in the apparent Km for GDP-Manose. Phosphorylation with [γ-32P]ATP resulted in the labeling of several polypeptides, one of which exhibited the molecular weight of the enzyme (30 kDa) and was also recognized using a specific anti-DPMS monoclonal antibody. This and the fact that the Phosphate label could be removed by an alkaline phosphatase indicate that Candida DPMS may be regulated by phosphorylation–dephosphorylation, a mechanism that has been proposed for the enzyme in other organisms.
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Partial purification and characterization of Dolichol Phosphate mannose synthase from Entamoeba histolytica
Glycobiology, 2000Co-Authors: Julio C. Villagómez-castro, Carlos Calvo-méndez, Arturo Flores-carreón, Everardo López-romeroAbstract:Dolichol Phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis, was partially purified from E.histolytica by hydrophobic interaction and affinity chromatography with octyl Sepharose CL-4B and Affi-Gel 501, respectively. Reducing agents, particularly dithiothreitol, positively influenced enzyme activity and stability, indicating a role of sulfhydryl groups on the transferase function. Activity did not depend on phospholipids; however, it was significantly stimulated by phosphatidylethanolamine and to a lower extent by other common phospholipids. Mixtures consisting of activating phospholipids did not exert an additive effect. In vitro phosphorylation with a cAMP-dependent protein kinase resulted in enzyme activation. This alteration was not associated with a change in the K(m) for the substrate but rather with a 2.6-fold increase in V(max). Phosphorylation in the presence of [gamma-(32)P]ATP resulted in strong labeling of two polypeptides, one of which exhibited the molecular mass reported for the enzyme from other organisms. Whether phosphorylation functions in vivo as a mechanism of regulation of Dolichol Phosphate mannose synthesis in E.histolytica remains to be determined.
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Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases.
Fungal genetics and biology : FG & B, 2000Co-Authors: Blanca L. Arroyo-flores, Carlos Calvo-méndez, Arturo Flores-carreón, Julio C. Villagómez-castro, Jesús Rodrı́guez-bonilla, Everardo López-romeroAbstract:The enzymes Dolichol Phosphate glucose synthase and Dolichol Phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to Dolichol Phosphate glucose and minor amounts of glucoproteins in the absence of exogenous Dolichol Phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.
Grazyna Palamarczyk - One of the best experts on this subject based on the ideXlab platform.
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Dolichol Phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme
Biochimica et biophysica acta, 2015Co-Authors: Mateusz Juchimiuk, Joanna S. Kruszewska, Grazyna PalamarczykAbstract:Dolichol Phosphate mannose synthase (DPMS) is a key enzyme in N- and O-linked glycosylations and glycosylphosphatidylinositol (GPI)-anchor synthesis. DPMS generates DPM, the substrate for mentioned processes, by the transfer of mannosyl residue from GDP-Man to Dolichol Phosphate. Here we describe the role of DPMS for Candida albicans physiology with emphasis on the cell wall composition and morphogenesis.C. albicans genes for DPMS subunits were cloned, tagged and expressed in Saccharomyces cerevisiae. The C. albicans strains with controlled expression of DPM genes were constructed and analyzed. Gene expression and enzyme activities were measured using RT-PCR and radioactive substrate. Sensitivities against chemical agents were tested with microdilution method. The composition of the cell wall was estimated by HPLC. Glycosylation status of the marker protein was analyzed by Western blot. Morphological differentiation of the strains was checked on the media promoting hyphae and chlamydospore formation.We demonstrate that C. albicans DPMS consists of three interacting subunits, among which Dpm1 and Dpm3 are indispensable, whereas Dpm2 increases enzymatic activity. Lowered expression of DPMS genes results in decreased DPMS activity, increased susceptibility to cell wall perturbing agents and in altered cell wall composition. Mutants Tetp-DPM1 and Tetp-DPM3 show defective protein glycosylation and are impaired in hyphae and chlamydospore formation.DPMS from C. albicans, opposite to S. cerevisiae, belongs to the family of DPMS with multimeric protein structure.This work provides important data about factors required for a proper protein glycosylation and for morphogenesis of pathogenic yeast C. albicans.
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Dolichol Phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme
Glycobiology, 2000Co-Authors: Joanna S. Kruszewska, Peter Orlean, Markku Saloheimo, A. Migdalski, Merja Penttilä, Grazyna PalamarczykAbstract:Dolichol Phosphate mannose (DPM) synthase activity, which is required in N-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway. We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase. The Trichoderma dpm1 (Trdpm1) gene complements a lethal null mutation in the S.pombe dpm1 + gene, but neither restores viability of a S.cerevisiae dpm1-disruptant nor complements the temperature-sensitivity of the S.cerevisiae dpm1-6 mutant. The T.reesei DPM synthase is therefore a member of the “human” class of enzyme. Overexpression of Trdpm1 in a dpm1 + ::his7/dpm1 + S.pombe diploid resulted in a 4-fold increase in specific DPM synthase activity. However, neither the wild type T.reesei DPM synthase, nor a chimera consisting of this protein and the hydrophobic COOH terminus of the S.cerevisiae DPM synthase, complemented an S.cerevisiae dpm1 null mutant or gave active enzyme when expressed in E.coli .T he level of the Trdpm1 mRNA in T.reesei QM9414 strain was dependent on the composition of the culture medium. Expression levels of Trdpm1 were directly correlated with the protein secretory capacity of the fungus.
Yusuke Maeda - One of the best experts on this subject based on the ideXlab platform.
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Glycosylphosphatidylinositol mannosyltransferase II is the rate-limiting enzyme in glycosylphosphatidylinositol biosynthesis under limited Dolichol-Phosphate mannose availability
Journal of Biochemistry, 2013Co-Authors: Tetsuya Hirata, Noriyuki Kanzawa, Yusuke Maeda, Morihisa Fujita, Yoshiko Murakami, Taroh KinoshitaAbstract:Although the genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI) are well characterized, the regulation of GPI biosynthesis remains unclear. We isolated and characterized a mutant cell line showing decreased surface expression of CD59 and the accumulation of GPI intermediates. The mutant cell line was partially defective in MPDU1, which encodes a protein required for the utilization of Dolichol-Phosphate mannose. Overexpression of PIGV, which encodes GPI mannosyltransferase II, restored the surface expression of CD59 and normalized the accumulation of GPI intermediates in the mutant cells. Among all known genes involved in GPI biosynthetic pathway, only PIGV had such suppressive activity. PIGV, however, did not restore the abnormality of N-glycosylation caused by MPDU1 mutation. Our results suggest that GPI mannosyltransferase II is the rate-limiting enzyme in GPI biosynthesis under limited Dolichol-Phosphate mannose availability.
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Dolichol-Phosphate mannose synthase: Structure, function and regulation
Biochimica et biophysica acta, 2008Co-Authors: Yusuke Maeda, Taroh KinoshitaAbstract:Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and Dolichol-Phosphate-sugars, in which the sugar moiety is mannose or glucose. The sugar transfer from Dolichol-Phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of Dolichol-Phosphate-mannose, and particularly on the mammalian enzyme complex involved in the reaction.
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DPM1, the Catalytic Subunit of Dolichol-Phosphate Mannose Synthase, Is Tethered to and Stabilized on the Endoplasmic Reticulum Membrane by DPM3
The Journal of biological chemistry, 2005Co-Authors: Hisashi Ashida, Yusuke Maeda, Taroh KinoshitaAbstract:Abstract Dolichol-Phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.
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mammalian pig x and yeast pbn1p are the essential components of glycosylphosphatidylinositol mannosyltransferase i
Molecular Biology of the Cell, 2005Co-Authors: Hisashi Ashida, Yusuke Maeda, Yoshiko Murakami, Yeongjin Hong, Nobue Shishioh, Nakaba Sugimoto, Taroh KinoshitaAbstract:Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from Dolichol-Phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using Dolichol-Phosphate-monosaccharide.
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Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2.
The EMBO journal, 2000Co-Authors: Reika Watanabe, Yusuke Maeda, Yoshiko Murakami, Jun Hino, Kenji Kangawa, Norimitsu Inoue, Mina D. Marmor, Michael Julius, Taroh KinoshitaAbstract:Glycosylphosphatidylinositols (GPIs) are attached to the C-termini of many proteins, thereby acting as membrane anchors. Biosynthesis of GPI is initiated by GPI-N-acetylglucosaminyltransferase (GPI-GnT), which transfers N-acetylglucosamine from UDP- N-acetylglucosamine to phosphatidylinositol. GPI-GnT is a uniquely complex glycosyltransferase, consisting of at least four proteins, PIG-A, PIG-H, PIG-C and GPI1. Here, we report that GPI-GnT requires another component, termed PIG-P, and that DPM2, which regulates Dolichol-Phosphate-mannose synthase, also regulates GPI-GnT. PIG-P, a 134-amino acid protein having two hydrophobic domains, associates with PIG-A and GPI1. PIG-P is essential for GPI-GnT since a cell lacking PIG-P is GPI-anchor negative. DPM2, but not two other components of Dolichol-Phosphate-mannose synthase, associates with GPI-GnT through interactions with PIG-A, PIG-C and GPI1. Lec15 cell, a null mutant of DPM2, synthesizes early GPI intermediates, indicating that DPM2 is not essential for GPI-GnT; however, the enzyme activity is enhanced 3-fold in the presence of DPM2. These results reveal new essential and regulatory components of GPI-GnT and imply co-regulation of GPI-GnT and the Dolichol-Phosphate-mannose synthase that generates a mannosyl donor for GPI.
Ralph T. Schwarz - One of the best experts on this subject based on the ideXlab platform.
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Plasmodium falciparum Dolichol Phosphate mannose synthase represents a novel clade
Biochemical and Biophysical Research Communications, 2008Co-Authors: Hosam Shams-eldin, C. De Macedo, Sebastian Niehus, C. Dorn, J. Kimmel, N. Azzouz, Ralph T. SchwarzAbstract:Dolichol Phosphate mannose synthase (DPM) catalyzes the reaction between Dolichol Phosphate (Dol-P) and guanosine diPhosphate mannose (GDP-Man) to form Dolichol-Phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker's yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.
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Dolichol Phosphate GlcNAc-1-P Transferase
Handbook of Glycosyltransferases and Related Genes, 2002Co-Authors: Andreas Hübel, Ralph T. SchwarzAbstract:Asparagine-linked glycosylation is a covalent modification of secretory and integral membrane proteins in eukaryotes that modulates the structure and function of these proteins. Dolichol-linked oligosaccharides are transferred en bloc to selected asparagine residues of nascent polypeptides. Biosynthesis of the Dolichol-bound oligosaccharides is initiated by formation of GlcNAc-P-P-Dolichol from UDP-GlcNAc and Dolichol Phosphate. This reaction is catalyzed by the endoplasmic reticulum (ER) residential enzyme UDP-GlcNAc:Dolichol Phosphate N-acetylglucosamine-1- Phosphate transferase (GPT). The two substrates, Dolichol Phosphate and UDP- GlcNAc, can serve other pathways, but the product of GPT activity can only participate in lipid-bound oligosaccharide biosynthesis. Thus, GPT catalyzes the committed step of the Dolichol cycle involved in asparagine-linked glycosylation.
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Regulation of Paramecium primaurelia glycosylphosphatidyl-inositol biosynthesis via Dolichol Phosphate mannose synthesis.
Biochimie, 2001Co-Authors: Nahid Azzouz, Hosam Shams-eldin, Peter Gerold, Mamdouh H. Kedees, Regina Werner, Yvonne Capdeville, Ralph T. SchwarzAbstract:Abstract A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-Phosphate-6Manα1-2Manα1-6(mannosylPhosphate) Manα1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl Phosphate substitution, we observed that the use of the nucleotide triPhosphate analogue GTPγS (guanosine 5ˈ- O -(thiotriPhosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTPγS inhibits the synthesis of Dolichol-Phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory ‘G’ proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through Dolichol-Phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTPγS interferes with synthesis of Dolichol monoPhosphate, indicating that the Dolichol kinase is regulated by the heterotrimeric G proteins.
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Cloning and functional expression of glycosyltransferases from parasitic protozoans by heterologous complementation in yeast: the Dolichol Phosphate mannose synthase from Trypanosoma brucei brucei *
Biochemical Journal, 1996Co-Authors: Ramin Mazhari-tabrizi, Volker Eckert, Michaela Blank, Rolf Müller, Dominik Mumberg, Martin Funk, Ralph T. SchwarzAbstract:The gene for the enzyme Dolichol Phosphate mannose (Dol-P-Man) synthase from the parasitic protozoan Trypanosoma brucei brucei (T. brucei) was cloned by screening a T. brucei cDNA library and then sequenced. The library was constructed in a yeast expression vector and the positive clone was identified by complementation of a temperature-sensitive defect in the yeast strain DPM 1-6 [Orlean, Albright and Robbins (1988) J. Biol. Chem. 263, 17499-17507]. The insert of this clone displayed an open reading frame of 801 nucleotides coding for a putative protein of 267 amino acids. The deduced protein sequence showed an identity of 49% and a similarity of 69% with the published yeast sequence. Additional features of the T. brucei sequence are the presence of a putative signal sequence, a C-terminal transmembrane domain, a consensus sequence for phosphorylation by cAMP-dependent protein kinase and a stretch of five nucleotides immediately upstream from the putative initiation codon that could function as a prokaryotic ribosome binding site. A consensus sequence for Dolichol binding (FI/VXF/YXXIPFXF/Y) found in the yeast protein could not be detected in the putative transmembrane domain of the T. brucei sequence. Biochemical characterization of the recombinant protein showed that it is functionally expressed in the yeast strain DPM 1-6 and Escherichia coli. In both constructs Dol-P-Man synthesis was shown in a cell-free system. Synthesis was stimulated by exogenous Dolichol Phosphate and inhibited by amphomycin. These results confirm that we have cloned the T. brucei Dol-P-Man synthase by heterologous complementation in yeast, an approach that might be applicable for other glycosyltransferases from various sources.