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Jung-do Choi - One of the best experts on this subject based on the ideXlab platform.
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Cloning and characterization of phosphoglucomutase and phosphomannomutase derived from Sphingomonas chungbukensis DJ77.
Journal of biochemistry, 2008Co-Authors: Sung-sook Yoon, Sung Ha Park, Young-chang Kim, Mal-shik Shin, Chom-kyu Chong, Jung-do ChoiAbstract:The enzymes phosphoglucomutase (PGM) and phosphomannomutase (PMM) play an important role in the synthesis of extracellular polysaccharide. By colony hybridization of the fosmid library of Sphingomonas chungbukensis DJ77, an open reading frame (ORF-1) of 1,626 nucleotides, whose predicted product is highly homologous with other PGM proteins from several bacterial species, was identified. An additional open reading frame (ORF-2) of 1,437 nucleotides was identified, and its encoded protein shows a high level of similarity with the PGM/PMM protein family. The two genes were cloned into a bacterial expression vector pET-15b (+) and expressed in Escherichia coli as fusion proteins with (His) 6 -tag. Both recombinant proteins (designated as SP-1 and SP-2 for ORF-1 and ORF-2, respectively) exhibited PGM and PMM activities. The molecular masses of subunits of SP-1 and SP-2 were estimated to be around 58 and 51 kDa from SDS-PAGE, respectively. However, molecular masses of SP-1 and SP-2 in their native condition were determined to be approximately 59.5 and 105.4 kDa, according to non-denaturing PAGE, respectively. The SP-1 protein has a preference for glucose-1-Phosphate rather than Mannose-1-Phosphate, while the preferred substrate of SP-2 is Mannose-1-Phosphate. Thus, the existence of two proteins with bifunctional PGM/PMM activities was first found S. chungbukensis DJ77.
Terry K Smith - One of the best experts on this subject based on the ideXlab platform.
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GDP-Mannose pyrophosphorylase is essential in the bloodstream form of Trypanosoma brucei
Biochemical Journal, 2010Co-Authors: Helen Denton, Stewart Fyffe, Terry K SmithAbstract:A putative guanidine diphosphoMannose pyrophophosphorylase (GDP-Man PP) gene from Trypanosoma brucei was identified in the genome and subsequently cloned, sequenced, and recombinantly expressed and shown to be a catalytically active dimer. Kinetic analysis revealed a Vmax of 0.34 μmol / min/ mg and Km's of 67 μM and 12 μM for GTP and Mannose-1-Phosphate respectively. Further kinetic studies showed GDP-Man was a potent product feedback inhibitor. RNAi of the cytosolic TbGDP-Man PP showed mRNA levels were reduced to ~20% of wild type levels, causing the cells to die after 3-4 days, demonstrating TbGDP-Man PP is essential in the bloodstream form of T.brucei and thus a potential drug target. The RNAi induced parasites have a greatly reduced capability to form GDP-Man, leading ultimately to a reduction in their ability to synthesize their essential glycosylphosphatidylinositol anchors. The RNAi induced parasites also showed aberrant N-glycosylation of their major cell-surface glycoprotein, variant surface glycoprotein, with loss of the high Mannose Man9GlcNAc2 N-glycosylation at Asn-428 and formation of complex N-glycans at the Asn-263.
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GDP-Mannose pyrophosphorylase is essential in the bloodstream form of Trypanosoma brucei
The Biochemical journal, 2010Co-Authors: Helen Denton, Stewart Fyffe, Terry K SmithAbstract:A putative GDP-Man PP (guanidine diphosphoMannose pyrophosphorylase) gene from Trypanosoma brucei (TbGDP-Man PP) was identified in the genome and subsequently cloned, sequenced and recombinantly expressed, and shown to be a catalytically active dimer. Kinetic analysis revealed a Vmax of 0.34 mumol/min per mg of protein and Km values of 67 muM and 12 muM for GTP and Mannose 1-Phosphate respectively. Further kinetic studies showed GDP-Man was a potent product feedback inhibitor. RNAi (RNA interference) of the cytosolic TbGDP-Man PP showed that mRNA levels were reduced to ~20% of wild-type levels, causing the cells to die after 3-4 days, demonstrating that TbGDP-Man PP is essential in the bloodstream form of T. brucei and thus a potential drug target. The RNAi-induced parasites have a greatly reduced capability to form GDP-Man, leading ultimately to a reduction in their ability to synthesize their essential GPI (glycosylphosphatidylinositol) anchors. The RNAi-induced parasites also showed aberrant N-glycosylation of their major cell-surface glycoprotein, variant surface glycoprotein, with loss of the high-Mannose Man9GlcNAc2 N-glycosylation at Asn428 and formation of complex N-glycans at Asn263.
Kurt Von Figura - One of the best experts on this subject based on the ideXlab platform.
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Targeted disruption of the mouse phosphomannomutase 2 gene causes early embryonic lethality
Molecular and cellular biology, 2006Co-Authors: Christian Thiel, Gert Matthijs, Torben Lübke, Kurt Von Figura, Christian KornerAbstract:Mutations in the cytosolic enzyme phosphomannomutase 2 (PMM2), which catalyzes the conversion of Mannose-6-phosphate to Mannose-1-Phosphate, cause the most common form of congenital disorders of glycosylation, termed CDG-Ia. It is an inherited multisystemic disease with severe neurological impairment. To study the pathophysiology of CDG-Ia and to investigate possible therapeutic approaches, we generated a mouse model for CDG-Ia by targeted disruption of the Pmm2 gene. Heterozygous mutant mice appeared normal in development, gross anatomy, and fertility. In contrast, embryos homozygous for the Pmm2-null allele were recovered in embryonic development at days 2.5 to 3.5. These results indicate that Pmm2 is essential for early development of mice. Mating experiments of heterozygous mice with wild-type mice could further show that transmission of the female Pmm2-null allele is impaired.
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abnormal synthesis of Mannose 1 phosphate derived carbohydrates in carbohydrate deficient glycoprotein syndrome type i fibroblasts with phosphomannomutase deficiency
Glycobiology, 1998Co-Authors: Christian Korner, Ludwig Lehle, Kurt Von FiguraAbstract:In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] Mannose into Mannose phosphates, GDP-Mannose, GDP-fucose, dolichol-P-Mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into Mannose 1-Phosphate, GDP-Mannose, GDP-fucose, dolichol-P-Mannose, and nascent glycoproteins. The incorporation of radioactivity into Mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (
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Abnormal synthesis of Mannose 1-Phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency
Glycobiology, 1998Co-Authors: Christian Korner, Ludwig Lehle, Kurt Von FiguraAbstract:In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] Mannose into Mannose phosphates, GDP-Mannose, GDP-fucose, dolichol-P-Mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into Mannose 1-Phosphate, GDP-Mannose, GDP-fucose, dolichol-P-Mannose, and nascent glycoproteins. The incorporation of radioactivity into Mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (
Anette Henriksen - One of the best experts on this subject based on the ideXlab platform.
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structural and biochemical characterization of the n terminal domain of flocculin lg flo1p from saccharomyces pastorianus reveals a unique specificity for phosphorylated Mannose
FEBS Journal, 2013Co-Authors: Lyann Sim, Magnus Groes, Kjeld Olesen, Anette HenriksenAbstract:UNLABELLED The mechanism of yeast flocculation is generally considered to be mediated through the interaction of cell surface flocculins and mannan carbohydrates. In the present study, the crystal structure of the soluble 25-kDa lectin domain of flocculin 1 from brewer's yeast (Lg-Flo1p) was resolved to 2.5 A, and its binding specificity towards oligosaccharides was investigated by fluorescence spectroscopy. Lg-Flo1p displays broad specificity towards sugars and has a 14-fold higher affinity for Mannose 1-Phosphate and glucose 1-Phosphate compared to their unphosphorylated counterparts. Based on the results of a structural analysis, we propose that this higher affinity is the result of a charge interaction with a lysine residue in a carbohydrate-binding loop region, NAKAL, unique to NewFlo type flocculins. This raises the possibility of a unique mechanism of flocculation in NewFlo type yeast, which recognizes phosphorylated cell surface mannans. DATABASE Structural data have been deposited in the Protein Data Bank under accession number 4GQ7.
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Structural and biochemical characterization of the N‐terminal domain of flocculin Lg‐Flo1p from Saccharomyces pastorianus reveals a unique specificity for phosphorylated Mannose
The FEBS journal, 2013Co-Authors: Lyann Sim, Magnus Groes, Kjeld Olesen, Anette HenriksenAbstract:UNLABELLED The mechanism of yeast flocculation is generally considered to be mediated through the interaction of cell surface flocculins and mannan carbohydrates. In the present study, the crystal structure of the soluble 25-kDa lectin domain of flocculin 1 from brewer's yeast (Lg-Flo1p) was resolved to 2.5 A, and its binding specificity towards oligosaccharides was investigated by fluorescence spectroscopy. Lg-Flo1p displays broad specificity towards sugars and has a 14-fold higher affinity for Mannose 1-Phosphate and glucose 1-Phosphate compared to their unphosphorylated counterparts. Based on the results of a structural analysis, we propose that this higher affinity is the result of a charge interaction with a lysine residue in a carbohydrate-binding loop region, NAKAL, unique to NewFlo type flocculins. This raises the possibility of a unique mechanism of flocculation in NewFlo type yeast, which recognizes phosphorylated cell surface mannans. DATABASE Structural data have been deposited in the Protein Data Bank under accession number 4GQ7.
Christian Korner - One of the best experts on this subject based on the ideXlab platform.
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Targeted disruption of the mouse phosphomannomutase 2 gene causes early embryonic lethality
Molecular and cellular biology, 2006Co-Authors: Christian Thiel, Gert Matthijs, Torben Lübke, Kurt Von Figura, Christian KornerAbstract:Mutations in the cytosolic enzyme phosphomannomutase 2 (PMM2), which catalyzes the conversion of Mannose-6-phosphate to Mannose-1-Phosphate, cause the most common form of congenital disorders of glycosylation, termed CDG-Ia. It is an inherited multisystemic disease with severe neurological impairment. To study the pathophysiology of CDG-Ia and to investigate possible therapeutic approaches, we generated a mouse model for CDG-Ia by targeted disruption of the Pmm2 gene. Heterozygous mutant mice appeared normal in development, gross anatomy, and fertility. In contrast, embryos homozygous for the Pmm2-null allele were recovered in embryonic development at days 2.5 to 3.5. These results indicate that Pmm2 is essential for early development of mice. Mating experiments of heterozygous mice with wild-type mice could further show that transmission of the female Pmm2-null allele is impaired.
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abnormal synthesis of Mannose 1 phosphate derived carbohydrates in carbohydrate deficient glycoprotein syndrome type i fibroblasts with phosphomannomutase deficiency
Glycobiology, 1998Co-Authors: Christian Korner, Ludwig Lehle, Kurt Von FiguraAbstract:In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] Mannose into Mannose phosphates, GDP-Mannose, GDP-fucose, dolichol-P-Mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into Mannose 1-Phosphate, GDP-Mannose, GDP-fucose, dolichol-P-Mannose, and nascent glycoproteins. The incorporation of radioactivity into Mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (
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Abnormal synthesis of Mannose 1-Phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency
Glycobiology, 1998Co-Authors: Christian Korner, Ludwig Lehle, Kurt Von FiguraAbstract:In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] Mannose into Mannose phosphates, GDP-Mannose, GDP-fucose, dolichol-P-Mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into Mannose 1-Phosphate, GDP-Mannose, GDP-fucose, dolichol-P-Mannose, and nascent glycoproteins. The incorporation of radioactivity into Mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (