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D242A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows about 10% increased activity compared to the wild-type enzyme
D244A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
D309N
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
F199A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
F261A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
F357A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
G19Q
site-directed mutagenesis, the mutation affects both secretion and fucosylation
G19R
site-directed mutagenesis, the mutation affects both secretion and fucosylation
G20H
site-directed mutagenesis, the mutation affects both secretion and fucosylation
N43A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows highly reduced activity compared to the wild-type enzyme
R240A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant
R240K
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant
R298K/R299K
site-directed mutagenesis, the mutant enzyme is stable against proteolysis and similarly active as the wild-type. The mutant enzymes is capable of fucosylating TSRs not only of group 1 but also of group 2, it not only recognizes and reacts with TSRs showing slightly different structures but also accepts TSRs with very low sequence identity. Residue Glu52 of mutant CePOFUT is the catalytic base
R40A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
S15D
site-directed mutagenesis, the mutant shows increased secretion, but a significant reduction in fucosylation, suggesting that HsPOFUT2 is highly selective for amino acids in the Xa position
S15Q
site-directed mutagenesis, the mutant shows increased secretion, but a significant reduction in fucosylation, suggesting that HsPOFUT2 is highly selective for amino acids in the Xa position
V16H
site-directed mutagenesis, the mutation affects both secretion and fucosylation
W245A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
R254A
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expression of a mutant, Ofut1R245A, lacking fucosyltransferase activity, rescues the requirement for Ofut1 in embryonic neurogenesis. Lack of requirement for O-fucosylation is further supported by the absence of embryonic phenotypes in Gmd mutants, which lack all forms of fucosylation. Requirements for O-fucose during imaginal development are evaluated by characterizing clones of cells expressing only Ofut1R245A. These clones phenocopy fringe mutant clones, indicating that the absence of O-fucose is functionally equivalent to the absence of elongated O-fucose
R275A
Drosophila sp. (in: flies)
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is catalytically dead. Ofut1 and mutant both bind the Notch extracellular domain, and expression of the mutant in cultured cells can increase both the amount and the ligand-binding activity of secreted Notch extracellular domain. Complete loss of ofut1 activity results in a strong phenotype mimicking a loss of Notch activity that is rescued to larval viability by the expression of mutant R275A, it rescues Notch receptor activity in these ofut1 mutant cells and leads to phenotypes mimicking a loss of fringe activity
D297A
site-directed mutagenesis, the mutant shows 15% activity compared to the wild-type enzyme
E396A
site-directed mutagenesis, the mutant shows 8% activity compared to the wild-type enzyme
E54A
site-directed mutagenesis, inactive mutant
R294A
site-directed mutagenesis, inactive mutant
W273a
site-directed mutagenesis, W273 is involved in controlling movements of the N- and C-terminal domain relative to each other during the catalytic cycle, and 90% activity is lost in mutant W273A
S1726A
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mutant protein shows no activity, loss of O-fucosylation causes a gain of function for muscle agrin such that it stimulates acetylcholine receptors, clustering and MuSK phosphorylation in cultured myotubes at levels normally only found with the neural splice form
R245A
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is fucosylation-defective, has a dominant negative effect on wild-type Pofut1, since Pofut1 activity in Pofut1-/- cells expressing Pofut1 R245A is markedly reduced in the in vitro assay
R245A
mutation disrupts O-fucosyltransferase activity destabilizes the protein and abolishes Notch1 signaling during mouse somitogenesis
additional information
mutants R40A, N43A and R240A/K are more stable while F199A, D309N, D242A, D244A, W245A, F261A and F357A are less stable than the wild-type. Mutants R40A, R240A/K, W245A and F357A show a decrease in binding to GDP, from this group, R40A and W245A bind better to GDP than F357A, R240K and R240A, with the latter being impaired in binding
additional information
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down-regulation of enzyme by RNA interference in Notch-secreting cells inhibits both Delta-Notch and Serrate-Notch binding. Overexpression of enzyme in cultured cells increases Serrate-Notch binding but inhibits Delta-Notch binding
additional information
enzyme disruption mutant, enzyme is essential for Notch signaling, Fringe function, for physical interaction of Notch with its ligand Delta, and lateral inhibition during neuroblast segregation
additional information
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analysis of O-fut 1 homozygous mutant cells shows that O-fut1 is required for the endocytic transportation of Notch to the early endosome which is shown to be independent of the O-fucosyltransferase activity of O-fut1
additional information
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O-fut 1 overexpression and analysis of O-fut 1 homozygous mutant cells indicates that O-fut 1 promotes the turnover of Notch, which consequently downregulates Notch signaling
additional information
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O-fut1 protein added to conditioned medium and endocytosed is sufficient to rescue normal Notch transportation to the early endosome in O-fut1 knockdown cells
additional information
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using O-fut I knock out mutants it is shown that the localization of Notch in the region from the subapical complex (SAC) to the apical portion of the adherens junctions (AJs) depends on its O-fucosylation
additional information
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an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae is generated. The in vivo O-fucosylation system is constructed via expression of genes that encode protein O-fut1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose
additional information
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enzyme deletion mutant, mouse embryose lacking enzyme die at midgestation with severe defects in somitogenesis, vasculinogenesis, cardiogenesis, and neurogenesis
additional information
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deletion of Pofut1 in cultured primary myotubes and in adult skeletal muscle increases acetylcholine receptor aggregation
additional information
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generation of Pofut1+/-/FX-/- mutant mice
additional information
generation of murine myoblastic Pofut1 knockdown C2C12 cell line, and analysis of the C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affects the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7/MyoD- cells and earlier myogenic program entrance are observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells, by generation of Pofut1 knockdown C2C12 cell lines reexpressing shRNA-resistant Pofut1, restores Notch signaling activation and a normal course in C2C12 differentiation. Downregulation of gene Pofut1 significantly lowers the quantity of cleaved Notch intracellular domain and the expression levels of genes Rbpj and Hes1 but without modification of the cell surface expression pattern of Notch1, phenotype, overview
additional information
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generation of murine myoblastic Pofut1 knockdown C2C12 cell line, and analysis of the C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affects the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7/MyoD- cells and earlier myogenic program entrance are observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells, by generation of Pofut1 knockdown C2C12 cell lines reexpressing shRNA-resistant Pofut1, restores Notch signaling activation and a normal course in C2C12 differentiation. Downregulation of gene Pofut1 significantly lowers the quantity of cleaved Notch intracellular domain and the expression levels of genes Rbpj and Hes1 but without modification of the cell surface expression pattern of Notch1, phenotype, overview