Information on EC 2.4.1.221 - peptide-O-fucosyltransferase

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The expected taxonomic range for this enzyme is: Bilateria

EC NUMBER
COMMENTARY
2.4.1.221
-
RECOMMENDED NAME
GeneOntology No.
peptide-O-fucosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
transfers an alpha-L-fucosyl residue from GDP-beta-L-fucose to the serine hydroxy group of a protein acceptor
show the reaction diagram
-
-
-
-
transfers an alpha-L-fucosyl residue from GDP-beta-L-fucose to the serine hydroxy group of a protein acceptor
show the reaction diagram
catalytic mechanism and substrate specificity, overview
Q9Y2G5
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
glycosyl group transfer
-
-
-
-
transglycosylation
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Other types of O-glycan biosynthesis
-
SYSTEMATIC NAME
IUBMB Comments
GDP-beta-L-fucose:polypeptide O-alpha-L-fucosyltransferase
Involved in the biosynthesis of O-fucosylated epidermal growth factor (EGF) and thrombospondin type 1 repeats. The attachment of O-linked fucose to serine or threonine occurs on EGF domains within the sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
alpha-6-fucosyltransferase
-
-
-
-
core alpha6FucT
-
-
-
-
fucosyltransferase, guanosine diphosphofucose-glycoprotein
-
-
-
-
GDP-fucose glycoprotein fucosyltransferase
-
-
-
-
GDP-fucose protein O-fucosyltransferase
-
-
-
-
GDP-fucose:polypeptide fucosyltransferase
-
-
-
-
GDP-L-fucose-glycoprotein fucosyltransferase
-
-
-
-
GDP-L-fucose:polypeptide fucosyltransferase
-
-
-
-
glycoprotein fucosyltransferase
-
-
-
-
guanosine diphosphofucose-glycoprotein fucosyltransferase
-
-
-
-
N-acetyl-beta-D-glucosaminide alpha1-->6-fucosyltransferase
-
-
-
-
N-glycan alpha-6-fucosyltransferase
-
-
-
-
O-fucosyltransferase
-
-
O-fucosyltransferase 1
-
-
O-fucosyltransferase 1
-
-
O-fucosyltransferase 1
-
-
O-fucosyltransferase 1
-
-
O-fucosyltransferase 1
-
-
O-fucosyltransferase O-fut 1
-
-
O-fucT-1
-
-
-
-
O-fucT-1
-
-
Pofut1
Q18014
-
POFUT2
Q9Y2G5
-
POFUT2
-
-
protein O-fucosyltransferase 1
Q18014
-
protein O-fucosyltransferase 1
-
-
protein O-fucosyltransferase 1
-
-
protein O-fucosyltransferase 1
-
-
protein O-fucosyltransferase 2
Q9Y2G5
-
protein O-fucosyltransferases 1
-
-
protein O-fucosyltransferases 2
-
-
CAS REGISTRY NUMBER
COMMENTARY
9033-08-3
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Candida elegans
-
-
-
Manually annotated by BRENDA team
Chinese hamster, CHO cells
-
-
Manually annotated by BRENDA team
enzyme has a distinct chaperone activity for Notch proteins independent of its enzymic activity
-
-
Manually annotated by BRENDA team
recombinant protein expressed in COS-1 cells
-
-
Manually annotated by BRENDA team
wild-type POFUT2 and high mannose type DelTA21-POFUT2
UniProt
Manually annotated by BRENDA team
floxed Pofut1 (Pofut1F/F) mice and Pofut1 heterozygous mice (Pofut1+/-)
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
evolution
Q9Y2G5
POFUT2 belongs to the classical GT-B fold family of glycosyltransferases with two closely interacting Rossmann-like domains. POFUT2 shows a variation of the classical GT-B fold
evolution
Q18014
CePOFUT1 is a member of the GT65 family and contains four conserved disulfide bridges through the GT65 family
malfunction
-
neural crest cell-specific Pofut1-knockout mice die within 1 day of birth, accompanied by a defect of enteric nervous system development. Sox10 expression is decreased in Pofut1-null enteric neural crest cells, whereas the number of enteric neural crest cells that express Mash1, a potent repressor of Sox10, is increased in the Pofut1-null mouse. Enteric neural crest cells lacking Pofut1 show premature neurogenesis and a decrease in the number of glial progenitors
malfunction
-
Pofut1 deletion inactivates Notch signaling, giving rise to smaller but viable mice. Dysplastic foci in Pofut1-deficient small intestine with occasional progression to tumor formation. Inactivation of Pofut1 leads to intestinal inflammation. Mucus hypersecretion upon Pofut1 inactivation is accompanied by alteration of the mucus-associated flora, which likely contributes to the development of enterocolitis
malfunction
-
CHO cells lacking Pofut1 express Notch receptors on the cell surface at similar levels to wild-type cells
malfunction
-
deletion of Pofut1 leads to global defects in Notch signaling and death of mice at E9.5, with a phenotype consistent with inactivation of signaling by the four Notch receptors. Embryonic stem cells lacking Pofut1 express Notch receptors on the cell surface at similar levels to wild-type cells. In mouse somites, there is evidence of altered Notch trafficking in the absence of Pofut1, consistent with reduced cell surface expression
malfunction
-
loss of OFUT1 results in the phenotype that is characteristic of Notch loss of function. Loss of OFUT1 leads to the loss of cell surface expression and the intracellular accumulation of Notch receptors. Secretion of Notch is impaired in OFUT1-depleted S2 cells and the abnormal endoplasmic reticulum accumulation of Notch receptors is observed in Ofut1 mutant clones in Drosophila wing imaginal discs
malfunction
-
loss of Pofut1 results in the phenotype that is characteristic of Notch loss of function
malfunction
-
siRNAs eliminating Pofut1 transcripts in CHO cells. CHO cells deficient in Pofut1 have reduced Notch signaling and ligand binding. Under certain conditions, mammalian Notch receptors can bind Notch ligands and transduce a Notch signal in the absence of Pofut1 and O-fucose
malfunction
-
embryonic stem cells lacking Pofut1 are deficient in Notch ligand binding, have wild-type levels of cell surface Notch receptors. Pofut1-/- embryonic stem cells do not bind Notch ligands or exhibit Notch signaling. Overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1-/- cells partially rescues ligand binding and Notch signaling, but this effect is not specific. Under certain conditions, mammalian Notch receptors can bind Notch ligands and transduce a Notch signal in the absence of Pofut1 and O-fucose
malfunction
-
in ofut1 mutant cells, Notch goes to the membrane, is internalized and accumulates in an uncharacterized endocytic compartment. Knockdown of Ofut1 using doublestranded RNA in cultured cells inhibits the secretion of a soluble version of the Notch extracellular domain. In ofut1 mutant cells, Notch does not accumulate at adherens junctions but instead accumulates into intracellular dots. A low level of Notch is also present at the surface of ofut1 mutant cells
malfunction
-
Pofut1-null mouse shows a similar phenotype to the RBP-Jk null mouse. Cardiac development in the Pofut1-/- mice is severely affected in valve formation, which is characterized in the lack of mesenchymal cells as seen in RBP-Jk null embryos and embryonic development is arrested at E9.0. Pofut1-null cells do not possess normally localized Notch1 receptors. Abnormal accumulation of the Notch1 receptor in the endoplasmic reticulum and cytoplasm in Pofut1-null mouse embryos
malfunction
-
mice lacking Pofut1 show myeloid hyperplasia and impaired lymphopoiesis. Mx-Cre/Pofut1F/F mice splenomegaly show in the bone marrow a decrease in T and B lymphocytes and an expansion of mature granulocytes and myeloid progenitors, mutant mice phenotypes, detailed overview. Mx-Cre/Pofut1F/F marrow progenitors have defective T lymphopoiesis and enhanced myeloid development in vitro that is correlated with decreased Notch ligand binding.. Defective T-cell development and myeloid hyperplasia are rescued by reinstating Notch1 signaling. Heterozygosity of Pofut1 affects rescue of FX-/- mice myeloid hyperplasia by exogenous fucose
malfunction
-
elimination of Pofut1 in mice has a profound effect on ligand binding in both embryonic stem cells and lymphoid cells. A small decrease in cell surface expression of Notch proteins is seen in embryonic stem cells lacking Pofut1 and in somites from mice with a hypomorphic allele of Pofut1, cax
physiological function
-
is essential for Notch signalling. Ability of OFUT1 to promote Notch secretion does not depend on its enzyme activity, suggesting the chaperon-like role of OFUT1
physiological function
-
is essential for Notch signalling
physiological function
-
Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but Pofut1 is not required for stable cell surface expression of Notch
physiological function
-
roles in the folding of Notch in the endoplasmic reticulum, in Notch-ligand binding and in endocytic trafficking of Notch. The carboxy-terminal extremity of Ofut1 contains a Lys-Asp-Glu-Leu (KDEL)-like motif that is dispensable for its catalytic activity but is required for both endoplasmic reticulum retention and function. Ofut1 is required only for Fringe-dependent signaling events
physiological function
-
Pofut1 is required for Notch signaling upstream of NICD1
physiological function
-
O-fucosylation is universally required for all Notch signaling. O-Fucose and O-glucose glycans on Notch occur at specific consensus sequences within the context of EGF repeats, which make up the majority of the Notch extracellular domain. Ofut1 might have a chaperone-like activity in Drosophila that is required for cell-surface expression of Notch in flies. Molecular mechanisms by which O-fucose and O-glucose glycans affect Notch function, overview
physiological function
-
O-fucosylation is universally required for all Notch signaling. O-Fucose and O-glucose glycans on Notch occur at specific consensus sequences within the context of EGF repeats, which make up the majority of the Notch extracellular domain. Molecular mechanisms by which O-fucose and O-glucose glycans affect Notch function
physiological function
Q9Y2G5
protein O-fucosyltransferase 2 catalyzes the protein O-fucosylation, a post-translational modification found on serine/threonine residues of thrombospondin type 1 repeats
physiological function
-
O-fucosylation of Notch is essential for its function
physiological function
-
Notch is a transmembrane receptor that shares homology with proteins containing epidermal growth factor-like repeats and mediates the cell-cell interactions necessary for many cell fate decisions. O-fucosyltransferase 1 catalyzes the O-fucosylation of these epidermal growth factor-like repeats. This O-fucose elongates, resulting in an O-linked tetrasaccharide that regulates the signaling activities of Notch. Fucosylation occurs in the lumen of the endoplasmicreticulum and Golgi. GDP-fucose uptake into the ER and Golgi is essential for fucosylation, Efr, a multifunctional nucleotide sugar transporter, and Golgi GDP-fucose transporter Gfr are involved in the biosynthesis of heparan sulfate-glycosaminoglycan chains and the O-fucosylation of Notch. Gfr but not Efr is crucial for the fucosylation of N-glycans, overview
malfunction
-
elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes, elimination of Pofut2 results in an early embryonic lethal phenotype in mice
additional information
-
O-glycome of Drosophila melanogaster by mass spectrometry, using beta-elimination to release the O-linked sugar modifications from total protein extracts of fly embryos, overview
additional information
Q9Y2G5
recognition of a small conserved 3D structural motif and mechanism of enzyme-protein substrate specificity. POFUT2 features a unique loop, residues 265-285, located on the opposite side of the large cleft, which protrudes from the C-terminal domain and which is attached to the N-terminal domain via a completely conserved tryptophan residue, 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. Structure-function analysis of POFUT2 and comparison to POFUT1
additional information
Q18014
GDP-fucose is bound in a conserved cavity formed mainly by amino acids from the C-terminal domain, it is localised in the interface where the two domains face each other, localisation of EGF repeat binding site in CePOFUT1, active site and ligand binding structure analysis, detailed overview
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
GDP-6-alkynyl fucose + protein
?
show the reaction diagram
-
6-alkynyl fucose is efficiently incorporated onto EGF repeats, TSRs, and N-glycan on Lfng and N-glycans on a number of proteins in crude lysates of CHO cells, e.g. the O-fucosylation site in EGF3 of mouse Notch1 and elongated by Lfng, mass spectrometry analysis, overview. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or click reaction, azido-biotin allows tagging and detection of 6AF-modified proteins
-
-
?
GDP-Fuc + biotin-DHPCTQALGNPCLNGGSCVPREATYECLCPGGFSGLHCEKG
?
show the reaction diagram
-
peptide of the fourth EGF domain of agrin (EGF4) is used as an acceptor substrate with biotin conjugated at the N-terminus
-
-
?
GDP-fucose + TSR1
GDP + fucosyl-TSR1
show the reaction diagram
-
-
-
-
?
GDP-fucose + TSR2
GDP + fucosyl-TSR2
show the reaction diagram
-
-
-
-
?
GDP-fucose + TSR3
GDP + fucosyl-TSR3
show the reaction diagram
-
-
-
-
?
GDP-fucose + TSR4
GDP + fucosyl-TSR4
show the reaction diagram
Q9Y2G5
-, GDP-fucose binding mode, overview. Activity with recombinant TSR4 mutants expressed in HEK293Tcells
-
-
?
GDP-L-fucose + Notch
?
show the reaction diagram
-
O-fucosylation of Notch, catalytic and non-catalytic activities
-
-
-
GDP-L-fucose + Notch
?
show the reaction diagram
-
Pofut1 transfers fucose in the endoplasmic reticulum, transfers fucose to Ser or Thr residues of epidermal growth factor-like repeats
-
-
-
additional information
?
-
-
fucosylates various synthetic peptides of EGF-1 domain of human factor VII, GDP-mannose, UDP-glucose, UDP-N-acetylglucosamine, UDP-galactose, UDP-H-acetylgalactosamine and UDP-xylose can replace GDP-fucose
-
-
-
additional information
?
-
-
O-fucosylation of thrombospondin-1 at Ser 377, Thr 432 and Thr 489
-
-
-
additional information
?
-
-
links fucose through an O-glycosidic linkage to a conserved serine or threonine residue in of the EGF-1 domain of human factor VII, various synthetic peptides serve as substrates
-
-
-
additional information
?
-
Q9H488
adds o-fucose to epidermal growth factor-like repeats
-
-
-
additional information
?
-
-
enzyme is an essential core member of Notch signaling pathways in mammals
-
-
-
additional information
?
-
-
may be involved in intracellular quality control
-
-
-
additional information
?
-
Q9V6X7
essential for Notch signaling
-
-
-
additional information
?
-
-
enzyme that glycosylates epidermal growth factor–like domains of Notch, also has a distinct Notch chaperone activity
-
-
-
additional information
?
-
Q18014
POFUT1s bind GDP-fucose and EGF repeats, and transfer this monosaccharide into small EGF repeats producing GDP during the reaction
-
-
-
additional information
?
-
-
Pofut1 adds fucose to Ser or Thr in the C2-x-x-x-x-(S/T)-C3 consensus sequence. Eliminating any of three highly conserved O-fucose sites at EGF 12, 26, or 27 within mouse Notch1 alters activity.
-
-
-
additional information
?
-
-
protein O-fucosyltransferase 1 and protein O-fucosyltransferase 2 add O-linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
GDP-fucose + TSR4
GDP + fucosyl-TSR4
show the reaction diagram
Q9Y2G5
-
-
-
?
additional information
?
-
-
enzyme is an essential core member of Notch signaling pathways in mammals
-
-
-
additional information
?
-
-
may be involved in intracellular quality control
-
-
-
additional information
?
-
Q9V6X7
essential for Notch signaling
-
-
-
additional information
?
-
Q18014
POFUT1s bind GDP-fucose and EGF repeats, and transfer this monosaccharide into small EGF repeats producing GDP during the reaction
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Ca2+
-
2fold increase at 10 mM
Ca2+
Q9Y2G5
activates, but less effectiv than Mg2+ and Mn2+
Mg2+
Q9Y2G5
activates
Mn2+
-
10fold increase at 50 mM, 4fold increase at 10 mM
Mn2+
-
17fold increase at 50 mM
Mn2+
Q9Y2G5
activates, but less effectiv than Mg2+
Mn2+
Q18014
activates transfer of fucose from DP-fucose to small EGF repeats
additional information
Q18014
Mg2+ is not required for catalytic activity by POFUT1, glycosyltransferases adopting GT-B folds are metal-independent
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
EDTA
Q9Y2G5
-
factor VII EGF-1
-
inhibition above 0.015 mM
-
Zn2+
Q9Y2G5
complete inhibition
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.006
-
factor VII EGF
Q9H488
-
-
0.015
-
factor VII EGF
-
-
-
0.004
-
GDP-fucose
Q9H488
-
0.006
-
GDP-fucose
-
-
0.0295
-
TSR4
Q9Y2G5
pH and temperature not specified in the publication
-
0.0098
-
GDP-fucose
Q9Y2G5
pH and temperature not specified in the publication
additional information
-
additional information
Q9Y2G5
steady-state kinetic measurements of wild-type and mutant POFUT2
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
2.4
-
GDP-fucose
Q9Y2G5
pH and temperature not specified in the publication
2.4
-
TSR4
Q9Y2G5
pH and temperature not specified in the publication
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
-
-
assay at
7
-
-
assay at
7.5
-
Q18014
assay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
Q18014
assay at
37
-
-
assay at
37
-
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
fucosylation-deficient Lec13 chinese hamster ovary cells have wild-type levels of Pofut1 and cell surface Notch receptors
Manually annotated by BRENDA team
-
presomitic mesoderm
Manually annotated by BRENDA team
-
derived from blastocyst outgrowths
Manually annotated by BRENDA team
additional information
Q9H488
gene is widely expressed in human tissues
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
OFUT1 may be a chaperone of Notch that is necessary for retaining Notch at the cell surface once it has traversed the secretory pathway
Manually annotated by BRENDA team
-
OFUT1 associates with Notch at the cell surface and promotes the constitutive endocytosis of Notch receptors
Manually annotated by BRENDA team
-
cell surface accumulation of Notch in ofut1 mutant cells
Manually annotated by BRENDA team
-
retention in the ER is required for normal OFUT1 function in vivo
Manually annotated by BRENDA team
-
OFUT1 may be a chaperone of Notch that is necessary for escorting Notch out of the endoplasmic reticulum
Manually annotated by BRENDA team
-
OFUT1 is required for the correct folding of Notch receptors in the endoplasmic reticulum and their subsequent trafficking to the cell surface
Manually annotated by BRENDA team
-
Ofut1 acts as a chaperone in the endoplasmic reticulum to promote the proper folding of the epidermal growth factor-like repeats of Notch, thereby ensuring its correct cell-surface localization and ligand-binding activity
Manually annotated by BRENDA team
-
OFUT1 promotes transcytosis of Notch from the apical plasma membrane to the adherens junctions
Manually annotated by BRENDA team
additional information
-
adherens junction
-
Manually annotated by BRENDA team
additional information
-
Ofut1 is required for the proper distribution of Notch at adherens junction
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
44000
-
-
SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
Q18014
the N- and the C-terminal domains adopt Rossmann-like folds, which are formed by a central beta-sheet surrounded by alpha-helices on both sides and these constitute the typical signature of a GT-B fold. The donor sugar, GDP-fucose, is localised in the interface where the two domains face each other
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
glycoprotein
-
glycoprotein with more than one high mannose type oligosaccharide chain
additional information
-
contains a KDEL-like sequence at the C-terminus for retention in the endoplasmic reticulum
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
purified recombinant truncated enzyme in apoform or complex with GDP-fucose, or GDP, or GDP and Mn2+, sitting drop method, 0.001 ml of 30 mg/ml protein is mixed with 0.001 ml of precipitant solution containing 100 mM HEPES, 100 mM MgCl2, 20% PEG 3350, pH 7.5, with or without 5 mM GDP, or 100 mM HEPES, 2% PEG 400, 1.8 M ammonium sulfate, pH 6.5, or 100 mM BIS-TRIS, 2 M ammonium sulfate, pH 6.0, 18°C, two crystals forms, X-ray diffraction structure determination and analysis at 1.54-2.60 A resolution
Q18014
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
50
55
Q18014
dependent on ligands present, Mn2+ shifts the optimum to 50°C, GDP to 55°C, overview
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant truncated POFUT1_26-382 from Pichia pastoris by affinity and ion exchange chromatography and gel filtration
Q18014
-
Q9H488
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression of a truncated form of CePOFUT1, comprising amino acids 26-382, excluding the signal sequence and the retention endoplasmic reticulum localisation sequence, as a secreted protein in Pichia pastoris
Q18014
-
Candida elegans, Drosophila melanogaster
-
-
Q9H488
-
Q91ZW2
embryonic stem cells transfected with wild-type or mutant R245A
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D242A
Q18014
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
Q18014
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
Q18014
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
Q18014
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
Q18014
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
N43A
Q18014
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
Q18014
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant
R240K
Q18014
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant
R40A
Q18014
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
-
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
-
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
Q9Y2G5
site-directed mutagenesis, the mutant shows 15% activity compared to the wild-type enzyme
E396A
Q9Y2G5
site-directed mutagenesis, the mutant shows 8% activity compared to the wild-type enzyme
R294A
Q9Y2G5
site-directed mutagenesis, inactive mutant
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
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
F357A
Q18014
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
additional information
Q18014
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
W245A
Q18014
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
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
Q9V6X7
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; 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; 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
E54A
Q9Y2G5
site-directed mutagenesis, inactive mutant
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
W273a
Q9Y2G5
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
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
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
molecular biology
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O-fucosylation is dispensable for many Notch signaling events during Drosophila development
molecular biology
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engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains