Information on EC 3.1.4.45 - N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
3.1.4.45
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RECOMMENDED NAME
GeneOntology No.
N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
glycoprotein N-acetyl-D-glucosaminyl-phospho-D-mannose + H2O = N-acetyl-D-glucosamine + glycoprotein phospho-D-mannose
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
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-
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SYSTEMATIC NAME
IUBMB Comments
glycoprotein-N-acetyl-D-glucosaminyl-phospho-D-mannose N-acetyl-D-glucosaminylphosphohydrolase
Acts on a variety of compounds in which N-acetyl-D-glucosamine is alpha-linked to a phosphate group, including the biosynthetic intermediates of the high mannose oligosaccharide components of some lysosomal enzymes and the products of EC 2.7.8.17 UDP-N-acetylglucosamine---lysosomal-enzyme N-acetylglucosaminephosphotransferase.
CAS REGISTRY NUMBER
COMMENTARY hide
75788-84-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
a human gut bacterium
UniProt
Manually annotated by BRENDA team
a human gut bacterium
UniProt
Manually annotated by BRENDA team
strain ATCC 13124
-
-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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fibroblasts of individuals with persistent stuttering have decreased enzyme activity, the stuttering phenotype is presumed to arise from neuron dysfunction, overview
metabolism
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the enzyme is a Golgi enzyme that mediates the second step in the synthesis of the D-mannose 6-phosphate lysosomal targeting signal on acid hydrolases
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,4-dinitrophenyl alpha-N-acetyl-D-glucosaminide + H2O
2,4-dinitrophenol + alpha-N-acetyl-D-glucosamine
show the reaction diagram
-
-
-
-
?
4-nitrophenyl alpha-N-acetyl-D-glucosaminide + H2O
4-nitrophenol + alpha-N-acetyl-D-glucosamine
show the reaction diagram
-
-
-
-
?
6-O-(N-acetyl-alpha-D-glucosaminyl)phosphono-D-mannopyranose + H2O
N-acetyl-D-glucosamine + 6-O-phosphono-D-mannopyranose
show the reaction diagram
-
-
-
?
6-O-(N-acetyl-D-glucosaminyl)phospho-D-mannose + H2O
N-acetyl-D-glucosamine + 6-phospho-D-mannose
show the reaction diagram
GlcNAc-phosphomannose alpha-methyl + H2O
GlcNAc + phosphomannose alpha-methyl
show the reaction diagram
glycoprotein N-acetyl-D-glucosaminyl-phospho-D-mannose + H2O
N-acetyl-D-glucosamine + glycoprotein phospho-D-mannose
show the reaction diagram
methyl 6-O-(N-acetyl-D-glucosaminyl)phospho-alpha D-mannose + H2O
N-acetyl-D-glucosamine + methyl 6-phospho-alpha-D-mannopyranoside
show the reaction diagram
methyl N-acetyl-D-glucosaminyl-6-phospho-D-mannopyranoside + H2O
N-acetyl-D-glucosamine + methyl-6-phospho-alpha-D-mannopyranoside
show the reaction diagram
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low activity
-
-
?
N-acetyl-D-glucosamine-6-phospho-D-mannose + H2O
N-acetyl-D-glucosamine + 6-phospho-D-mannose
show the reaction diagram
-
-
-
-
?
N-acetyl-D-glucosaminyl-alpha-methyl-phospho-D-mannose + H2O
N-acetyl-D-glucosamine + alpha-methyl-phospho-D-mannose
show the reaction diagram
N-acetylglucosamine-phosphate
N-acetylglucosamine + phosphate
show the reaction diagram
N-acetylglucosamine-phosphomannose-uteroferrin + H2O
N-acetylglucosamine + phosphomannose-uteroferrin
show the reaction diagram
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-
-
-
?
UDP-N-acetyl-alpha-D-glucosamine + H2O
N-acetyl-D-glucosamine + UDP
show the reaction diagram
UDP-N-acetyl-D-glucosamine + H2O
UDP + N-acetyl-D-glucosamine
show the reaction diagram
[alpha-L-iduronidase]-N-acetyl-D-glucosaminyl-phospho-D-mannose + H2O
N-acetyl-D-glucosamine + [alpha-L-iduronidase]-phospho-D-mannose
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
glycoprotein N-acetyl-D-glucosaminyl-phospho-D-mannose + H2O
N-acetyl-D-glucosamine + glycoprotein phospho-D-mannose
show the reaction diagram
[alpha-L-iduronidase]-N-acetyl-D-glucosaminyl-phospho-D-mannose + H2O
N-acetyl-D-glucosamine + [alpha-L-iduronidase]-phospho-D-mannose
show the reaction diagram
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-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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unaffected by divalent cation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-acetamido-1,2-dideoxynojirimycin
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competitive inhibitor
6-acetamido-6-deoxycastanospermine
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competitive inhibitor
acetoamino-deoxycastanospermine
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CdSO4
Cu2+
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reduces activity by 30-40%
CuSO4
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D-glucosamine 6-phosphate
D-glucose 1-phosphate
D-glucose 6-phosphate
D-mannose 1-phosphate
D-mannose 6-phosphate
diphosphate
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dithiothreitol
FeCl2
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FeCl3
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GDP-mannose
Hg2+
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reduces activity by 30-40%
mannose-6-phosphate
MgCl2
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inhibits activity by 30%
MnCl2
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inhibits activity by 30%
N-acetyl-L-galactosamine
N-acetyl-L-glucosamine
N-acetyl-L-glucosamine 1-phosphate
N-acetyl-L-mannosamine
N-acetylglucosamine
N-acetylglucosamine phosphate
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O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate
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competitive inhibitor
phosphate
PO43-
SDS
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0.02% SDS inhibits activity by 14%
UDP-galactose
UDP-GlcNAc
UDP-glucose
UDP-N-acetyl-galactosamine
UDP-N-acetyl-glucosamine
Zn2+
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inhibits by 50%
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
furin
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UCE is synthesized as an inactive proenzyme that is specifically activated by the endoprotease furin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0011
4-nitrophenyl alpha-N-acetyl-D-glucosaminide
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in 0.1 M sodium phosphate, pH 7.3, at 30C
0.74
dinitrophenyl alpha-N-acetyl-Dglucosaminide
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in 0.1 M sodium phosphate, pH 7.3, at 30C
0.4 - 0.74
GlcNAc-phosphomannose alpha-methyl
1.6
N-acetylglucosamine 1-phosphate
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31
N-acetylglucosamine-phosphate
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-
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0.76
N-acetylglucosamine-phospho-alpha-methylmannoside
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0.19
N-acetylglucosamine-phospho-methylmannoside
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0.04
N-acetylglucosamine-phosphomannose-uteroferrin
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0.78
N-acetylglucosamine-UDP
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0.734 - 0.94
UDP-GlcNAc
1.3
UDP-N-acetylglucosamine
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.26
4-nitrophenyl alpha-N-acetyl-D-glucosaminide
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8.5
dinitrophenyl alpha-N-acetyl-Dglucosaminide
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.92
6-acetamido-6-deoxycastanospermine
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0.062
O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.2
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recombinant UCE expressed in CHO-K1 cells
4.75
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recombinant UCE expressed in Sf9 insect cells
15000
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purified enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
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6.5
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UDP-GlcNAc
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
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4.5 - 8.8
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at pH 4.5 and pH 8.8 activity more than 50% of activity at pH 6.8
6 - 9.5
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serum
6.5 - 9
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liver
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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soluble enzyme
Manually annotated by BRENDA team
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mouse WT 1-38 L-cells expressing human wild-type UCE
Manually annotated by BRENDA team
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derived from a lymph node metastasis of a human colon adenocarcinoma, CCL-229, extremely low UCE activity due to a lack of the activating endoprotease furin, addition of furin restores UCE activity
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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in tubulovesicular regions of the trans-Golgi network and in clathrin-coated trans Golgi network vesicles and buds, GFP-fusion protein expressed in HeLa cells, localization of mutants E502stop and Y488A/Q492A/E502stop like wild-type
Manually annotated by BRENDA team
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recombinant UCE expressed in CHO-K1 cells and in Sf9 insect cells
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
68000
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liver, reducing SDS-PAGE
77000
liver, reducing SDS-PAGE
80000
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x * 80000, sialylated UCE, SDS-PAGE
114000
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lymphoblasts, nonreducing SDS-PAGE
118000
serum
121000
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liver, nonreducing SDS-PAGE
129000
136000
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liver, nonreducing SDS-PAGE
156000
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radiation inactivation, skin fibroblast
204600
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liver, Svedberg equation
270000
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fetal calf serum, gel filtration
272000
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liver, gel filtration
additional information
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1885 Da, peptides of amino acid position 80 to 84 and 268 to 274, determined by LC/ESI-MS/MS analysis, soluble form; 2797 Da, peptide of amino acid position 159 to 183, determined by MALDI-TOF and by LC/MS, soluble form; 2878 Da, peptide of amino acid position 331 to 355, determined by LC/MS, soluble form; 2925 Da, peptide of amino acid position 305 to 355, determined by MALDI-TOF and by LC/MS, soluble form; 3124 Da, peptides of amino acid position 54 to 69 and 94 to 108, determined by LC/ESI-MS/MS analysis, soluble form; 3230 Da, peptides of amino acid position 1 to 4 and 159 to 182, determined by LC/ESI-MS/MS analysis, soluble form; 4023 Da, peptide of amino acid position 228 to 264, determined by MALDI-TOF and by LC/MS, soluble form; 4337 Da, peptides of amino acid position 228 to 264 and 265 to 267, determined by LC/ESI-MS/MS analysis, soluble form; 880 Da, peptide of amino acid position 369 to 376, determined by MALDI-TOF, soluble form
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 80000, sialylated UCE, SDS-PAGE
homotetramer
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monomer
tetramer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
proteolytic modification
sialoprotein
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addition of sialic acid in the trans-Golgi network
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant detagged wild-type and selenomethionine-labeled enzyme, sitting nanodroplet vapor diffusion method, mixing of 200 nl of 21 mg/ml protein in 20 mM HEPES, pH 8.0, 200 mM NaCl, 40 mM imidazole, and 1 mM TCEP, with 200 nl of reservoir solution containing 0.2 M Li2SO4, 30% PEG 4000, and 0.1 M Tris, pH 8.5, and equilibration against 0.05 ml of reservoir solution, 4C, 10% v/v ethylene glycol as a cryoprotectant, X-ray diffraction structure determination and analysis at 1.80 A resolution, modeling, the crystal structure of the BACOVA_00430 protein from Bacteroides ovatus is used to model the luminal portion of the human enzyme N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase
hanging drop vapour diffusion method, in 2.0 M (NH4)2SO4 with 3% (v/v) glycerol
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
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at pH 4, 54% of activity, at pH 10, 83% of activity
135493
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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after 3 min 80% of activity
60
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stable for 10 min
70
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activity diminished to 20% and 10%
75
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after 3 min only 7.6% activity
80
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activity diminished to 20% and 10%
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
loss of activity by repeated freezing and thawing, or dialysis
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, superose 6 eluate, 3 months
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-80C, 20% glycerol, pH 7.4, 6 months
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27% loss of activity after 120 days at -80C
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4C, 1 month
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50% loss of activity after 120 days at 4C
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
499fold: recombinant UCE expressed in CHO-K1 cells, 1392fold: recombinant UCE expressed in Sf9 insect cells
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affinity chromatography on HPC4 monoclonal antibody column
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recombinant enzyme with TEV cleavage site and His6-tag by nickel affinity chromatography, tag cleavage by tobacco etch virus protease, another step of nickel affinity chromatography, and centrifugal ultrafiltration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as GFP-fusion protein in HeLa cells
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expressed in Escherichia coli BL21 Star (DE3) cells
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expression in COS-cells
expression in mouse L-cells, expression of UCE containing green fluorescent protein in place of the luminal domain in HeLa cells
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expression in mouse L-cells, in CHO-K1 cells and in Sf9 insect cells
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expression of the soluble form in COS cells
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polymerase incomplete primer extension cloning method
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polymerase incomplete primer extension cloning method, recombinant expression of enzyme with TEV cleavage site and His6-tag in Escherichia coli as wild-type and selenomethionine-labeled enzyme
two splice forms, one of these isoforms lacks 102-base pairs corresponding to exon 8 of the 10 exons present in the genomic DNA and is predominantly expressed in brain. Expression of wild-type and mutant enzymes in HeLa cells
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wild-type and mutant UCE expression in mouse L-cells, expression of various green fluorescent protein-UCE fusion proteins in HeLa cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C115X
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site-directed mutagenesis, the mutation greatly impairs folding of the enzyme, the mutant is retained in the endoplasmic reticulum
C132X
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site-directed mutagenesis, the mutation greatly impairs folding of the enzyme, the mutant is retained in the endoplasmic reticulum
C221L
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site-directed mutagenesis, the mutation leads to loss of enzymatic activity
C51M
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site-directed mutagenesis, the mutant shows 65% of wild-type activity
C51M/C221L
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site-directed mutagenesis, the mutant folds adequately and is trafficked to the Golgi where it is cleaved by furin, the mutant shows only 9.7% of wild-type activity
E502stop
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similar localization to the trans Golgi network as the wild-type, lumenal domain of UCE was replaced with monomeric GFP
F513S
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naturally occuring Phe513SerfsX113 frameshift mutation that adds 113 amino acids to the C-terminus of the cytoplasmic tail of the protein including a VWLL sequence, causing rapid degradation via the proteasomal system
H510stop
H84Q
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naturally occuring mutation that impairs folding and in addition, decreases the specific activity of the enzyme, to about half the relative specific activity of wild-type enzyme, but folds sufficiently to traffic to the Golgi
M494stop
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mutant without increased cell surface expression
N137A
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site-directed mutagenesis, the mutant shows 11% of wild-type activity
N281A
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site-directed mutagenesis, the mutant is recombinantly expressed but retained in the endoplasmic reticulum
Q225H
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site-directed mutagenesis, exchange of the human residue for the residue of protein BACOVA_00430 from Bacteroides ovatus, the mutant shows 5.8% of wild-type activity
R247A
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site-directed mutagenesis, the mutant shows 87% of wild-type activity
R328C
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naturally occuring mutation that impairs folding in the endoplasmic reticulum, resulting in degradation of a significant portion by the proteasomal system. The mutation leads to lower cellular UCE activity, and might be involved in persistent stuttering phenomena. The R328C mutant forms oligomers with the wild-type enzyme
T227R
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site-directed mutagenesis, exchange of the human residue for the residue of protein BACOVA_00430 from Bacteroides ovatus, the mutant shows 0.1% of wild-type activity
T320A
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site-directed mutagenesis, the mutant shows 43% of wild-type activity
V318A
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site-directed mutagenesis, the mutant is recombinantly expressed but retained in the endoplasmic reticulum
V322A
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site-directed mutagenesis, the mutant shows 67% of wild-type activity
Y486A
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mutated residue is required for efficient return of the enzyme from endosomes to the trans-Golgi network, mutant UCE is internalized normally but accumulates on the cell surface, 26-36% of activity on the cell surface compared with 1-3% of wild-type activity, because of increased recycling to the plasma membrane, kinetics of internalization
Y486stop
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mutant with 16% of total enzyme activity on the cell surface at steady state compared with 1-3% of wild-type activity, kinetics of internalization
Y488A/E493A/E502stop
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localized to a large extent to the cell surface, lumenal domain of UCE was replaced with monomeric GFP
Y488A/E502stop
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localized to a large extent to the cell surface, lumenal domain of UCE was replaced with monomeric GFP
Y488A/G496A/E497A/E502stop
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localized to a large extent to the cell surface, lumenal domain of UCE was replaced with monomeric GFP
Y488A/M494A/E502stop
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localization shows endosome pattern, not at the cell surface, lumenal domain of UCE was replaced with monomeric GFP
Y488A/M494A/N495A/E502stop
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intermediate phenotype, localized to the cell surface and to the trans Golgi network, lumenal domain of UCE was replaced with monomeric GFP
Y488A/N495A/E502stop
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localization shows endosome pattern, not at the cell surface, lumenal domain of UCE was replaced with monomeric GFP
Y488A/P498A/L499A/E502stop
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localized to a large extent to the cell surface, lumenal domain of UCE was replaced with monomeric GFP
Y488A/Q492A/E493A/E502stop
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intermediate phenotype, localized to the cell surface and to the trans Golgi network, lumenal domain of UCE was replaced with monomeric GFP
Y488A/Q492A/E493A/M494A/N495A/E502stop
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localization shows endosome pattern and localized to the Golgi, lumenal domain of UCE was replaced with monomeric GFP
Y488A/Q492A/E502stop
-
similar localization to the trans Golgi network as the wild-type, lumenal domain of UCE was replaced with monomeric GFP
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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the uncovering enzyme is generated as soluble enzyme and so can be used to conduct the in vitro phosphorylation of purified recombinant lysosomal enzymes