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Effects of mutations and glycosylations on STS activity: a site-directed mutagenesis study

Stengel, C.; Newman, S.P.; Day, J.M.; Tutill, H.J.; Reed, M.J.; Purohit, A.; Mol. Cell. Endocrinol. 283, 76-82 (2008)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
3.1.6.2
His6-tagged wild-type STS cDNA subcloned into pCEP4, transfected into 293-EBNA cells, cell line with stable overexpression of STS-His6 selected for production of tagged wildtype STS, subsequent specific antibody production, mutated STS is transfected into human MCF-7 cells and monkey COS-1 cells to test for STS activity by immunoblotting
Homo sapiens
Engineering
EC Number
Amino acid exchange
Commentary
Organism
3.1.6.2
DELTA1-21
lacks 21 N-terminal amino acids, that encode the signal peptide, reduced STS activity
Homo sapiens
3.1.6.2
DELTA185-583
lacks 399 C-terminal amino acids, reduced STS activity
Homo sapiens
3.1.6.2
H136F
point mutation in histidine 136, reduced STS activitiy
Homo sapiens
3.1.6.2
H136W
point mutation in histidine 136, reduced STS activity
Homo sapiens
3.1.6.2
H136Y
point mutation in histidine 136, reduced STS activity
Homo sapiens
3.1.6.2
additional information
site directed mutagenisis is introduced using pALTERMax kit
Homo sapiens
3.1.6.2
N259Q
point mutation in asparagine 259 glycosylation site, reduced STS activity
Homo sapiens
3.1.6.2
N333Q
point mutation in other potential glycosylation site asparagine 333, no reduced STS activity
Homo sapiens
3.1.6.2
N459Q
point mutation in other potential glycosylation site asparagine 459, no reduced STS activity
Homo sapiens
3.1.6.2
N47Q
point mutation in asparagine 47 glycosylation site, reduced STS activity
Homo sapiens
3.1.6.2
N47Q/N259Q
double mutation in asparagine 47 and 259 glycosylation sites, reduced STS activity
Homo sapiens
3.1.6.2
N54Q
point mutation in in asparagine 54 non-glycosylation site , no reduced STS activity
Homo sapiens
3.1.6.2
P212G
point mutation in proline 212, no reduced STS activity
Homo sapiens
Localization
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
3.1.6.2
endoplasmic reticulum
-
Homo sapiens
5783
-
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
3.1.6.2
61000
-
SDS-PAGE, immunoblot analysis with specific antibody
Homo sapiens
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
3.1.6.2
estrone sulfate
Homo sapiens
-
estrone + sulfate
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
3.1.6.2
Homo sapiens
P08842
-
-
Posttranslational Modification
EC Number
Posttranslational Modification
Commentary
Organism
3.1.6.2
glycoprotein
4 potential glycosylation sites
Homo sapiens
Purification (Commentary)
EC Number
Commentary
Organism
3.1.6.2
affinity chromatography with NiCl2-primed HisTrap Chelating columns and concentrated 6-fold using ICON concentrators
Homo sapiens
Source Tissue
EC Number
Source Tissue
Commentary
Organism
Textmining
3.1.6.2
HEK-293-EBNA cell
human embryonic kidney carcinoma cell line selected for overexpression of transfected His6-tagged STS cDNA to aid purification of the His6-tagged STS protein and subsequent antibody production in mice and rabbits
Homo sapiens
-
3.1.6.2
JEG-3 cell
choriocarcinoma cell line used for expression test for transfection experiments
Homo sapiens
-
3.1.6.2
MCF-7 cell
ER positive human breast cancer cells, used for transfection and activity experiments, shows STS activity by itself in contrast to monkey COS-1 cells used for the same experiments
Homo sapiens
-
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.1.6.2
estrone sulfate
-
694168
Homo sapiens
estrone + sulfate
-
-
-
?
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
3.1.6.2
His6-tagged wild-type STS cDNA subcloned into pCEP4, transfected into 293-EBNA cells, cell line with stable overexpression of STS-His6 selected for production of tagged wildtype STS, subsequent specific antibody production, mutated STS is transfected into human MCF-7 cells and monkey COS-1 cells to test for STS activity by immunoblotting
Homo sapiens
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
3.1.6.2
DELTA1-21
lacks 21 N-terminal amino acids, that encode the signal peptide, reduced STS activity
Homo sapiens
3.1.6.2
DELTA185-583
lacks 399 C-terminal amino acids, reduced STS activity
Homo sapiens
3.1.6.2
H136F
point mutation in histidine 136, reduced STS activitiy
Homo sapiens
3.1.6.2
H136W
point mutation in histidine 136, reduced STS activity
Homo sapiens
3.1.6.2
H136Y
point mutation in histidine 136, reduced STS activity
Homo sapiens
3.1.6.2
additional information
site directed mutagenisis is introduced using pALTERMax kit
Homo sapiens
3.1.6.2
N259Q
point mutation in asparagine 259 glycosylation site, reduced STS activity
Homo sapiens
3.1.6.2
N333Q
point mutation in other potential glycosylation site asparagine 333, no reduced STS activity
Homo sapiens
3.1.6.2
N459Q
point mutation in other potential glycosylation site asparagine 459, no reduced STS activity
Homo sapiens
3.1.6.2
N47Q
point mutation in asparagine 47 glycosylation site, reduced STS activity
Homo sapiens
3.1.6.2
N47Q/N259Q
double mutation in asparagine 47 and 259 glycosylation sites, reduced STS activity
Homo sapiens
3.1.6.2
N54Q
point mutation in in asparagine 54 non-glycosylation site , no reduced STS activity
Homo sapiens
3.1.6.2
P212G
point mutation in proline 212, no reduced STS activity
Homo sapiens
Localization (protein specific)
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
3.1.6.2
endoplasmic reticulum
-
Homo sapiens
5783
-
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
3.1.6.2
61000
-
SDS-PAGE, immunoblot analysis with specific antibody
Homo sapiens
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
3.1.6.2
estrone sulfate
Homo sapiens
-
estrone + sulfate
-
-
?
Posttranslational Modification (protein specific)
EC Number
Posttranslational Modification
Commentary
Organism
3.1.6.2
glycoprotein
4 potential glycosylation sites
Homo sapiens
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
3.1.6.2
affinity chromatography with NiCl2-primed HisTrap Chelating columns and concentrated 6-fold using ICON concentrators
Homo sapiens
Source Tissue (protein specific)
EC Number
Source Tissue
Commentary
Organism
Textmining
3.1.6.2
HEK-293-EBNA cell
human embryonic kidney carcinoma cell line selected for overexpression of transfected His6-tagged STS cDNA to aid purification of the His6-tagged STS protein and subsequent antibody production in mice and rabbits
Homo sapiens
-
3.1.6.2
JEG-3 cell
choriocarcinoma cell line used for expression test for transfection experiments
Homo sapiens
-
3.1.6.2
MCF-7 cell
ER positive human breast cancer cells, used for transfection and activity experiments, shows STS activity by itself in contrast to monkey COS-1 cells used for the same experiments
Homo sapiens
-
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.1.6.2
estrone sulfate
-
694168
Homo sapiens
estrone + sulfate
-
-
-
?