Information on EC 2.4.99.3 - alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.4.99.3
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RECOMMENDED NAME
GeneOntology No.
alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
CMP-N-acetylneuraminate + glycano-(1->3)-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein = CMP + glycano-[(2->6)-alpha-N-acetylneuraminyl]-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycosyl group transfer
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
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Mucin type O-glycan biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
CMP-N-acetylneuraminate:glycano-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein alpha-2,6-N-acetylneuraminyltransferase
alpha-N-Acetylgalactosamine linked to threonine or serine is also an acceptor, when substituted at the 3-position.
CAS REGISTRY NUMBER
COMMENTARY hide
71124-50-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
strain OF1
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Manually annotated by BRENDA team
strain OF1
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Manually annotated by BRENDA team
rainbow trout
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
CMP-9-acetamidoneuraminate + R-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + R-(2,6-9-acetamidoneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
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less effective than CMP-N-acetylneuraminate
-
-
?
CMP-9-aminoacetylneuraminate + R-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + R-(2,6-9-aminoacetylneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
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CMP-9-aminoacetylneuraminate can replace CMP-N-acetylneuraminate only with asialofetuin as acceptor
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-
?
CMP-9-azido-N-acetylneuraminate + R-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + R-(2,6-9-azido-N-acetylneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
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CMP-9-azido-N-acetylneuraminate can replace CMP-N-acetylneuraminate with asialofetuin or antifreeze glycoprotein as acceptor
-
-
?
CMP-9-benzamidoneuraminate + R-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + R-(2,6-9-benzamidoneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
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less effective than CMP-N-acetylneuraminate
-
-
?
CMP-9-hexanoylamidoneuraminate + R-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + R-(2,6-9-hexanoylamidoneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
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less effective than CMP-N-acetylneuraminate
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-
?
CMP-9-O-acetyl-N-acetylneuraminate + R-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + R-(2,6-9-O-acetyl-N-acetylneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
CMP-N-acetylneuraminate + antifreeze glycoprotein
?
show the reaction diagram
CMP-N-acetylneuraminate + asialo-fetuin
CMP + fetuin
show the reaction diagram
CMP-N-acetylneuraminate + asialo-milk glycopeptide
CMP + milk glycopeptide
show the reaction diagram
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-
-
-
?
CMP-N-acetylneuraminate + asialo-orosomucoid
CMP + orosomucoid
show the reaction diagram
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-
-
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?
CMP-N-acetylneuraminate + asialo-polysialoglycoprotein
CMP + alpha-sialosyl-(2-6)-asialo-polysialoglycoprotein
show the reaction diagram
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from egg, Sia residue is alpha-2,6-linked to the proximal GalNAc residue in asialo-polysialoglycoprotein, acceptor carbohydrate unit: Galbeta(1-3)GalNAcalpha(1-O)-Ser/Thr, but not GalNAc1 to O-Ser/Thr
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?
CMP-N-acetylneuraminate + erythrocyte hemagglutination inhibitor
?
show the reaction diagram
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-
-
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?
CMP-N-acetylneuraminate + Galbeta(1-3)GalNAc-R
CMP + Galbeta(1-3)(NeuAcalpha(2-6))GalNAc-R
show the reaction diagram
CMP-N-acetylneuraminate + GalNAc-R
CMP + NeuAcalpha(2-6)GalNAc-R
show the reaction diagram
CMP-N-acetylneuraminate + glycano-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + glycano-(2,6-alpha-N-acetylneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
CMP-N-acetylneuraminate + GTTPSPVPT[GalNAc]TSTTSAP
CMP + GTTPSPVPT[Neu5Acalpha(2-6)GalNAc]TSTTSAP
show the reaction diagram
hexadecaglycopeptide acceptor, corresponding to the MUC5AC tandem repeat sequence substituted by a GalNAc residue on Thr-9, hST6GalNAc-I
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?
CMP-N-acetylneuraminate + lactose
CMP + 6'-sialyllactose
show the reaction diagram
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poor acceptor
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?
CMP-N-acetylneuraminate + native fetuin
?
show the reaction diagram
CMP-N-acetylneuraminate + native polysialoglycoprotein
?
show the reaction diagram
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from egg, almost identical amount of Neu5Ac is incorporated into native and asialo-polysialoglycoprotein acceptor, acceptor carbohydrate unit: Galbeta(1-3)GalNAcalpha(1-O)-Ser/Thr, but not GalNAc1 to O-Ser/Thr
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-
?
CMP-N-acetylneuraminate + NeuAcalpha(2-3)Galbeta(1-3)GalNAc-R
CMP + NeuAcalpha(2-3)Galbeta(1-3)(NeuAcalpha(2-6))GalNAc-R
show the reaction diagram
CMP-N-acetylneuraminate + p-nitrophenyl-beta-D-galactoside
CMP + alpha-sialosyl-(2-6)-p-nitrophenyl-beta-D-galactoside
show the reaction diagram
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-
-
?
CMP-N-acetylneuraminate + protein MUC1-MFP6
?
show the reaction diagram
CMP-N-acetylneuraminate + R-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + R-(2,6-alpha-N-acetylneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
CMP-N-acetylneuraminate + submaxillary asialo-mucin
CMP + submaxillary mucin
show the reaction diagram
CMP-N-acetylneuraminate + VPSTPPTPSPSTPPTPSPSCCHPR-GalNAc
CMP + ?
show the reaction diagram
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-
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?
CMP-N-glycolylneuraminate + R-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + R-(2,6-alpha-N-glycolylneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
CMP-Neu5Ac + asialofetuin
CMP + ?
show the reaction diagram
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-
-
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?
CMP-Neu5Ac + fetuin
CMP + ?
show the reaction diagram
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isoform ST6GalNAc-I, 84% of reactivity with asialofetuin, isoform ST6GalNAc-II, 88% of reactivity with asialofetuin
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?
CMP-Neu5Ac + HIV 120gp (17-mer)
CMP + ?
show the reaction diagram
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isoform ST6GalNAc-I, 34% of reactivity with asialofetuin, isoform ST6GalNAc-II, 38% of reactivity with asialofetuin
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?
CMP-Neu5Ac + HIV 120gp (21-mer)
CMP + ?
show the reaction diagram
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isoform ST6GalNAc-I, 62% of reactivity with asialofetuin, isoform ST6GalNAc-II, 58% of reactivity with asialofetuin
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?
CMP-sialic acid-PEG4-biotin + desialyated bovine serum albumin
?
show the reaction diagram
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bacterially produced ST6 is active and capable of catalyzing the transfer of sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin
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?
additional information
?
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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
CMP-N-acetylneuraminate + glycano-1,3-(N-acetyl-alpha-D-galactosaminyl)-glycoprotein
CMP + glycano-(2,6-alpha-N-acetylneuraminyl)-(N-acetyl-D-galactosaminyl)-glycoprotein
show the reaction diagram
CMP-N-acetylneuraminate + protein MUC1-MFP6
?
show the reaction diagram
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recombinant protein substrate expressed in MDA-MB-231 breast cancer cells, glycoprofiling, overview
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Antifreeze glycoprotein
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competitive to ovine submaxillary asialo-mucin, non-competitive to CMP-NeuAc
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CDP
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less effective than CTP
CMP
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less effective than CTP
dithioerythritol
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PCMB
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weak
Triton X-100
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above critical micelle concentration of 0.016%, 15% inhibition, reversible
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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ST6 activity yield produced in Sf9 insect cells is higher as compared to bacterial cells
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.02
Antifreeze glycoprotein
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antifreeze glycoprotein 8, pH 6.5, 37C
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0.08 - 0.1
CMP-9-O-acetyl-N-acetylneuraminate
0.15 - 2.8
CMP-N-acetylneuraminate
0.21 - 0.52
CMP-N-glycolylneuraminate
1.9 - 4.7
submaxillary asialo-mucin
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.92
Antifreeze glycoprotein
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pH 6.5, 37C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0003 - 0.005
0.00435
28.3
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37C
44.6
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.9
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acceptor: asialo-fetuin
6 - 7
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acceptor: asialo-polysialoglycoprotein
6 - 6.1
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with cacodylate-acetate buffer
6 - 6.5
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7.5
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acceptor: asialo-fetuin
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2 - 8.5
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about half-maximal activity at pH 6.2 and 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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assay at
25
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broad
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17 - 38
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about half-maximal activity at 17C and 38C
20 - 40
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about half-maximal activity at 20C and 40C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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unfertilized mature eggs
Manually annotated by BRENDA team
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malignant hematopoietic cell. Sialylated histo-blood group carbohydrate CD175 is present in all hematopoietic cell lines and native lymphocytes examined. CD175 and sialylated CD175 are preferentially expressed on erythroblastic leukemia cell lines. Presence of sialylated CD175 is consistent with the expression of the gene encoding alpha2,6-sialyltransferase ST6GalNAc-1
Manually annotated by BRENDA team
colon cancer cells, hST6GalNAc-I
Manually annotated by BRENDA team
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hematopoietic cell line, high ST6GalNAc-I expression
Manually annotated by BRENDA team
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human colon cancer cell line, recombinant ST6GalNAc-II
Manually annotated by BRENDA team
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sialylated histo-blood group carbohydrate CD175 is present in all hematopoietic cell lines and native lymphocytes examined. CD175 and sialylated CD175 are preferentially expressed on erythroblastic leukemia cell lines. Presence of sialylated CD175 is consistent with the expression of the gene encoding alpha2,6-sialyltransferase ST6GalNAc-1
Manually annotated by BRENDA team
normal human mammary epithelial cell strains 70N, 76N and 81N, STM RNA expression is highest in 76N, followed by 81N and 70N
Manually annotated by BRENDA team
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human colon carcinoma cells, recombinant ST6GalNAc-II
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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less than 4% of sialyltransferase activity
Manually annotated by BRENDA team
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in ovaries 96% of sialyltransferase activity is found in the Golgi-derived immature cortical vesicles or as soluble enzyme released from the cortical vesicles
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
39000
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x * 65000, isoform ST6GalNAc-I, x * 39000, isoform ST6GalNAc-II, SDS-PAGE
42000
x * 42000, predicted from the amino acid sequence
100000 - 172000
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enzyme exists in several active molecular weight forms from 100000-172000, gel filtration or PAGE
104000
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maltose-binding protein-ST6, Western blotting
120000
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x * 120000, SDS-PAGE
160000
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x * 160000, enzyme consists of several electrophoretic forms, 160 kDa: largest one, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 6.5
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maximal stability
636800
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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t1/2: about 60 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
50 mM NaCl stabilizes during purification
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bovine serum albumin, 1 mg/ml, stabilizes purified enzyme
glycerol, 25% w/v, stabilizes during purification
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glycerol, 50% w/v, stabilizes during storage
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 10 mM sodium cacodylate, pH 6.5, 50 mM NaCl, 50% glycerol, 0.02% NaN3, at least 6 months, stable
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4C, partially purified, at least 6 months
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frozen, crude preparation, at least 2 months, stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA agarose column chromatography
partial
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partial, 44fold
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recombinant ST6GalNAc-II, immobilized metal-affinity chromatography
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solubilized with Triton X-100, CDP-agarose affinity chromatography, 117000fold
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solubilized with Triton X-100, CDP-agarose affinity chromatography, 4500fold
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a codon-optimized gene encoding the catalytic domain of ST6 (amino acids Lys36-end) cloned into pTrc99a downstream from the tac promoter, FLAG tag and an octahistidine tag fused at the N- and C-termini, respectively. Catalytic domain of ST6 cloned into the high-copy number plasmid pCWin2MBP, which expresses an N-terminal fusion of ST6 with the Escherichia coli maltose-binding protein under the control of a dual tac promoter. Expression of soluble ST6 in a variety of Escherichia coli strains with oxidizing cytoplasm. Fusion of the ST6 enzyme to maltose-binding protein expressed in Origami 2 cells
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cDNA encoding the full-length hST6GalNAc-I from HT-29-MTX cells is cloned, stable transfection of MDA-MB-231 breast cancer cells expressing enzyme induces the expression of sialyl-Tn antigen at the cell surface associated with morphological changes, decreased growth and increased migration of the cells
expressed in HEK-293 cells
expressed in Sf9 insect cells
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expressed in Sf9 insect cells or HEK-293T cells
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overexpression in baculovirus system
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SIATL1 gene encoding STM is mapped to the long arm of chromosome 17 at q23-qter, full-length cDNA from normal human mammary epithelial cell strain 76N is cloned, sequenced and encodes a protein of 374 amino acids, expression in COS-7 cells
ST6GalNAc-II cDNA is cloned, LS147T and SW-480 human colorectal carcinoma cells are transfected with human ST6GalNAc-II cDNA leading to an increased cell surface expression of Thomsen-Friedenreich-related blood group antigen TF, which depends in SW-480 cells on the ratio of core 2 beta-1,6-N-acetylglucosaminyltransferase and ST6GalNAc-II
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stable transfection of the breast cancer cell line MDA-MB-231 with ST6GalNAc I cDNA induces sialyl-Tn antigen expression, modifications of the O-glycosylation pattern of a MUC1-related recombinant protein secreted by MDA-MB-231 sialyl-Tn positive cells occurs, sialyl-Tn expression and concomitant changes in the overall O-glycan profiles induce a decrease of adhesion and an increase of migration of MDA-MB-231, overview
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
while wild-type Escherichia coli strain DHB4 produces very small amounts of soluble ST6, engineered strains of Escherichia coli (strains SMG96, FA113, MJF277.2, and DR611), which possess certain types of oxidative cytoplasm or which co-express specific molecular chaperones (DnaK/DnaJ, GroEL/GroES, and Skp), can result in significant yields of ST6. DR611 cells are able to produce dramatically increased amounts of soluble ST6. Fusion of the ST6 enzyme to maltose-binding protein results in a marked increase in the amount of soluble ST6 in Origami 2 cells, as well as in the DR611 strain
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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Escherichia coli system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities
medicine
synthesis
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use of glycan microarrays for high-throughput acceptor specificity screening of recombinant sialyltransferases. Donor substrate is cytidine 5'-monophospho-N-acteylneuraminate, which has been biotinylated at position 9. human alpha2,6sialyltransferase-I also sialylates chitobiose structures including N-glycans