HNK-1 (human natural killer-1) epitope, comprising HSO3-3GlcAβ1-3Galβ1-4GlcNAc-, is expressed in the nervous system and functions in learning and memory. The key step of its biosynthesis is glucuronic acid transfer which is catalyzed by one of two glucuronyltransferases, GlcAT-P and GlcAT-S. Here we describe the method how to measure the glucuronyltransferase activity in vitro. |
Category | Glycosyltransferases & related proteins |
Protocol Name | Enzyme assay of glucuroyltransferase for HNK-1 epitope |
Authors
|
Kizuka, Yasuhiko
Disease Glycomics Team, Advanced Science Institute, RIKEN
Oka, Shogo
*
Department of Biological Chemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University
*To whom correspondence should be addressed.
|
KeyWords |
|
Reagents
|
● |
Asialo-orosomucoid (ASOR) as a glycoprotein acceptor |
● |
ASOR-coupled Sepharose beads (optional) |
● |
N-acetyllastosamine as an oligosaccharide acceptor |
● |
[14C]-UDP-Glucuronic acid (GlcA) (PerkinElmer, Waltham, MA) as a donor |
● |
|
● |
Enzyme source (recombinant GlcAT-P, GlcAT-S or cell- or brain-extract) |
● |
|
● |
|
● |
Anion exchange resin AG1x4 (Bio-Rad Laboratories, Hercules, CA) |
|
Instruments
|
● |
|
● |
2.5-cm Whatman No.1 paper disc |
● |
|
● |
10-cm Petri-dish (BD, Franklin Lakes, NJ) |
● |
|
● |
Liquid scintillation counter (LS-6000: Beckman Coulter, Inc., Brea, CA) |
● |
Muromac column S (Muromachi Technos Co., Ltd., Tokyo, Japan) (Optional) |
|
Methods |
1. |
Preparation of enzyme source
|
1) |
Recombinant GlcAT-P (or –S)
A) ProteinA-tagged GlcAT-P from COS-1 cells
Please see the reference1) for the detailed method of purification. 50 ng of purified enzyme is sufficient for one assay.
B) FLAG-tagged GlcAT-P from E. coli
Please see the reference2) for the detailed method of purification. 3 ng of purified enzyme is enough for one assay. |
Comment 0
|
|
2) |
Extract of mammalian cells transfected with GlcAT-P cDNA
A) Transfect 6 μg of plasmid using FUGENE6 encoding full-length GlcAT-P into COS-1 cells (10-cm dish).
B) Culture the cells for 24 h to 48h.
C) Lyse the cells in 500 μL of buffer (TBS containing 1% Triton X-100, protease inhibitor cocktail).
D) Centrifuge at 14,000 rpm for 10 min.
E) Use 5 to 20 μL of the supernatant for one assay. |
Comment 0
|
|
3) |
Extract of mouse brain
A) Excise a brain from young mouse (2-4-week-old).
B) Homogenize in 4 mL of buffer (TBS containing protease cocktail).
C) Centrifuge at 1,000 x g for 10 min at 4˚C to remove nuclei and debris.
D) Take 2 mL of the supernatant.
E) Ultracentrifuge at 105,000 x g for 1 h at 4˚C.
F) Solubilize the pellet in 2 mL of buffer (TBS containing 1% Triton X-100, protease inhibitor cocktail).
G) Ultracentrifuge at 105,000 x g for 30 min at 4˚C.
H) Use 1 mL of the supernatant for one assay. |
Comment 0
|
|
|
2. |
Preparation of acceptor substrate (ASOR)
|
1) |
Sialic acid was chemically removed from orosomucoid by the treatment with 50 mM H2SO4 at 80˚C for 1 h.
Please see the reference3) for detailed method. |
Comment 0
|
|
|
3. |
|
1) |
Toward glycoprotein (ASOR)
A) Prepare a reaction mixture (Final volume: 50 μL after addition of enzyme (B) and substrate (C), 100 mM MES pH 6.5, 0.2% NP40, 10 mM MnCl2, 2.5 mM ATP, 20 μg of ASOR, at final concentrations).
B) Add 5 to 20 μL of enzyme solution.
C) Add 5 μL of [14C]-UDP-GlcA (200,000 dpm, 5 nmol).
D) Incubate at 37˚C for 1 h.
E) Spot the mixture onto a Whatman paper disc (see Comment 1).
F) Transfer the disc to 10% TCA solution in a petri-dish (see Comment 2) and incubate for 10 min.
G) Transfer the disc into new 10% TCA solution in a new dish and incubate for 10 min.
H) Repeat G) once.
I) Transfer the disc to ethanol/ether (20 mL/10 mL) solution in a glass dish (see Comment 3) followed by incubation for 5 min.
J) Transfer the disc to ether (30 mL) solution in another glass dish followed by incubation for 5 min.
K) Dry the disc on aluminum foil for 1 or 2 min.
L) Measure radioactivity of the disc using liquid scintillation counter. |
Comment 1
|
|
2) |
Toward glycoprotein (ASOR)-coupled beads using mouse brain extract as an enzyme source (see Comment 4).
A) Prepare ASOR-conjugating beads using purified ASOR and CNBr-activated sepharose (see the reference3)).
B) Add 50 μL of ASOR-beads (50% slurry) into 1 mL of extract from mouse brain (see above).
C) Rotate at 4˚C for 1 h to trap GlcAT-P.
D) Centrifuge at 3,000 x g for 1 min and discard the sup.
E) Wash the beads with 300 μL of buffer (TBS containing 0.1% Triton X-100).
F) Repeat D) and E) twice.
G) Centrifuge at 3,000 xg for 1 min and discard the sup.
H) Add 45 μL of reaction mixture (100 mM MES pH 6.5, 0.2% NP40, 10 mM MnCl2, 2.5 mM ATP, at final concentrations. Final volume: 50 μL after addition of the substrate).
I) Add 5 μL of [14C]-UDP-GlcA (200,000 dpm, 5 nmol).
J) Incubate at 37˚C for 1 h with shaking.
K) Repeat D) and E) three times.
L) Centrifuge at 3,000 x g for 1 min and discard the sup.
M) Measure radioactivity of the beads using liquid scintillation counter. |
Comment 1
|
|
3) |
Toward oligosaccharide (N-acetyllactosamine, LacNAc)
A) Equilibrate AG1x4 resin with 5 mM phosphate buffer (PB), pH 6.8.
B) Prepare a reaction mixture (Final volume: 50 μL after addition of enzyme (C) and substrate (D), 100 mM MES pH 6.5, 0.2% NP40, 10 mM MnCl2, 2.5 mM ATP, 20 μg of ASOR, at final concentrations).
C) Add 5 to 20 μL of an enzyme solution.
D) Add 5 μL of [14C]-UDP-GlcA (200,000 dpm, 5 nmol).
E) Incubate for 2 h at 37˚C (see Comment 5).
F) Fill the column (Muromac S) with 0.5 mL of pre-equilibrated AG1x4 resin.
G) Add 1 mL of 5 mM PB pH 6.8 to the reaction mixture to stop reaction.
H) Apply the mixture onto column.
I) Collect the flow through.
I) Add 3 mL of 5 mM pH 6.8 onto the column.
J) Collect the flow through and combine it with the first flow through (total 4 mL).
K) Measure the radioactivity of 1 mL of the combined flow through using liquid scintillation counter. |
Comment 1
|
|
|
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
|
Date of registration:2013-12-24 16:36:14 |
- Kakuda, S., Sato, Y., Tonoyama, Y., Oka, S., and Kawasaki, T. (2005) Different acceptor specificities of two glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate. Glycobiology 15, 203-210 [PMID : 15470230]
- Kakuda, S., Oka, S., and Kawasaki, T. (2004) Purification and characterization of two recombinant human glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate in Escherichia coli. Protein Expr Purif. 35, 111-119 [PMID : 15039073]
- Oka, S., Terayama, K., Kawashima, C., and Kawasaki, T. (1992) A novel glucuronyltransferase in nervous system presumably associated with the biosynthesis of HNK-1 carbohydrate epitope on glycoproteins. J Biol Chem. 267, 22711-22714 [PMID : 1385405]
|
This work is licensed under Creative Commons Attribution-Non-Commercial Share Alike. Please include the following citation
How to Cite this Work in an article:
Kizuka, Yasuhiko,
Oka, Shogo,
(2013). GlycoPOD https://jcggdb.jp/GlycoPOD.
Web.18,4,2024 .
How to Cite this Work in Website:
Kizuka, Yasuhiko,
Oka, Shogo,
(2013).
Enzyme assay of glucuroyltransferase for HNK-1 epitope.
Retrieved 18,4,2024 ,
from https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t80.
html source
Kizuka, Yasuhiko,
Oka, Shogo,
(2013).
<b>Enzyme assay of glucuroyltransferase for HNK-1 epitope</b>.
Retrieved 4 18,2024 ,
from <a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t80" target="_blank">https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t80</a>.
Including references that appeared in the References tab in your work is
much appreciated.
For those who wish to reuse the figures/tables, please contact JCGGDB
management office (jcggdb-ml@aist.go.jp).
|
|