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 final biosynthetic step of this unique structure is sulfation of glucuronic acid which is catalyzed by a specific sulfotransferase, HNK-1ST. Functionally, it was reported that sulfate group is required for binding of HNK-1 mAb and laminin to this glycan. Here we describe the method how to measure the sulfotransferase activity in vitro. |
Category | Glycosyltransferases & related proteins |
Protocol Name | Enzyme assay of sulfotransferase for HNK-1 epitope |
Authors
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Kizuka, Yasuhiko
Disease Glycomics Team, Advanced Science Institute, RIKEN
Oka, Shogo
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Department of Biological Chemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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Asialo-orosomucoid for acceptor synthesis |
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Recombinant GlcAT-P and GlcAT-S purified from E. coli1) |
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[14C]-UDP-Glucuronic acid (GlcA) (Perkin Elmer, Waltham, MA) (Optional) |
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[35S]-3’-phospho-adenosine-5’-phosphosulfate (PAPS) |
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Enzyme source (recombinant HNK-1ST) |
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Blue-sepharose (GE Healthcare, Little Chalfont, UK) |
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Instruments
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2.5-cm Whatman No.1 paper disc |
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10-cm Petri-dish (BD, Franklin Lakes, NJ) |
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Liquid scintillation counter (Beckman LS-6000) |
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Methods |
1. |
Preparation of enzyme source (protein A-tagged HNK-1ST)
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Please see the reference2) for the detailed method of purification. 100 ng of recombinant proteinA-tagged HNK-1ST is sufficient for one assay. |
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2. |
Preparetion of acceptor substrate (Glucuronylation of ASOR and removal of GlcAT-P (S) by blue-column)
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Incubate 3 mg of ASOR overnight in 3 mL of reaction buffer (100 mM MES pH 6.5, 0.2% NP40, 2.5 mM ATP, 10 mM MnCl2, 750 ng of purified FLAG-tagged GlcAT-P and 750 ng of purified FLAG-GlcAT-S, 0.4 mM UDP-GlcA).
(Optional) We took a part of the mixture (100 μL) and added radioactive UDP-GlcA (20,000 dpm) to estimate the amount of transferred GlcA to ASOR. |
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2) |
Dialyze the mixture against water (ASOR is not aggregated). |
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Add MES pH 6.5 and NaCl (Final conc. are 10 mM and 150 mM, respectively). |
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Pack blue-Sepharose to a column and equilibrate with a buffer (10 mM MES pH 6.5, 150 mM NaCl, 0.2% NP40). |
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Apply the dialyzed sample to the column and pass through twice. |
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Check whether GlcAT-P and GlcAT-S were correctly removed by western blotting with anti-FLAG antibody. |
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Dialyze the sample against water. |
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3. |
Preparetion of acceptor substrate (Sulfotransferase assay)
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Prepare a reaction mixture (Final volume: 50 μL after addition of enzyme (B) and substrate (C), 20 mM BisTris-HCl pH 6.6, 0.1% Triton X-100, 10 mM MnCl2, 2.5 mM ATP, 20 μg of GlcA-ASOR, at final concentrations). |
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2) |
Add 5 to 20 μL of enzyme solution. |
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Add 5 μL of [35S]-PAPS (300,000 dpm, 5 nmol). |
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5) |
Spot the mixture onto a Whatman paper disc (see Comment). |
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Transfer the disc to 10% TCA solution in a petri-dish (see Comment) and incubate for 10 min. |
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Transfer the disc into new 10% TCA solution in a new dish and incubate for 10 min. |
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Transfer the disc to ethanol/ether (20 mL/10 mL) solution in a glass dish (see Comment) followed by incubation for 5 min. |
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Transfer the disc to ether (30 mL) solution in another glass dish followed by incubation for 5 min. |
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Dry the disc on aluminum foil for 1 or 2 min. |
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12) |
Measure radioactivity of the disc using liquid scintillation counter. |
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Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2013-12-26 11:42:54 |
- 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]
- Kizuka, Y., Matsui, T., Takematsu, H., Kozutsumi, Y., Kawasaki, T., and Oka, S. (2006) Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis. J Biol Chem. 281, 13644-13651 [PMID : 16543228]
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How to Cite this Work in an article:
Kizuka, Yasuhiko,
Oka, Shogo,
(2013). GlycoPOD https://jcggdb.jp/GlycoPOD.
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How to Cite this Work in Website:
Kizuka, Yasuhiko,
Oka, Shogo,
(2013).
Enzyme assay of sulfotransferase for HNK-1 epitope.
Retrieved 20,4,2024 ,
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Oka, Shogo,
(2013).
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