Enzyme assay of sulfotransferase for HNK-1 epitope
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.
Asialo-orosomucoid for acceptor synthesis
Recombinant GlcAT-P and GlcAT-S purified from E. coli1)
[14C]-UDP-Glucuronic acid (GlcA) (PerkinElmer) (Optional)
Enzyme source (recombinant HNK-1ST)
Blue-sepharose (GE healthcare)
2.5-cm Whatman No.1 paper disc
10-cm Petri-dish (BD Falcon)
10-cm Glass dish
Liquid scintillation counter (Beckman LS-6000)
Preparation of enzyme source (protein A-tagged HNK-1ST)
Please see the reference2) for the detailed method of purification. 100 ng of recombinant proteinA-tagged HNK-1ST is sufficient for one assay.
Preparetion of acceptor substrate (Glucuronylation of ASOR and removal of GlcAT-P (S) by blue-column)
Incubate 3 mg of ASOR overnight in 3 ml of reaction buffer (100 mM MES pH6.5, 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.
Dialyze the mixture against water (ASOR is not aggregated).
Add MES pH 6.5 and NaCl (Final conc. are 10 mM and 150 mM, respectively).
Pack blue-Sepharose to a column and equilibrate with a buffer (10 mM MES pH 6.5, 150 mM NaCl, 0.2% NP40).
Apply the dialyzed sample to the column and pass through twice.
Collect the flow through.
Check whether GlcAT-P and GlcAT-S were correctly removed by western blotting with anti-FLAG antibody.
Dialyze the sample against water.
Preparetion of acceptor substrate (Sulfotransferase assay)
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).
Add 5 to 20 μl of enzyme solution.
Add 5 μl of [35S]-PAPS (300,000 dpm, 5 nmol).
Incubate at 37˚C for 1 h.
Spot the mixture onto a Whatman paper disc (see Comment 1).
Transfer the disc to 10% TCA solution in a petri-dish (see Comment 2) and incubate for 10 min.
Transfer the disc into new 10% TCA solution in a new dish and incubate for 10 min.
Repeat step 7) once.
Transfer the disc to ethanol/ether (20 ml/10 ml) solution in a glass dish (see Comment 3) followed by incubation for 5 min.
Transfer the disc to ether (30 ml) solution in another glass dish followed by incubation for 5 min.
Dry the disc on aluminum foil for 1 or 2 min.
Measure radioactivity of the disc using liquid scintillation counter.