Enzyme assay of glucuroyltransferase for HNK-1 epitope

 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.

 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.

 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.

 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.

 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.

 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.

 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.