Enzyme assay of N-acetylglucosamine-6-sulfotransferases for selectin ligands

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Category

Glycosyltransferases & related proteins

Protocol Name

Authors

Kenji Uchimura
-, National Center for Geriatrics and Gerontology

Keywords

Sulfated carbohydrate

L-selectin

Sulfotransferase

Enzyme assay

Thin-layer chromatography

Reagents

●	Phosphate buffered saline (PBS)
●	Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen)
●	Fetal bovine serum (FBS; Invitrogen)
●	LipofectAMINE PLUS (Invitrogen)
●	IgG-Sepharose (GE Healthcare Life Sciences)
●	50 mM Tris-HCl, pH 7.5
●	Thin-layer chromatography (TLC) plates (Merck)
●	[³⁵S] PAPS (1.9 Ci/mmol; PerkinElmer Life Sciences)

Instruments

●	Water bath incubator, 37 °C
●	Tube rotator
●	BAS2000 bioimaging analyzer (Fuji Film)

Methods

Expression and Purification of GlcNAc6STs Fused with Protein A

1)	Construction of the plasmids encoded fusion proteins of GlcNAc6STs to the IgM signal sequence and protein A in the N-terminal region are as described¹⁾.	Comment 0

2)	Culture COS-7 cells in DMEM containing 10% FBS on a 10-cm dish and then transfect 4 µg of a GlcNAc6ST expression plasmid using LipofectAMINE PLUS according to manufacturer instructions.	Comment 0

3)	Replace the medium with DMEM containing 2% IgG-free FBS after 24 h. Culture cells for another 48h.	Comment 0

4)	Collect the culture medium and then mix with IgG-Sepharose (10 µl of resin/10 ml of culture).	Comment 0

5)	Incubate the mixture at 4 °C for 3 h with gently rocking to adsorb the protein A fusion GlcNAc6STs expressed in the medium to the resin.	Comment 0

6)	Collect the resin by centrifugation and wash three times with PBS	Comment 0

Suspend the resin in 30 µl of 50 mM Tris-HCl, pH 7.5, and used as enzymes.

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Assay of GlcNAc6ST activities toward oligosaccharides

1)	The standard reaction mix in a 1.5 mL tube contains 1 µmol Tris-HCl, pH 7.5, 0.2 µmol MnCl2, 0.04 µmol adenosine 5’-monophosphate, 2 µmol sodium fluoride, 20 nmol oligosaccharides, 150 pmol [³⁵S]-PAPS (1.5 x 10⁶ cpm), 0.05% of Triton-X and 1 µl of fusion protein suspension in a final volume of 20 µl.	Comment 0

2)	Incubate the tube containing the reaction mix at 30 °C for 1h.	Comment 0

3)	Apply aliquots of 2 µl of the reaction mix to TLC plates. The plates are pre-coated with cellulose 0.1-mm thick.	Comment 0

4)	Develop the plates with a developing solvent, ethanol/pyridine/n-butyl alcohol/water/acetic acid (100:10:10:30:3, by volume).	Comment 0

5)	End the development when the solvent front reaches to the top of the plates. The ³⁵S-labeled oligosaccharides migrate faster than [³⁵S]-PAPS.	Comment 0

Visualize and measure the radioactivity of the ³⁵S-labeled oligosaccharides with a BAS2000 bioimaging analyzer.

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Notes

Oligosaccharide substrates tested are GlcNAcβ1-3Galβ1-4GlcNAc, GlcNAcβ1-6Man-O-methyl, GlcNAcβ1-2Man, GlcNAcβ1-6[Galβ1-3]GalNAc-p-nitrophenyl (Core2-pNP) and GlcNAcβ1-3GalNAc-p-nitrophenyl (Core3-pNP)¹⁾.
TLC is suitable to handle a large number of samples.

Figure & Legends

Figure. 1. Sulfation of various oligosaccharides by human GlcNAc6ST-1, -2 , and -3.

Various oligosaccharides were incubated with the protein A-fused GlcNAc6ST-1, -2, and -3 under standard assay conditions. Aliquots of 2 µl of each reaction were applied to a TLC plate and then developed with ethanol-pyridine-n-butyl alcohol-water-acetate (100:10:10:30:3 (v/v), respectively). ³⁵S-Labeled products were visualized, and the radioactivity was measured with a BAS2000 bioimaging analyzer.

This figure was originally published in J Biol Chem. Uchimura K. et al. "Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression" 2002, 277(6):3979-84. © the American Society for Biochemistry and Molecular Biology.

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Date of registration：2012-11-13 15:37:19