Enzyme assay of N-acetylglucosamine-6-sulfotransferases for selectin ligands
N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) transfers a sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to an N-acetylglucosamine (GlcNAc) residue, which is usually located at the non-reducing end of glycans. Five members of the GlcNAc6ST family have been cloned in humans, four of which have orthologs in mice. Among the GlcNAc6STs, GlcNAc6ST-1 and GlcNAc6ST-2 have been confirmed to be involved in the synthesis of sialyl 6-sulfo Lewis X, which is a major class of L-selectin ligands in lymphocyte homing. GlcNAc-6-sulfation mainly regulates the rolling velocity of lymphocytes in high endothelial venules of peripheral lymph nodes. In this section, we describe an in vitro assay for GlcNAc6STs. GlcNAc6STs fused with Protein A are expressed in mammalian cell lines and then purified from the culture media. Various oligosaccharides derived from N-linked or O-linked glycans are applicable to the assay.
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)
Water bath incubator, 37 °C
BAS2000 bioimaging analyzer (Fuji Film)
Expression and Purification of GlcNAc6STs Fused with Protein A
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).
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.
Replace the medium with DMEM containing 2% IgG-free FBS after 24 h. Culture cells for another 48h.
Collect the culture medium and then mix with IgG-Sepharose (10 µl of resin/10 ml of culture).
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.
Collect the resin by centrifugation and wash three times with PBS
Suspend the resin in 30 µl of 50 mM Tris-HCl, pH 7.5, and used as enzymes.
Assay of GlcNAc6ST activities toward oligosaccharides
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.
Incubate the tube containing the reaction mix at 30 °C for 1h.
Apply aliquots of 2 µl of the reaction mix to TLC plates. The plates are pre-coated with cellulose 0.1-mm thick.
Develop the plates with a developing solvent, ethanol/pyridine/n-butyl alcohol/water/acetic acid (100:10:10:30:3, by volume).
End the development when the solvent front reaches to the top of the plates. The 35S-labeled oligosaccharides migrate faster than [35S]-PAPS.
Visualize and measure the radioactivity of the 35S-labeled oligosaccharides with a BAS2000 bioimaging analyzer.
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.