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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.
Introduction:
N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) transfers a sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to C-6 of N-acetylglucosamine (GlcNAc) exposed at the nonreducing end of glycans (1). The discovery of the GlcNAc6ST gene was reported in 1998 (1). Five members of the GlcNAc6ST family have been identified in humans, four of which have mouse orthologs so far (2). GlcNAc6ST1 (encoded by Chst2) (1, 3) and GlcNAc6ST2 (Chst4) (4–6) are involved in the synthesis of sialyl 6-sulfo Lewis X, a major class of L-selectin ligands (7, 8). GlcNAc6ST3 (Chst5), originally found as an intestinal GlcNAc6ST (9), is also known as a keratan sulfate (KS) GlcNAc6ST in adult brains (10). GlcNAc6ST1 mediates synthesis of KS in developing brains (11, 12) and microglial KS-related N-acetyllactosamine (LacNAc) oligosaccharides in neurodegenerative diseases (13–15). GlcNAc6ST4 (Chst7) has the highest homology with GlcNAc6ST1 compared to other members and is capable of sulfating GalNAc with a weak activity (16–18). GlcNAc6ST5 (CHST6) is a human corneal enzyme synthesizing KS. Defective CHST6 causes macular corneal dystrophy (19). We describe here an in vitro assay for GlcNAc6STs. Recombinant GlcNAc6STs and various oligosaccharides derived from N-linked or O-linked glycans can be used in this assay.
Protocol
The following protocol of enzyme assay is based on thin-layer chromatography (TLC) system. TLC is suitable for handling numerous samples. Oligosaccharide substrates tested are GlcNAcβ1-6Man-O-methyl, GlcNAcβ1-2Man, GlcNAcβ1-6[Galβ1-3]GalNAc-p-nitrophenyl (Core2-pNP), GlcNAcβ1-3GalNAc-p-nitrophenyl (Core3-pNP), and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc (17, 20) (Table 1).
Materials
- 1.
Phosphate-buffered saline (PBS)
- 2.
Ham’s F12 modified medium (Thermo Fisher Scientific, Waltham, MA, USA)
- 3.
Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific)
- 4.
Fetal bovine serum (FBS; Thermo Fisher Scientific)
- 5.
Chinese Hamster ovary (CHO) cells (CHO-K1; ATCC, Manassas, VA, USA)
- 6.
COS-7 cells (simian kidney cell line; ATCC)
- 7.
Lipofectamine™ 2000 (Thermo Fisher Scientific)
- 8.
1M Tris-HCl, pH 7.5
- 9.
3M Sucrose
- 10.
Immunoglobulin G (IgG)-Sepharose 6 Fast Flow (Cytiva, Marlborough, MA, USA)
- 11.
10% (wt/vol) Triton X-100 solution
- 12.
80% (vol/vol) glycerol solution
- 13.
50 mM of adenosine 5’-monophosphate (AMP)
- 14.
1M sodium fluoride (NaF)
- 15.
0.2M MnCl2
- 16.
Thick-wall ultracentrifuge open tubes (Beckman Coulter, Brea, CA, USA)
- 17.
TLC aluminum plates coated with cellulose (0.1-mm thick; Merck, Darmstadt, Germany)
- 18.
TLC-developing solvent: ethanol/pyridine/n-butyl alcohol/water/acetate (100:10:10:30:3 by volume)
- 19.
[35S]-PAPS (1.9 Ci/mmol; PerkinElmer, Waltham, MA, USA)
Instruments
- 1.
Tube rotator (Thermo Fisher Scientific)
- 2.
Optima TLX Ultracentrifuge (Beckman Coulter)
- 3.
TLC developing tank (Merck)
- 4.
BAS2000 bioimaging analyzer (Fuji Film, Tokyo, Japan) (Note 1)
Methods
- 1.
Expression of GlcNAc6ST in cultured cells and collect the enzyme as a microsome fraction
- a.
Construction of expression plasmids for full-length GlcNAc6STs is described previously (1, 4, 6, 9, 17, 21).
- b.
Culture CHO cells on 10-cm dishes and passage them until they reach ~70% confluence with Ham’s F12 modified medium containing 10% FBS.
- c.
Rinse the cells with warm PBS and then transfect transiently with a GlcNAc6ST expression plasmid using Lipofectamine™ 2000 according to the manufacturer’s instructions.
- d.
Culture the cells in Ham’s F12 modified medium containing 10% FBS for 48 h.
- e.
Wash the cell layer with PBS, scrape the cells off the dish, and homogenize them in 3 mL of 0.25 M sucrose and 20 mM of Tris-HCl, pH 7.5.
- f.
Centrifuge the homogenates briefly and then transfer the supernatant to an ultracentrifuge tube.
- g.
Ultracentrifuge the tube at 100,000 ×g for 1 h.
- h.
Discard the supernatant and then dissolve the precipitate in 50 µL of 20 mM of Tris-HCl, pH 7.5, 0.5% Triton X-100, and 10% glycerol.
- i.
Use this microsome fraction as the enzyme source.
- 2.
Expression and purification of GlcNAc6STs fused with protein A
- a.
Construction of the plasmids encoding fusion proteins of GlcNAc6STs to the IgM signal sequence and protein A in the N-terminal region are as described (20).
- b.
Culture COS-7 cells on 10-cm dishes and passage them until they reach ~70% confluence with DMEM containing 10% FBS.
- c.
Rinse the cells with warm PBS and then transfect transiently with protein A-fused GlcNAc6ST expression plasmid using Lipofectamine™ 2000 according to the manufacturer’s instructions.
- d.
Replace the medium with DMEM containing 2% IgG-free FBS (or IgG ultra-low level FBS), and then culture the cells for 48 h (Note 2).
- e.
Collect 10 mL of conditioned medium (CM) and mix with 10 µL of IgG-Sepharose resin in a tube.
- f.
Incubate the tube at 4°C for 3 h with gently rocking to adsorb the protein A fused-GlcNAc6STs in CM to the resin.
- g.
Collect the resin by centrifugation at 12,000 ×g and then wash three times with PBS.
- h.
Suspend the resin in 50 µL of 50 mM of Tris-HCl, pH 7.5, and used as enzymes.
- 3.
Assay of GlcNAc6ST activities toward oligosaccharides
- a.
Prepare the standard reaction mix in a 1.5-mL tube containing 1 µmol of Tris-HCl, pH7.5, 0.2 µmol of MnCl2, 0.04 µmol of AMP, 2 µmol of NaF, 20 nmol of oligosaccharides, 150 pmol of [35S]-PAPS (1.5 × 106 cpm), 0.05% Triton X-100, and 1 µL of microsome fraction or fusion protein suspension in a final volume of 20 µL.
- b.
Incubate the tube containing the reaction mix at 30°C for 1 h.
- c.
Apply aliquots of 2 µL of the reaction mix to TLC plates.
- d.
Develop the plates with the developing solvent.
- e.
End the development when the solvent front reaches the top of the plates. The 35S-labeled oligosaccharides migrate faster than [35S]-PAPS.
- f.
Visualize and measure the radioactivity of the 35S-labeled oligosaccharides with a BAS2000 bioimaging analyzer (Note 3).
Notes
- 1.
Other devices that allow for conducting auto-radiography with imaging plates and quantify the radioactivity of the image recorded on a plate are also useful instead of the BAS system.
- 2.
IgG-free or IgG ultra-low level FBS can be prepared by applying FBS to protein-G affinity column chromatography. IgG ultra-low level FBS is also commercially available (Thermo Fisher Scientific).
- 3.
The reaction product can also be analyzed using Superdex 30 chromatography (1, 17).
References
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- Hiraoka N, Petryniak B, Nakayama J, Tsuboi S, Suzuki M, Yeh JC, Izawa D, Tanaka T, Miyasaka M, Lowe JB, Fukuda M. A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewis(x), an L-selectin ligand displayed by CD34. Immunity. 1999;11(1):79–89. [PubMed: 10435581] [CrossRef]
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- Narentuya, Takeda-Uchimura Y, Foyez T, Zhang Z, Akama TO, Yagi H, Kato K, Komatsu Y, Kadomatsu K, Uchimura K. GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain. Sci Rep. 2019;9(1):4387. [PMC free article: PMC6416290] [PubMed: 30867513] [CrossRef]
- 11.
- Hoshino H, Foyez T, Ohtake-Niimi S, Takeda-Uchimura Y, Michikawa M, Kadomatsu K, Uchimura K. KSGal6ST is essential for the 6-sulfation of galactose within keratan sulfate in early postnatal brain. J Histochem Cytochem. 2014;62(2):145–56. [PMC free article: PMC3902094] [PubMed: 24152993] [CrossRef]
- 12.
- Takeda-Uchimura Y, Uchimura K, Sugimura T, Yanagawa Y, Kawasaki T, Komatsu Y, Kadomatsu K. Requirement of keratan sulfate proteoglycan phosphacan with a specific sulfation pattern for critical period plasticity in the visual cortex. Exp Neurol. 2015 274(Pt B):145-55. doi: 10.1016/j.expneurol.2015.08.005. PMID: 26277687. [PubMed: 26277687] [CrossRef]
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- Hirano K, Ohgomori T, Kobayashi K, Tanaka F, Matsumoto T, Natori T, Matsuyama Y, Uchimura K, Sakamoto K, Takeuchi H, Hirakawa A, Suzumura A, Sobue G, Ishiguro N, Imagama S, Kadomatsu K. Ablation of keratan sulfate accelerates early phase pathogenesis of ALS. PLoS One. 2013;8(6):e66969. [PMC free article: PMC3692529] [PubMed: 23825599] [CrossRef]
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- Foyez T, Takeda-Uchimura Y, Ishigaki S. Narentuya, Zhang Z, Sobue G, Kadomatsu K, Uchimura K. Microglial keratan sulfate epitope elicits in central nervous tissues of transgenic model mice and patients with amyotrophic lateral sclerosis. Am J Pathol. 2015;185(11):3053–65. [PubMed: 26362733] [CrossRef]
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- Zhang Z, Takeda-Uchimura Y, Foyez T, Ohtake-Niimi S. Narentuya, Akatsu H, Nishitsuji K, Michikawa M, Wyss-Coray T, Kadomatsu K, Uchimura K. Deficiency of a sulfotransferase for sialic acid-modified glycans mitigates Alzheimer's pathology. Proc Natl Acad Sci U S A. 2017;114(14):E2947–E54. [PMC free article: PMC5389269] [PubMed: 28320965] [CrossRef]
- 16.
- Kitagawa H, Fujita M, Ito N, Sugahara K. Molecular cloning and expression of a novel chondroitin 6-O-sulfotransferase. J Biol Chem. 2000;275(28):21075–80. [PubMed: 10781596] [CrossRef]
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- Uchimura K, Fasakhany F, Kadomatsu K, Matsukawa T, Yamakawa T, Kurosawa N, Muramatsu T. Diversity of N-acetylglucosamine-6-O-sulfotransferases: molecular cloning of a novel enzyme with different distribution and specificities. Biochem Biophys Res Commun. 2000;274(2):291–6. [PubMed: 10913333] [CrossRef]
- 18.
- Bhakta S, Bartes A, Bowman KG, Kao WM, Polsky I, Lee JK, Cook BN, Bruehl RE, Rosen SD, Bertozzi CR, Hemmerich S. Sulfation of N-acetylglucosamine by chondroitin 6-sulfotransferase 2 (GST-5). J Biol Chem. 2000;275(51):40226–34. [PubMed: 10956661] [CrossRef]
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- Akama TO, Nishida K, Nakayama J, Watanabe H, Ozaki K, Nakamura T, Dota A, Kawasaki S, Inoue Y, Maeda N, Yamamoto S, Fujiwara T, Thonar EJ, Shimomura Y, Kinoshita S, Tanigami A, Fukuda MN. Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene. Nat Genet. 2000;26(2):237–41. [PubMed: 11017086] [CrossRef]
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- Uchimura K, El-Fasakhany FM, Hori M, Hemmerich S, Blink SE, Kansas GS, Kanamori A, Kumamoto K, Kannagi R, Muramatsu T. Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression. J Biol Chem. 2002;277(6):3979–84. [PubMed: 11726653] [CrossRef]
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- Akama TO, Nakayama J, Nishida K, Hiraoka N, Suzuki M, McAuliffe J, Hindsgaul O, Fukuda M, Fukuda MN. Human corneal GlcNac 6-O-sulfotransferase and mouse intestinal GlcNac 6-O-sulfotransferase both produce keratan sulfate. J Biol Chem. 2001;276(19):16271–8. [PubMed: 11278593] [CrossRef]
Footnotes
The authors declare no competing or financial interests.
Tables
Table 1:
Comparison of the substrate specificity of GlcNAc6ST1, GlcNAc6ST2, GlcNAc6ST3, and GlcNAc6ST4.
| Enzyme activity¶ | ||||
|---|---|---|---|---|
| Acceptor | GlcNAc6ST1 | GlcNAc6ST2 | GlcNAc6ST3 | GlcNAc6ST4 |
| GlcNAcβ1-6ManOMe | 20.6 (100) | 6.8 (100) | N.D. | 2.8 (100) |
| GlcNAcß1-2Man | 26.3 (128) | 10.3 (151) | N.D. | 2.5 (91) |
| GlcNAcβ1-6[Galβ1-3]GalNAc-pNP (core 2) | 39.3 (191) | 9.9 (145) | 5.7‡ | 0.7 (25) |
| GlcNAcβ1-3GalNAc-pNP (core 3) | N.D.§ | 12.5 (184) | N.D. | -† |
| GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc | 4.3‡ (21) | 5.0‡ (73) | N.D. | 0.14 (5) |
- ¶
The values for GlcNAc6ST1, 2, and 3 were obtained with their protein-A fused versions [pmol/h/ml of medium]. For GlcNAc6ST4, the values were obtained with microsome fractions [pmol/min/mg protein]. The percentage of the activity compared with that of GlcNAcβ1-6ManOMe is also shown. §N.D., less than 0.1 pmol/h/ml of medium. ‡The actually observed radioactivities were around 30,000 cpm, while the assay without the enzyme or with IgG-Sepharose exposed to culture supernatant of mock-transfected cells gave values less than 500 cpm. † -, not tested. Data in previous reports (17, 20) were adopted.