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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
Keratan sulfate is a major glycosaminoglycan and comprises poly-N-acetyllactosamine backbone structure [(Galβ1-4GlcNAcβ1-3)n] with modification of sulfates at the 6-OH of Gal and GlcNAc residues. Based on the substrate specificities of glycosyl/sulfotransferases involved in keratan sulfate biosynthesis, keratan sulfate can be elongated as follows (Figure 1):
- 1.
Nonreducing terminal GlcNAc residues on specific branches of N-linked/O-linked glycan chains are 6-O-sulfated by CHST2 (also known as GlcNAc 6-O-sulfotransferase-1 and GlcNAc6ST1), CHST5 (GlcNAc 6-O-sulfotransferase-3, GlcNAc6ST3, I-GlcNAc6ST, and GST4α), or CHST6 (GlcNAc 6-O-sulfotransferase-5, GlcNAc6ST5, CGn6ST, and GST4β) (see Notes 1 and 2).
- 2.
GlcNAc 6-O-sulfate residues are next galactosylated by B4GALT4 (also known as β4GalT-IV), which is only the β4GalT specific for GlcNAc 6-O-sulfate.
- 3.
6-O-Sulfated LacNAc, Galβ1-4(SO3−-6)GlcNAc, is elongated by B3GNT7 (β3Gn-T7), which is specific for the sulfated moiety.
- 4.
Repeating 1, 2, and 3 results in the formation of [Galβ1-4(SO3−-6)GlcNAcβ1-3]n.
- 5.
CHST1 (also known as Gal6ST, KSGal6ST, and GST-1) catalyzes 6-O-sulfation of both the nonreducing terminal and internal Gal residues.
As described above, at least four sulfotransferases, CHST1, 2, 5, and 6, are involved in the biosynthesis of keratan sulfate. The enzymatic activities of these enzymes can be measured using a donor substrate, adenosine 3’-phosphate 5’-phospho[35S]sulfate ([35S]PAPS) and acceptor substrates bearing appropriate nonreducing terminal sugar residues.
The first step, GlcNAc 6-O-sulfation, can be catalyzed using CHST2, 5, or 6. So far, CHST2, 5, and 6 mainly work on GlcNAc 6-O-sulfation in the developmental stage of the brain in mice, in the adult brain in mice, and in the cornea in mice and humans, respectively (Note 2). The difference in the substrate specificities for glycan or protein moieties in each organ is not fully understood.
Protocol
In this chapter, the methods for assaying CHST6 and CHST1 activities and preparing the acceptor substrate for CHST6 are shown (1-3).
Materials
- 1.
Adenosine 3’-phosphate 5’-phospho[35S]sulfate (PerkinElmer, Waltham, MA)
- 2.
Keratan sulfate (from bovine cornea, Seikagaku Corp., Tokyo, Japan)
- 3.
UDP-GlcNAc and GlcNAc (Sigma-Aldrich Co., St. Louis, MO)
- 4.
L2L2 [Galβ1-4(SO3−-6)GlcNAcβ1-3Galβ1-4(SO3−-6)GlcNAc] (prepared from bovine articular cartilage KS according to Ref. 4)
- 5.
RCA-I agarose (4 mg/mL of gel, J-OIL MILLS, Inc., Tokyo, Japan)
- 6.
Recombinant B3GNT7 (prepared according to Ref. 5) (Note 3)
Instruments
- 1.
High-voltage paper electrophoresis unit (Advantec, Co., Ltd., Ehime, Japan) (Note 4)
- 2.
Radiochromatogram scanner (RITA, Raytest, Straubenhardt, Germany)
- 3.
Whatman No.1 paper (Cytiva, Boston, MA)
- 4.
Vacuum evaporator (Sakuma Co. Ltd., Tokyo, Japan)
- 5.
Sephadex G-25 and Sephadex G-50 (superfine, Cytiva, Boston, MA)
Methods
- 1.
Preparation of an intermediate analog of keratan sulfate, GlcNAcβ1-3Galβ1-4(SO3--6)GlcNAcβ1-3Galβ1-4(SO3−-6)GlcNAc (GlcNAcβ1-3L2L2)
- a.
0.1 mL of the reaction mixture containing 50 mM of HEPES-NaOH (pH 7.2), 10 mM of MnCl2, 0.05%(v/v) Triton X-100, 50 μg/mL of protamine chloride, 1 mM of L2L2, 0.1 M GlcNAc, 1.25 mM of UDP-GlcNAc, and recombinant B3GNT7 (5 nmol/h. The activity is assayed using 0.5 mM of L2L2 and 0.5 mM of UDP-GlcNAc), is incubated at 37°C for 1 d.
- b.
The mixture is applied to a Sephadex G-50 gel filtration (1.3 × 68 cm, equilibrated and eluted with 0.1 M NaCl).
- c.
The hexose-positive fractions (phenol-sulfuric acid method) are applied to RCA-I-agarose lectin chromatography to remove residual L2L2.
- d.
The pass-through fractions are desalted using Sephadex G-25 gel filtration (1.3 × 68 cm, equilibrated and eluted with EtOH/water 1:19). Finally, 27 nmol of GlcNAcβ1-3L2L2 is obtained.
- 2.
Assay of CHST6
- a.
A total of 20 μL of the reaction mixture containing 50 mM of sodium cacodylate (pH 6.8), 10 mM of MnCl2, 0.1% (w/v) digitonin (if enzyme sources are membrane fractions), 50 μg/mL of protamine chloride, 2 mM of dithiothreitol, 0.1 M NaF, 2 mM of ATP-Na2, 6.5 μM of [35S]PAPS (4.9 × 105 dpm), 0.1 mM of GlcNAcβ1-3L2L2, and the enzyme fractions [in 20 mM of HEPES-NaOH (pH 7.2); prepared according to Ref. 3], is incubated at 37°C for 1 h.
- b.
Add 0.5 mL of 0.01 N HCl and heat at 100°C for 10 min to destroy residual [35S]PAPS. After cooling, the mixture is neutralized and concentrated by vacuum evaporator.
- c.
Spot the mixture and bromophenol blue as a marker on a Whatman No. 1 paper (46 × 57 cm).
- d.
Wet the paper with pyridine/acetic acid/water = 3:1:387 (pH 5.4).
- e.
Set the paper into the high-voltage paper electrophoresis unit and perform electrophoresis at 4,000 V with the same solvent as Step 2d), until the marker moves 10 cm from the origin.
- f.
Dry the paper in the draft, and measure the radioactivity using a radiochromatogram scanner. Generally, [35S]-labeled reaction product moves near the marker, while free sulfate moves about 2.5-fold forward from the marker.
- 3.
Assay of CHST1
- a.
Prepare the reaction mixture as above, except for adding 50 mM of sodium cacodylate (pH 6.4), 0.1% (v/v) Triton X-100, and 0.5 mg/mL of keratan sulfate in place of sodium cacodylate (pH 6.8), digitonin, and GlcNAcβ1-3L2L2, respectively.
- b.
Same as Steps 2b-f. [35S]-labeled reaction products move as a smear profile between the origin and the marker.
Notes
- 1.
CHST6 and CHST1 belong to the GlcNAc6ST family, which comprises 7 members in humans. If enzyme fractions to be analyzed are crude, it is difficult to determine the individual enzymatic activities of CHST6 and CHST1 because some of the other members can utilize keratan sulfate or GlcNAcβ1-3L2L2 as a donor substrate. For example, not only CHST1 but also CHST3 (C6ST-1) can act on keratan sulfate. Similarly, GlcNAcβ1-3L2L2 can be utilized by CHST1, CHST2 (GlcNAc6ST1), CHST6, and CHST7 (GlcNAc6ST4). CHST2, 7, and 6 can catalyze the sulfation of the 6-OH of nonreducing terminal GlcNAc of GlcNAcβ1-3L2L2, although the ratio of Vmax/Km values of the three enzymes for the substrate is 26:4:100, indicating the preference of CHST6 for the substrate. CHST1 can catalyze the sulfation of internal Gal residues of the substrate.
- 2.
Akama et al. showed that CHST6 is a cause gene of macular corneal dystrophy and responsible for keratan sulfate biosynthesis in the human cornea (6). Conversely, Zhang et al. showed that the deficiency of CHST2 causes loss of keratan sulfate in the mouse brain (7). Recently, Narentuya et al. showed that CHST5 is a major enzyme as KS sulfotransferase in oligodendrocytes in adult mice (8).
- 3.
Their recombinant enzymes produced in E. coli would be generally inactive, probably due to their insolubility or improper folding. Other hosts, such as Pichia pastoris or culture cells (CHO, COS, and so on), are recommended.
- 4.
There is a danger of electric shock when using a high-voltage paper electrophoresis unit. The power source must be turned off when papers would be placed into the unit or taken out from it.
References
- 1.
- Torii T, Fukuta M, Habuchi O. Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate Gal-6-sulfotransferase. Glycobiology. 2000 Feb;10(2):203–11. [PubMed: 10642612] [CrossRef]
- 2.
- Akama TO, Misra AK, Hindsgaul O, Fukuda MN. Enzymatic synthesis in vitro of the disulfated disaccharide unit of corneal keratin sulfate. J Biol Chem. 2002 Nov 8;277(45):42505–13. [PubMed: 12218059] [CrossRef]
- 3.
- Seko A, Nagata K, Yonezawa S, Yamashita K. Ectopic expression of a GlcNAc 6-O-sulfotransferase, GlcNAc6ST-2, in colonic mucinous adenocarcinoma. Glycobiology. 2002 Jun;12(6):379–88. [PubMed: 12107080] [CrossRef]
- 4.
- Brown GM, Huckerby TN, Nieduszynski IA. Oligosaccharides derived by keratanase II digestion of bovine articular cartilage keratin sulphates. Eur J Biochem. 1994 Sep 1;224(2):281–308. [PubMed: 7925342] [CrossRef]
- 5.
- Seko A, Yamashita K. β1,3-N-Acetylglucosaminyltransferase-7 (β3Gn-T7) acts efficiently on keratan sulfate-related glycans. FEBS Lett. 2004 Jan 2;556(1-3):216–20. [PubMed: 14706853] [CrossRef]
- 6.
- 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 sulphotranseferase gene. Nat Genet. 2000 Oct;26(2):237–41. [PubMed: 11017086] [CrossRef]
- 7.
- Zhang H, Muramatsu T, Murase A, Yuasa S, Uchimura K, Kadomatsu K. N-acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury. Glycobiology. 2006 Aug;16(8):702–10. [PubMed: 16624895] [CrossRef]
- 8.
- 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 Mar 13;9(1):4387. [PMC free article: PMC6416290] [PubMed: 30867513] [CrossRef]
Footnotes
The authors declare no competing or financial interests.
Figures
Figure 1.
Biosynthesis of keratan sulfate.
See Introduction for each step in detail.