Any fingerprinting method for analyzing the structure of keratin sulfate (KS) requires both fragmentation usually with KS degrading enzymes and subsequent separation. At present, KS-degrading enzymes are classified into three groups based on their cleavage sites and substrate specificity. The first is endo-β-galactosidases identified in Escherichia freundii, Coccobacterium sp., Flavobacterium keratolyticus, and Bacteroides fragilis, which hydrolyze the internal non-sulfated galactosidic bonds of KS. The second is the keratanase identified in Pseudomonas sp. IFO-13309. This enzyme also cleaves internal β1-4 galactosidic linkages in KS, but differs in substrate specificity from the endo-β-galactosidase. It requires at least one N-acetylglucosamine 6-sulfate residue and does not hydrolyze the desulfated KS-polymer. The third group is the keratanase II in Bacillus sp. Ks 36, endo-β-N-acetylglucosaminidase, which cleaves N-acetylglucosamine linkages of the KS chain, releasing Galβ1-4GlcNAc disaccharides with mono- or di-sulfates, or tetrasaccharides of Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc. Fucose residues on adjacent N-acetylglucosamines interfere with digestion of KS by keratanase, resulting in a larger number of oligosaccharides. In contrast, keratanase II efficiently digests KS chains even with fucose residues, and therefore is usually used for structural analyses of KS. In this protocol, the methods of KS digestion with KS degrading enzymes and detection of reducing sugar are shown. |
| Category | Glycosaminoglycans |
| Protocol Name | Keratan sulfate-degrading enzymes from bacteria |
Authors
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Watanabe, Hideto
Institute for Molecular Science of Medicine, Aichi Medical University
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| KeyWords |
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Reagents
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bovine corneal keratin sulfate (KS) |
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keratanase from Pseudomonas sp. |
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keratanase II from Bacillus sp. |
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| Methods |
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1. |
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| 1) |
• Keratanase digestion
KS (0.2 μmol galactose) and keratanase (5–50 mU) are reacted in 200 μL of 10 mM Tris-HCl, pH 7.4, for 15 min at 37°C.
• Keratanase II digestion
KS (0.2 μmol galactose) and keratanase II (0.1–1 mU) are reacted in 200 μL of 10 mM sodium acetate buffer, pH 6.0, for 15 min at 37°C. |
Comment 0
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2. |
Detection of reducing sugar by the method of Park and Johnson |
| 1) |
Add 200 μL of carbonate/KCN solution (50 mM Na2CO3, 10 mM KCN) to the reaction mixture. |
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| 3) |
Boil for 10 min and cool down to room temperature. |
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| 4) |
Add 1 mL Alum solution (3.1 mM Fe(NH4SO4)2・12H2O, 0.05 M H2SO4, 0.1%SDS), mix well, and incubate for 15 min at RT. |
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| 5) |
Measure the absorbance at 690 nm. |
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| Notes | Keratanase II of Bacillus sp. Ks36 is not thermo-stable, and may contain β-galactosidase activity. In contrast, keratanase II from Bacillus circulans has a higher stability with an optimal reaction temperature at 55°C, although it is not commercially available.
Seikagaku biobusiness discontinued the coreneal KS and the enzymes. Thus, other manufacturers and distributors should be found.
For endo-β-galactosidase, the protocol by Michiko N. Fukuda is referred to. |
| Copyrights |
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This work is released underCreative Commons licenses
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| Date of registration：2015-07-06 13:59:51 |
- Nakazawa, K., and Suzuki, S. (1975) Sequential degradation of keratan sulfate by bacterial enzymes and purification of a sulfatase in the enzymatic system. J. Biol. Chem. 250, 905–911 [PMID : 1112796]
- Hashimoto, N., Morikawa, K., Kikuchi, H., Yoshida, K., and Tokuyasu, K. (1988) Seikagaku. 60, 935.
- Park, J. T., and Johnson, M. J. (1949) A submicrodetermination of glucose. J Biol. Chem. 181, 149–151 [PMID : 15390401]
- Yamagishi, K., Suzuki, K., Imai, K., Mochizuki, H., Morikawa, K., Kyogashima, M., Kimata, K., and Watanabe, H. (2003) Purification, characterization, and molecular cloning of a novel keratan sulfate hydrolase, endo-β-N-acetylglucosaminidase, from Bacillus circulans. J. Biol. Chem. 278, 25766–25772 [PMID : 12732618]
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