Abstract
Although the polysaccharide backbone Glycosaminoglycans (GAGs) have been demonstrated to interact with a variety of proteins and such interactions are thought to be involved in the regulation of the physiological functions of these proteins (1). Although the polysaccharide backbone of GAGs is a linear polymer composed of alternating amino sugar and hexuronic acid residues, this simple repeat structure acquires a considerable degree of variability by extensive modifications involving sulfations and uronate epimerization (2). The structural variability is the basis for the wide variety of domain structures with biological activities (1). Investigation of the structure-function relationship of GAGs has been hindered by the difficulty in microanalyzing their complicated structure. GAG molecules are so heterogeneous that sequence analysis on unfractionated GAG chains can give only statistical structural information. Actual sequencing is possible, however, on oligosaccharide fragments, which can be obtained by chemical or enzymatic degradation of GAG chains followed by separation by means of various chromatographies. In this chapter, we describe the methods for enzymatic degradation of GAG chains and fractionation of the oligosaccharide products. Because many kinds of highly purified GAG-degrading enzymes, which are not contaminated by sulfatases, are commercially available and cleave polysaccharides with high specificities under mild conditions, enzymatic cleavage is a useful method for the preparation of GAG oligosaccharides.
Keywords
- Testicular Hyaluronidase
- Oligosaccharide Fraction
- Glucuronic Acid Residue
- Streptococcus Dysgalactiae
- Unsaturated Uronic Acid
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kjellén, L. and Lindahl, U. (1991) Proteoglycans: sructures and interactions. Annu. Rev. Biochem. 60, 443–475.
Fransson, L.-Å. (1985) Mammalian glycosaminoglycans, in The Polysaccharides (Aspinall, G. O., ed.), Academic Press, New York, pp. 337–415.
Linker, A., Hoffman, P., Meyer, K., Sampson, P., and Korn, E. D. (1960) The formation of unsaturated disacharides from mucopolysaccarides and their cleavage to α-keto acid by bacterial enzymes. J. Biol. Chem. 235, 3061–3065.
Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. (1968) Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem. 243, 1523–1535.
Kresse, H. and GlöSsl, J. (1987) Glycosaminoglycan degradation. Adv. Enzymol. Relat. Areas Mol. Biol. 60, 217–314.
Yamada, S. and Sugahara, K. (1998) Structure of oligosaccharides isolated from heparan sulfate/heparin and substrate specificities of the degrading enzymes of bacterial origin. Trends Glycosci. Glycotech. 10, 95–123.
Sasisekharan, R., Bulmer, M., Moreman, K. W., Cooney, C. L., and Langer, R. (1993) Cloning and expression of heparinase I gene from Flavobacterium heparinum. Proc. Natl. Acad. Sci. USA 90, 3660–3664.
Godavarti, R., Davis, M., Venkataraman, G., Cooney, C, Langer, R., and Sasisekharan, R. (1996) Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli. Biochem. Biophys. Res. Commun. 225, 751–758.
Su, H., Blain, F., Musil, R. A., Zimmerman, J. J. F., Gu, K., and Bennett, D. C. (1996) Isolation and expression in Escherichia coli of hepB and hepC, genes coding for the glycosaminoglycan-degrading enzymes heparinase II and heparinase III, respectively, from Flavobacterium heparinum. Appl. Environ. Microbiol. 62, 2723–2734.
Yoshida, K., Miyazono, H., Tawada, A., Kikuchi, H., and Morikawa, K. (1989) Comparison of the substrate specificities of heparan sulfate-lyases from new bacterium with those from F. heparinum. In Proc. Xth Int. Symp. Glycoconjugates (Sharon, N., Lis, H., Duskin, D., and Kahane I., eds.), Jerusalem, Israel, pp. 330–331.
Sugahara, K., Okumura, Y., and Yamashina, I. (1989) The Engelbreth-Holm-Swarm mouse tumor produces undersulfated heparan sulfate and oversulfated galactosamionoglycans. Biochem. Biophys. Res. Commun. 162, 189–197.
Yoshida, K., Miyauchi, S., Kikuchi, H., Tawada, A., and Tokuyasu, K. (1989) Analysis of unsaturated disaccharides from glycosaminoglycan by high-performance liquid chromatography. Analyt. Biochem. 177, 327–332.
Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart, J. F. G. (1992) A novel sulfated structure in the carbohydrate-protein linkage region isolated from porcine intestinal heparin. J. Biol. Chem. 267, 1528–1533.
Yamada, S., Murakami, T., Tsuda, H., Yoshida, K., and Sugahara, K. (1995) Isolation of the porcine heparin tetrasaccharides with glucuronate 2-O-sulfate. J. Biol. Chem. 270, 8696–8705.
Sugahara, K., Shigeno, K., Masuda, M., Fujii, N., Kurosaka, A., and Takeda, K. (1994) Structural studies on the chondroitinase ABC-resistant sulfated tetrasaccharides isolated from various chondroitin sulfate isomers. Carbohydr. Res. 255, 145–163.
Sugahara, K., Nadanaka, S., Takeda, K., and Kojima, T. (1996) Structural analysis of unsaturated hexasaccharides isolated from shark cartilage chondroitin sulfate D: substrates for exolytic action of chondroitinase ABC. Eur. J. Biochem. 239, 871–880.
Sugahara, K., Tanaka, Y., and Yamada, S. (1996) Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz 1H NMR spectroscopy. Glycoconjugate J. 13, 609–619.
Yamada, S., Sakamoto, K., Tsuda, H., Yoshida, K., Sugiura, M., and Sugahara, K. (1999) Structural studies of octasaccharides derived from the low-sulfated repeating disaccharide region and octasaccharide serines derived from the protein linkage region of porcine intestinal heparin. Biochemistry 38, 838–847.
Lohse, D. L. and Linhardt, R. J. (1992) Purification and characterization of heparin lyases from Flavobacterium heparinum. J. Biol. Chem. 267, 24,347–24,355.
Di Ferrante, N. (1956) Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J. Biol. Chem. 220, 303–306.
Poh, C. H., Yuen, R., Chung, M. C. M., and Khoo, H. E. (1992) Purification and partial characterization of hyaluronidase from stonefish (Synanceja horrida) venom. Comp. Biochem. Physiol. 101B, 159–163.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc.
About this protocol
Cite this protocol
Yamada, S., Sugahara, K. (2003). Preparation of Oligosaccharides from Sulfated Glycosaminoglycans Using Bacterial Enzymes. In: Thibault, P., Honda, S. (eds) Capillary Electrophoresis of Carbohydrates. Methods in Molecular Biology™, vol 213. Humana Press. https://doi.org/10.1385/1-59259-294-5:71
Download citation
DOI: https://doi.org/10.1385/1-59259-294-5:71
Publisher Name: Humana Press
Print ISBN: 978-0-89603-826-4
Online ISBN: 978-1-59259-294-4
eBook Packages: Springer Protocols