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Glycosaminoglycan Chain Analysis and Characterization (Glycosylation/Epimerization)

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Proteoglycans

Part of the book series: Methods in Molecular Biology ((MIMB,volume 836))

Abstract

Glycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate/heparin (HS/Hep) are linear polysaccharides and involved in the regulation of various biological events through interaction with functional proteins. GAGs are modified by sulfation at various positions of each saccharide residue and the epimerization of uronic acid residues during the chain’s biosynthesis, resulting in enormous structural diversity. This structural diversity is the basis for the wide range of biological activities of GAGs. Thus, the structural analysis of GAGs is key to understanding their biological functions. This chapter describes detailed instructions for the extraction and structural analysis of GAGs from cultured cells and tissues using a combination of GAG-degrading enzymes and high-performance liquid chromatography.

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References

  1. Iozzo, R. V. (1998) Matrix proteoglycans: From molecular design to cellular function. Annu. Rev. Biochem. 67, 609–652.

    Article  PubMed  CAS  Google Scholar 

  2. Sugahara, K., Mikami, T., Uyama, T., Mizuguchi, S., Nomura, K., and Kitagawa, H. (2003) Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Curr. Opin. Struct. Biol. 13, 612–620.

    Article  PubMed  CAS  Google Scholar 

  3. Sugahara, K. and Mikami, T. (2007) Chondroitin/dermatan sulfate in the central nervous system. Curr. Opin. Struct. Biol. 17, 536–545.

    Article  PubMed  CAS  Google Scholar 

  4. Bishop, J. R., Schuksz, M., and Esko, J. D. (2007) Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030–1037.

    Article  PubMed  CAS  Google Scholar 

  5. Sugahara, K. and Kitagawa, H. (2000) Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans. Curr. Opin. Struct. Biol. 10, 518–527.

    Article  PubMed  CAS  Google Scholar 

  6. Petit, E., Delattre, C., Papy-Garcia, D., and Michaud, P. (2006) Chondroitin sulfate lyases: Applications in analysis and glycobiology, in Advanced in Pharmacology, vol, 53 “Chondroitin sulfate: structure, role and pharmacological activity” (Volpi, N., ed.), Academic Press, London, UK, pp. 337–356.

    Google Scholar 

  7. 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 in Glycosci. and Glycotechnol. 10, 95–123.

    Article  CAS  Google Scholar 

  8. Yoshida, K., Arai, M., Kohno, Y., Maeyama, K., Miyazono, H., Kikuchi, H., Morikawa, K., Tawada, A., and Suzuki, S. (1993) Activity of bacterial eliminases towards dermatan sulphates and dermatan sulphate proteoglycan, in Dermatan sulphate proteoglycans: chemistry, biology, chemical pathology (Scott, J. E., ed.), Portland Press, London, pp. 55–80.

    Google Scholar 

  9. Conrad, H. E. (2001) Degradation of heparan sulfate by nitrous acid, in Methods in Molecular Biology, vol. 171 “Proteoglycan protocols” (Iozzo, R. V., ed.), Humana Press, Totowa, New Jersey, pp. 347–351.

    Google Scholar 

  10. Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. (1968) Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem. 243, 1523–1535.

    PubMed  CAS  Google Scholar 

  11. Hiyama, K. and Okada, S. (1975) Crystallization and some properties of chondroitinase from Arthrobacter aurescens. J. Biol. Chem. 250, 1824–1828.

    PubMed  CAS  Google Scholar 

  12. Michelacci, Y. M. and Dietrich, C. P. (1974) Isolation and partial characterization of an induced chondroitinase B from Flavobacterium heparinum. Biochem. Biophys. Res. Commun. 56, 973–980.

    Article  PubMed  CAS  Google Scholar 

  13. Ototani, N., Kikuchi, M., and Yosizawa, Z. (1981) Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound AH-sepharose 4B. Carbohydr. Res. 88, 291–303.

    Article  PubMed  CAS  Google Scholar 

  14. Uyama, T., Ishida, M., Izumikawa, T., Trybala, E., Tufaro, F., Bergstrom, T., et al. (2006) Chondroitin 4-O-sulfotransferase-1 regulates “E” disaccharide expression of chondroitin sulfate required for herpes simplex virus infectivity. J. Biol. Chem. 281, 38668–38674.

    Article  PubMed  CAS  Google Scholar 

  15. Mizumoto, S., Mikami, T., Yasunaga, D., Kobayashi, N., Yamauchi, H., Miyake, A., et al. (2009) Chondroitin 4-O-sulfotransferase-1 is required for somitic muscle development and motor axon guidance in zebrafish. Biochem. J. 419, 387–399.

    Article  PubMed  CAS  Google Scholar 

  16. Sakaguchi, H., Watanabe, M., Ueoka, C., Sugiyama, E., Taketomi, T., Yamada, S., and Sugahara, K. (2001) Isolation of oligosaccharides from the chondroitin/dermatan sulfate-protein linkage region as reducing sugar chains and preparation of the analytical probes by fluorescent labeling with 2- aminobenzamide. J. Biochem. 129, 107–118

    PubMed  CAS  Google Scholar 

  17. Hashiguchi, T., Mizumoto, S., Yamada, S., and Sugahara, K. (2010) Analysis of the structure and neuritogenic activity of chondroitin sulfate/dermatan sulfate hybrid chains from porcine fetal membranes. Glycoconjugate J. 27, 49–60.

    Article  CAS  Google Scholar 

  18. Bitter, T. and Muir, H. (1962) A modified uronic acid carbazole reaction. Anal. Biochem. 4, 330–334.

    Article  PubMed  CAS  Google Scholar 

  19. Kinoshita, A. and Sugahara, K. (1999) Microanalysis of glycosaminoglycan -derived oligosaccharides labeled with the fluorophore 2- aminobenzamide by high-performance liquid chromatography: Application to disaccharide composition analysis and exo-sequencing of oligosaccharides. Anal. Biochem. 269, 367–378.

    Article  PubMed  CAS  Google Scholar 

  20. Kawashima, H., Atarashi, K., Hirose, M., Hirose, J., Yamada, S., Sugahara, K., and Miyasaka, M. (2002) Oversulfated chondroitin/dermatan sulfates containing GlcAβ1/IdoAα1-3GalNAc(4,6-O-disulfate)    interact with L- and P-selectin and chemokines. J. Biol. Chem. 277, 12921–12930.

    Article  PubMed  CAS  Google Scholar 

  21. Nakagawa, H., Hama, Y., Sumi, T., Li, S. C., Maskos, K., Kalayanamitra, K., et al. (2007) Occurrence of a non-sulfated chondroitin proteoglycan in the dried saliva of Collocalia swiftlets (edible bird’s nest). Glycobiology 17, 157–164.

    Article  PubMed  CAS  Google Scholar 

  22. Yamada, S., Okada, Y., Ueno, M., Iwata, S., Deepa, S.S., Nishimura, S., et al. (2002) Determination of the glycosaminoglycan-protein linkage region oligosaccharide structures of proteoglycans from Drosophila melanogaster and Caenorhabditis elegans. J. Biol. Chem. 277, 31877–31886.

    Article  PubMed  CAS  Google Scholar 

  23. Bao, X., Muramatsu, T., and Sugahara, K. (2005) Demonstration of the pleiotrophin-binding oligosaccharide sequences isolated from chondroitin sulfate/dermatan sulfate hybrid chains of embryonic pig brains. J. Biol. Chem. 280, 35318–35328.

    Article  PubMed  CAS  Google Scholar 

  24. Deepa, S. S., Kalayanamitra, K., Ito, Y., Kongtawelert, P., Fukui, S., Yamada, S., et al. (2007) Novel sulfated octa- and decasaccharides from squid cartilage chondroitin sulfate-E: Sequencing and their application for determination of the epitope structure of monoclonal antibody MO-225. Biochemistry 46, 2453–2465.

    Article  PubMed  CAS  Google Scholar 

  25. Deepa, S. S., Yamada, S., Fukui, S., and Sugahara, K. (2007) Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes. Glycobiology 17, 631–645.

    Article  PubMed  CAS  Google Scholar 

  26. Pothacharoen, P., Kalayanamitra, K., Deepa, S. S., Fukui, S., Hattori, T., Fukushima, N., et al. (2007) Two related but distinct chondroitin sulfate mimetope octasaccharide sequences recognized by monoclonal antibody WF6. J. Biol. Chem. 282, 35232–35246.

    Article  PubMed  CAS  Google Scholar 

  27. Li, F., Nandini, C. D., Hattori, T., Bao, X., Murayama, D., Nakamura, T., et al. (2010) Structure of pleiotrophin- and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches. J. Biol. Chem. 285, 27673–27685.

    Article  PubMed  CAS  Google Scholar 

  28. Malavaki, C., Mizumoto, S., Karamanos, N., and Sugahara, K. (2008) Recent advances in the structural study of functional chondroitin sulfate and dermatan sulfate in health and disease. Connect. Tissue Res. 49, 133–139.

    Article  PubMed  CAS  Google Scholar 

  29. McLean M. W., Bruce, J. S., Long, W. F., and Williamson, F. B. (1984) Flavobacterium heparinum 2-O-sulphatase for 2-O-sulphato -∆4,5-glycuronate-terminated oligosaccharides from heparin. Eur. J. Biochem. 145, 607–615.

    Article  PubMed  CAS  Google Scholar 

  30. Warnick, C. T. and Linker, A. (1972) Purification of an unusual α-glycuronidase from Flavobacteria. Biochemistry 11, 568–572.

    Article  PubMed  CAS  Google Scholar 

  31. Hamai, A., Morikawa, K., Horie, K., and Tokuyasu, K. (1989) Purification and characterization of hyaluronidase from Streptococcus dysgalactiae. Agric. Biol. Chem. 53, 2163–2168.

    Article  CAS  Google Scholar 

  32. Ohya, T. and Kaneko, Y. (1970) Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acta 198, 607–609.

    PubMed  CAS  Google Scholar 

  33. Nakazawa, K., Suzuki, N., 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.

    PubMed  CAS  Google Scholar 

  34. Hashimoto, N., Morikawa, K., Kikuchi, H., Yoshida. K., and Tokuyasu, K. (1988) Purification and charac-terization of kerdtanase. Seikagaku 60, 935.

    Google Scholar 

  35. Nakagawa, H., Yamada, T., Chien, J. L., Gardas, A., Kitamikado, M., Li, S. C., and Li, Y. T. (1980) Isolation and characterization of an endo-β-galactosidase from a new strain of Escherichia freundii. J. Biol. Chem. 255, 5955–5959.

    PubMed  CAS  Google Scholar 

  36. Nandini, C.D., Mikami, T., Ohta, M., Itoh, N., Akiyama-Nambu, F., and Sugahara, K. (2004) Structural and functional characterization of oversulfated chondroitin sulfate/dermatan sulfate hybrid chains from the notochord of hagfish: Neuritogenic activity and binding activities toward growth factors and neurotrophic factors. J. Biol. Chem. 279, 50799–50809.

    Article  PubMed  CAS  Google Scholar 

  37. Yanagishita, M. (2001) Isolation of proteoglycans from cell cultures and tissues, in Methods in Molecular Biology, vol. 171 “Proteoglycan protocols” (Iozzo, R. V., ed.), Humana Press, Totowa, New Jersey, pp. 3–10.

    Google Scholar 

  38. Brückner, J. (1955) Estimation of monosaccharides by the orcinol-sulphuric acid reaction. Biochem. J. 60, 200–205.

    PubMed  Google Scholar 

  39. Chandrasekhar, S., Esterman, M. A., and Hoffman, H. A. (1987) Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal. Biochem. 161, 103–108.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported in part by Grants-in-Aid for Young Scientists (B) 23790066 (to S.M.) from Japan Society for the Promotion of Science, Japan, and Future Drug Discovery and Medical Care Innovation Program (to K.S.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).

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Authors and Affiliations

  1. Laboratory of Proteoglycan Signaling and Therapeutics, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Sapporo, Japan

    Shuji Mizumoto & Kazuyuki Sugahara

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  1. Shuji Mizumoto
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  2. Kazuyuki Sugahara
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Correspondence to Kazuyuki Sugahara .

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Editors and Affiliations

  1. , Faculté de Médicine, INSERM U957 - EA 3822, rue Gaston Veil 1, Nantes, 44035, France

    Françoise Rédini

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Mizumoto, S., Sugahara, K. (2012). Glycosaminoglycan Chain Analysis and Characterization (Glycosylation/Epimerization). In: Rédini, F. (eds) Proteoglycans. Methods in Molecular Biology, vol 836. Humana Press. https://doi.org/10.1007/978-1-61779-498-8_7

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  • DOI: https://doi.org/10.1007/978-1-61779-498-8_7

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-497-1

  • Online ISBN: 978-1-61779-498-8

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