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Determination of iduronic acid and glucuronic acid in sulfated chondroitin/dermatan hybrid chains by 1H-nuclear magnetic resonance spectroscopy

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Abstract

The relative proportion of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) is of great importance for the structure–function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. 1H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the 1H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D-galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) 1H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D 1H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains.

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Abbreviations

1D:

one-dimensional

CSase:

chondroitinase

CS:

chondroitin sulfate

DS:

dermatan sulfate

GalNAc:

N-acetyl-D-galactosamine

GlcA:

D-glucuronic acid

IdoA:

L-iduronic acid

ΔHexA:

4-deoxy-L-threo-hex-4-enepyranosyluronic acid

HPLC:

high performance liquid chromatography

NMR:

nuclear magnetic resonance

References

  1. Rodén, L.: Structure and metabolism of connective tissue proteoglycans. In: Lennarz, W.J. (ed.) The Biochemistry of Glycoproteins and Proteoglycans, pp. 267–371. Plenum, New York (1980)

    Google Scholar 

  2. Poole, A.R.: Proteoglycans in health and disease: structures and functions. Biochem. J. 236, 1–14 (1986)

    PubMed  CAS  Google Scholar 

  3. Bandtlow, C.E., Zimmermann, D.R.: Proteoglycans in the developing brain: new conceptual insights for old proteins. Physiol. Rev. 80, 1267–1290 (2000)

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  5. Silbert, J.E., Sugumaran, G.: Biosynthesis of chondroitin/dermatan sulfate. IUBMB Life 54, 177–180 (2002)

    Article  PubMed  CAS  Google Scholar 

  6. Sugahara, K., Yamada, S.: Structure and function of oversulfated chondroitin sulfate variants: unique sulfation patterns and neuroregulatory activities. Trends Glycosci. Glycotechnol. 12, 321–349 (2000)

    CAS  Google Scholar 

  7. Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C., Schachner, M.: Isolation of a neural chondroitin sulfate proteoglycan with neurite outgrowth promoting properties. J. Cell Biol. 126, 783–799 (1994)

    Article  PubMed  CAS  Google Scholar 

  8. Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., Sugahara, K.: Chondroitin sulfate/dermatan sulfate hybrid chains from embryonic pig brain, which contain a higher proportion of L-iduronic acid than those from the adult pig brain, exhibit neuritogenic and growth factor-binding activities. J. Biol. Chem. 279, 9765–9776 (2004)

    Article  PubMed  CAS  Google Scholar 

  9. Nandini, C.D., Mikami, T., Ohta, M., Itoh, N., Akiyama-Nambu, F., Sugahara, K.: 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 (2004)

    Article  PubMed  CAS  Google Scholar 

  10. Nandini, C.D., Itoh, N., Sugahara, K.: Novel 70 kDa chondroitin sulfate/dermatan sulfate hybrid chains with a unique heterogenous sulfation pattern from shark skin, which exhibit neuritogenic activity and binding activities for growth factors and neurotrophic factors. J. Biol. Chem. 280, 4058–4069 (2005)

    Article  PubMed  CAS  Google Scholar 

  11. Li, F., Shetty, A.K., Sugahara, K.: Neuritogenic activity of chondroitin/dermatan sulfate hybrid chains of embryonic pig brain and their mimicry from shark liver: involvement of the pleiotrophin and hepatocyte growth factor signaling pathways. J. Biol. Chem. 282, 2956–2966 (2007)

    Article  PubMed  CAS  Google Scholar 

  12. Bao, X., Muramatsu, T., Sugahara, K.: 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 (2005)

    Article  PubMed  CAS  Google Scholar 

  13. von Holst, A., Sirko, S., Faissner, A.: The unique 473HD-chondroitinsulfate epitope is expressed by radial glia and involved in neural precursor cell proliferation. J. Neurosci 26, 4082–4094 (2006)

    Article  CAS  Google Scholar 

  14. Akita, K., von Holst, A., Furukawa, Y., Mikami, T., Sugahara, K., Faissner, A.: Expression of multiple chondroitin/dermatan sulfotransferases in the neurogenic regions of the embryonic and adult CNS suggests that complex chondroitin sulfates function in neural stem cell maintenance. Stem Cells (in press)

  15. Saito, H., Yamagata, T., Suzuki, S.: Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243, 1536–1542 (1968)

    PubMed  CAS  Google Scholar 

  16. Yoshida, K., Miyauchi, S., Kikuchi, H., Tawada, A., Tokuyasu, K.: Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography. Anal. Biochem. 177, 327–332 (1989)

    Article  PubMed  CAS  Google Scholar 

  17. Radhakrishnamurthy, B., Berenson, G.S.: Identification of uronic acids in mucopolysaccharides. Arch. Biochem. Biophys. 101, 360–362 (1963)

    Article  PubMed  CAS  Google Scholar 

  18. Fransson, L.Å., Rodén, L., Spach, M.L.: Automated ion-exchange chromatography of uronic acids and uronic acid containing oligosaccharides. Anal. Biochem. 23, 317–330 (1968)

    Article  PubMed  CAS  Google Scholar 

  19. Inoue, S., Miyawaki, M.: Quantitative analysis of iduronic acid and glucuronic acid in sulfated galactosaminoglycuronans by gas chromatography. Anal. Biochem. 65, 164–174 (1975)

    Article  PubMed  CAS  Google Scholar 

  20. Spiro, M.J.: Uronic acid analysis by automated anion exchange chromatography. Anal. Biochem. 82, 348–352 (1977)

    Article  PubMed  CAS  Google Scholar 

  21. Whitfield, D.M., Stojkovski, S., Pang, H., Baptista, J., Sarkar, B.: Diagnostic methods for the determination of iduronic acid in oligosaccharides. Anal. Biochem. 194, 259–267 (1991)

    Article  PubMed  CAS  Google Scholar 

  22. Shaklee, P.N., Conrad, H.E.: The disaccharides formed by deaminative cleavage of N-deacetylated glycosaminoglycans. Biochem. J. 235, 225–236 (1986)

    PubMed  CAS  Google Scholar 

  23. Sudo, M., Sato, K., Chaidedgumjorn, A., Toyoda, H., Toida, T., Imanari, T.: 1H nuclear magnetic resonance spectroscopic analysis for determination of glucuronic and iduronic acids in dermatan sulfate, heparin, and heparan sulfate. Anal. Biochem. 297, 42–51 (2001)

    Article  PubMed  CAS  Google Scholar 

  24. Tabeur, C., Machetto, F., Mallet, J.M., Duchaussoy, P., Petitou, M., Sinaÿ, P.: L-Iduronic acid derivatives as glycosyl donors. Carbohydr. Res. 281, 253–276 (1996)

    Article  PubMed  CAS  Google Scholar 

  25. Yamada, S., Sugahara, K.: 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), pp. 71–78. Humana, Totowa (2003)

    Chapter  Google Scholar 

  26. Sugahara, K., Okumura, Y., Yamashina, I.: The Engelbreth–Holm–Swarm mouse tumor produces undersulfated heparan sulfate and oversulfated galactosaminoglycans. Biochem. Biophys. Res. Commun. 162, 189–197 (1989)

    Article  PubMed  CAS  Google Scholar 

  27. Sugahara, K., Tohno-oka, R., Yamada, S., Khoo, K.H., Morris, H.R., Dell, A.: Structural studies on the oligosaccharides isolated from bovine kidney heparan sulphate and characterization of bacterial heparitinases used as substrates. Glycobiology 4, 535–544 (1994)

    Article  PubMed  CAS  Google Scholar 

  28. Sugahara, K., Ohkita, Y., Shibata, Y., Yoshida, K., Ikegami, A.: Structural studies on the hexasaccharide alditols isolated from the carbohydrate- protein linkage region of dermatan sulphate proteoglycans of bovine aorta. Demonstration of iduronic acid-containing components. J. Biol. Chem. 270, 7204–7212 (1995)

    Article  PubMed  CAS  Google Scholar 

  29. Nandini, C.D., Sugahara, K.: Role of the sulfation pattern of chondroitin sulfate in its biological activities in the binding of growth factors. In: Volpi, N. (ed.) Advances in Pharmacology, vol 53, pp. 253–279. Elsevier, Oxford (2006)

    Google Scholar 

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

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The authors thank X. Bao for the preparation of E-CS/DS. This work was supported by HAITEKU (2004–2008) from the Japan Private School Promotion Foundation, Grant-in-aid for Scientific Research C-19590052 (to S. Y.) and Scientific Research (B) 18390030 (to K. S.) from MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan), The Human Frontier Science Program RGP0018/2005 (to K. S.), the Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology (JST) agency (to K. S.), and a National Project “Knowledge Cluster Initiative” (2nd stage “Sapporo Bio-cluster Bio-S”) from MEXT.

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Authors and Affiliations

  1. Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Frontier Research Center for Post-Genomic Science and Technology, Nishi 11-choume, Kita 21-jo, Kita-ku, Sapporo, Hokkaido, 001-0021, Japan

    Fuchuan Li, Shuhei Yamada,  Basappa, Ajaya K. Shetty & Kazuyuki Sugahara

  2. Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe, 658-8558, Japan

    Fuchuan Li, Shuhei Yamada & Kazuyuki Sugahara

  3. NMR Laboratory, Kobe Pharmaceutical University, Higashinada-ku, Kobe, 658-8558, Japan

    Makiko Sugiura

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  1. Fuchuan Li
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  2. Shuhei Yamada
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  3. Basappa
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  4. Ajaya K. Shetty
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  5. Makiko Sugiura
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  6. Kazuyuki Sugahara
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Correspondence to Kazuyuki Sugahara.

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The contributions of Fuchuan Li and Shuhei Yamada should be considered equal.

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Li, F., Yamada, S., Basappa et al. Determination of iduronic acid and glucuronic acid in sulfated chondroitin/dermatan hybrid chains by 1H-nuclear magnetic resonance spectroscopy. Glycoconj J 25, 603–610 (2008). https://doi.org/10.1007/s10719-008-9124-x

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