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Preparation of heparin/heparan sulfate oligosaccharides with internal N-unsubstituted glucosamine residues for functional studies

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Abstract

The rare N-unsubstituted glucosamine (GlcNH +3 ) residues in heparan sulfate (HS) have important biological and pathophysiological roles. However, it is difficult to prepare naturally-occuring, GlcNH +3 -containing oligosaccharides from HS because of their low abundance, as well as the inherent problems in both excising and identifying them. Therefore, the ability to chemically generate a series of structurally-defined oligosaccharides containing GlcNH +3 residues would greatly contribute to investigating their natural role in HS. In this study, a series of heparin/HS oligosaccharides, from dp6 up to dp16 in length that possess internal GlcNH +3 residues were prepared by a combination of chemical modification and heparinase I digestion. Purification and structural analysis of the major species derived from the octa- to dodeca-saccharide size fractions indicated the introduction of between 1 and 3 internal GlcNH +3 residues per oligosaccharide. In addition, a GlcNH +3 residue was selectively introduced into an internal position in a tetrasaccharide species by direct chemical modification. This selectivity has potential as an alternative procedure for preparing internally-modified oligosaccharides of various lengths. The utility of such oligosaccharides was demonstrated by a comparison of the binding of three different tetrasaccharide species containing 0, 1 and 2 free amino groups to the NK1 truncated variant of hepatocyte growth factor/scatter factor.

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Abbreviations

AMAC:

2-amino-acridone

CyPB:

Cyclophilin-B

DMSO:

Dimethyl sulfoxide

dp:

Degree of polymerization (i.e. number of monosaccharide units e.g. dp2 is disaccharide)

GAG:

Glycosaminoglycan

GlcA:

β-D-glucuronic acid

GlcNAc:

β-D-N-acetylglucosamine

GlcNH +3 :

β-D-N-unsubstituted glucosamine

GlcNS:

β-D-N-sulfoglucosamine

GMSA:

Gel mobility shift assay

∆HexA:

∆4–5-unsaturated hexuronic acid

HS:

Heparan sulfate

HSV:

Herpes simplex virus

HS 3-OST:

Heparan sulfate 3-O-sulfotransferase

IdoA:

α-L-iduronic acid

NDST:

N-deacetylase/N-sulfotransferase

NK1:

Truncated variant of hepatocyte growth factor/scatter factor containing only the N-terminal and the first Kringle domain

S:

Sulfate

SAX-HPLC:

Strong anion-exchange HPLC

References

  1. Perrimon, N., Bernfield, M.: Specificities of heparan sulfate proteoglycans in developmental processes. Nature 404, 725–728 (2000)

    Article  PubMed  CAS  Google Scholar 

  2. Lander, A.D., Selleck, S.B.: The elusive functions of proteoglycans: in vivo veritas. J. Cell Biol. 148, 227–232 (2000)

    Article  PubMed  CAS  Google Scholar 

  3. Robinson, C.J., Stringer, S.E.: The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J. Cell Sci. 114, 853–865 (2001)

    PubMed  CAS  Google Scholar 

  4. Marcum, J.A., McKenney, J.B., Galli, S.J., Jackman, R.W., Rosenberg, R.D.: Anticoagulantly active heparin-like molecules from mast cell-deficient mice. Am. J. Physiol. 250, H879–H888 (1986)

    PubMed  CAS  Google Scholar 

  5. Couchman, J.R.: Syndecans: proteoglycan regulators of cell-surface microdomains? Nat. Rev. Mol. Cell Biol. 4, 926–937 (2003)

    Article  PubMed  CAS  Google Scholar 

  6. Liu, D., Shriver, Z., Venkataraman, G., El Shabrawi, Y., Sasisekharan, R.: Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis. Proc. Natl. Acad. Sci. U.S.A. 99, 568–573 (2002)

    Article  PubMed  CAS  Google Scholar 

  7. Bernfield, M., Cotte, M., Park, P.W., Reizes, O., Fitzgerald, M.L., Lincecum, J., Zako, M.: Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68, 729–777 (1999)

    Article  PubMed  CAS  Google Scholar 

  8. Casu, B., Lindahl, U.: Structure and biological interactions of heparin and heparan sulfate. Adv. Carbohydr. Chem. Biochem. 57, 159–206 (2001)

    Article  PubMed  CAS  Google Scholar 

  9. Conrad, H.E.: Heparin binding proteins. Academic, San Diego (1998)

    Google Scholar 

  10. Esko, J.D., Selleck, S.B.: Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435–471 (2002)

    Article  PubMed  CAS  Google Scholar 

  11. Liu, J., Thorp, S.C.: Cell surface heparan sulfate and its roles in assisting viral infections. Med. Res. Rev. 22, 1–25 (2002)

    Article  PubMed  Google Scholar 

  12. Salmivirta, M., Lidholt, K., Lindahl, U.: Heparan sulfate: a piece of information. FASEB J. 10, 1270–1279 (1996)

    PubMed  CAS  Google Scholar 

  13. Lindahl, U., Kusche-Gullberg, M., Kjellén, L.: Regulated diversity of heparan sulfate. J. Biol. Chem. 273, 24979–24982 (1998)

    Article  PubMed  CAS  Google Scholar 

  14. Esko, J.D., Lindahl, U.: Molecular diversity of heparan sulfate. J. Clin. Invest. 108, 169–173 (2001)

    PubMed  CAS  Google Scholar 

  15. Murphy, K.J., Merry, C.L., Lyon, M., Thompson, J.E., Roberts, I.S., Gallagher, J.T.: A new model for the domain structure of heparan sulfate based on the novel specificity of K5 lyase. J. Biol. Chem. 279, 27239–27245 (2004)

    Article  PubMed  CAS  Google Scholar 

  16. Toida, T., Yoshida, H., Toyoda, H., Koshiishi, I., Imanari, T., Hileman, R.E., Fromm, J.R., Linhardt, R.J.: Structural differences and the presence of unsubstituted amino groups in heparan sulphates from different tissues and species. Biochem. J. 322, 499–506 (1997)

    PubMed  CAS  Google Scholar 

  17. Westling, C., Lindahl, U.: Location of N-unsubstituted glucosamine residues in heparan sulfate. J. Biol. Chem. 277, 49247–49255 (2002)

    Article  PubMed  CAS  Google Scholar 

  18. Norgard-Sumnicht, K., Varki, A.: Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups. J. Biol. Chem. 270, 12012–12024 (1995)

    Article  PubMed  CAS  Google Scholar 

  19. Liu, J., Shriver, Z., Blaiklock, P., Yoshida, K., Sasisekharan, R., Rosenberg, R.D.: Heparan sulfate D-glucosaminyl 3-O-sulfotransferase 3A sulfates N-unsubstituted glucosamine. J. Biol. Chem. 274, 38155–38162 (1999)

    Article  PubMed  CAS  Google Scholar 

  20. Liu, J., Shriver, Z., Pope, M., Thorp, S.C., Duncan, M.B., Copeland, R.J., Sasisekharan, R.: Characterization of a heparan sulfate octasaccharide that binds to herpes simplex viral type 1 glycoprotein D. J. Biol. Chem. 277, 33456–33467 (2002)

    Article  PubMed  CAS  Google Scholar 

  21. Shukla, D., Liu, J., Blaiklock, P., Shworak, N.W., Bai, X., Esko, J.D., Cohen, G.H., Eisenberg, R.J., Rosenberg, R.D., Spear, P.G.: A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell 99, 13–22 (1999)

    Article  PubMed  CAS  Google Scholar 

  22. Vanpouille, C., Deligny, A., Delehedde, M., Denys, A., Melchior, A., Liénard, X., Lyon, M., Mazurier, J., Fernig, D.G., Allain, F.: The heparin/heparan sulfate sequence that interacts with cyclophilin B contains a 3-O-sulfated N-unsubstituted glucosamine residue. J. Biol. Chem. 282, 24416–24429 (2007)

    Article  PubMed  CAS  Google Scholar 

  23. Van den Born, J., Gunnarsson, K., Bakker, M.A., Kjellén, L., Kusche-Gullberg, M., Maccarana, M., Berden, J.H., Lindahl, U.: Presence of N-unsubstituted glucosamine units in native heparan sulfate revealed by a monoclonal antibody. J. Biol. Chem. 270, 31303–31309 (1995)

    Article  PubMed  Google Scholar 

  24. Leteux, C., Chai, W., Nagai, K., Herbert, C.G., Lawson, A.M., Feizi, T.: 10E4 antigen of Scrapie lesions contains an unusual nonsulfated heparan motif. J. Biol. Chem. 276, 12539–12545 (2001)

    Article  PubMed  CAS  Google Scholar 

  25. McBride, P.A., Wilson, M.I., Eikelenboom, P., Tunstall, A., Bruce, M.E.: Heparan sulfate proteoglycan is associated with amyloid plaques and neuroanatomically targeted PrP pathology throughout the incubation period of scrapie-infected mice. Exp. Neurol. 149, 447–454 (1998)

    Article  PubMed  CAS  Google Scholar 

  26. Cheng, F., Mani, K., van den Born, J., Ding, K., Belting, M., Fransson, L.-A.: Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1-containing endosomes. J. Biol. Chem. 277, 44431–44439 (2002)

    Article  PubMed  CAS  Google Scholar 

  27. Carlsson, P., Presto, J., Spillmann, D., Lindahl, U., Kjellén, L.: Heparin/heparan sulfate biosynthesis: processive formation of N-sulfated domains. J. Biol. Chem. 283, 20008–20014 (2008)

    Article  PubMed  CAS  Google Scholar 

  28. Rees, M.D., Pattison, D.I., Davies, M.J.: Oxidation of heparan sulphate by hypochlorite: role of N-chloro derivatives and dichloramine-dependent fragmentation. Biochem. J. 391, 125–134 (2005)

    Article  PubMed  CAS  Google Scholar 

  29. Wei, Z., Lyon, M., Gallagher, J.T.: Distinct substrate specificities of bacterial heparinases against N-unsubstituted glucosamine residues in heparan sulfate. J. Biol. Chem. 280, 15742–15748 (2005)

    Article  PubMed  CAS  Google Scholar 

  30. Shi, X., Zaia, J.: Organ-specific heparan sulfate structural phenotypes. J. Biol. Chem. 284, 11806–11814 (2009)

    Article  PubMed  CAS  Google Scholar 

  31. Shaka, A.J., Lee, C.J., Pines, A.: Iterative schemes for bilinear operators—application to spin decoupling. J. Magn. Reson. 77, 274 (1988)

    Google Scholar 

  32. Stott, K., Stonehouse, J., Keeler, J., Hwang, T.L., Shaka, A.J.: Excitation sculpting in high-resolution nuclear magnetic resonance spectroscopy—application to selective NOE experiments. J. Am. Chem. Soc. 117, 4199 (1995)

    Article  CAS  Google Scholar 

  33. Chirgadze, D.Y., Hepple, J.P., Zhou, H., Byrd, A., Blundell, T.L., Gherardi, E.: Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding. Nat. Struct. Biol. 6, 72–79 (1999)

    Article  PubMed  CAS  Google Scholar 

  34. Lyon, M., Deakin, J.A., Lietha, D., Gherardi, E., Gallagher, J.T.: The interactions of hepatocyte growth factor/scatter factor and its NK1 and NK2 variants with glycosaminoglycans using a modified gel mobility shift assay. Elucidation of the minimal size of binding and activatory oligosaccharides. J. Biol. Chem. 279, 43560–43567 (2004)

    Article  PubMed  CAS  Google Scholar 

  35. Brisson, J.-R., Sue, S.C., Wu, W.G., McManus, G., Nghia, P.T., Uhrín, D.: NMR of Carbohydrates: 1D Homonuclear Selective Methods. In: Jiménez-Barbero, J., Peters, T. (eds.) NMR of Glycoconjugates, Ch. 4, pp. 59–93. Wiley-VCH, Weinheim (2002)

    Chapter  Google Scholar 

  36. Yates, E.A., Santini, F., Guerrini, M., Naggi, A., Torri, G., Casu, B.: 1H and 13C NMR spectral assignments of the major sequences of twelve systematically modified heparin derivatives. Carbohydr. Res. 294, 15–27 (1996)

    PubMed  CAS  Google Scholar 

  37. Catlow, K.R., Deakin, J.A., Wei, Z., Delehedde, M., Fernig, D.G., Gherardi, E., Gallagher, J.T., Pavão, M.S., Lyon, M.: Interactions of hepatocyte growth factor/scatter factor with various glycosaminoglycans reveal an important interplay between the presence of iduronate and sulfate density. J. Biol. Chem. 283, 5235–5248 (2008)

    Article  PubMed  CAS  Google Scholar 

  38. Deakin, J.A., Blaum, B.S., Gallagher, J.T., Uhrín, D., Lyon, M.: The binding properties of minimal oligosaccharides reveal a common heparan sulfate/dermatan sulfate-binding site in hepatocyte growth factor/scatter factor that can accommodate a wide variety of sulfation patterns. J. Biol. Chem. 284, 6311–6321 (2009)

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported by a European Union Project Grant QLK3-CT-2001-01976 and Chinese National Funding Grants 20773023 and XSJRC2007-01 (to Z.W.), a Cancer Research UK Programme Grant (to J.A.D., J.T.G. & M.L.), and an EaStCHEM Studentship (to B.S.B. & D.U.)

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Authors and Affiliations

  1. Institute of Glycobiochemistry, National Engineering Research Centre of Chemical Fertilizer Catalyst, Fu Zhou University, Fu Zhou, 350002, People’s Republic of China

    Zheng Wei

  2. Cancer Research UK Glyco-Oncology Group, School of Cancer and Enabling Science, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK

    Zheng Wei, Jon Andrew Deakin, John Thomas Gallagher & Malcolm Lyon

  3. EaStCHEM, School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, UK

    Bärbel Sabine Blaum & Dušan Uhrín

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  1. Zheng Wei
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  2. Jon Andrew Deakin
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Correspondence to Zheng Wei.

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Wei, Z., Deakin, J.A., Blaum, B.S. et al. Preparation of heparin/heparan sulfate oligosaccharides with internal N-unsubstituted glucosamine residues for functional studies. Glycoconj J 28, 525–535 (2011). https://doi.org/10.1007/s10719-011-9352-3

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