Glycoconjugate Journal

, 26:1247 | Cite as

S-Nitrosylation of secreted recombinant human glypican-1



Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to internalization and further modifications during recycling. By using SDS-PAGE, Western blotting and radiolabeling experiments we show that the glypican-1 can be S-nitrosylated. We have measured the level of S-nitrosylation in the glypican-1 core protein by biotin switch assay and find that the core protein can be S-nitrosylated in the presence of copper II ions and NO donor. Furthermore the glypican-1 proteoglycan produced in the presence of polyamine synthesis inhibitor, α-difluoromethylornithine, was endogenously S-nitrosylated and release of nitric oxide induced deaminative autocleavage of the HS side chains of glypican-1. We also show that the N-unsubstituted glucosamine residues are formed during biosynthesis of glypican-1 and that the content increased upon inhibition of polyamine synthesis. It cannot be excluded that endogenous glypican-1 can become further S-nitrosylated during recycling.


Ascorbate Proteoglycans Heparan sulfate Glycosaminoglycans Nitric oxide Difluoromethylornithine Nitrosothiols 







N-unsubstituted glucosamine






Heparan sulfate


Monoclonal antibody


Nitric oxide






Sodium nitroprusside dihydrate



We thank Prof. Lars-Åke Fransson, Department of Experimental Medical Science, Lund University, for advice. The work was supported by grants from the Swedish Science Council (VR-M), The Royal physiographic society, and the Crafoord, Hedborg, Kock, Segerfalk, Zoegas and Jeanssons Foundations.

Supplementary material

10719_2009_9243_MOESM1_ESM.pdf (59 kb)
ESM 1 (PDF 58 kb)


  1. 1.
    Belting, M.: Heparan sulfate proteoglycan as a plasma membrane carrier. Trends. Biochem. Sci. 28, 145–151 (2003). doi: 10.1016/S0968-0004(03)00031-8 CrossRefPubMedGoogle Scholar
  2. 2.
    Fransson, L.-Å., Belting, M., Cheng, F., Jonsson, M., Mani, K., Sandgren, S.: Novel aspects of glypican glycobiology. Cell Mol. Life Sci. 61, 1016–1024 (2004). doi: 10.1007/s00018-004-3445-0 CrossRefPubMedGoogle Scholar
  3. 3.
    Bernfield, M., Gotte, 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). doi: 10.1146/annurev.biochem.68.1.729 CrossRefPubMedGoogle Scholar
  4. 4.
    David, G., Lories, V., Decock, B., Marynen, P., Cassiman, J.J., Van den Berghe, H.: Molecular cloning of a phosphatidylinositol-anchored membrane heparan sulfate proteoglycan from human lung fibroblasts. J. Cell Biol. 111, 3165–3176 (1990). doi: 10.1083/jcb.111.6.3165 CrossRefPubMedGoogle Scholar
  5. 5.
    Hess, D.T., Matsumoto, A., Kim, S.O., Marshall, H.E., Stamler, J.S.: Protein S-nitrosylation: purview and parameters. Nat. Rev. Mol. Cell Biol. 6, 150–166 (2005). doi: 10.1038/nrm1569 CrossRefPubMedGoogle Scholar
  6. 6.
    Greco, T.M., Hodara, R., Parastatidis, I., Heijnen, H.F., Dennehy, M.K., Liebler, D.C., Ischiropoulos, H.: Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells. Proc. Natl. Acad. Sci. U. S. A. 103, 7420–7425 (2006). doi: 10.1073/pnas.0600729103 CrossRefPubMedGoogle Scholar
  7. 7.
    Ding, K., Mani, K., Cheng, F., Belting, M., Fransson, L.Å.: Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols. J. Biol. Chem. 277, 33353–33360 (2002). doi: 10.1074/jbc.M203383200 CrossRefPubMedGoogle Scholar
  8. 8.
    Mani, K., Cheng, F., Havsmark, B., Jonsson, M., Belting, M., Fransson, L.-Å.: Prion, amyloid beta-derived Cu(II) ions, or free Zn(II) ions support S-nitroso-dependent autocleavage of glypican-1 heparan sulfate. J. Biol. Chem. 278, 38956–38965 (2003). doi: 10.1074/jbc.M300394200 CrossRefPubMedGoogle Scholar
  9. 9.
    Cappai, R., Cheng, F., Ciccotosto, G.D., Needham, B.E., Masters, C.L., Multhaup, G., Fransson, L.-Å., Mani, K.: The amyloid precursor protein (APP) of Alzheimer disease and its paralog, APLP2, modulate the Cu/Zn-Nitric Oxide-catalyzed degradation of glypican-1 heparan sulfate in vivo. J. Biol. Chem. 280, 13913–13920 (2005). doi: 10.1074/jbc.M409179200 CrossRefPubMedGoogle Scholar
  10. 10.
    Cheng, F., Lindqvist, J., Haigh, C.L., Brown, D.R., Mani, K.: Copper-dependent co-internalization of the prion protein and glypican-1. J. Neurochem. 98, 1445–1457 (2006). doi: 10.1111/j.1471-4159.2006.03981.x CrossRefPubMedGoogle Scholar
  11. 11.
    Mani, K., Jonsson, M., Edgren, G., Belting, M., Fransson, L.-Å.: A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells. Glycobiology 10, 577–586 (2000). doi: 10.1093/glycob/10.6.577 CrossRefPubMedGoogle Scholar
  12. 12.
    Cheng, F., Mani, K., van den Born, J., Ding, K., Belting, M., Fransson, L.-Å.: 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). doi: 10.1074/jbc.M205241200 CrossRefPubMedGoogle Scholar
  13. 13.
    Mani, K., Cheng, F., Fransson, L.-Å.: Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann-Pick C1 fibroblasts. Glycobiology 16, 711–718 (2006). doi: 10.1093/glycob/cwj121 CrossRefPubMedGoogle Scholar
  14. 14.
    Ding, K., Sandgren, S., Mani, K., Belting, M., Fransson, L.-Å.: Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites. J. Biol. Chem. 276, 46779–46791 (2001). doi: 10.1074/jbc.M105419200 CrossRefPubMedGoogle Scholar
  15. 15.
    Westling, C., Lindahl, U.: Location of N-unsubstituted glucosamine residues in heparan sulfate. J. Biol. Chem. 277, 49247–49255 (2002). doi: 10.1074/jbc.M209139200 CrossRefPubMedGoogle Scholar
  16. 16.
    Belting, M., Mani, K., Jonsson, M., Cheng, F., Sandgren, S., Jonsson, S., Ding, K., Delcros, J.G., Fransson, L.-Å.: Glypican-1 is a vehicle for polyamine uptake in mammalian cells: a pivital role for nitrosothiol-derived nitric oxide. J. Biol. Chem. 278, 47181–47189 (2003). doi: 10.1074/jbc.M308325200 CrossRefPubMedGoogle Scholar
  17. 17.
    Bengtsson, E., Aspberg, A., Heinegard, D., Sommarin, Y., Spillmann, D.: The amino-terminal part of PRELP binds to heparin and heparan sulfate. J. Biol. Chem. 275, 40695–40702 (2000). doi: 10.1074/jbc.M007917200 CrossRefPubMedGoogle Scholar
  18. 18.
    Mani, K., Cheng, F., Sandgren, S., Van Den Born, J., Havsmark, B., Ding, K., Fransson, L.-Å.: The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1. Glycobiology 14, 599–607 (2004). doi: 10.1093/glycob/cwh067 CrossRefPubMedGoogle Scholar
  19. 19.
    Jaffrey, S.R., Snyder, S.H.: The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE., Issue 86 p. pl 1 (2001). doi: 10.1126/stke.2001.86.pl1
  20. 20.
    Shively, J.E., Conrad, H.E.: Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15, 3932–3942 (1976). doi: 10.1021/bi00663a005 CrossRefPubMedGoogle Scholar
  21. 21.
    Lindahl, U., Bäckström, G., Jansson, L., Hallen, A.: Biosynthesis of heparin. II. Formation of sulfamino groups. J. Biol. Chem. 248, 7234–7241 (1973)PubMedGoogle Scholar
  22. 22.
    Ramamurthy, P., Hocking, A.M., McQuillan, D.J.: Recombinant decorin glycoforms. Purification and structure. J. Biol. Chem. 271, 19578–19584 (1996). doi: 10.1074/jbc.271.32.19578 CrossRefPubMedGoogle Scholar
  23. 23.
    Ding, K., Jonsson, M., Mani, K., Sandgren, S., Belting, M., Fransson, L.-Å.: N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and nitrite-deprived endothelial cells. Mapping of nitric oxide/nitrite-susceptible glucosamine residues to clustered sites near the core protein. J. Biol. Chem. 276, 3885–3894 (2001). doi: 10.1074/jbc.M005238200 CrossRefPubMedGoogle Scholar
  24. 24.
    Sessa, W.C., Garcia-Cardena, G., Liu, J., Keh, A., Pollock, J.S., Bradley, J., Thiru, S., Braverman, I.M., Desai, K.M.: The Golgi association of endothelial nitric oxide synthase is necessary for the efficient synthesis of nitric oxide. J. Biol. Chem. 270, 17641–17644 (1995). doi: 10.1074/jbc.270.30.17641 CrossRefPubMedGoogle Scholar
  25. 25.
    Ghosh, D.K., Rashid, M.B., Crane, B., Taskar, V., Mast, M., Misukonis, M.A., Weinberg, J.B., Eissa, N.T.: Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: a critical role for Asp-280 in substrate binding and subunit interactions. Proc. Natl Acad. Sci. USA 98, 10392–10397 (2001). doi: 10.1073/pnas.181251298 CrossRefPubMedGoogle Scholar
  26. 26.
    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). doi: 10.1006/exnr.1997.6740 CrossRefPubMedGoogle Scholar
  27. 27.
    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). doi: 10.1074/jbc.M010291200 CrossRefPubMedGoogle Scholar
  28. 28.
    Liu, J., Shriver, Z., Pope, R.M., Thorp, S.C., Duncan, M.B., Copeland, R.J., Raska, C.S., Yoshida, K., Eisenberg, R.J., Cohen, G., et al.: Characterization of a heparan sulfate octasaccharide that binds to herpes simplex virus type 1 glycoprotein D. J. Biol. Chem. 277, 33456–33467 (2002). doi: 10.1074/jbc.M202034200 CrossRefPubMedGoogle Scholar
  29. 29.
    Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.P., Malmström, A., Shukla, D., Liu, J.: Heparan sulfate 3-O-sulfotransferase isoform 5 generates both an antithrombin-binding site and an entry receptor for herpes simplex virus, type 1. J. Biol. Chem. 277, 37912–37919 (2002). doi: 10.1074/jbc.M204209200 CrossRefPubMedGoogle Scholar
  30. 30.
    Löfgren, K., Cheng, F., Fransson, L.-Å., Bedecs, K., Mani, K.: Involvement of glypican-1 autoprocessing in scrapie infection. Eur. J. NeuroSci. 28, 964–972 (2008). doi: 10.1111/j.1460-9568.2008.06386.x CrossRefPubMedGoogle Scholar
  31. 31.
    Mani, K., Cheng, F., Fransson, L.-Å.: Heparan sulfate degradation products can associate with oxidized proteins and proteasomes. J. Biol. Chem. 282, 21934–21944 (2007). doi: 10.1074/jbc.M701200200 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Experimental Medical Science, Division of Neuroscience, Glycobiology GroupLund UniversityLundSweden

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