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The proteoglycan bikunin has a defined sequence

Nature Chemical Biology volume 7, pages 827–833 (2011)Cite this article

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Abstract

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan, with a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike nucleic acids and proteins, whose synthesis is template driven, Golgi-synthesized glycosaminoglycans are not believed to have predictable or deterministic sequences. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size-similar and charge-similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular ions corresponding to monocompositional peptidoglycosaminoglycans. Fragmentation using collision-induced dissociation unexpectedly afforded a single sequence for each monocompositional parent ion, unequivocally demonstrating the presence of a defined sequence. The biosynthetic pathway common to all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment in the understanding of information storage in complex glycans.

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Figure 1: Biosynthetic pathway for chondroitin sulfate A GAG.
Figure 2: FT-ICR-MS analysis of polydisperse and heterogeneous bikunin peptidoglycosaminoglycan.
Figure 3: FT-ICR-MS analysis of a bikunin peptidoglycosaminoglycan fraction.
Figure 4: Bikunin sequencing flow chart.

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References

  1. Feero, W.G., Guttmacher, A.E. & Collins, F.S. Genomic medicine–an updated primer. N. Engl. J. Med. 362, 2001–2011 (2010).

    Article  CAS  PubMed  Google Scholar 

  2. Ly, M., Laremore, T.N. & Linhardt, R.J. Proteoglycomics: recent progress and future challenges. OMICS 14, 389–399 (2010)10.1089/omi.2009.0123.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kreuger, J., Spillmann, D., Li, J. & Lindahl, U. Interactions between heparan sulfate and proteins: the concept of specificity. J. Cell Biol. 174, 323–327 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ball, S. et al. From glycogen to amylopectin: a model for the biogenesis of the plant starch granule. Cell 86, 349–352 (1996).

    Article  CAS  PubMed  Google Scholar 

  5. Perez, S. & Mazeau, K. Conformations, structures, and morphologies of celluloses. in Polysaccharides: Structural diversity and functional versatility 2nd edn (ed. Dumitiu, S.) 41–68 (Marcel Dekker, 1998).

  6. 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  CAS  PubMed  Google Scholar 

  7. Silbert, J.E. & Sugumaran, G. Biosynthesis of chondroitin/dermatan sulfate. IUBMB Life 54, 177–186 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Nairn, A.V. et al. Glycomics of proteoglycan biosynthesis in murine embryonic stem cell differentiation. J. Proteome Res. 6, 4374–4387 (2007)10.1021/pr070446f.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Couchman, J.R. Transmembrane signaling proteoglycans. Annu. Rev. Cell Dev. Biol. 26, 89–114 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Linhardt, R.J. & Toida, T. Role of glycosaminoglycans in cellular communication. Acc. Chem. Res. 37, 431–438 (2004)10.1021/ar030138x.

    Article  CAS  PubMed  Google Scholar 

  11. Atha, D.H., Lormeau, J.C., Petitou, M., Rosenberg, R.D. & Choay, J. Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III. Biochemistry 26, 6454–6461 (1987).

    Article  CAS  PubMed  Google Scholar 

  12. Petitou, M. & van Boeckel, C.A. A synthetic antithrombin III binding pentasaccharide is now a drug! What comes next? Angew. Chem. Int. Edn Engl. 43, 3118–3133 (2004).

    Article  CAS  Google Scholar 

  13. Fries, E. & Blom, A.M. Bikunin–not just a plasma proteinase inhibitor. Int. J. Biochem. Cell Biol. 32, 125–137 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Fries, E. & Kaczmarczyk, A. Inter-alpha-inhibitor, hyaluronan and inflammation. Acta Biochim. Pol. 50, 735–742 (2003).

    CAS  PubMed  Google Scholar 

  15. Zhuo, L., Salustri, A. & Kimata, K. A physiological function of serum proteoglycan bikunin: the chondroitin sulfate moiety plays a central role. Glycoconj. J. 19, 241–247 (2002)10.1023/A:1025331929373.

    Article  CAS  PubMed  Google Scholar 

  16. Michalski, C. et al. Preparation and properties of a therapeutic inter-alpha-trypsin inhibitor concentrate from human plasma. Vox Sang. 67, 329–336 (1994)10.1111/j.1423-0410.1994.tb01269.x.

    Article  CAS  PubMed  Google Scholar 

  17. Enghild, J.J. et al. Chondroitin 4-sulfate covalently cross-links the chains of the human blood protein pre-alpha-inhibitor. J. Biol. Chem. 266, 747–751 (1991).

    CAS  PubMed  Google Scholar 

  18. Morelle, W. et al. Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-alpha-trypsin inhibitor. Eur. J. Biochem. 221, 881–888 (1994).

    Article  CAS  PubMed  Google Scholar 

  19. Zhuo, L., Hascall, V.C. & Kimata, K. Inter-alpha-trypsin inhibitor, a covalent protein-glycosaminoglycan-protein complex. J. Biol. Chem. 279, 38079–38082 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Enghild, J.J. et al. Organization of the inter-alpha-inhibitor heavy chains on the chondroitin sulfate originating from Ser(10) of bikunin: posttranslational modification of IalphaI-derived bikunin. Biochemistry 38, 11804–11813 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. Josic, D. et al. Proteomic characterization of inter-alpha inhibitor proteins from human plasma. Proteomics 6, 2874–2885 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Delaria, K.A. et al. Characterization of placental bikunin, a novel human serine protease inhibitor. J. Biol. Chem. 272, 12209–12214 (1997)10.1074/jbc.272.18.12209.

    Article  CAS  PubMed  Google Scholar 

  23. Chi, L. et al. Structural analysis of bikunin glycosaminoglycan. J. Am. Chem. Soc. 130, 2617–2625 (2008)10.1021/ja0778500.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Conrad, H.E. Beta-elimination for release of O-linked glycosaminoglycans from proteoglycans. Curr. Protoc. Mol. Biol. 17, 15.1–15.3 (2001)10.1002/0471142727.mb1715as31.

    Article  Google Scholar 

  25. Venkataraman, G., Shriver, Z., Raman, R. & Sasisekharan, R. Sequencing complex polysaccharides. Science 286, 537–542 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Laremore, T.N. et al. Domain structure elucidation of human decorin glycosaminoglycans. Biochem. J. 431, 199–205 (2010)10.1042/BJ20100788.

    Article  CAS  PubMed  Google Scholar 

  27. Wolff, J.J., Amster, I.J., Chi, L. & Linhardt, R.J. Electron detachment dissociation of glycosaminoglycan tetrasaccharides. J. Am. Soc. Mass Spectrom. 18, 234–244 (2007)10.1016/j.jasms.2006.09.020.

    Article  CAS  PubMed  Google Scholar 

  28. Wolff, J.J., Laremore, T.N., Aslam, H., Linhardt, R.J. & Amster, I.J. Electron-induced dissociation of glycosaminoglycan tetrasaccharides. J. Am. Soc. Mass Spectrom. 19, 1449–1458 (2008)10.1016/j.jasms.2008.06.024.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wolff, J.J. et al. Negative electron transfer dissociation of glycosaminoglycans. Anal. Chem. 82, 3460–3466 (2010)10.1021/ac100554a.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Turnbull, J.E., Hopwood, J.J. & Gallagher, J.T. A strategy for rapid sequencing of heparan sulfate and heparin saccharides. Proc. Natl. Acad. Sci. USA 96, 2698–2703 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Merry, C.L.R., Lyon, M., Deakin, J.A., Hopwood, J.J. & Gallagher, J.T. Highly sensitive sequencing of the sulfated domains of heparan sulfate. J. Biol. Chem. 274, 18455–18462 (1999).

    Article  CAS  PubMed  Google Scholar 

  32. Turnbull, J.E. & Gallager, J.T. Sequence analysis of heparan sulphate indicates defined location of N-sulphated glucosamine and iduronate 2-sulphate residues proximal to the protein linkage region. Biochem. J. 277, 297–303 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Liu, J., Desai, U.R., Han, X.-J., Toida, T. & Linhardt, R.J. Strategy for the sequence analysis of heparin. Glycobiology 5, 765–774 (1995)10.1093/glycob/5.8.765.

    Article  CAS  PubMed  Google Scholar 

  34. Zaia, J., Li, X.Q., Chan, S.Y. & Costello, C.E. Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides. J. Am. Soc. Mass Spectrom. 14, 1270–1281 (2003)10.1016/S1044-0305(03)00541-5.

    Article  CAS  PubMed  Google Scholar 

  35. McClellan, J.E., Costello, C.E., O'Connor, P.B. & Zaia, J. Influence of charge state on product ion mass spectra and the determination of 4S/6S sulfation sequence of chondroitin sulfate oligosaccharides. Anal. Chem. 74, 3760–3771 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Hitchcock, A.M., Yates, K.E., Costello, C.E. & Zaia, J. Comparative glycomics of connective tissue glycosaminoglycans. Proteomics 8, 1384–1397 (2008)10.1002/pmic.200700787.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Toyoda, H., Kobayashi, S., Sakamoto, S., Toida, T. & Imanari, T. Structural analysis of a low-sulfated chondroitin sulfate chain in human urinary trypsin inhibitor. Biol. Pharm. Bull. 16, 945–947 (1993).

    Article  CAS  PubMed  Google Scholar 

  38. Yamada, S. et al. The sulphated carbohydrate-protein linkage region isolated from chondroitin 4-sulphate chains of inter-alpha-trypsin inhibitor in human plasma. Glycobiology 5, 335–341 (1995).

    Article  CAS  PubMed  Google Scholar 

  39. Laremore, T.N., Ly, M., Solakyildirim, K., Zagorevski, D.V. & Linhardt, R.J. High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis. Anal. Biochem. 401, 236–241 (2010)10.1016/j.ab.2010.03.004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ly, M. et al. Analysis of E. coli K5 capsular polysaccharide heparosan. Anal. Bioanal. Chem. 399, 737–745 (2011)10.1007/s00216-010-3679-7.

    Article  CAS  PubMed  Google Scholar 

  41. Wolff, J.J., Laremore, T.N., Busch, A.M., Linhardt, R.J. & Amster, I.J. Influence of charge state and sodium cationization on the electron detachment dissociation and infrared multiphoton dissociation of glycosaminoglycan oligosaccharides. J. Am. Soc. Mass Spectrom. 19, 790–798 (2008)10.1016/j.jasms.2008.03.010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Laremore, T.N., Leach, F.E. III, Amster, I.J. & Linhardt, R.J. Electrospray ionization Fourier transform mass spectrometric analysis of intact bikunin glycosaminoglycan from normal human plasma. Int. J. Mass. Spectrom. 305, 109–115 (2011)10.1016/j.ijms.2010.09.020.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ly, M. Glycosaminoglycan Sequencing of Proteoglycan. PhD thesis, Rensselaer Polytechnic Institute (2011).

  44. Gu, K., Liu, J., Pervin, A. & Linhardt, R.J. Comparison of the activity of two chondroitin AC lyases on dermatan sulfate. Carbohydr. Res. 244, 369–377 (1993).

    Article  CAS  PubMed  Google Scholar 

  45. Zaia, J. Principles of mass spectrometry of glycosaminoglycans. J. Biomacromol. Mass. Spec. 1, 3–36 (2005).

    Google Scholar 

  46. Gunay, N.S., Tadano-Aritomi, K., Toida, T., Ishizuka, I. & Linhardt, R.J. Evaluation of counterions for electrospray ionization mass spectral analysis of a highly sulfated carbohydrate, sucrose octasulfate. Anal. Chem. 75, 3226–3231 (2003)10.1021/ac034053l.

    Article  CAS  PubMed  Google Scholar 

  47. Capon, C., Mizon, C., Lemoine, J., Rodié-Talbère, P. & Mizon, J. In acute inflammation, the chondroitin-4 sulphate carried by bikunin is not only longer; it is also undersulphated. Biochimie 85, 101–107 (2003)10.1016/S0300-9084(03)00066-X.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  49. Ceroni, A. et al. GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J. Proteome Res. 7, 1650–1659 (2008).

    Article  CAS  PubMed  Google Scholar 

  50. Domon, B. & Costello, C. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 5, 397–409 (1988).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank D. Zagorevski for his expertise in the proteomics core at Rensselaer Polytechnic Institute and the US National Institutes of Health for support (GM38060).

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York, USA

    Mellisa Ly, Tatiana N Laremore & Robert J Linhardt

  2. Department of Chemistry, University of Georgia, Athens, Georgia, USA

    Franklin E Leach III & I Jonathan Amster

  3. Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

    Toshihiko Toida

  4. Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA

    Robert J Linhardt

  5. Department of Biology, Rensselaer Polytechnic Institute, Troy, New York, USA

    Robert J Linhardt

  6. Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, USA

    Robert J Linhardt

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  1. Mellisa Ly
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Contributions

M.L. and F.E.L. III contributed experiments and data interpretation. T.N.L. contributed the fractions for analysis and assisted in writing. T.T. contributed the bikunin and assisted in writing. R.J.L. and I.J.A. contributed experimental planning, result interpretation and wrote the paper.

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Correspondence to Robert J Linhardt.

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Ly, M., Leach, F., Laremore, T. et al. The proteoglycan bikunin has a defined sequence. Nat Chem Biol 7, 827–833 (2011). https://doi.org/10.1038/nchembio.673

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