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Fast Screening of Glycosaminoglycan Disaccharides by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE): Applications to Biologic Samples and Pharmaceutical Formulations

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Glycosaminoglycans

Abstract

Hyaluronan (HA), chondroitin sulfate (CS), and heparan sulfate (HS) are glycosaminoglycans (GAGs) with a great importance in biological processes as they participate in functional cell properties, such as migration, adhesion, and proliferation. A perturbation of the quantity and/or the sulfation of GAGs is often associated with pathological conditions. In this chapter, we present valuable and validated protocols for the analysis of HA-, CS-, and HS-derived disaccharides after derivatization with 2-aminoacridone and by using the fluorophore-assisted carbohydrate electrophoresis (FACE). FACE is a well-known technique and a reliable tool for a fast screening of GAGs, as it is possible to analyze 16 samples at the same time with one electrophoretic apparatus. The protocols for the gel preparation are based on the variations of the acrylamide/bisacrylamide and buffer concentrations. Different approaches for the extraction and purification of the disaccharides of various biologic samples and pharmaceutical preparations are also stressed.

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References

  1. Iozzo RV (2005) Basement membrane proteoglycans: from cellar to ceiling. Nat Rev Mol Cell Biol 6:646–656

    Article  CAS  PubMed  Google Scholar 

  2. Afratis N, Gialeli C, Nikitovic D, Tsegenidis T, Karousou E, Theocharis AD, Pavao MS, Tzanakakis GN, Karamanos NK (2012) Glycosaminoglycans: key players in cancer cell biology and treatment. FEBS J 279:1177–1197

    Article  CAS  PubMed  Google Scholar 

  3. Mizumoto S, Sugahara K (2013) Glycosaminoglycans are functional ligands for receptor for advanced glycation end-products in tumors. FEBS J 280:2462–2470

    Article  CAS  PubMed  Google Scholar 

  4. Karousou E, Stachtea X, Moretto P, Viola M, Vigetti D, D’Angelo ML, Raio L, Ghezzi F, Pallotti F, De Luca G, Karamanos NK, Passi A (2013) New insights into the pathobiology of Down syndrome—hyaluronan synthase-2 overexpression is regulated by collagen VI alpha2 chain. FEBS J 280:2418–2430

    Article  CAS  PubMed  Google Scholar 

  5. Karousou EG, Porta G, De Luca G, Passi A (2004) Analysis of fluorophore-labelled hyaluronan and chondroitin sulfate disaccharides in biological samples. J Pharm Biomed Anal 34:791–795

    Article  CAS  PubMed  Google Scholar 

  6. Karousou EG, Viola M, Vigetti D, Genasetti A, Rizzi M, Clerici M, Bartolini B, De Luca G, Passi A (2008) Analysis of glycosaminoglycans by electrophoretic approach. Curr Pharmaceut Anal 4:78–89

    Article  CAS  Google Scholar 

  7. Karousou EG, Militsopoulou M, Porta G, De Luca G, Hascall VC, Passi A (2004) Polyacrylamide gel electrophoresis of fluorophore-labeled hyaluronan and chondroitin sulfate disaccharides: application to the analysis in cells and tissues. Electrophoresis 25:2919–2925

    Article  CAS  PubMed  Google Scholar 

  8. Viola M, Vigetti D, Karousou E, Bartolini B, Genasetti A, Rizzi M, Clerici M, Pallotti F, De Luca G, Passi A (2008) New electrophoretic and chromatographic techniques for analysis of heparin and heparan sulfate. Electrophoresis 29:3168–3174

    Article  CAS  PubMed  Google Scholar 

  9. Viola M, Karousou EG, Vigetti D, Genasetti A, Pallotti F, Guidetti GF, Tira E, De Luca G, Passi A (2006) Decorin from different bovine tissues: study of glycosaminoglycan chain by PAGEFS. J Pharm Biomed Anal 41:36–42

    Article  CAS  PubMed  Google Scholar 

  10. Sugahara K, Kitagawa H (2002) Heparin and heparan sulfate biosynthesis. IUBMB Life 54:163–175

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Heldin P (2003) Importance of hyaluronan biosynthesis and degradation in cell differentiation and tumor formation. Braz J Med Biol Res 36:967–973

    Article  CAS  PubMed  Google Scholar 

  13. Vigetti D, Viola M, Karousou E, Rizzi M, Moretto P, Genasetti A, Clerici M, Hascall VC, De Luca G, Passi A (2008) Hyaluronan-CD44-ERK1/2 regulate human aortic smooth muscle cell motility during aging. J Biol Chem 283:4448–4458

    Article  CAS  PubMed  Google Scholar 

  14. Karamanos NK, Syrokou A, Vanky P, Nurminen M, Hjerpe A (1994) Determination of 24 variously sulfated galactosaminoglycan- and hyaluronan-derived disaccharides by high-performance liquid chromatography. Anal Biochem 221:189–199

    Article  CAS  PubMed  Google Scholar 

  15. Karamanos NK, Vanky P, Syrokou A, Hjerpe A (1995) Identity of dermatan and chondroitin sequences in dermatan sulfate chains determined by using fragmentation with chondroitinases and ion-pair high-performance liquid chromatography. Anal Biochem 225:220–230

    Article  CAS  PubMed  Google Scholar 

  16. Karamanos NK, Vanky P, Tzanakakis GN, Hjerpe A (1996) High performance capillary electrophoresis method to characterize heparin and heparan sulfate disaccharides. Electrophoresis 17:391–395

    Article  CAS  PubMed  Google Scholar 

  17. Karamanos NK, Vanky P, Tzanakakis GN, Tsegenidis T, Hjerpe A (1997) Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate. J Chromatogr A 765:169–179

    Article  PubMed  Google Scholar 

  18. Karousou EG, Viola M, Genasetti A, Vigetti D, Luca GD, Karamanos NK, Passi A (2005) Application of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides for analysis of hyaluronan and chondroitin sulfate in human and animal tissues and cell cultures. Biomed Chromatogr 19:761–765

    Article  CAS  PubMed  Google Scholar 

  19. Lamari FN, Kuhn R, Karamanos NK (2003) Derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis. J Chromatogr B Analyt Technol Biomed Life Sci 793:15–36

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Nikos Karamanos .

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Karousou, E. et al. (2015). Fast Screening of Glycosaminoglycan Disaccharides by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE): Applications to Biologic Samples and Pharmaceutical Formulations. In: Balagurunathan, K., Nakato, H., Desai, U. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 1229. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1714-3_14

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  • DOI: https://doi.org/10.1007/978-1-4939-1714-3_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1713-6

  • Online ISBN: 978-1-4939-1714-3

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