Skip to main content
SpringerLink
Log in

Identification and Analysis of O-Acetylated Sialoglycoproteins

  • Protocol
  • First Online:
Protein Acetylation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 981))

Abstract

5-N-acetylneuraminic acid, commonly known as sialic acid (Sia), constitutes a family of N- and O-substituted 9-carbon monosaccharides. Frequent modification of O-acetylations at positions C-7, C-8, or C-9 of Sias generates a family of O-acetylated sialic acid (O-AcSia) and plays crucial roles in many cellular events like cell–cell adhesion, proliferation, migration, etc. Therefore, identification and analysis of O-acetylated sialoglycoproteins (O-AcSGPs) are important. In this chapter, we describe several approaches for successful identification of O-AcSGPs. We broadly divide them into two categories, i.e., invasive and noninvasive methods. Several O-AcSias-binding probes are used for this purpose. Detailed methodologies for step-by-step identification using these probes have been discussed. We have also included a few invasive analytical methods for identification and quantitation of O-AcSias. Several indirect methods are also elaborated for such purpose, in which O-acetyl group from sialic acids is initially removed followed by detection of Sias by several approaches. For molecular identification, we have described methods for affinity purification of O-AcSGPs using an O-AcSias-binding lectin as an affinity matrix followed by sequencing using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF) mass spectroscopy (MS). In spite of special attention, loss of O-acetyl groups due to its sensitivity towards alkaline pH and high temperature along with migration of labile O-acetyl groups from C7–C8–C9 during sample preparation is difficult to avoid. Therefore there is always a risk for underestimation of O-AcSias.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Schauer R (2000) Achievements and challenges of sialic acid research. Glycoconj J 17:485–499

    Article  PubMed  CAS  Google Scholar 

  2. Klein A, Roussel P (1998) O-Acetylation of sialic acids. Biochimie 80:49–57

    Article  PubMed  CAS  Google Scholar 

  3. Angata T, Varki A (2002) Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective. Chem Rev 102:439–469

    Article  PubMed  CAS  Google Scholar 

  4. Schauer R (2004) Sialic acids: fascinating sugars in higher animals and man. Zoology 107:49–64

    Article  PubMed  CAS  Google Scholar 

  5. Kamerling JP, Schauer R, Shukla AK, Stoll S, VanHalbeek H, Vliegenthart JF (1987) Migration of O-acetyl groups in N, O-acetylneuraminic acids. Eur J Biochem 162:601–607

    Article  PubMed  CAS  Google Scholar 

  6. Vandamme-Feldhaus V, Schauer R (1998) Characterization of the enzymatic 7-O-acetylation of sialic acids and evidence for enzymatic O-acetyl migration from C-7 to C-9 in bovine submandibular gland. J Biochem (Tokyo) 124:111–121

    Article  CAS  Google Scholar 

  7. Ghosh S, Bandhyopadhyay S, Pal S, Das B, Bhattacharya DK, Mandal C (2005) Increased interferon gamma production by peripheral blood mononuclear cells in response to stimulation of overexpressed disease-specific 9-O-acetylated sialoglycoconjugates in children suffering from acute lymphoblastic leukaemia. Br J Haematol 128:35–41

    Article  PubMed  CAS  Google Scholar 

  8. Ghosh S, Bandyopadhyay S, Mukherjee K, Mallick A, Pal S, Mandal C, Bhattacharya DK, Mandal C (2007) O-Acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL). Glycoconj J 24:17–24

    Article  PubMed  CAS  Google Scholar 

  9. Sen G, Mandal C (1995) A unique specificity of the binding site of Achatinin-H, a sialic acid binding lectin from Achatina fulica. Carbohydr Res 268:115–125

    Article  PubMed  CAS  Google Scholar 

  10. Fragkiadakis GA, Stratakis EK (1997) The lectin from the crustacean Liocarcinus depurator recognizes O-acetyl sialic acids. Comp Biochem Physiol 117:545–552

    CAS  Google Scholar 

  11. Ravindranath MH, Higa HH, Cooper EL, Paulson JC (1985) Purification and characterization of an O-acetyl sialic acid-specific lectin from a marine crab Cancer antennarius. J Biol Chem 260:8850–8856

    PubMed  CAS  Google Scholar 

  12. Ravindranath MH, Morton DL, Irie RF (1989) An epitope common to gangliosides 0-acetyl-GD3 and GD3 recognized by antibodies in melanoma patients after active specific immunotherapy. Cancer Res 49:3891–3897

    PubMed  CAS  Google Scholar 

  13. Denis M, Palatty PD, Bai NR, Suriya SJ (2003) Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii. Eur J Biochem 270:4348–4355

    Article  PubMed  CAS  Google Scholar 

  14. Ahmed H, Gabius HJ (1989) Purification and properties of a Ca2+-independent sialic acid-binding lectin from human placenta with preferential affinity to O-acetylsialic acids. J Biol Chem 264:18673–18678

    PubMed  CAS  Google Scholar 

  15. Van Damme EJM, Bane A, Rouge P, Van Leuven F, Peumans WJ (1996) The NeuAc(a-2,6)Gal/GaINAc-binding lectin from elderberry (Sanbucus nigra) bark, a type-2 ribosome-inactivating pmtein with an unusual specificity and structure. Eur J Biochem 235:128–137

    Article  PubMed  Google Scholar 

  16. Kaku H, Mon Y, Goldstein IJ, Shibuya N (1993) Monomeric, monovalent derivative of Maackio arnurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry. J Biol Chem 268:13237–13241

    PubMed  CAS  Google Scholar 

  17. Mandal C, Basu S (1987) A unique specificity of a sialic acid binding lectin Achatinin-H, from the hemolymph of a Achatina fulica snail. Biochem Biophys Res Commun 148:795–801

    Article  PubMed  CAS  Google Scholar 

  18. Mandal C, Mandal C (1990) Sialic acid ­binding lectins—a review. Cell Mol Life Sci 46:433–441

    Article  PubMed  CAS  Google Scholar 

  19. Chava AK, Chatterjee M, Sundar S, Mandal C (2002) Development of an assay for quantification of linkage-specific O-acetylated sialoglycans on erythrocytes; its application in Indian visceral leishmaniasis. J Immunol Methods 270:1–10

    Article  PubMed  CAS  Google Scholar 

  20. Chava AK, Chatterjee M, Sharma V, Sundar S, Mandal C (2004) Variable degree of alternative complement pathway–mediated hemolysis in Indian visceral leishmaniasis induced by differential expression of 9-O–acetylated sialoglycans. J Infect Dis 189:1257–1264

    Article  PubMed  CAS  Google Scholar 

  21. Pal S, Ghosh S, Bandhyopadhyay S, Mandal C, Bandhyopadhyay S, Bhattacharya DK, Mandal C (2004) Differential expression of 9-O-acetylated sialoglycoconjugates on leukemic blasts: a potential tool for long-term monitoring of children with acute lymphoblastic leukaemia. Int J Cancer 111:270–277

    Article  PubMed  CAS  Google Scholar 

  22. Pal S, Ghosh S, Mandal C, Kohla G, Brossmer R, Isecke R, Merling A, Schauer R, Schwartz-Albiez R, Bhattacharya DK, Mandal C (2004) Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukaemia. Glycobiology 14:859–870

    Article  PubMed  CAS  Google Scholar 

  23. Sinha D, Mandal C, Bhattacharya DK (1999) A novel method for prognostic evaluation of childhood acute lymphoblastic leukemia. Leukemia 13:309–312

    Article  PubMed  CAS  Google Scholar 

  24. Sinha D, Mandal C, Bhattacharya DK (1999) Identification of 9-O acetyl sialoglycoconjugates (9-OAcSGs) as biomarkers in childhood acute lymphoblastic leukemia using a lectin, AchatininH, as a probe. Leukemia 13:119–125

    Article  PubMed  CAS  Google Scholar 

  25. Chowdhury S, Bandyopadhyay S, Mandal C, Chandra S, Mandal C (2008) Flow-cytometric monitoring of disease-associated expression of 9-O-acetylated sialoglycoproteins in combination with known CD antigens, as an index for MRD in children with acute lymphoblastic leukaemia: a two-year longitudinal follow-up study. BMC Cancer 8:40

    Article  PubMed  Google Scholar 

  26. Sinha D, Mandal C, Bhattacharya DK (1999) A colorimetric assay to evaluate the chemotherapeutic response of children with Acute Lymphoblastic Leukemia (ALL) employing achatinin: a 9-O acetylated sialic acid binding lectin. Leukemia Res 23:803–809

    Article  CAS  Google Scholar 

  27. Sinha D, Mandal C, Bhattacharya DK (1999) Development of a simple, blood based lymphoproliferation assay to assess the clinical status of patients with acute lymphoblastic leukemia. Leukemia Res 23:433–439

    Article  CAS  Google Scholar 

  28. Scatchard G (1949) The attraction of protein for small molecules and ions. Ann NY Acad Sci 51:660–672

    Article  CAS  Google Scholar 

  29. Ghoshal A, Mukhopadhyay S, Demine R, Forgber M, Jarmalavicius S, Saha B, Sundar S, Walden P, Mandal C, Mandal C (2009) Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis. Glycoconj J 26:675–689

    Article  PubMed  CAS  Google Scholar 

  30. Harms G, Reuter G, Corfield AP, Schauer R (1996) Binding specificity of influenza C-virus to variably O-acetylated glycoconjugates and its use for histochemical detection of N-acetyl-9-O-acetylneuraminic acid in mammalian tissues. Glycoconj J 13:621–630

    Article  PubMed  CAS  Google Scholar 

  31. Hubl U, Ishida H, Kiso M, Hasegawa A, Schauer R (2000) Studies on the specificity and sensitivity of the influenza C virus binding assay for 9-O-acetylated sialic acids and its application to human melanomas. J Biochem (Tokyo) 127:1021–1031

    Article  CAS  Google Scholar 

  32. Herrler G, Rott R, Klenk HD, Muller HP, Shukla AK, Schauer R (1985) The receptor-destroying enzyme of influenza C virus neuraminate-O-acetylesterase. EMBO J 4:1503–1506

    PubMed  CAS  Google Scholar 

  33. Rogers GN, Herrler G, Paulson JC, Klenk HD (1986) Influenza C virus uses 9-O-acetyl-N-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells. J Biol Chem 261:5947–5951

    PubMed  CAS  Google Scholar 

  34. Vlasak R, Krystal M, Nacht M, Palese P (1987) The influenza C virus glycoprotein (HE) exibits receptor-binding (hemagglutinin and receptor-destoying (esterase)). Virology 160:419–425

    Article  PubMed  CAS  Google Scholar 

  35. Klein A, Krishna M, Varki NM, Varki A (1994) 9-O-Acetylated sialic acids have widespread but selective expression: analysis using a chimeric dual-function probe derived from influenza C hemagglutinin-esterase. Proc Natl Acad Sci U S A 91:7782–7786

    Article  PubMed  CAS  Google Scholar 

  36. Shi WX, Chammas R, Varki A (1996) Regulation of sialic acid 9-O-acetylation during the growth and differentiation of murine erythroleukemia cells. J Biol Chem 271:31517–31525

    Article  PubMed  CAS  Google Scholar 

  37. Siebert HC, von der Lieth CW, Dong X, Reuter G, Schauer R, Gabius HJ, Vliegenthart JF (1996) Molecular dynamics-derived conformation and intramolecular interaction analysis of the N-acetyl-9-O-acetylneuraminic acid-containing ganglioside GD1a and NMR-based analysis of its binding to a human polyclonal immunoglobulin G fraction with selectivity for O-acetylated sialic acids. Glycobiology 6:561–572

    Article  PubMed  CAS  Google Scholar 

  38. Bandyopadhyay S, Bhattacharyya A, Sen AK, Das T, Sa G, Bhattacharya DK, Mandal C (2005) Over expressed IgG2 antibodies against O-acetylated sialoglycoconjugates incapable of proper effector functioning in childhood acute lymphoblastic leukemia (ALL). Int Immunol 17:177–191

    Article  PubMed  CAS  Google Scholar 

  39. Bandyopadhyay S, Chatterjee M, Pal S, Waller RF, Sundar S, McConville MJ, Mandal C (2004) Purification, characterization of O-acetylated sialoglycoconjugates-specific IgM and development of an enzyme-linked immunosorbent assay for diagnosis and follow-up of Indian visceral leishmaniasis patients. Diagn Microbiol Infect Dis 50:15–24

    Article  PubMed  CAS  Google Scholar 

  40. Pal S, Chatterjee M, Bhattacharya DK, Bandhyopadhyay S, Mandal C (2000) Identification and purification of cytolytic antibodies directed against O-acetylated sialic acid in childhood acute lymphoblastic leukemia. Glycobiology 10:539–549

    Article  PubMed  CAS  Google Scholar 

  41. Pal S, Chatterjee M, Bhattacharya DK, Bandhyopadhyay S, Mandal C, Mandal C (2001) O-acetyl sialic acid specific IgM as a diagnostic marker in childhood acute lymphoblastic leukaemia. Glycoconj J 18:529–537

    Article  PubMed  CAS  Google Scholar 

  42. Hara S, Yamaguchi M, Takemori Y, Furuhata K, Ogura H, Nakamura M (1989) Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorimetric highperformance liquid chromatography. Anal Biochem 179:162–166

    Article  PubMed  CAS  Google Scholar 

  43. Mandal C, Srinivasan GV, Chowdhury S, Chandra S, Mandal C, Schauer R, Mandal C (2009) High level of sialate-O-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (ALL): enzyme characterization and correlation with disease status. Glycoconj J 26:57–73

    Article  PubMed  CAS  Google Scholar 

  44. Ghoshal A, Gerwig GJ, Kamerling JP, Mandal C (2010) Sialic acids in different Leishmania sp., its correlation with nitric oxide resistance and host responses. Glycobiology 20:553–566

    Article  PubMed  CAS  Google Scholar 

  45. Varki A (1997) Sialic acids as ligands in recognition phenomena. FASEB J 11:248–255

    PubMed  CAS  Google Scholar 

  46. Crocker PR, Varki A (2001) Siglecs, sialic acids and innate immunity. Trends Immunol 22:337–342

    Article  PubMed  CAS  Google Scholar 

  47. Chatterjee M, Chava AK, Kohla G, Pal S, Merling A, Hinderlich S, Unger U, Trasser P, Gerwig GJ, Kamerling JP, Vlasak R, Crocker PR, Schauer R, Schwartz-Albiez R, Mandal C (2003) Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes. Glycobiology 13:351–361

    Article  PubMed  CAS  Google Scholar 

  48. Brinkman-Van der Linden ECM, Varki A (2000) New Aspects of Siglec Binding Specificities, Including the Significance of Fucosylation and of the Sialyl-Tn Epitope. J Biol Chem 275:8625–8632

    Article  PubMed  CAS  Google Scholar 

  49. Khatua B, Ghoshal A, Bhattacharya K, Mandal C, Saha B, Crocker PR, Mandal C (2010) Sialic acids acquired by Pseudomonas aeruginosa are involved in reduced complement deposition and siglec mediated host-cell ­recognition. FEBS Lett 584:555–561

    Article  PubMed  CAS  Google Scholar 

  50. Bandyopadhyay S, Chatterjee M, Sundar S, Mandal C (2004) Identification of 9-O-acetylated sialoglycans on peripheral blood mononuclear cells in Indian visceral leishmaniasis. Glycoconj J 20:531–536

    Article  PubMed  CAS  Google Scholar 

  51. Ghoshal A, Mukhopadhyay S, Saha B, Mandal C (2009) 9-O-Acetylated sialoglycoproteins: Important immunomodulators in Indian visceral leishmaniasis. Clin Vaccine Immunol 18:889–898

    Article  Google Scholar 

  52. Haverkamp J, Schauer R, Wember M, Kamerling JP, Vliegenthart JF (1975) Synthesis of 9-O-acetyl- and 4,9-di-O-acetyl derivatives of the methyl ester of N-acetyl-beta-D-neuraminic acid methylglycoside. Their use as models in periodate oxidation studies. Hoppe Seylers Z Physiol Chem 356:1575–1583

    Article  PubMed  CAS  Google Scholar 

  53. Veh RW, Meessen D, Kuntz D, May B (1982) A new method for histochemical demonstration of side chain substituted sialic acids. In: Malt RA, Williamson RCN (eds) Colonic carcinogenesis. MTP, Lancaster, pp 355–365

    Google Scholar 

  54. Jass JR, Sugihara K, Love SB (1988) Basis of sialic acid heterogeneity in ulcerative colitis. J Clin Pathol 41:388–392

    Article  PubMed  CAS  Google Scholar 

  55. Campbell F, Puller CE, Williams GT, Williams ED (1994) Human colonic stem cell mutation frequency with and without irradiation. J Pathol 174:175–182

    Article  PubMed  CAS  Google Scholar 

  56. Reuter G, Schauer R (1984) Determination of sialic acids. Methods Enzymol 230:168–199

    Article  Google Scholar 

  57. Blum AS, Barnstable CJ (1987) O-acetylation of a cell-surface carbohydrate creates discrete molecular patterns during neural development. Proc Natl Acad Sci U S A 84:8716–8720

    Article  PubMed  CAS  Google Scholar 

  58. Kniep B, Peter-Katalinic J, Flegel W, Northoff H, Rieber EP (1992) CDw 60 antibodies bind to acetylated forms of ganglioside GD3. Biochem Biophys Res Commun 187:1343–1349

    Article  PubMed  CAS  Google Scholar 

  59. Kniep B, Claus C, Peter-Katalinic J, Monner DA, Dippold W, Nimtz M (1995) 7-O-acetyl-GD3 in human T-lymphocytes is detected by a specific T-cell-activating monoclonal antibody. J Biol Chem 270:30173–30180

    Article  PubMed  CAS  Google Scholar 

  60. Cerato E, Birkle S, Portoukalian J, Mezazigh A, Chatal JF, Aubry J (1997) Variable region gene segments of nine monoclonal antibodies specific to disialogangliosides (GD2, GD3) and their O-acetylated derivatives. Hybridoma 16:307–316

    Article  PubMed  CAS  Google Scholar 

  61. Murphy WH, Gottschalk A (1961) Studies on mucoproteins. VII. The linkage of the prosthetic group to aspartic and glutamic acid residues in bovine submaxillary gland mucoprotein. Biochim Biophys Acta 52:349–360

    Article  PubMed  CAS  Google Scholar 

  62. Bertolini M, Pigman W (1970) The existence of oligosaccharides in bovine and ovine submaxillary mucins. Carbohydr Res 14:53–63

    Article  CAS  Google Scholar 

  63. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

Download references

Acknowledgement

This work received financial support from the Council of Scientific and Industrial Research (CSIR, TLP-006, IAP-001, NWP-004, HCP-004), Department of Biotechnology (GAP-235), Indian Council of Medical Research (GAP-266), New Delhi, India. CM acknowledges support from Sir J.C. Bose National Fellowship, Department of Science and Technology, Govt. of India.

Author information

Authors and Affiliations

  1. Cancer and Cell Biology Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology, Kolkata, India

    Chandan Mandal & Chitra Mandal

Authors
  1. Chandan Mandal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chitra Mandal
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Adolf-Butenandt-Institut, Department of Molecular Biology, Ludwig-Maximilians-Universität München, Schillerstr. 44, München, 80336, Germany

    Sandra B. Hake

  2. , Dep. of Cell & Developmental Biology, Biocenter University of Würzburg, Am Hubland, Würzburg, 97074, Germany

    Christian J. Janzen

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Mandal, C., Mandal, C. (2013). Identification and Analysis of O-Acetylated Sialoglycoproteins. In: Hake, S., Janzen, C. (eds) Protein Acetylation. Methods in Molecular Biology, vol 981. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-305-3_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-305-3_6

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-304-6

  • Online ISBN: 978-1-62703-305-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation