Abstract
Few solution NMR pulse sequences exist that are explicitly designed to characterize carbohydrates (glycans). This is despite the essential role carbohydrate motifs play in cell–cell communication, microbial pathogenesis, autoimmune disease progression and cancer metastasis, and despite that fact that glycans, often shed to extra-cellular fluids, can be diagnostic of disease. Here we present a suite of two dimensional coherence experiments to measure three different correlations (H3–C2, H3–C1, and C1–C2) on sialic acids, a group of nine-carbon carbohydrates found on eukaryotic cell surfaces that often play a key role in disease processes. The chemical shifts of the H3, C2, and C1 nuclei of sialic acids are sensitive to carbohydrate linkage, linkage conformation, and ionization state of the C1 carboxylate. The experiments reported include rigorous filter elements to enable detection and characterization of isotopically labeled sialic acids with high sensitivity in living cells and crude isolates with minimal interference from unwanted signals arising from the ~1% 13C-natural abundance of cellular metabolites. Application is illustrated with detection of sialic acids on living cells, in unpurified mixtures, and at the terminus of the N-glycan on the 55 kDa immunoglobulin G Fc.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aubin Y, Prestegard JH (1993) Structure and dynamics of sialic-acid at the surface of a magnetically oriented membrane system. Biochemistry 32:3422–3428
Azurmendi HF, Vionnet J, Wrightson L, Trinh LB, Shiloach J, Freedberg DI (2007) Extracellular structure of polysialic acid explored by on cell solution NMR. Proc Natl Acad Sci USA 104:11557–11561
Barb AW, Prestegard JH (2011) NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic. Nat Chem Biol 7:147–153
Barb AW, Brady EK, Prestegard JH (2009) Branch-specific sialylation of IgG-Fc glycans by ST6Gal-I. Biochemistry 48:9705–9707
Bhattacharjee AK, Jennings HJ, Kenny CP, Martin A, Smith IC (1975) Structural determination of the sialic acid polysaccharide antigens of neisseria meningitidis serogroups B and C with carbon 13 nuclear magnetic resonance. J Biol Chem 250:1926–1932
Bodenhausen G, Ruben DJ (1980) Natural abundance N-15 Nmr by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189
Brockhausen I (2006) Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO Rep 7:599–604
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) Nmrpipe—a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293
Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141
Gerken TA, Dearborn DG (1984) Carbon-13 NMR studies of native and modified ovine submaxillary mucin. Biochemistry 23:1485–1497
Liu Y, Prestegard JH (2010) A device for the measurement of residual chemical shift anisotropy and residual dipolar coupling in soluble and membrane-associated proteins. J Biomol NMR 47:249–258
Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2d Nmr-spectra without phase cycling—Application to the study of Hydrogen-Exchange in Proteins. J Magn Reson 85:393–399
Melrose J, Fuller ES, Roughley PJ, Smith MM, Kerr B, Hughes CE, Caterson B, Little CB (2008) Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues. Arthritis Res Ther 10:R79
Morris GA, Freeman R (1979) Enhancement of nuclear magnetic-resonance signals by polarization transfer. J Am Chem Soc 101:760–762
Muramatsu T, Muramatsu H (2004) Carbohydrate antigens expressed on stem cells and early embryonic cells. Glycoconj J 21:41–45
Nacher J, Guirado R, Varea E, Alonso-Llosa G, Rockle I, Hildebrandt H (2010) Divergent impact of the polysialyltransferases ST8SiaII and ST8SiaIV on polysialic acid expression in immature neurons and interneurons of the adult cerebral cortex. Neuroscience 167:825–837
Nasirikenari M, Segal BH, Ostberg JR, Urbasic A, Lau JT (2006) Altered granulopoietic profile and exaggerated acute neutrophilic inflammation in mice with targeted deficiency in the sialyltransferase ST6Gal I. Blood 108:3397–3405
Nelson RW, Bates PA, Rutishauser U (1995) Protein determinants for specific polysialylation of the neural cell adhesion molecule. J Biol Chem 270:17171–17179
Otting G (2008) Prospects for lanthanides in structural biology by NMR. J Biomol NMR 42:1–9
Parekh RB, Dwek RA, Sutton BJ, Fernandes DL, Leung A, Stanworth D, Rademacher TW, Mizuochi T, Taniguchi T, Matsuta K et al (1985) Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316:452–457
Prestegard JH, Bougault CM, Kishore AI (2004) Residual dipolar couplings in structure determination of biomolecules. Chem Rev 104:3519–3540
Reis CA, Osorio H, Silva L, Gomes C, David L (2010) Alterations in glycosylation as biomarkers for cancer detection. J Clin Pathol 63:322–329
Simon ES, Bednarski MD, Whitesides GM (1988) Synthesis of Cmp-Neuac from N-Acetylglucosamine—generation of Ctp from Cmp using adenylate kinase. J Am Chem Soc 110:7159–7163
Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69:531–569
Steenbergen SM, Wrona TJ, Vimr ER (1992) Functional analysis of the sialyltransferase complexes in Escherichia coli K1 and K92. J Bacteriol 174:1099–1108
Takeuchi K, Frueh DP, Hyberts SG, Sun ZY, Wagner G (2010a) High-resolution 3D CANCA NMR experiments for complete mainchain assignments using C(alpha) direct detection. J Am Chem Soc 132:2945–2951
Takeuchi K, Heffron G, Sun ZY, Frueh DP, Wagner G (2010b) Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments. J Biomol NMR 47:271–282
Theocharis AD, Skandalis SS, Tzanakakis GN, and Karamanos NK (2010) Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting. FEBS J 277:3904–3923
Tugarinov V, Kay LE (2003) Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J Am Chem Soc 125:13868–13878
Varki A (2007) Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 446:1023–1029
Varki A (2009) Essentials of glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Vimr ER (1992) Selective synthesis and labeling of the polysialic acid capsule in Escherichia coli K1 strains with mutations in nanA and neuB. J Bacteriol 174:6191–6197
Witsch-Prehm P, Miehlke R, Kresse H (1992) Presence of small proteoglycan fragments in normal and arthritic human cartilage. Arthritis Rheum 35:1042–1052
Acknowledgments
We thank Dr. Eric Vimr (U. Illinois) for the E. coli EV239 cells, and Dr. Yizhou Liu and Dr. Xu Wang for helpful discussions regarding pulse sequence design. This work was financially supported by the grants RO1GM033225 and P41RR005351 from the National Institutes of Health. A.W.B. was supported by a NIH Kirschstein National Research Service Award (F32AR058084). The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Barb, A.W., Freedberg, D.I., Battistel, M.D. et al. NMR detection and characterization of sialylated glycoproteins and cell surface polysaccharides. J Biomol NMR 51, 163 (2011). https://doi.org/10.1007/s10858-011-9550-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s10858-011-9550-0