Abstract
This report describes a novel method for overexpression of 13C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly 13C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man8GlcNAc2 oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, 13C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific 13C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The 13C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides.
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References
Aebi M, Bernasconi R, Clerc S, Molinari M (2010) N-glycan structures: recognition and processing in the ER. Trends Biochem Sci 35:74–82
Amano K, Chiba Y, Kasahara Y, Kato Y, Kaneko MK, Kuno A, Ito H, Kobayashi K, Hirabayashi J, Jigami Y, Narimatsu H (2008) Engineering of mucin-type human glycoproteins in yeast cells. Proc Natl Acad Sci USA 105:3232–3237
Angulo J, Rademacher C, Biet T, Benie AJ, Blume A, Peters H, Palcic M, Parra F, Peters T (2006) NMR analysis of carbohydrate-protein interactions. Methods Enzymol 416:12–30
Arya R, Bhattacharya A, Saini KS (2008) Dictyostelium discoideum—a promising expression system for the production of eukaryotic proteins. FASEB J 22:4055–4066
Berman E, Walters DE, Allerhand A (1981) Structure and dynamic behavior of the oligosaccharide side chain of bovine pancreatic ribonuclease B. Application of carbon 13 nuclear magnetic resonance spectroscopy. J Biol Chem 256:3853–3857
Blanchard V, Gadkari RA, George AVE, Roy S, Gerwig GJ, Leeflang BR, Dighe RR, Boelens R, Kamerling JP (2008) High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains–selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG. Glycoconj J 25:245–257
Bose B, Zhao S, Stenutz R, Cloran F, Bondo PB, Bondo G, Hertz B, Carmichael I, Serianni AS (1998) Three-bond C–O–C–C spin-coupling constants in carbohydrates: development of a Karplus relationship. J Am Chem Soc 120:11158–11173
Chiba Y, Akeboshi H (2009) Glycan engineering and production of ‘humanized’ glycoprotein in yeast cells. Biol Pharm Bull 32:786–795
Chiba Y, Suzuki M, Yoshida S, Yoshida A, Ikenaga H, Takeuchi M, Jigami Y, Ichishima E (1998) Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae. J Biol Chem 273:26298–26304
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
Duker JM, Serianni AS (1993) (13C)-substituted sucrose: 13C–1H and 13C–13C spin coupling constants to assess furanose ring and glycosidic bond conformations in aqueous solution. Carbohydr Res 249:281–303
Fadda E, Woods RJ (2010) Molecular simulations of carbohydrates and protein-carbohydrate interactions: motivation, issues and prospects. Drug Discov Today 15:596–609
Gabius HJ, André S, Jiménez-Barbero J, Romero A, Solís D (2011) From lectin structure to functional glycomics: principles of sugar code. Trends Biochem Sci 36:298–313
González L, Bruix M, Díaz-Mauriño T, Feizi T, Rico M, Solís D, Jiménez-Barbero J (2000) Conformational studies of the Man8 oligosaccharide on native ribonuclease B and on the reduced and denatured protein. Arch Biochem Biophys 383:17–27
Hamilton SR, Gerngross TU (2007) Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol 18:387–392
Herscovics A, Orlean P (1993) Glycoprotein biosynthesis in yeast. FASEB J 7:540–550
Jonsson KHM, Pendrill R, Widmalm G (2011) NMR analysis of conformationally dependent nJC, H and nJC, C in the trisaccharide α-L-Rhap-(1 → 2)[α-L-Rhap-(1 → 3)]-α-L-Rhap-OMe and a site-specifically labeled isotopologue thereof. Magn Reson Chem 49:117–124
Kamiya Y, Yamaguchi Y, Takahashi N, Arata Y, Kasai K, Ihara Y, Matsuo I, Ito Y, Yamamoto K, Kato K (2005) Sugar-binding properties of VIP36, an intracellular animal lectin operating as a cargo receptor. J Biol Chem 280:37178–37182
Kamiya Y, Kamiya D, Urade R, Suzuki T, Kato K (2009) Sophisticated modes of sugar recognition by intracellular lectins involved in quality control of glycoproteins. In: Powell G, McCabe O (eds) Glycobiology research trends. Nova Scirnce Publisher, Inc., NY, pp 27–40
Kamiya Y, Yagi-Utsumi M, Yagi H, Kato K (2011) Structural and molecular basis of carbohydrate-protein interaction systems as potential therapeutic targets. Curr Pharma Des (in press)
Kato K, Kamiya Y (2007) Structural views of glycoprotein-fate determination in cells. Glycobiology 17:1031–1044
Kato K, Sasakawa H, Kamiya Y, Utsumi M, Nakano M, Takahashi N, Yamaguchi Y (2008) 920 MHz ultra-high field NMR approaches to structural glycobiology. Biochim Biophys Acta 1780:619–625
Kato K, Yamaguchi Y, Arata Y (2010) Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system. Prog Nucl Magn Reson Spectrosc 56:346–359
Lederkremer GZ (2009) Glycoprotein folding, quality control and ER-associated degradation. Curr Opin Struct Biol 19:515–523
Lustbader JW, Birken S, Pollak S, Pound A, Chait BT, Mirza UA, Ramnarain S, Canfield RE, Brown JM (1996) Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: an advance toward rapid determination of glycoprotein structures. J Biomol NMR 7:295–304
Matsuo I, Wada M, Manabe S, Yamaguchi Y, Otake K, Kato K, Ito Y (2003) Synthesis of monoglucosylated high-mannose-type dodecasaccharide, a putative ligand for molecular chaperone, calnexin, and calreticulin. J Am Chem Soc 125:3402–3403
Nakanishi-Shindo Y, Nakayama K, Tanaka A, Toda Y, Jigami Y (1993) Structure of the N-linked oligosaccharides that show the complete loss of α-1, 6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae. J Biol Chem 268:26338–26345
Olsson U, Serianni AS, Stenutz R (2008) Conformational analysis of beta-glycosidic linkages in 13C-labeled glucobiosides using inter-residue scalar coupling constants. J Phys Chem B 112:4447–4453
Peat S, Whelan WJ, Edwards TE (1961) Polysaccharides of Baker’s yeast. Part IV. Mannan. J Chem Soc:29–34
Säwén E, Massad T, Landersjö C, Damberg P, Widmalm G (2010) Population distribution of flexible molecules from maximum entropy analysis using different priors as background information: application to the ϕ, ψ-conformational space of the α-(1 → 2)-linked mannose disaccharide present in N- and O-linked glycoproteins. Org Biomol Chem 8:3684–3695
Sharon N (2007) Lectins: carbohydrate-specific reagents and biological recognition molecules. J Biol Chem 282:2753–2764
Skrisovska L, Schubert M, Allain FHA (2010) Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins. J Biomol NMR 46:51–65
Strauss A, Bitsch F, Fendrich G, Graff P, Knecht R, Meyhack B, Jahnke W (2005) Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells. J Biomol NMR 31:343–349
Takahashi N, Kato K (2003) GALAXY (Glycoanalysis by the Three Axes of MS and Chromatography): a web application that assists structural analyses of N-glycans. Trends Glycosci Glycotech 14:235–251
Takamatsu S, Chiba Y, Ishii T, Nakayama K, Yokomatsu-Kubota T, Makino T, Fujibayashi Y, Jigami Y (2004) Monitoring of the tissue distribution of fibroblast growth factor containing a high mannose-type sugar chain produced in mutant yeast. Glycoconj J 20:385–397
Tomiya N, Lee YC, Yoshida T, Wada Y, Awaya J, Kurono M, Takahashi N (1991) Calculated two-dimensional sugar map of pyridylaminated oligosaccharides: elucidation of the jack bean alpha-mannosidase digestion pathway of Man9GlcNAc2. Anal Biochem 193:90–100
Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3:97–130
Vliegenthart JFG (1980) High resolution 1H-NMR spectroscopy of carbohydrate structures. Adv Exp Med Biol 125:77–91
von der Lieth CW, Siebert HC, Kožár T, Burchert M, Frank M, Gilleron M, Kaltner H, Kayser G, Tajkhorshid E, Bovin NV, Vliegenthart JFG, Gabius HJ (1998) Lectin ligands: new insights into their conformations and their dynamic behavior and the discovery of conformer selection by lectins. Acta Anat (Basel) 161:91–109
Walton WJ, Kasprzak AJ, Hare JT, Logan TM (2006) An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells. J Biomol NMR 36:225–233
Weller CT, Lustbader J, Seshadri K, Brown JM, Chadwick CA, Kolthoff CE, Ramnarain S, Pollak S, Canfield R, Homans SW (1996) Structural and conformational analysis of glycan moieties in situ on isotopically 13C, 15N-enriched recombinant human chorionic gonadotropin. Biochemistry 35:8815–8823
Yamaguchi Y (2008) Development and applications of stable-isotope-labeling methods oriented to structural glycobiology. Trends Glycosci Glycotech 20:117–130
Yamaguchi Y, Kato K (2010) Dynamics and interactions of glycoconjugates probed by stable-isotope-assisted NMR spectroscopy. Methods Enzymol 478:305–322
Yamaguchi Y, Nishimura M, Nagano M, Yagi H, Sasakawa H, Uchida K, Shitara K, Kato K (2006) Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy. Biochim Biophys Acta 1760:693–700
Zhang W, Zhao H, Carmichael I, Serianni AS (2009) An NMR investigation of putative interresidue H-bonding in methyl α-cellobioside in solution. Carbohydr Res 344:1582–1587
Acknowledgments
We thank Yukiko Isono (IMS) for her help in preparation of the oligosaccharide and Michiko Nakano (IMS) for her help with NMR measurements. This work was supported, in part, by the Nanotechnology Network Project and Grants in Aid for Scientific Research, (20107004, 21370050, and 22020039) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the CREST project from the Japan Science and Technology Agency.
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Kamiya, Y., Yamamoto, S., Chiba, Y. et al. Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain. J Biomol NMR 50, 397–401 (2011). https://doi.org/10.1007/s10858-011-9525-1
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DOI: https://doi.org/10.1007/s10858-011-9525-1