Glycobiology
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  • Glycobiology

    New England Biolabs (NEB) offers a selection of endoglycosidases, exoglycosidases, and heparinases for glycobiology research. Many of these reagents are recombinant, and all undergo several quality control assays, enabling us to provide products with lower unit cost, high purity and reduced lot-to-lot variation. All of our glycosidases are tested for contaminants. Since p-nitrophenyl-glycosides are not hydrolyzed by some exoglycosidases, we use only fluorescently-labeled oligosaccharides to screen for contaminating glycosidases.

    1. Overview of Glycobiology

      Learn about the core sequences and common modifications of N-linked and O-linked glycans in this video. Analysis of these glycans and/or peptide portions of the glycoprotein can be accomplished with the use of deglycosylation enzymes, which are explained in detail. Unlike other chemical deglycosylation methods, enzymatic treatment is much gentler and can provide complete sugar removal with no protein degradation.

    2. Identification and Characterization of Protein Glycosylation

      Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries both N-glycans and O-glycans in this video. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.

    3. Elucidating the Complexity of Heparin Oligosaccharide Analysis

      Learn more about the structural elucidation of complex and diverse heparin oligosaccharides using Bacteroides Heparinase I, II and III in combination with downstream mass spec analysis.

    Glycobiology includes these subcategories:

    Endoglycosidases
    Exoglycosidases
    Heparinases
    Heparin Hexasaccharide Mass Spectrometry Standards

      Publications related to Glycobiology:

    1. Stech M, Quast RB, Sachse R, Schulze C, Wüstenhagen DA, Kubick S (2014)A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells PLoS One 9(5), e96635. PubMedID: 24804975, DOI: 10.1371/journal.pone.0096635
    2. Kwon HM, Lee KH, Han BW, Han MR, Kim DH, Kim DE (2014)An RNA aptamer that specifically binds to the glycosylated hemagglutinin of avian influenza virus and suppresses viral infection in cells PLoS One 9(5), e97574. PubMedID: 24835440, DOI: 10.1371/journal.pone.0097574
    3. Haller G, Li P, Esch C, Hsu S, Goate AM, Steinbach JH (2014)Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior PLoS One 9(5), e96753. PubMedID: 24804708, DOI: 10.1371/journal.pone.0096753
    4. Wright CR, Brown EL, Della-Gatta PA, Ward AC, Lynch GS, Russell AP (2014)G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle Front Physiol 5, 170. PubMedID: 24822049, DOI: 10.3389/fphys.2014.00170
    5. Zhao H, Blazanovic K, Choi Y, Bailey-Kellogg C, Griswold KE (2014)Gene and protein sequence optimization for high-level production of fully active and aglycosylated lysostaphin in Pichia pastoris Appl Environ Microbiol 80(9), 2746-53. PubMedID: 24561590, DOI: 10.1128/AEM.03914-13
    6. Wicht O, Burkard C, de Haan CA, van Kuppeveld FJ, Rottier PJ, Bosch BJ (2014)Identification and Characterization of a Proteolytically Primed Form of the Murine Coronavirus Spike Proteins after Fusion with the Target Cell J Virol 88(9), 4943-52. PubMedID: 24554652, DOI: 10.1128/JVI.03451-13
    7. Rosenbaum EE, Vasiljevic E, Brehm KS, Colley NJ (2014)Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster PLoS Genet 10(5), e1004349. PubMedID: 24785692, DOI: 10.1371/journal.pgen.1004349
    8. Rosenbaek LL, Kortenoeven ML, Aroankins TS, Fenton RA (2014)Phosphorylation decreases ubiquitylation of the thiazide-sensitive cotransporter NCC and subsequent clathrin-mediated endocytosis J Biol Chem 289(19), 13347-61. PubMedID: 24668812, DOI: 10.1074/jbc.M113.543710
    9. Botto L, Cunati D, Coco S, Sesana S, Bulbarelli A, Biasini E, Colombo L, Negro A, Chiesa R, Masserini M, Palestini P (2014)Role of lipid rafts and GM1 in the segregation and processing of prion protein PLoS One 9(5), e98344. PubMedID: 24859148, DOI: 10.1371/journal.pone.0098344
    10. Möykkynen T, Coleman SK, Semenov A, Keinänen K (2014)The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization J Biol Chem 289(19), 13197-205. PubMedID: 24652293, DOI: 10.1074/jbc.M113.526301
    11. Itahana Y, Han R, Barbier S, Lei Z, Rozen S, Itahana K (2014)The uric acid transporter SLC2A9 is a direct target gene of the tumor suppressor p53 contributing to antioxidant defense Oncogene PubMedID: 24858040, DOI: 10.1038/onc.2014.119
    12. Arakel EC, Brandenburg S, Uchida K, Zhang H, Lin YW, Kohl T, Schrul B, Sulkin MS, Efimov IR, Nichols CG, Lehnart SE, Schwappach B (2014)Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes J Cell Sci 127(Pt 9), 2106-19. PubMedID: 24569881, DOI: 10.1242/jcs.141440
    13. Magnelli P, Bielik A, Guthrie E.  (2012)Identification and characterization of protein glycosylation using specific endo- and exoglycosidases Methods Mol Biol. 801, 189-211. PubMedID: 21987255
    14. Magnelli PE, Bielik AM, Guthrie EP. (2011)Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases J Vis Exp. 58, pii: 3749. PubMedID: 21987255, DOI: 10.3791/3749
    15. Rasmussen, T.N., Plenge, P., Bay, T., Egebjerg, J., Gether, U. (2009)A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation Neuropharmacology 57, 287-94. PubMedID: 19500602
    16. Gong, B., Cukan, M., Fisher, R., Li, H., Stadheim, T.A., Gerngross, T. (2009)Characterization of N-linked glycosylation on recombinant glycoproteins produced in Pichia pastoris using ESI-MS and MALDI-TOF Methods Mol. Biol. 534, 213-23.
    17. Gefter, J.V., Shaufl, A.L., Fink, M.P., Delude, R.L. (2009)Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines Cell Tissue Res. 337, 79-89. PubMedID: 19415334
    18. Lee, K.J., Jung, J.H., Lee, J.M., So, Y., Kwon, O., Callewaert, N., Kang, H.A., Ko, K., Oh, D.B. (2009)High-throughput quantitative analysis of plant N-glycan using a DNA sequencer Biochem. Biophys. Res. Commun. 380, 223-9. PubMedID: 19167352
    19. Lee, K.J., Jung, J.H., Lee, J.M,, So, Y., Kwon, O., Callewaert, N., Kang, H.A., Ko, K., Oh, D.B. (2009)High-throughput quantitative analysis of plant N-glycan using a DNA sequencer Biochem. Biophys. Res. Commun.  380, 223-9. PubMedID: 19167352
    20. Piro, J.R., Harris, B.T., Nishina, K., Soto, C., Morales, R., Rees, J.R., Supattapone, S. (2009)Prion protein glycosylation is not required for strain-specific neurotropism  J Virol.  83, 5321-8.
    21. Boeggeman, E., Ramakrishnan, B., Pasek, M., Manzoni, M., Puri, A., Loomis, K.H., Waybright, T.J., Qasba, P.K. (2009)Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection Bioconjug. Chem. 20, 1228-36. PubMedID: 19425533
    22. Velho, A.M., Jarvis, S.M. (2009)Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting Exp. Cell Res. 315, 2312-21. PubMedID: 19379732
    23. Macnaughtan, M.A., Tian, F., Liu, S., Meng, L., Park, S., Azadi, P., Moremen, K.W., Prestegard, J.H. (2008)13C-sialic acid labeling of glycans on glycoproteins using ST6Gal-I J. Am. Chem Soc. 130, 11864-5. PubMedID: 18700760
    24. Banala J. et al. (2008)Caged substrates for protein labeling and immobilization Chembiochem 4, 38-41. PubMedID: 18033718
    25. Baudino L, Shinohara Y, Nimmerjahn F, Furukawa J, Nakata M, Martínez-Soria E, Petry F, Ravetch JV, Nishimura S, Izui S. (2008)Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions J Immunol. 181(9), 6664-9. PubMedID: 18941257
    26. Oh, D.B., Park, J.S., Kim, M.W., Cheon, S.A., Kim, E.J., Moon, H.Y., Kwon, O., Rhee, S.K., Kang, H.A. (2008)Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly Biotechnol. J. 3, 659-68. PubMedID: 18320567
    27. Zhuo, Y., Chammas, R., Bellis, S.L. (2008)Sialylation of beta1 integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis J. Biol. Chem. 283, 22177-85. PubMedID: 18676377
    28. Fujita, N., Tamura, A., Higashidani, A., Tonozuka, T., Freeze, H.H., Nishikawa, A. (2008)The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells FEBS J.  275, 788-98. PubMedID: 18215164
    29. Wagner-Rousset, E., Bednarczyk, A., Bussat, M.C., Colas, O., Corvaïa, N., Schaeffer, C., Van Dorsselaer, A., Beck, A. (2008)The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 872, 23-37. PubMedID: 18672411
    30. Graham, D.R., Mitsak, M.J., Elliott, S.T., Chen, D., Whelan, S.A., Hart, G.W., Van Eyk, J.E. (2008)Two-dimensional gel-based approaches for the assessment of N-Linked and O-GlcNAc glycosylation in human and simian immunodeficiency viruses Proteomics 8, 4919-30. PubMedID: 19072736
    31. Park, Y., Zhang, Z., Laremore, T.N., Li, B., Sim, J.S., Im, A.R., Ahn, M.Y., Kim, Y.S., Linhardt, R.J., Glycoconj, J. (2008)Variation of acharan sulfate and monosaccharide composition and analysis of neutral N-glycans in African giant snail (Achatina fulica) Glycoconjugate Journal 25, 863-77. PubMedID: 18670878
    32. Aoki, K., Perlman, M., Lim, J.M., Cantu, R., Wells, L., Tiemeyer, M. (2007)Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo J. Biol. Chem. 282, 9127-42. PubMedID: 17264077
    33. Song, Y., Choi, M.H., Park, J.N., Kim, M.W., Kim, E.J., Kang, H.A., Kim, J.Y. (2007)Engineering of the yeast Yarrowia lipolytica for the production of glycoproteins lacking the outer-chain mannose residues of N-glycans Appl. Environ. Microbiol.  73, 4446-54. PubMedID: 17513593
    34. Teuton, J.R., Brandt, C.R. (2007)Sialic acid on herpes simplex virus type 1 envelope glycoproteins is required for efficient infection of cells  J. Virol. 81, 3731-9. PubMedID: 17229687
    35. Hagen, B.M., Sanders, K.M. (2006)Deglycosylation of the beta1-subunit of the BK channel changes its biophysical properties Am. J. Physiol.Cell. Physiol.  1, 750-6. PubMedID: 16738006
    36. O’Reilly, M.K., Zhang, G., Imperiali, B. (2006)In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis Biochemistry 8, 9593-603. PubMedID: 16878994
    37. Sielaff I. et al. (2006)Protein function microarrays based on self-immobilizing and self-labeling fusion proteins ChemBioChem. 7, 194-202. PubMedID: 16342318
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    39. Lüftenegger, D., Picard-Maureau, M., Stanke, N., Rethwilm, A., Lindemann, D. (2005)Analysis and function of prototype foamy virus envelope N glycosylation J. Virol.  79, 7664-72. PubMedID: 15919919
    40. Cipollo, J.F., Awad, A.M., Costello, C.E., Hirschberg, C.B. (2005)N-Glycans of Caenorhabditis elegans are specific to developmental stages J. Biol. Chem. 280, 26063-72. PubMedID: 15899899
    41. Srikrishna, G., Brive, L., Freeze, H.H. (2005)Novel carboxylated N-glycans contain oligosaccharide-linked glutamic acid Biochem. Biophys. Res. Commun. 332, 1020-7. PubMedID: 15922305
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    43. Kalidas, C., Joshi, L., Batt, C. (2001)Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris Protein Eng. 14, 201-7. PubMedID: 11342718
    44. Wong-Madden, S.T., Landry, D., and Guthrie, E.P. (1997)Discovery and Uses of Novel Glycosidases Techniques in Glycobiology 401-408.