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Emerging methods for the production of homogeneous human glycoproteins

Nature Chemical Biology volume 5, pages 206–215 (2009)Cite this article

Abstract

Most circulating human proteins exist as heterogeneously glycosylated variants (glycoforms) of an otherwise homogeneous polypeptide. Though glycan heterogeneity is most likely important to glycoprotein function, the preparation of homogeneous glycoforms is important both for the study of the consequences of glycosylation and for therapeutic purposes. This review details selected approaches to the production of homogeneous human N- and O-linked glycoproteins with human-type glycans. Particular emphasis is placed on recent developments in the engineering of glycosylation pathways within yeast and bacteria for in vivo production, and on the in vitro remodeling of glycoproteins by enzymatic means. The future of this field is very exciting.

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Figure 1: Mammalian glycoproteins.
Figure 2: N-glycan synthesis.

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References

  1. Apweiler, R., Hermjakob, H. & Sharon, N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta 1473, 4–8 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Varki, A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130 (1993).

    Article  CAS  PubMed  Google Scholar 

  3. Gabius, H.J. et al. Chemical biology of the sugar code. ChemBioChem 5, 740–764 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Solá, R.J., Rodríguez-Martinez, J.A. & Griebenow, K. Modulation of protein biophysical properties by chemical glycosylation: biochemical insights and biomedical implications. Cell. Mol. Life Sci. 64, 2133–2152 (2007).

    Article  PubMed  CAS  Google Scholar 

  5. Mitra, N., Sinha, S., Ramya, T.N.C. & Surolia, A. N-Linked oligosaccharides as outfitters for glycoprotein folding, form and function. Trends Biochem. Sci. 31, 156–163 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Spiro, R.G. Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12, 43R–56R (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Rudd, P.M. & Dwek, R.A. Glycosylation: heterogeneity and the 3D structure of proteins. Crit. Rev. Biochem. Mol. Biol. 32, 1–100 (1997).

    Article  CAS  PubMed  Google Scholar 

  8. Hossler, P., Mulukutla, B.C. & Hu, W.-S. Systems analysis of N-glycan processing in mammalian cells. PLoS ONE 2, e713 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Julenius, K., Molgaard, A., Gupta, R. & Brunak, S. Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 15, 153–164 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Rudd, P.M. et al. The glycosylation of the complement regulatory protein, human erythrocyte CD59. J. Biol. Chem. 272, 7229–7244 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. Rudd, P.M. et al. Glycoforms modify the dynamic stability and functional activity of an enzyme. Biochemistry 33, 17–22 (1994).

    Article  CAS  PubMed  Google Scholar 

  12. Sethuraman, N. & Stadheim, T.A. Challenges in therapeutic glycoprotein production. Curr. Opin. Biotechnol. 17, 341–346 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Brooks, S.A. Appropriate glycosylation of recombinant proteins for human use - implications of choice of expression system. Mol. Biotechnol. 28, 241–255 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Hamilton, S.R. & Gerngross, T.U. Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr. Opin. Biotechnol. 18, 387–392 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Chiba, Y. & Jigami, Y. Production of humanized glycoproteins in bacteria and yeasts. Curr. Opin. Chem. Biol. 11, 670–676 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Bennett, C.S. & Wong, C.H. Chemoenzymatic approaches to glycoprotein synthesis. Chem. Soc. Rev. 36, 1227–1238 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Brik, A., Ficht, S. & Wong, C.H. Strategies for the preparation of homogenous glycoproteins. Curr. Opin. Chem. Biol. 10, 638–644 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Davis, B.G. Synthesis of glycoproteins. Chem. Rev. 102, 579–601 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Grogan, M.J., Pratt, M.R., Marcaurelle, L.A. & Bertozzi, C.R. Homogeneous glycopeptides and glycoproteins for biological investigation. Annu. Rev. Biochem. 71, 593–634 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Guo, Z. & Shao, N. Glycopeptide and glycoprotein synthesis involving unprotected carbohydrate building blocks. Med. Res. Rev. 25, 655–678 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Wang, L.X. Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation. Carbohydr. Res. 343, 1509–1522 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Czlapinski, J.L. & Bertozzi, C.R. Synthetic glycobiology: exploits in the Golgi compartment. Curr. Opin. Chem. Biol. 10, 645–651 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Jigami, Y. Yeast glycobiology and its application. Biosci. Biotechnol. Biochem. 72, 637–648 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Callewaert, N., Vervecken, W., Geysens, S. & Contreras, R. N-Glycan engineering in yeasts and fungi: progress toward human-like glycosylation. in Handbook of Carbohydrate Bioengineering (ed. Yarema, K.J.) 431–477 (Taylor & Francis Group, New York, 2005).

    Chapter  Google Scholar 

  25. Grinna, L.S. & Tschopp, J.F. Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast 5, 107–115 (1989).

    Article  CAS  PubMed  Google Scholar 

  26. Kornfeld, R. & Kornfeld, S. Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631–664 (1985).

    Article  CAS  PubMed  Google Scholar 

  27. Kukuruzinska, M.A., Bergh, M.L.E. & Jackson, B.J. Protein glycosylation in Yeast. Annu. Rev. Biochem. 56, 915–944 (1987).

    Article  CAS  PubMed  Google Scholar 

  28. Gemmill, T.R. & Trimble, R.B. Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta 1426, 227–237 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Nakayama, K. et al. Och1 encodes a novel membrane-bound mannosyltransferase - outer chain elongation of asparagine-linked oligosaccharides. EMBO J. 11, 2511–2519 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chiba, Y. et al. Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae. J. Biol. Chem. 273, 26298–26304 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Choi, B.-K. et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. USA 100, 5022–5027 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Nakanishi-Shindo, Y. et al. 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 (1993).

    Article  CAS  PubMed  Google Scholar 

  33. Hamilton, S.R. et al. Production of complex human glycoproteins in yeast. Science 301, 1244–1246 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Aebi, M., Gassenhuber, J., Domdey, H. & Heesen, S.T. Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae. Glycobiology 6, 439–444 (1996).

    Article  CAS  PubMed  Google Scholar 

  35. Davidson, R.C. et al. Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of Pichia pastoris. Glycobiology 14, 399–407 (2004).

    Article  CAS  PubMed  Google Scholar 

  36. Bobrowicz, P. et al. Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology 14, 757–766 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Byrne, B., Donohoe, G.G. & O'Kennedy, R. Sialic acids: carbohydrate moieties that influence the biological and physical properties of biopharmaceutical proteins and living cells. Drug Discov. Today 12, 319–326 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Hamilton, S.R. et al. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313, 1441–1443 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Rothman, R.J., Perussia, B., Herlyn, D. & Warren, L. Antibody-dependent cytotoxicity mediated by natural killer cells is enhanced by castanospermine-induced alterations of IgG glycosylation. Mol. Immunol. 26, 1113–1123 (1989).

    Article  CAS  PubMed  Google Scholar 

  40. Arnold, J.N. et al. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21–50 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Li, H. et al. Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat. Biotechnol. 24, 210–215 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Choi, B.K. et al. Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response. Glycoconj. J. 25, 581–593 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. van Berkel, P.H. et al. Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants. Biochem. J. 319, 117–122 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ghosh, P., Dahms, N.M. & Kornfeld, S. Mannose 6-phosphate receptors: new twists in the tale. Nat. Rev. Mol. Cell Biol. 4, 202–213 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Chiba, Y. et al. Production in yeast of α-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease. Glycobiology 12, 821–828 (2002).

    Article  CAS  PubMed  Google Scholar 

  46. Akeboshi, H. et al. Production of recombinant β-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta. Appl. Environ. Microbiol. 73, 4805–4812 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chigira, Y., Oka, T., Okajima, T. & Jigami, Y. Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains. Glycobiology 18, 303–314 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. van den Steen, P., Rudd, P.M., Dwek, R.A. & Opdenakker, G. Concepts and principles of O-linked glycosylation. Crit. Rev. Biochem. Mol. Biol. 33, 151–208 (1998).

    Article  CAS  PubMed  Google Scholar 

  49. Orchard, M.G. et al. Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1). Bioorg. Med. Chem. Lett. 14, 3975–3978 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Kuroda, K. et al. Efficient antibody production upon suppression of O-mannosylation in the yeast Ogataea minuta. Appl. Environ. Microbiol. 74, 446–453 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Gentzsch, M. & Tanner, W. The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital. EMBO J. 15, 5752–5759 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Amano, K. et al. Engineering of mucin-type human glycoproteins in yeast cells. Proc. Natl. Acad. Sci. USA 105, 3232–3237 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Abu-Qarn, M., Eichler, J. & Sharon, N. Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea. Curr. Opin. Struct. Biol. 18, 544–550 (2008).

    Article  CAS  PubMed  Google Scholar 

  54. Weerapana, E. & Imperiali, B. Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology 16, 91R–101R (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Aebi, M. N-linked protein glycosylation: a comparison of the bacterial and eukaryotic pathways. in Comprehensive Glycoscience Vol. 3 (ed. Kamerling, J.P.) 1–9 (Elsevier, San Francisco, 2007).

    Google Scholar 

  56. Szymanski, C.M., Logan, S.M., Linton, D. & Wren, B.W. Campylobacter - a tale of two protein glycosylation systems. Trends Microbiol. 11, 233–238 (2003).

    Article  CAS  PubMed  Google Scholar 

  57. Young, N.M. et al. Structure of the N-linked glycan present on multiple glycoproteins in the gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277, 42530–42539 (2002).

    Article  CAS  PubMed  Google Scholar 

  58. Wacker, M. et al. N-Linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790–1793 (2002).

    Article  CAS  PubMed  Google Scholar 

  59. Wacker, M. et al. Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems. Proc. Natl. Acad. Sci. USA 103, 7088–7093 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Feldman, M.F. et al. Engineering N-linked protein glycosylation with diverse O-antigen lipopolysaccharide structures in Escherichia coli. Proc. Natl. Acad. Sci. USA 102, 3016–3021 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kowarik, M. et al. N-Linked glycosylation of folded proteins by the bacterial oligosaccharyltransferase. Science 314, 1148–1150 (2006).

    Article  CAS  PubMed  Google Scholar 

  62. Faridmoayer, A. et al. Functional characterization of bacterial oligosaccharyltransferases involved in O-linked protein glycosylation. J. Bacteriol. 189, 8088–8098 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Faridmoayer, A. et al. Extreme substrate promiscuity of the Neisseria oligosaccharyltransferase involved in protein O-glycosylation. J. Biol. Chem. 283, 34596–34604 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Ajisaka, K., Miyasato, M. & Ishii-Karakasa, I. Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo α-N-acetylgalactosaminidase from Streptomyces sp. Biosci. Biotechnol. Biochem. 65, 1240–1243 (2001).

    Article  CAS  PubMed  Google Scholar 

  65. Wang, L. & Schultz, P.G. Expanding the genetic code. Angew. Chem. Int. Ed. 44, 34–66 (2005).

    Article  CAS  Google Scholar 

  66. Arslan, T., Mamaev, S.V., Mamaeva, N.V. & Hecht, S.M. Structurally modified firefly luciferase, effects of amino acid substitution at position 286. J. Am. Chem. Soc. 119, 10877–10887 (1997).

    Article  CAS  Google Scholar 

  67. Mamaev, S.V., Laikhter, A.L., Arslan, T. & Hecht, S.M. Firefly luciferase: alteration of the color of emitted light resulting from substitutions at position 286. J. Am. Chem. Soc. 118, 7243–7244 (1996).

    Article  CAS  Google Scholar 

  68. Fahmi, N.E. et al. Site-specific incorporation of glycosylated serine and tyrosine derivatives into proteins. J. Am. Chem. Soc. 129, 3586–3597 (2007).

    Article  CAS  PubMed  Google Scholar 

  69. Xu, R. et al. Site-specific incorporation of the mucin-type N-acetylgalactosamine-α-O-threonine into protein in Escherichia coli. J. Am. Chem. Soc. 126, 15654–15655 (2004).

    Article  CAS  PubMed  Google Scholar 

  70. Zhang, Z. et al. A new strategy for the synthesis of glycoproteins. Science 303, 371–373 (2004).

    Article  CAS  PubMed  Google Scholar 

  71. Xie, J. & Schultz, P.G. A chemical toolkit for proteins - an expanded genetic code. Nat. Rev. Mol. Cell Biol. 7, 775–782 (2006).

    Article  CAS  PubMed  Google Scholar 

  72. Tarp, M.A. & Clausen, H. Mucin-type O-glycosylation and its potential use in drug and vaccine development. Biochim. Biophys. Acta 1780, 546–563 (2008).

    Article  CAS  PubMed  Google Scholar 

  73. Hang, H.C. & Bertozzi, C.R. The chemistry and biology of mucin-type O-linked glycosylation. Bioorg. Med. Chem. 13, 5021–5034 (2005).

    Article  CAS  PubMed  Google Scholar 

  74. Ten Hagen, K.G., Fritz, T.A. & Tabak, L.A. All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology 13, 1R–16R (2003).

    Article  CAS  PubMed  Google Scholar 

  75. Raman, J. et al. The catalytic and lectin domains of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site Selection. J. Biol. Chem. 283, 22942–22951 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Wandall, H.H. et al. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation. Glycobiology 17, 374–387 (2007).

    Article  CAS  PubMed  Google Scholar 

  77. Pratt, M.R. et al. Deconvoluting the functions of polypeptide N-α-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling. Chem. Biol. 11, 1009–1016 (2004).

    Article  CAS  PubMed  Google Scholar 

  78. Gerken, T.A., Raman, J., Fritz, T.A. & Jamison, O. Identification of common and unique peptide substrate preferences for the UDP-GalNAc: polypeptide α-N-acetylgalactosaminyltransferases T1 and T2 derived from oriented random peptide substrates. J. Biol. Chem. 281, 32403–32416 (2006).

    Article  CAS  PubMed  Google Scholar 

  79. DeFrees, S. et al. GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli. Glycobiology 16, 833–843 (2006).

    Article  CAS  PubMed  Google Scholar 

  80. Yamamoto, N. et al. Chemical synthesis of a glycoprotein having an intact human complex-type sialyloligosaccharide under the Boc and Fmoc synthetic strategies. J. Am. Chem. Soc. 130, 501–510 (2008).

    Article  CAS  PubMed  Google Scholar 

  81. Shin, Y. et al. Fmoc-based synthesis of peptide-α-thioesters: application to the total chemical synthesis of a glycoprotein by native chemical ligation. J. Am. Chem. Soc. 121, 11684–11689 (1999).

    Article  CAS  Google Scholar 

  82. Marcaurelle, L.A. et al. Chemical synthesis of lymphotactin: a glycosylated chemokine with a C-terminal mucin-like domain. Chem. Eur. J. 7, 1129–1132 (2001).

    Article  CAS  PubMed  Google Scholar 

  83. Hackenberger, C.P.R., Friel, C.T., Radford, S.E. & Imperiali, B. Semisynthesis of a glycosylated lm7 analogue for protein folding studies. J. Am. Chem. Soc. 127, 12882–12889 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Hojo, H. & Nakahara, Y. Recent progress in the field of glycopeptide synthesis. Biopolymers 88, 308–324 (2007).

    Article  CAS  PubMed  Google Scholar 

  85. Buskas, T., Ingale, S. & Boons, G.-J. Glycopeptides as versatile tools for glycobiology. Glycobiology 16, 113R–136R (2006).

    Article  CAS  PubMed  Google Scholar 

  86. Wang, L.-X., Ni, J., Singh, S. & Li, H. Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12: implications for HIV-1 vaccine design. Chem. Biol. 11, 127–134 (2004).

    PubMed  Google Scholar 

  87. Seko, A. et al. Occurrence of a sialylglycopeptide and free sialylglycans in hen's egg yolk. Biochim. Biophys. Acta 1335, 23–32 (1997).

    Article  CAS  PubMed  Google Scholar 

  88. Yamamoto, K., Kadowaki, S., Watanabe, J. & Kumagai, H. Transglycosylation activity of Mucor hiemalis endo-β-N-acetylglucosaminidase which transfers complex oligosaccharides to the N-acetylglucosamine moieties of peptides. Biochem. Biophys. Res. Commun. 203, 244–252 (1994).

    Article  CAS  PubMed  Google Scholar 

  89. Takegawa, K. et al. Transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae. Biochem. Int. 24, 849–855 (1991).

    CAS  PubMed  Google Scholar 

  90. Fan, J.-Q. et al. Enhanced transglycosylation activity of Arthrobacter protophormiae endo-β-N-acetylglucosaminidase in media containing organic solvents. J. Biol. Chem. 270, 17723–17729 (1995).

    Article  CAS  PubMed  Google Scholar 

  91. Akaike, E. & Yamanoi, T. The transglycosylation activity of the recombinant endo-β-N-acetylglucosaminidase from Mucor hiemalis in media containing organic solvents. Trends Glycosci. Glyc. 18, 63–71 (2006).

    Article  CAS  Google Scholar 

  92. Fujita, M. et al. A novel disaccharide substrate having 1,2-oxazoline moiety for detection of transglycosylating activity of endoglycosidases. Biochim. Biophys. Acta 1528, 9–14 (2001).

    Article  CAS  PubMed  Google Scholar 

  93. Tews, I. et al. Substrate-assisted catalysis unifies two families of chitinolytic enzymes. J. Am. Chem. Soc. 119, 7954–7959 (1997).

    Article  CAS  Google Scholar 

  94. Mark, B.L. et al. Crystallographic evidence for substrate-assisted catalysis in a bacterial β-hexosaminidase. J. Biol. Chem. 276, 10330–10337 (2001).

    Article  CAS  PubMed  Google Scholar 

  95. Li, B. et al. Synthesis and inhibitory activity of oligosaccharide thiazolines as a class of mechanism-based inhibitors for endo-β-N-acetylglucosaminidases. Bioorg. Med. Chem. 16, 4670–4675 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Li, B. et al. Highly efficient endoglycosidase-catalyzed synthesis of glycopeptides using oligosaccharide oxazolines as donor substrates. J. Am. Chem. Soc. 127, 9692–9693 (2005).

    Article  CAS  PubMed  Google Scholar 

  97. Li, B., Song, H.J., Hauser, S. & Wang, L.X. A highly efficient chemoenzymatic approach toward glycoprotein synthesis. Org. Lett. 8, 3081–3084 (2006).

    Article  CAS  PubMed  Google Scholar 

  98. Rising, T.W.D.F. et al. Synthesis of N-glycan oxazolines: donors for endohexosaminidase catalysed glycosylation. Carbohydr. Res. 341, 1574–1596 (2006).

    Article  CAS  PubMed  Google Scholar 

  99. Heidecke, C.D. et al. Enhanced glycosylation with mutants of endohexosaminidase A (Endo A). ChemBioChem 9, 2045–2051 (2008).

    Article  CAS  PubMed  Google Scholar 

  100. Fujita, K. & Takegawa, K. Tryptophan-216 is essential for the transglycosylation activity of endo-β-N-acetylglucosaminidase A. Biochem. Biophys. Res. Commun. 283, 680–686 (2001).

    Article  CAS  PubMed  Google Scholar 

  101. Fujita, K. et al. Identification of the catalytic acid base residue of Arthrobacter endo-β-N-acetylglucosaminidase by chemical rescue of an inactive mutant. J. Biochem. 142, 301–306 (2007).

    Article  CAS  PubMed  Google Scholar 

  102. Umekawa, M. et al. Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J. Biol. Chem. 283, 4469–4479 (2008).

    Article  CAS  PubMed  Google Scholar 

  103. Wang, L.X. et al. Chemoenzymatic synthesis of HIV-1 gp41 glycopeptides: effects of glycosylation on the anti-HIV activity and α-helix bundle-forming ability of peptide C34. ChemBioChem 6, 1068–1074 (2005).

    Article  CAS  PubMed  Google Scholar 

  104. Ochiai, H., Huang, W. & Wang, L.-X. Expeditious chemoenzymatic synthesis of homogeneous N-glycoproteins carrying defined oligosaccharide ligands. J. Am. Chem. Soc. 130, 13790–13803 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Wei, Y. et al. Glycoengineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation. Biochemistry 47, 10294–10304 (2008).

    Article  CAS  PubMed  Google Scholar 

  106. Kaneko, Y., Nimmerjahn, F. & Ravetch, J. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670–673 (2006).

    Article  CAS  PubMed  Google Scholar 

  107. Anthony, R.M. et al. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320, 373–376 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Warnock, D. et al. In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase. Biotechnol. Bioeng. 92, 831–842 (2005).

    Article  CAS  PubMed  Google Scholar 

  109. Hodoniczky, J., Zheng, Y.Z. & James, D.C. Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol. Prog. 21, 1644–1652 (2005).

    Article  CAS  PubMed  Google Scholar 

  110. Ceroni, A. et al. GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J. Proteome Res. 7, 1650–1659 (2008).

    Article  CAS  PubMed  Google Scholar 

  111. Gamblin, D.P., Scanlan, E.M. & Davis, B.G. Glycoprotein synthesis: an update. Chem. Rev. 109, 131–163 (2009).

    Article  CAS  PubMed  Google Scholar 

  112. Huang, W. et al. Glycosynthases enable a highly efficient chemoenzymatic synthesis of N-glycoproteins carrying intact natural N-glycans. J. Am. Chem. Soc. 131, 2214–2223 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

J.R.R. is supported by a postdoctoral fellowship from the Michael Smith Foundation for Health Research. S.G.W. thanks the Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes for Health Research for financial support.

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    Jamie R Rich & Stephen G Withers

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Rich, J., Withers, S. Emerging methods for the production of homogeneous human glycoproteins. Nat Chem Biol 5, 206–215 (2009). https://doi.org/10.1038/nchembio.148

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