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N-Glyco-Engineering in Plants: Update on Strategies and Major Achievements

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Part of the Methods in Molecular Biology book series (MIMB,volume 1321)

Abstract

Plants are being developed as a cost-effective production system for biopharmaceuticals in large quantities. Although plants properly fold and assemble complex proteins from human origin, one issue that needs to be addressed is their glycan structure. In the past years we have been witnessing outstanding results in targeted manipulation of the plant N-glycosylation pathway allowing recombinant proteins to be produced with human-type oligosaccharides at large homogeneity. This opens new possibility in manufacturing next-generation biopharmaceuticals.

This review presents a variety of technologies and strategies that are being employed to engineer the plant N-glycosylation, thus pointing to the enormous potential of plants being used as a novel production system with unique features and possibilities.

Key words

  • Glyco-engineering
  • Plants
  • Recombinant protein expression
  • Glycosyltransferases
  • Glycosidases
  • Subcellular targeting

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Abbreviations

C1GalT1:

Drosophila melanogaster Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1

CMAS:

Human CMP-N-acetylneuraminic acid synthase

CST:

Mouse CMP-sialic acid transporter

CTS:

Cytoplasmic tail, transmembrane domain, and stem

FUT11:

A. thaliana α1,3-fucosyltransferase

FUT8:

Human α1,6-fucosyltransferase

FUT9a:

Human α1,3-fucosyltransferase IXa

GalNAc-T2:

Human polypeptide N-acetylgalactosaminyltransferase

GalT:

Human β1,4-galactosyltransferase

GE:

Pseudomonas aeruginosa or Yersinia enterocolitica GMI, Golgi mannosidase I

GMII:

Golgi mannosidase II

GNE:

Mouse UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase

GnT-I:

α1,3-Mannosyl-β1,2-N-acetylglucosaminyltransferase I

GnT-II:

α1,6-Mannosyl-β1,2-N-acetylglucosaminyltransferase II

GnT-III:

Human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase III

GnT-IV:

Human α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase IVa

GnT-V:

Human α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase V

HEXO1 and HEXO3:

Arabidopsis β-N-acetylhexosaminidases 1 and 3

NANS:

Human N-acetylneuraminic acid phosphate-synthase

P4H:

Prolyl-4-hydroxylase

ST:

Rat or human α2,6-sialyltransferase

ST3GalI:

Human α2,3-sialyltransferase

ST6GalNAc4:

Mouse α2,6-sialyltransferase

UDP-GlcNAc/UDP-GalNAc transporter:

UPD-GlcNAc 4-epimerase

UGT:

Caenorhabditis elegans

XylT:

β1,2-Xylosyltransferase

α1,4-FucT:

α1,4-Fucosyltransferase

β1,3-GalT:

β1,3-Galactosyltransferase

References

  1. Gomord V, Faye L (2004) Posttranslational modification of therapeutic proteins in plants. Curr Opin Plant Biol 7:171–181

    CAS  PubMed  CrossRef  Google Scholar 

  2. Sussman MR, Amasino RM, Young JC et al (2000) The Arabidopsis knockout facility at the University of Wisconsin-Madison. Plant Physiol 124:1465–1467

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  3. Strasser R, Altmann F, Mach L et al (2004) Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose. FEBS Lett 561:132–136

    CAS  PubMed  CrossRef  Google Scholar 

  4. Koprivova A, Stemmer C, Altmann F et al (2004) Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. Plant Biotechnol J 2:517–523

    CAS  PubMed  CrossRef  Google Scholar 

  5. Huether CM, Lienhart O, Baur A et al (2005) Glyco-engineering of moss lacking plant-specific sugar residues. Plant Biol (Stuttg) 7:292–299

    CAS  CrossRef  Google Scholar 

  6. Cox KM, Sterling JD, Regan JT et al (2006) Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24:1591–1597

    CAS  PubMed  CrossRef  Google Scholar 

  7. Strasser R, Stadlmann J, Schähs M et al (2008) Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol J 6:392–402

    CAS  PubMed  CrossRef  Google Scholar 

  8. Sourrouille C, Marquet-Blouin E, D'Aoust MA et al (2008) Down-regulated expression of plant-specific glycoepitopes in alfalfa. Plant Biotechnol J 6:702–721

    CAS  PubMed  CrossRef  Google Scholar 

  9. Shin YJ, Chong YJ, Yang MS et al (2011) Production of recombinant human granulocyte macrophage-colony stimulating factor in rice cell suspension culture with a human-like N-glycan structure. Plant Biotechnol J 9:1109–1119

    CAS  PubMed  CrossRef  Google Scholar 

  10. Betenbaugh MJ, Tomiya N, Narang S et al (2004) Biosynthesis of human-type N-glycans in heterologous systems. Curr Opin Struct Biol 14:601–606

    CAS  PubMed  CrossRef  Google Scholar 

  11. Gomord V, Fitchette AC, Menu-Bouaouiche L et al (2010) Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol J 8:564–587

    CAS  PubMed  CrossRef  Google Scholar 

  12. Liebminger E, Veit C, Pabst M et al (2011) Beta-N-acetylhexosaminidases HEXO1 and HEXO3 are responsible for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. J Biol Chem 286:10793–10802

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  13. Fitchette-Laine AC, Gomord V, Cabanes M et al (1997) N-glycans harboring the Lewis a epitope are expressed at the surface of plant cells. Plant J 12:1411–1417

    CAS  PubMed  CrossRef  Google Scholar 

  14. Strasser R, Bondili JS, Vavra U et al (2007) A unique beta1,3-galactosyltransferase is indispensable for the biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana. Plant Cell 19:2278–2292

    PubMed Central  PubMed  CrossRef  Google Scholar 

  15. Weise A, Altmann F, Rodriguez-Franco M et al (2007) High-level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Delta-fuc-t Delta-xyl-t mutant. Plant Biotechnol J 5:389–401

    CAS  PubMed  CrossRef  Google Scholar 

  16. Castilho A, Gattinger P, Grass J et al (2011) N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans. Glycobiology 21:813–823

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  17. Parsons J, Altmann F, Arrenberg CK et al (2012) Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants. Plant Biotechnol J 10:851–861

    CAS  PubMed  CrossRef  Google Scholar 

  18. Mori K, Iida S, Yamane-Ohnuki N et al (2007) Non-fucosylated therapeutic antibodies, the next generation of therapeutic antibodies. Cytotechnology 55:109–114

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  19. Yamane-Ohnuki N, Satoh M (2009) Production of therapeutic antibodies with controlled fucosylation. MAbs 1:230–236

    PubMed Central  PubMed  CrossRef  Google Scholar 

  20. Forthal DN, Gach JS, Landucci G et al (2010) Fc-glycosylation influences Fcγ receptor binding and cell-mediated anti-HIV activity of monoclonal antibody 2G12. J Immunol 185:6876–6882

    CAS  PubMed  CrossRef  Google Scholar 

  21. Castilho A, Bohorova N, Grass J et al (2011) Rapid high yield production of different glycoforms of Ebola virus monoclonal antibody. PLoS One 6, e26040

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  22. Rouwendal GJ, Florack DE, Hesselink T et al (2009) Synthesis of Lewis X epitopes on plant N-glycans. Carbohydr Res 344:1487–1493

    CAS  PubMed  CrossRef  Google Scholar 

  23. Wang J, Zhang Y, Wei J et al (2007) Lewis X oligosaccharides targeting to DC-SIGN enhanced antigen-specific immune response. Immunology 121:174–182

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  24. Gleeson PA, Schachter H (1983) Control of glycoprotein synthesis. J Biol Chem 258:6162–6173

    CAS  PubMed  Google Scholar 

  25. Schachter H (1986) Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Biochem Cell Biol 64:163–181

    CAS  PubMed  CrossRef  Google Scholar 

  26. Rouwendal GJ, Wuhrer M, Florack DE et al (2007) Efficient introduction of a bisecting GlcNAc residue in tobacco N-glycans by expression of the gene encoding human N-acetylglucosaminyltransferase III. Glycobiology 17:334–344

    CAS  PubMed  CrossRef  Google Scholar 

  27. Frey AD, Karg SR, Kallio PT (2009) Expression of rat beta(1,4)-N-acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant-specific N-glycosylation. Plant Biotechnol J 7:33–48

    CAS  PubMed  CrossRef  Google Scholar 

  28. Karg SR, Frey AD, Kallio PT (2010) Reduction of N-linked xylose and fucose by expression of rat beta1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression. J Biotechnol 146:54–65

    CAS  PubMed  CrossRef  Google Scholar 

  29. Nagels B, Van Damme EJ, Callewaert N et al (2012) Introduction of tri-antennary N-glycans in Arabidopsis thaliana plants. Plant Sci 185–186:161–168

    PubMed  CrossRef  Google Scholar 

  30. Nagels B, Van Damme EJ, Pabst M et al (2011) Production of complex multiantennary N-glycans in Nicotiana benthamiana plants. Plant Physiol 155:1103–1112

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  31. Palacpac NQ, Yoshida S, Sakai H et al (1999) Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc Natl Acad Sci U S A 96:4692–4697

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  32. Fujiyama K, Palacpac NQ, Sakai H et al (2001) In vivo conversion of a glycan to human compatible type by transformed tobacco cells. Biochem Biophys Res Commun 289:553–557

    CAS  PubMed  CrossRef  Google Scholar 

  33. Misaki R, Kimura Y, Palacpac NQ et al (2003) Plant cultured cells expressing human Î21,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans. Glycobiology 13:199–205

    CAS  PubMed  CrossRef  Google Scholar 

  34. Bakker H, Bardor M, Molthoff JW et al (2001) Galactose-extended glycans of antibodies produced by transgenic plants. Proc Natl Acad Sci U S A 98:2899–2904

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  35. Bakker H, Rouwendal GJ, Karnoup AS et al (2006) An antibody produced in tobacco expressing a hybrid beta-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes. Proc Natl Acad Sci U S A 103:7577–7582

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  36. Vézina LP, Faye L, Lerouge P et al (2009) Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants. Plant Biotechnol J 7:442–455

    PubMed  CrossRef  Google Scholar 

  37. Strasser R, Castilho A, Stadlmann J et al (2009) Improved virus neutralization by plant-produced anti-HIV antibodies with a homogeneous beta1,4-galactosylated N-glycan profile. J Biol Chem 284:20479–20485

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

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

    CAS  PubMed  CrossRef  Google Scholar 

  39. Erbayraktar S, Grasso G, Sfacteria A et al (2003) Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci U S A 100:6741–6746

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  40. Varki A (2007) Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 446:1023–1029

    CAS  PubMed  CrossRef  Google Scholar 

  41. Takashima S, Abe T, Yoshida S et al (2006) Analysis of sialyltransferase-like proteins from Oryza sativa. J Biochem 139:279–287

    CAS  PubMed  CrossRef  Google Scholar 

  42. Takashima S, Seino J, Nakano T et al (2009) Analysis of CMP-sialic acid transporter-like proteins in plants. Phytochemistry 70:1973–1981

    CAS  PubMed  CrossRef  Google Scholar 

  43. Shah MM, Fujiyama K, Flynn CR et al (2003) Sialylated endogenous glycoconjugates in plant cells. Nat Biotechnol 21:1470–1471

    CAS  PubMed  CrossRef  Google Scholar 

  44. Bakker H, Routier F, Ashikov A et al (2008) A CMP-sialic acid transporter cloned from Arabidopsis thaliana. Carbohydr Res 343:2148–2152

    CAS  PubMed  CrossRef  Google Scholar 

  45. Zeleny R, Kolarich D, Strasser R et al (2006) Sialic acid concentrations in plants are in the range of inadvertent contamination. Planta 224:222–227

    CAS  PubMed  CrossRef  Google Scholar 

  46. Wee EG, Sherrier DJ, Prime TA et al (1998) Targeting of active sialyltransferase to the plant Golgi apparatus. Plant Cell 10:1759–1768

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  47. Misaki R, Fujiyama K, Seki T (2006) Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell. Biochem Biophys Res Commun 339:1184–1189

    CAS  PubMed  CrossRef  Google Scholar 

  48. Kajiura H, Misaki R, Fujiyama K et al (2011) Stable coexpression of two human sialylation enzymes in plant suspension-cultured tobacco cells. J Biosci Bioeng 111:471–477

    CAS  PubMed  CrossRef  Google Scholar 

  49. Paccalet T, Bardor M, Rihouey C et al (2007) Engineering of a sialic acid synthesis pathway in transgenic plants by expression of bacterial Neu5Ac-synthesizing enzymes. Plant Biotechnol J 5:16–25

    CAS  PubMed  CrossRef  Google Scholar 

  50. Castilho A, Pabst M, Leonard R et al (2008) Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis. Plant Physiol 147:331–339

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  51. Castilho A, Strasser R, Stadlmann J et al (2010) In planta protein sialylation through overexpression of the respective mammalian pathway. J Biol Chem 285:15923–15930

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  52. Jez J, Castilho A, Grass J et al (2013) Expression of functionally active sialylated human erythropoietin in plants. Biotechnol J 8:371–382

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  53. Castilho A, Neumann L, Gattinger P et al (2013) Generation of biologically active multi-sialylated recombinant human EPOFc in plants. PLoS One 8, e54836

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  54. Schneider JD, Castilho A, Neumann L et al (2013) Expression of human butyrylcholinesterase with an engineered glycosylation profile resembling the plasma-derived orthologue. Biotechnol J 9:501–510

    PubMed Central  PubMed  CrossRef  Google Scholar 

  55. Parsons J, Altmann F, Graf M et al (2013) A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin. Sci Rep 3:3019

    PubMed Central  PubMed  CrossRef  Google Scholar 

  56. Strasser R (2013) Engineering of human-type O-glycosylation in Nicotiana benthamiana plants. Bioengineered 4:191–196

    PubMed Central  PubMed  CrossRef  Google Scholar 

  57. Yang Z, Drew DP, Jorgensen B et al (2012) Engineering mammalian mucin-type O-glycosylation in plants. J Biol Chem 287:11911–11923

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  58. Daskalova SM, Radder JE et al (2010) Engineering of N benthamiana L plants for production of N-acetylgalactosamine-glycosylated proteins-towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation. BMC Biotechnol 10:62

    PubMed Central  PubMed  CrossRef  Google Scholar 

  59. Yang Z, Bennett EP, Jorgensen B et al (2012) Toward stable genetic engineering of human O-glycosylation in plants. Plant Physiol 160:450–463

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  60. Castilho A, Neumann L, Daskalova S et al (2012) Engineering of sialylated mucin-type O-glycosylation in plants. J Biol Chem 287:36518–36526

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  61. Chung SM, Frankman EL, Tzfira T (2005) A versatile vector system for multiple gene expression in plants. Trends Plant Sci 10:357–361

    CAS  PubMed  CrossRef  Google Scholar 

  62. Dafny-Yelin M, Tzfira T (2007) Delivery of multiple transgenes to plant cells. Plant Physiol 145:1118–1128

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  63. Weber E, Engler C, Gruetzner R et al (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6, e16765

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  64. Werner S, Engler C, Weber E et al (2012) Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng Bugs 3:38–43

    PubMed  Google Scholar 

  65. Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J et al (2013) GoldenBraid 2.0, a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol 162:1618–1631

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  66. Peremarti A, Twyman RM, Gómez-Galera S et al (2010) Promoter diversity in multigene transformation. Plant Mol Biol 73:363–378

    CAS  PubMed  CrossRef  Google Scholar 

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Steinkellner, H., Castilho, A. (2015). N-Glyco-Engineering in Plants: Update on Strategies and Major Achievements. In: Castilho, A. (eds) Glyco-Engineering. Methods in Molecular Biology, vol 1321. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2760-9_14

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  • DOI: https://doi.org/10.1007/978-1-4939-2760-9_14

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2759-3

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