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Subcellular Targeting of Proteins Involved in Modification of Plant N- and O-Glycosylation

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Glyco-Engineering

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

Abstract

Plants are attractive expression hosts for the production of recombinant glycoprotein therapeutics. The quality and efficiency of these biopharmaceuticals are very often influenced by the glycosylation profile. Consequently, approaches are needed that enable the production of recombinant glycoproteins with customized and homogenous N- and O-glycan structures. Here, we describe convenient tools that allow targeting and retention of glycan-modifying enzymes in the early secretory pathway of plants. These protocols can be used to fine-tune the subcellular localization of glycosyltransferases and glycosidases in plants and consequently to increase the homogeneity of glycosylation on recombinant glycoproteins.

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References

  1. Liebminger E, Hüttner S, Vavra U et al (2009) Class I alpha-mannosidases are required for N-glycan processing and root development in Arabidopsis thaliana. Plant Cell 21:3850–3867

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Altmann F (2007) The role of protein glycosylation in allergy. Int Arch Allergy Immunol 142:99–115

    Article  CAS  PubMed  Google Scholar 

  3. Jin C, Altmann F, Strasser R et al (2008) A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits. Glycobiology 18:235–241

    Article  CAS  PubMed  Google Scholar 

  4. Bardor M, Faveeuw C, Fitchette A et al (2003) Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology 13:427–434

    Article  CAS  PubMed  Google Scholar 

  5. Lerouge P, Cabanes-Macheteau M, Rayon C et al (1998) N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol 38:31–48

    Article  CAS  PubMed  Google Scholar 

  6. Fitchette A, Cabanes-Macheteau M et al (1999) Biosynthesis and immunolocalization of Lewis a-containing N-glycans in the plant cell. Plant Physiol 121:333–344

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Strasser R, Bondili J, 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

    Article  PubMed Central  PubMed  Google Scholar 

  8. Schoberer J, Runions J, Steinkellner H et al (2010) Sequential depletion and acquisition of proteins during Golgi stack disassembly and reformation. Traffic 11:1429–1444

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Essl D, Dirnberger D, Gomord V et al (1999) The N-terminal 77 amino acids from tobacco N-acetylglucosaminyltransferase I are sufficient to retain a reporter protein in the Golgi apparatus of Nicotiana benthamiana cells. FEBS Lett 453:169–173

    Article  CAS  PubMed  Google Scholar 

  10. Dirnberger D, Bencúr P, Mach L et al (2002) The Golgi localization of Arabidopsis thaliana beta1,2-xylosyltransferase in plant cells is dependent on its cytoplasmic and transmembrane sequences. Plant Mol Biol 50:273–281

    Article  CAS  PubMed  Google Scholar 

  11. Saint-Jore-Dupas C, Nebenführ A, Boulaflous A et al (2006) Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway. Plant Cell 18:3182–3200

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Strasser R, Schoberer J, Jin C et al (2006) Molecular cloning and characterization of Arabidopsis thaliana Golgi alpha-mannosidase II, a key enzyme in the formation of complex N-glycans in plants. Plant J 45:789–803

    Article  CAS  PubMed  Google Scholar 

  13. Bretscher M, Munro S (1993) Cholesterol and the Golgi apparatus. Science 261:1280–1281

    Article  CAS  PubMed  Google Scholar 

  14. Nilsson T, Slusarewicz P, Hoe M et al (1993) Kin recognition. A model for the retention of Golgi enzymes. FEBS Lett 330:1–4

    Article  CAS  PubMed  Google Scholar 

  15. Nilsson T, Rabouille C, Hui N et al (1996) The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells. J Cell Sci 109:1975–1989

    CAS  PubMed  Google Scholar 

  16. Fenteany F, Colley K (2005) Multiple signals are required for alpha2,6-sialyltransferase (ST6Gal I) oligomerization and Golgi localization. J Biol Chem 280:5423–5429

    Article  CAS  PubMed  Google Scholar 

  17. Tu L, Banfield D (2010) Localization of Golgi-resident glycosyltransferases. Cell Mol Life Sci 67:29–41

    Article  CAS  PubMed  Google Scholar 

  18. Ali MF, Chachadi VB, Petrosyan A et al (2012) Golgi phosphoprotein 3 determines cell binding properties under dynamic flow by controlling Golgi localization of core 2 N-acetylglucosaminyltransferase 1. J Biol Chem 287:39564–39577

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Boevink P, Oparka K, Santa Cruz S et al (1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15:441–447

    Article  CAS  PubMed  Google Scholar 

  20. Wee E, Sherrier D, Prime T et al (1998) Targeting of active sialyltransferase to the plant Golgi apparatus. Plant Cell 10:1759–1768

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Palacpac N, 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Bakker H, Rouwendal G, Karnoup A 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. 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

    Article  PubMed  Google Scholar 

  26. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Bennett EP, Mandel U, Clausen H et al (2012) Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology 22:736–756

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Strasser R, Mucha J, Schwihla H et al (1999) Molecular cloning and characterization of cDNA coding for beta1,2 N-acetylglucosaminyltransferase I (GlcNAc-TI) from Nicotiana tabacum. Glycobiology 9:779–785

    Article  CAS  PubMed  Google Scholar 

  32. Schoberer J, Vavra U, Stadlmann J et al (2009) Arginine/lysine residues in the cytoplasmic tail promote ER export of plant glycosylation enzymes. Traffic 10:101–115

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. 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

    Article  CAS  PubMed  Google Scholar 

  34. Hamilton CM (1997) A binary-BAC system for plant transformation with high-molecular-weight DNA. Gene 200:107–116

    Article  CAS  PubMed  Google Scholar 

  35. Strasser R, Stadlmann J, Svoboda B et al (2005) Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans. Biochem J 387:385–391

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Schoberer J, Liebminger E, Botchway SW et al (2013) Time-resolved fluorescence imaging reveals differential interactions of N-glycan processing enzymes across the Golgi stack in planta. Plant Physiol 161:1737–1754

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. 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

    Article  CAS  PubMed  Google Scholar 

  38. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Sparkes I, Runions J, Kearns A et al (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025

    Article  CAS  PubMed  Google Scholar 

  40. Schähs M, Strasser R, Stadlmann J et al (2007) Production of a monoclonal antibody in plants with a humanized N-glycosylation pattern. Plant Biotechnol J 5:657–663

    Article  PubMed  Google Scholar 

  41. Stadlmann J, Pabst M, Kolarich D et al (2008) Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides. Proteomics 8:2858–2871

    Article  CAS  PubMed  Google Scholar 

  42. Henquet M, Heinhuis B, Borst JW et al (2010) Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants. Transgenic Res 19:535–547

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Grefen C, Donald N, Hashimoto K et al (2010) A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J 64:355–365

    Article  CAS  PubMed  Google Scholar 

  44. Boulaflous A, Saint-Jore-Dupas C, Herranz-Gordo MC et al (2009) Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER. BMC Plant Biol 9:144

    Article  PubMed Central  PubMed  Google Scholar 

  45. Pelham HR (1990) The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci 15:483–486

    Article  PubMed  Google Scholar 

  46. De Muynck B, Navarre C, Boutry M (2010) Production of antibodies in plants: status after twenty years. Plant Biotechnol J 8:529–563

    Article  PubMed  Google Scholar 

  47. Ko K, Tekoah Y, Rudd P et al (2003) Function and glycosylation of plant-derived antiviral monoclonal antibody. Proc Natl Acad Sci U S A 100:8013–8018

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Triguero A, Cabrera G, Cremata JA et al (2005) Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans. Plant Biotechnol J 3:449–457

    Article  CAS  PubMed  Google Scholar 

  49. Petruccelli S, Otegui MS, Lareu F et al (2006) A KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol J 4:511–527

    CAS  PubMed  Google Scholar 

  50. Fujiyama K, Misaki R, Sakai Y et al (2009) Change in glycosylation pattern with extension of endoplasmic reticulum retention signal sequence of mouse antibody produced by suspension-cultured tobacco BY2 cells. J Biosci Bioeng 107:165–172

    Article  CAS  PubMed  Google Scholar 

  51. Niemer M, Mehofer U, Torres Acosta JA et al (2014) The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms. Biotechnol J 9:493–500

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  52. von Heijne G (1989) Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature 341:456–458

    Article  Google Scholar 

  53. Nebenführ A, Gallagher L, Dunahay T et al (1999) Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol 121:1127–1142

    Article  PubMed Central  PubMed  Google Scholar 

  54. Saint-Jore C, Evins J, Batoko H et al (2002) Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks. Plant J 29:661–678

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Christiane Veit (Department of Applied Genetics and Cell Biology) for assistance in cloning and Friedrich Altmann and Daniel Maresch (both Department of Chemistry) for LC-ESI-MS-analysis. This work was supported by a grant from the Federal Ministry of Transport, Innovation and Technology (bmvit) and Austrian Science Fund (FWF): TRP 242-B20.

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Dicker, M., Schoberer, J., Vavra, U., Strasser, R. (2015). Subcellular Targeting of Proteins Involved in Modification of Plant N- and O-Glycosylation. 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_18

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  • DOI: https://doi.org/10.1007/978-1-4939-2760-9_18

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2759-3

  • Online ISBN: 978-1-4939-2760-9

  • eBook Packages: Springer Protocols

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