Biological activity of human interleukin-22 expressed in Nicotiana benthamiana is independent of its N-glycosylation

R.H.P. Wilbers, L.B. Westerhof, L.J. Reuter, A. Castilho, D.R. van Raaij, G. Smant, J. Bakker, A. Schots

Research output: Chapter in Book/Report/Conference proceedingAbstract

Abstract

In the last decade plants have emerged as a suitable expression platform for several therapeutic proteins, but the expression of cytokines in plants has been challenging so far. Interleukin-22 (IL-22) is a member of the IL-10 cytokine family and has only recently been shown to have therapeutic potential. IL-22 is an unusual cytokine, as it does not act directly on immune cells, but mainly on epithelial cells. IL-22 controls the differentiation, proliferation and expression of anti-microbial proteins of epithelial cells, thereby maintaining epithelial barrier function. IL-22 activity is mediated via IL-22R1 and IL-10R2, the latter being shared by other members of the IL-10 cytokine family. Furthermore, IL-22 activity is reported to be dependent on the glycosylation of asparagine 54 (Asn54) as core a1,6-fucose on this N-glycan enables binding to IL-10R2. Surprisingly, bacterial expressed IL-22 is active, while lacking N-glycans. In this study we investigated the possibility to express human IL-22 transiently in Nicotiana benthamiana and in more detail the role of N-glycosylation on IL-22 activity. Yield obtained for IL-22 was up to 90 µg IL-22/gram fresh weight and IL-22 could be efficiently purified from the leaf apoplast fluid. Plant-produced IL-22 was shown to be biologically active, but most strikingly as active as bacterial and human cell expressed IL-22. Further examination of the role of N-glycosylation of IL-22 by mutagenesis of Asn54, in vivo deglycosylation with PNGase F or glyco-engineering by co-expression of human a1,6-fucosyltransferase (Fut8) revealed that N-glycosylation of IL-22 is not required for biological activity. All in all our data demonstrate that plants are a promising platform for the expression of IL-22, but also offer a great toolbox for studying the role of N-glycans on glycoprotein function.
Original languageEnglish
Title of host publicationProceedings of The First Conference of the International Society for Plant Molecular Farming
Pages76-76
Publication statusPublished - 2014
EventThe First Conference of the International Society for Plant Molecular Farming, Berlin-Dahlem, Germany -
Duration: 17 Jun 201419 Jun 2014

Conference

ConferenceThe First Conference of the International Society for Plant Molecular Farming, Berlin-Dahlem, Germany
Period17/06/1419/06/14

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Glycosylation
Human Activities
Tobacco
Cytokines
Polysaccharides
interleukin-22
Asparagine
Interleukin-10
Epithelial Cells
Fucosyltransferases
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Fucose
Mutagenesis
Glycoproteins
Proteins

Cite this

Wilbers, R. H. P., Westerhof, L. B., Reuter, L. J., Castilho, A., van Raaij, D. R., Smant, G., ... Schots, A. (2014). Biological activity of human interleukin-22 expressed in Nicotiana benthamiana is independent of its N-glycosylation. In Proceedings of The First Conference of the International Society for Plant Molecular Farming (pp. 76-76)
Wilbers, R.H.P. ; Westerhof, L.B. ; Reuter, L.J. ; Castilho, A. ; van Raaij, D.R. ; Smant, G. ; Bakker, J. ; Schots, A. / Biological activity of human interleukin-22 expressed in Nicotiana benthamiana is independent of its N-glycosylation. Proceedings of The First Conference of the International Society for Plant Molecular Farming. 2014. pp. 76-76
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title = "Biological activity of human interleukin-22 expressed in Nicotiana benthamiana is independent of its N-glycosylation",
abstract = "In the last decade plants have emerged as a suitable expression platform for several therapeutic proteins, but the expression of cytokines in plants has been challenging so far. Interleukin-22 (IL-22) is a member of the IL-10 cytokine family and has only recently been shown to have therapeutic potential. IL-22 is an unusual cytokine, as it does not act directly on immune cells, but mainly on epithelial cells. IL-22 controls the differentiation, proliferation and expression of anti-microbial proteins of epithelial cells, thereby maintaining epithelial barrier function. IL-22 activity is mediated via IL-22R1 and IL-10R2, the latter being shared by other members of the IL-10 cytokine family. Furthermore, IL-22 activity is reported to be dependent on the glycosylation of asparagine 54 (Asn54) as core a1,6-fucose on this N-glycan enables binding to IL-10R2. Surprisingly, bacterial expressed IL-22 is active, while lacking N-glycans. In this study we investigated the possibility to express human IL-22 transiently in Nicotiana benthamiana and in more detail the role of N-glycosylation on IL-22 activity. Yield obtained for IL-22 was up to 90 µg IL-22/gram fresh weight and IL-22 could be efficiently purified from the leaf apoplast fluid. Plant-produced IL-22 was shown to be biologically active, but most strikingly as active as bacterial and human cell expressed IL-22. Further examination of the role of N-glycosylation of IL-22 by mutagenesis of Asn54, in vivo deglycosylation with PNGase F or glyco-engineering by co-expression of human a1,6-fucosyltransferase (Fut8) revealed that N-glycosylation of IL-22 is not required for biological activity. All in all our data demonstrate that plants are a promising platform for the expression of IL-22, but also offer a great toolbox for studying the role of N-glycans on glycoprotein function.",
author = "R.H.P. Wilbers and L.B. Westerhof and L.J. Reuter and A. Castilho and {van Raaij}, D.R. and G. Smant and J. Bakker and A. Schots",
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pages = "76--76",
booktitle = "Proceedings of The First Conference of the International Society for Plant Molecular Farming",

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Wilbers, RHP, Westerhof, LB, Reuter, LJ, Castilho, A, van Raaij, DR, Smant, G, Bakker, J & Schots, A 2014, Biological activity of human interleukin-22 expressed in Nicotiana benthamiana is independent of its N-glycosylation. in Proceedings of The First Conference of the International Society for Plant Molecular Farming. pp. 76-76, The First Conference of the International Society for Plant Molecular Farming, Berlin-Dahlem, Germany, 17/06/14.

Biological activity of human interleukin-22 expressed in Nicotiana benthamiana is independent of its N-glycosylation. / Wilbers, R.H.P.; Westerhof, L.B.; Reuter, L.J.; Castilho, A.; van Raaij, D.R.; Smant, G.; Bakker, J.; Schots, A.

Proceedings of The First Conference of the International Society for Plant Molecular Farming. 2014. p. 76-76.

Research output: Chapter in Book/Report/Conference proceedingAbstract

TY - CHAP

T1 - Biological activity of human interleukin-22 expressed in Nicotiana benthamiana is independent of its N-glycosylation

AU - Wilbers, R.H.P.

AU - Westerhof, L.B.

AU - Reuter, L.J.

AU - Castilho, A.

AU - van Raaij, D.R.

AU - Smant, G.

AU - Bakker, J.

AU - Schots, A.

PY - 2014

Y1 - 2014

N2 - In the last decade plants have emerged as a suitable expression platform for several therapeutic proteins, but the expression of cytokines in plants has been challenging so far. Interleukin-22 (IL-22) is a member of the IL-10 cytokine family and has only recently been shown to have therapeutic potential. IL-22 is an unusual cytokine, as it does not act directly on immune cells, but mainly on epithelial cells. IL-22 controls the differentiation, proliferation and expression of anti-microbial proteins of epithelial cells, thereby maintaining epithelial barrier function. IL-22 activity is mediated via IL-22R1 and IL-10R2, the latter being shared by other members of the IL-10 cytokine family. Furthermore, IL-22 activity is reported to be dependent on the glycosylation of asparagine 54 (Asn54) as core a1,6-fucose on this N-glycan enables binding to IL-10R2. Surprisingly, bacterial expressed IL-22 is active, while lacking N-glycans. In this study we investigated the possibility to express human IL-22 transiently in Nicotiana benthamiana and in more detail the role of N-glycosylation on IL-22 activity. Yield obtained for IL-22 was up to 90 µg IL-22/gram fresh weight and IL-22 could be efficiently purified from the leaf apoplast fluid. Plant-produced IL-22 was shown to be biologically active, but most strikingly as active as bacterial and human cell expressed IL-22. Further examination of the role of N-glycosylation of IL-22 by mutagenesis of Asn54, in vivo deglycosylation with PNGase F or glyco-engineering by co-expression of human a1,6-fucosyltransferase (Fut8) revealed that N-glycosylation of IL-22 is not required for biological activity. All in all our data demonstrate that plants are a promising platform for the expression of IL-22, but also offer a great toolbox for studying the role of N-glycans on glycoprotein function.

AB - In the last decade plants have emerged as a suitable expression platform for several therapeutic proteins, but the expression of cytokines in plants has been challenging so far. Interleukin-22 (IL-22) is a member of the IL-10 cytokine family and has only recently been shown to have therapeutic potential. IL-22 is an unusual cytokine, as it does not act directly on immune cells, but mainly on epithelial cells. IL-22 controls the differentiation, proliferation and expression of anti-microbial proteins of epithelial cells, thereby maintaining epithelial barrier function. IL-22 activity is mediated via IL-22R1 and IL-10R2, the latter being shared by other members of the IL-10 cytokine family. Furthermore, IL-22 activity is reported to be dependent on the glycosylation of asparagine 54 (Asn54) as core a1,6-fucose on this N-glycan enables binding to IL-10R2. Surprisingly, bacterial expressed IL-22 is active, while lacking N-glycans. In this study we investigated the possibility to express human IL-22 transiently in Nicotiana benthamiana and in more detail the role of N-glycosylation on IL-22 activity. Yield obtained for IL-22 was up to 90 µg IL-22/gram fresh weight and IL-22 could be efficiently purified from the leaf apoplast fluid. Plant-produced IL-22 was shown to be biologically active, but most strikingly as active as bacterial and human cell expressed IL-22. Further examination of the role of N-glycosylation of IL-22 by mutagenesis of Asn54, in vivo deglycosylation with PNGase F or glyco-engineering by co-expression of human a1,6-fucosyltransferase (Fut8) revealed that N-glycosylation of IL-22 is not required for biological activity. All in all our data demonstrate that plants are a promising platform for the expression of IL-22, but also offer a great toolbox for studying the role of N-glycans on glycoprotein function.

M3 - Abstract

SP - 76

EP - 76

BT - Proceedings of The First Conference of the International Society for Plant Molecular Farming

ER -

Wilbers RHP, Westerhof LB, Reuter LJ, Castilho A, van Raaij DR, Smant G et al. Biological activity of human interleukin-22 expressed in Nicotiana benthamiana is independent of its N-glycosylation. In Proceedings of The First Conference of the International Society for Plant Molecular Farming. 2014. p. 76-76