The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity

Ruud H.P. Wilbers, Lotte B. Westerhof, Lauri J. Reuter, Alexandra Castilho, Debbie R. van Raaij, Dieu Linh Nguyen, Jose L. Lozano-Torres, Geert Smant, Cornelis H. Hokke, Jaap Bakker, Arjen Schots

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

Human interleukin-22 (IL-22) is a member of the IL-10 cytokine family that has recently been shown to have major therapeutic potential. IL-22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL-22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL-22 in Nicotiana benthamiana plants and investigated the role of N-glycosylation on protein folding and biological activity. Expression levels of IL-22 were up to 5.4 μg/mg TSP, and N-glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human-like N-glycans on IL-22 by co-expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6-fucoylation did not improve the biological activity of IL-22. The combination of site-directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N-glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N-glycan on Asn54 contributes to the atypical N-glycan composition of plant-produced IL-22 and influences the N-glycan composition of N-glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N-glycosylation on folding and activity of recombinant glycoproteins, such as IL-22.

Original languageEnglish
Pages (from-to)670-681
Number of pages12
JournalPlant Biotechnology Journal
Volume14
Issue number2
DOIs
Publication statusPublished - 1 Feb 2016
MoE publication typeA1 Journal article-refereed

Fingerprint

chemical constituents of plants
interleukins
Polysaccharides
polysaccharides
glycosylation
Glycosylation
protein folding
Protein Folding
bioactive properties
cytokines
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Cytokines
antimicrobial proteins
interleukin-22
ergonomics
Human Engineering
Nicotiana benthamiana
site-directed mutagenesis
interleukin-10
Site-Directed Mutagenesis

Keywords

  • Core α1,6-fucose
  • Glyco-engineering
  • Interleukin-22
  • Lewis A
  • N-glycosylation
  • Nicotiana

Cite this

Wilbers, R. H. P., Westerhof, L. B., Reuter, L. J., Castilho, A., van Raaij, D. R., Nguyen, D. L., ... Schots, A. (2016). The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity. Plant Biotechnology Journal, 14(2), 670-681. https://doi.org/10.1111/pbi.12414
Wilbers, Ruud H.P. ; Westerhof, Lotte B. ; Reuter, Lauri J. ; Castilho, Alexandra ; van Raaij, Debbie R. ; Nguyen, Dieu Linh ; Lozano-Torres, Jose L. ; Smant, Geert ; Hokke, Cornelis H. ; Bakker, Jaap ; Schots, Arjen. / The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity. In: Plant Biotechnology Journal. 2016 ; Vol. 14, No. 2. pp. 670-681.
@article{38920f44f5754c2094b1245e355bc9ef,
title = "The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity",
abstract = "Human interleukin-22 (IL-22) is a member of the IL-10 cytokine family that has recently been shown to have major therapeutic potential. IL-22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL-22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL-22 in Nicotiana benthamiana plants and investigated the role of N-glycosylation on protein folding and biological activity. Expression levels of IL-22 were up to 5.4 μg/mg TSP, and N-glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human-like N-glycans on IL-22 by co-expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6-fucoylation did not improve the biological activity of IL-22. The combination of site-directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N-glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N-glycan on Asn54 contributes to the atypical N-glycan composition of plant-produced IL-22 and influences the N-glycan composition of N-glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N-glycosylation on folding and activity of recombinant glycoproteins, such as IL-22.",
keywords = "Core α1,6-fucose, Glyco-engineering, Interleukin-22, Lewis A, N-glycosylation, Nicotiana",
author = "Wilbers, {Ruud H.P.} and Westerhof, {Lotte B.} and Reuter, {Lauri J.} and Alexandra Castilho and {van Raaij}, {Debbie R.} and Nguyen, {Dieu Linh} and Lozano-Torres, {Jose L.} and Geert Smant and Hokke, {Cornelis H.} and Jaap Bakker and Arjen Schots",
year = "2016",
month = "2",
day = "1",
doi = "10.1111/pbi.12414",
language = "English",
volume = "14",
pages = "670--681",
journal = "Plant Biotechnology Journal",
issn = "1467-7644",
publisher = "Wiley",
number = "2",

}

Wilbers, RHP, Westerhof, LB, Reuter, LJ, Castilho, A, van Raaij, DR, Nguyen, DL, Lozano-Torres, JL, Smant, G, Hokke, CH, Bakker, J & Schots, A 2016, 'The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity', Plant Biotechnology Journal, vol. 14, no. 2, pp. 670-681. https://doi.org/10.1111/pbi.12414

The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity. / Wilbers, Ruud H.P.; Westerhof, Lotte B.; Reuter, Lauri J.; Castilho, Alexandra; van Raaij, Debbie R.; Nguyen, Dieu Linh; Lozano-Torres, Jose L.; Smant, Geert; Hokke, Cornelis H.; Bakker, Jaap; Schots, Arjen.

In: Plant Biotechnology Journal, Vol. 14, No. 2, 01.02.2016, p. 670-681.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity

AU - Wilbers, Ruud H.P.

AU - Westerhof, Lotte B.

AU - Reuter, Lauri J.

AU - Castilho, Alexandra

AU - van Raaij, Debbie R.

AU - Nguyen, Dieu Linh

AU - Lozano-Torres, Jose L.

AU - Smant, Geert

AU - Hokke, Cornelis H.

AU - Bakker, Jaap

AU - Schots, Arjen

PY - 2016/2/1

Y1 - 2016/2/1

N2 - Human interleukin-22 (IL-22) is a member of the IL-10 cytokine family that has recently been shown to have major therapeutic potential. IL-22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL-22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL-22 in Nicotiana benthamiana plants and investigated the role of N-glycosylation on protein folding and biological activity. Expression levels of IL-22 were up to 5.4 μg/mg TSP, and N-glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human-like N-glycans on IL-22 by co-expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6-fucoylation did not improve the biological activity of IL-22. The combination of site-directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N-glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N-glycan on Asn54 contributes to the atypical N-glycan composition of plant-produced IL-22 and influences the N-glycan composition of N-glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N-glycosylation on folding and activity of recombinant glycoproteins, such as IL-22.

AB - Human interleukin-22 (IL-22) is a member of the IL-10 cytokine family that has recently been shown to have major therapeutic potential. IL-22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL-22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL-22 in Nicotiana benthamiana plants and investigated the role of N-glycosylation on protein folding and biological activity. Expression levels of IL-22 were up to 5.4 μg/mg TSP, and N-glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human-like N-glycans on IL-22 by co-expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6-fucoylation did not improve the biological activity of IL-22. The combination of site-directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N-glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N-glycan on Asn54 contributes to the atypical N-glycan composition of plant-produced IL-22 and influences the N-glycan composition of N-glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N-glycosylation on folding and activity of recombinant glycoproteins, such as IL-22.

KW - Core α1,6-fucose

KW - Glyco-engineering

KW - Interleukin-22

KW - Lewis A

KW - N-glycosylation

KW - Nicotiana

UR - http://www.scopus.com/inward/record.url?scp=84930713458&partnerID=8YFLogxK

U2 - 10.1111/pbi.12414

DO - 10.1111/pbi.12414

M3 - Article

VL - 14

SP - 670

EP - 681

JO - Plant Biotechnology Journal

JF - Plant Biotechnology Journal

SN - 1467-7644

IS - 2

ER -