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 language | English |
|---|---|
| Pages (from-to) | 670-681 |
| Number of pages | 12 |
| Journal | Plant Biotechnology Journal |
| Volume | 14 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 1 Feb 2016 |
| MoE publication type | A1 Journal article-refereed |
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Keywords
- Core α1,6-fucose
- Glyco-engineering
- Interleukin-22
- Lewis A
- N-glycosylation
- Nicotiana
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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 journal › Article › Scientific › peer-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 -