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

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    6 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

    Keywords

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

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  • Cite this

    Wilbers, R. H. P., Westerhof, L. B., Reuter, L. J., Castilho, A., van Raaij, D. R., Nguyen, D. L., Lozano-Torres, J. L., Smant, G., Hokke, C. H., 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, 14(2), 670-681. https://doi.org/10.1111/pbi.12414