Open AccessThe N‐glycan on Asn54 affects the atypical N‐glycan composition of plant‐produced interleukin‐22, but does not influence its activity
Author(s) -
Wilbers Ruud H. P.,
Westerhof Lotte B.,
Reuter Lauri J.,
Castilho Alexandra,
Raaij Debbie R.,
Nguyen DieuLinh,
LozanoTorres Jose L.,
Smant Geert,
Hokke Cornelis H.,
Bakker Jaap,
Schots Arjen
Publication year - 2016
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.12414
Subject(s) - glycan , glycosylation , biology , glycoprotein , n linked glycosylation , biochemistry , mutagenesis , microbiology and biotechnology , mutation , gene
Summary 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 FUT 8 in Δ XT / 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 PNG ase 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.