Journal
PLANT BIOTECHNOLOGY JOURNAL
Volume 15, Issue 2, Pages 197-206Publisher
WILEY-BLACKWELL
DOI: 10.1111/pbi.12602
Keywords
alpha 1-antitrypsin; glyco-engineering; N-glycosylation; Nicotiana benthamiana; plant-made pharmaceuticals
Funding
- Federal Ministry of Transport, Innovation and Technology (bmvit)
- Austrian Science Fund (FWF) [TRP 242-B20]
- Austrian Research Promotion Agency (FFG) in the frame of Laura Bassi Centres of Expertise [822757]
- Austrian Science Fund (FWF) [TRP242] Funding Source: Austrian Science Fund (FWF)
- Austrian Science Fund (FWF) [TRP 242] Funding Source: researchfish
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Plants are attractive hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco-engineering facilitate the elimination of nonmammalian-type glycosylation and introduction of missing pathways for customized N-glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) N-glycans that lack GlcNAc residues at the nonreducing terminal end. These paucimannosidic N-glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, beta-hexosaminidases (HEXOs) that account for the formation of paucimannosidic N-glycans in Nicotiana benthamiana, a widely used expression host for recombinant proteins. Subcellular localization studies showed that HEXO1 is a vacuolar protein and HEXO3 is mainly located at the plasma membrane in N. benthamiana leaf epidermal cells. Both enzymes are functional and can complement the corresponding HEXO-deficient Arabidopsis thaliana mutants. In planta expression of HEXO3 demonstrated that core alpha 1,3-fucose enhances the trimming of GlcNAc residues from the Fc domain of human IgG. Finally, using RNA interference, we show that suppression of HEXO3 expression can be applied to increase the amounts of complex N-glycans on plant-produced human alpha 1-antitrypsin.
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