Reduced paucimannosidic N‐glycan formation by suppression of a specific β‐hexosaminidase from Nicotiana benthamiana

Abstract

SummaryPlants 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, β‐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 α1,3‐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 α1‐antitrypsin.

Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 Cells by a Multiplex CRISPR/Cas9 Strategy Results in Glycoproteins without Plant-Specific Glycans

“…This correlates with a recent study (Shin et al, 2016) that demonstrated that in N. benthamiana , the presence of the core α(1,3)-fucose on the N -glycans enhances the trimming of GlcNAc residues. These authors showed that this trimming is due to the activity of β-hexosaminidases, mainly located in the plasma membrane in leaf epidermal cells.…”

CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

“…The absence of these sugar residues from the endogenous N-glycans of the FX-KO line thus results in less trimming and consequently a higher content of GnGn. For the 2G12 glycovariants, on the other hand, the main glycoforms were GnGn(F/X) variants in all tested samples (Table 3) as was the case for the 2G12 glycovariants expressed in the RNAi N. benthamiana lines by Strasser et al (2008), likely because the antibody heavy chain is not a substrate for the bhexosaminidases present in the cell (Shin et al, 2017).…”

“…For the 2G12 glycovariants, on the other hand, the main glycoforms were GnGn(F/X) variants in all tested samples (Table ) as was the case for the 2G12 glycovariants expressed in the RNAi N. benthamiana lines by Strasser et al . (), likely because the antibody heavy chain is not a substrate for the β‐hexosaminidases present in the cell (Shin et al ., ).…”

“…The main glycoform in the FX‐KO line was GnGn in contrast to MMF in X‐KO and MMX in F‐KO. This difference can be explained with the findings of a recent study (Shin et al ., ), which showed that the trimming of terminal GlcNAc residues in GnGn(X/F) glycofoms by a specific β‐hexosaminidase is promoted by the presence of either β‐1,2‐xylose or α‐1,3‐fucose. The absence of these sugar residues from the endogenous N‐glycans of the FX‐KO line thus results in less trimming and consequently a higher content of GnGn.…”

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

“…Unlike in humans and higher organisms in which paucimannosylation is now being considered as an immunecentric N-glycan type (Loke et al, 2016) important for cellcell immune communication, for fighting pathogenic infections and for the proliferation of neural progenitor cells (Dahmen et al, 2015;Thaysen-Andersen et al, 2015), it has been established that plants constitutively and abundantly express plant-specific paucimannosidic N-glycan structures (Man 1-3 Xyl 0-1 Fuc 0-1 GlcNAc 2 ) as a common part of their glycoprotein repertoire (Dam et al, 2013;Shin et al, 2017). Trimming of GlcNAc caps of the developing complex/hybrid N-glycan intermediates to paucimannosidic structures is catalysed by the hexosaminidases (HEXO I, II, and III) localised in the plasma or vacuole membranes.…”