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

Summary 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 Glc NA c 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 ( HEXO s) 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 HEXO 1 is a vacuolar protein and HEXO 3 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 HEXO 3 demonstrated that core α1,3‐fucose enhances the trimming of Glc NA c residues from the Fc domain of human IgG. Finally, using RNA interference, we show that suppression of HEXO 3 expression can be applied to increase the amounts of complex N ‐glycans on plant‐produced human α1‐antitrypsin.