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

Yun‐Ji Shin,Flavio Sádio,Alexandra Castilho,Friedrich Altmann,Herta Steinkellner,Martina Dicker,Tae‐Ho Kwon,Richard Strasser,Clemens Grünwald‐Gruber,Ulrike Vavra

doi:10.1111/pbi.12602

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.

Highlights

The majority of therapeutic proteins including monoclonal antibodies, hormones and lysosomal enzymes are glycoproteins
HEXO1 and HEXO3 coding regions were PCR amplified using cDNA derived from N. benthamiana leaf RNA
The obtained DNA sequence information suggested the amplification of a clone corresponding to a putative full-length HEXO1 homolog from N. benthamiana that was annotated in different N. benthamiana sequence databases (Figure S2)

Summary

Introduction

The majority of therapeutic proteins including monoclonal antibodies, hormones and lysosomal enzymes are glycoproteins. Recent progress in glyco-engineering of plants has shown that Nicotiana benthamiana is highly suitable for the production of recombinant glycoproteins with tailor-made N- and O-glycan structures (Steinkellner and Castilho, 2015; Strasser et al, 2014). The glyco-engineered DXT/FT mutant that exhibits stable down-regulation of the plant enzymes b1,2-xylosyl- and core a1,3-fucosyltransferase has been used for the transient expression of different therapeutically relevant glycoproteins with custom-made glycosylation (Castilho et al, 2014; Dicker et al, 2016; Jez et al, 2013; Loos et al, 2014, 2015; Schneider et al, 2014; Strasser et al, 2008, 2009; Wilbers et al, 2016). The DXT/FT plants are used to manufacture ZMapp the experimental antibody cocktail for treatment of acute Ebola virus infections (Qiu et al, 2014) and for the production of other monoclonal antibodies against infectious diseases that are currently under development (Loos et al, 2015; Zeitlin et al, 2016)

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