Summary A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N‐glycosylation (i.e. the presence of β1,2‐xylosylation and core α1,3‐fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down‐regulation of the endogenous β1,2‐xylosyltransferase (XylT) and α1,3‐fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco‐related plant species widely used for recombinant protein expression. Three glyco‐engineered lines with significantly reduced xylosylated and/or core α1,3‐fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild‐type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core α1,3‐fucose residues in their N‐glycans were produced. Notably, 2G12 produced in XylT/FucT‐RNAi plants was found to contain an almost homogeneous N‐glycan species without detectable xylose and α1,3‐fucose residues. Plant‐derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)‐derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N‐glycan structure.

Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals and yeast. In contrast, late N-glycan maturation steps in the Golgi differ significantly in plants giving rise to complex N-glycans with β1,2-linked xylose, core α1,3-linked fucose and Lewis A-type structures. While the essential role of N-glycan modifications on distinct mammalian glycoproteins is already well documented, we have only begun to decipher the biological function of this ubiquitous protein modification in different plant species. In this review, I focus on the biosynthesis and function of different protein N-linked glycans in plants. Special emphasis is given on glycan-mediated quality control processes in the ER and on the biological role of characteristic complex N-glycan structures.

The plant glycosyltransferases, L L1,2-xylosyltransferase (XylT) and core K K1,3-fucosyltransferase (FucT), are responsible for the transfer of L L1,2-linked xylose and core K K1,3-linked fucose residues to glycoprotein N-glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modi¢cation of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete de¢ciency of XylT and FucT. These plants lack antigenic protein-bound Nglycans and instead synthesise predominantly structures with two terminal L LN-acetylglucosamine residues (GlcNAc 2 Man 3 -GlcNAc 2 ).

In eukaryotes, class I a-mannosidases are involved in early N-glycan processing reactions and in N-glycan-dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ERtype a-mannosidase I (MNS3) and the two Golgi-a-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected a1,2-mannose residue from Man 9 GlcNAc 2 , MNS1/2 readily removed three a1,2-mannose residues from Man 8 GlcNAc 2 . Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man 8 GlcNAc 2 accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man 9 GlcNAc 2 , demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I a-mannosidases in wildtype seedlings resulted in a similar root phenotype. These findings show that class I a-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuca1-4(Galb1-3) GlcNAc-R] structures. This process involves the sequential attachment of b1,3-galactose and a1,4-fucose residues by b1,3-galactosyltransferase and a1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, b1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana b1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring b1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with a1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of b1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.