Down‐regulated expression of plant‐specific glycoepitopes in alfalfa

Abstract

Summary Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N‐glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N‐glycosylation in alfalfa. The first was a knock‐down of two plant‐specific N‐glycan maturation enzymes, β1,2‐xylosyltransferase and α1,3‐fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human β1,4‐galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N‐acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock‐down and knock‐in strategies strongly, but not completely, inhibited the biosynthesis of α1,3‐fucose‐ and β1,2‐xylose‐containing glycoepitopes in transgenic alfalfa. However, recombinant human β1,4‐galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N‐glycans.

Reduction of N-linked xylose and fucose by expression of rat β1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression

“…Furthermore, plant glycoproteins contain paucimannosidic glycans, produced by the action of vacuolar or extracellular exoglycosidases, as well as a small proportion of Lewis a structures, caused by the addition of ␤1,3-linked galactose and ␣1,4-linked fucose to the terminal GlcNAc, which is thought to happen in the trans-golgi network (Saint-Jore-Dupas et al, 2006). Plant N-linked glycosylation has been remodeled using two main approaches: reducing or eliminating endogenous enzymatic activities of ␤1,2-xylosyltransferase (␤1,2-XylT) and ␣1,3-fucosyltransferase (␣1,3-FucT) activities in the plant glycosylation pathway or introducing new recombinant enzymatic activities such as ␤1,4-galactosyltransferase or ␤1,4-Nacetylglucosaminyltransferase III (GnTIII) activities into the cells (Bakker et al, 2001;Cox et al, 2006;Koprivova et al, 2004;Palacpac et al, 1999;Rouwendal et al, 2007;Sourrouille et al, 2008;Strasser et al, 2004Strasser et al, , 2008.…”

Plant glycans: friend or foe in vaccine development?

“…However, given the presence of gene families in plants, combined with the fact that homologous recombination in plants is very rare, the implementation of these approaches to other plant species is very difficult. As an alternative, gene silencing by RNAi of xylosyl-and fucosyltranferases has been established in several plant species, including Nicotiana benthamiana [33], Medicago sativa [34] and Lemna minor [35]. Through yet another approach, the addition of xylose and fucose was strongly inhibited in Nicotiana tabacum [36] via the expression of mutant galactosyltransferases, which appear to result in intermediate galactosylated N-glycan structures that are not the substrates for the xylosyl-and fucosyltransferases (Figure 1, Structure 9).…”

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

“…plants of A. thaliana were viable and exhibited no obvious phenotype under standard growth conditions (Strasser et al, 2004). In addition, the transgenic plants (L. minor, N. benthamiana, and M. sativa), in which Xyl and Fuc residues were eliminated by RNAi, did not exhibit any obvious morphological phenotype (Cox et al, 2006;Sourrouille et al, 2008;Strasser et al, 2008). In our study, we also found that the deficiency of XylT and FucT did not affect the morphology, growth rate, or growth status of tobacco BY2 cells.…”

“…More importantly, the glycan-optimized mAb exhibited high effector activities as compared to its Chinese hamster ovary (CHO)-derived homologs. Recently, several studies also showed that RNAi effectively prevented the function of β1,2-XylT and core α1,3-FucT in the whole plant of N. benthamiana and alfalfa, respectively (Sourrouille et al, 2008;Strasser et al, 2008). As compared to the whole plant, tobacco BY2 cells possess the merits of precise control over growth conditions, easy downstream processing, and no pollen contamination (Hellwig et al, 2004).…”