Galactosylation in Plants

The majority of therapeutic proteins are glycoproteins and require the attachment of sugar structures (i.e. glycosylation) to attain full therapeutic activity. Current manufacturing methods based on mammalian cell culture do not allow for the control of glycosylation and produce a mixture of different glycoforms; some of which are more active then others and some of which have no activity at all. This proposal aims at the generation of a plant based expression system that allows an efficient production of recombinant proteins with high glycoform uniformity. We aim at the generation of proteins with consistent beta 1,4-galactosylation. The presence of this terminal mammalian-type N-glycan residue, naturally absent in plants, may not only influence the efficacy of a protein, galactosylated structures also serve as acceptor substrate for sialylation, the final step of human glycosylation. Recently we generated a glycosylation mutant of Nicotiana benthamiana dXT/FT (a tobacco related species) that lacks unwanted plant specific N-glycan residues and allows for the generation of mammalian proteins with a human like glycan structure required for efficient beta 1,4-galactosylation. Preliminary results obtained by transient expression of the mammalian enzyme beta 1,4 galactosyltransferase (GalT) in dXT/FT exhibit a quantitative galactosylation of a reporter protein. Based on these results dXT/FT will serve as parental line for the stable transformation of various GalT constructs along with other endogenous glycosylation enzymes that might negatively interfere with efficient galactosylation, to generate a line enabling the production of recombinant glycoproteins with consistent galactosylated structures. To this end a highly galactosylated therapeutic glycoprotein (a monoclonal antibody) will be expressed in these lines and tested with regard to its biological activity. The availability of glyco-engineered plants enabling the generation of recombinant mammalian glycoproteins with a consistent galactosylation proved an alternative for mammalian cell culture and allow for the production of therapeutic glycoproteins in plants with increased in vivo efficacy. Moreover, consistent galactosylation of glycoproteins, which is not achieved by mammalian cell based expression systems, will expand our ability to conduct other biological important studies e.g. structure-function investigations and will be a further step towards the reconstruction of protein sialylation in plants.

The major achievement of this project was to further establish plants as a versatile production platform for the expression of pharmaceutically relevant proteins with enhanced efficacy compared to the established mammalian cell based systems. This was achieved by the modulation of the N-glycosylation in plants towards human structures. Glycosylation, the attachment of sugar to the protein backbone, significantly contributes to the biological activity of many proteins. In a series of publication we have demonstrated the enormous plasticity of plants which allows them to tolerate modifications in their protein N-glycosylation. In parallel we showed both in vitro and in vivo the influence of single N-glycan residues to the function/efficacy of a protein. Plant N-glycan processing enzymes are arranged along the early secretory pathway, forming an assembly line to facilitate the step-by-step modification of oligosaccharides on glycoproteins. Thus, these enzymes provide excellent tools to study the so far largely unknown intracellular transport of proteins in plants. In depths studies of targeting sequences which are typical for glycosylation enzymes exhibited that certain amino acids are responsible for sub-Golgi protein targeting. Our investigations led to a better understanding of basic intracellular protein transport mechanisms and shed new light in the assembly of cellular organels in plants (i.e. Golgi apparatus). The success of the project is evidenced by a series of publications in international high ranking scientific journal. Importantly, the results of the project attracted the pharmaceutical industry and were the basis for novel larger Consortium project with industrial participation (Laura Bassi Center of Expertise).