N. benthamiana ß-galactosidases acting on glycoproteins

The use of plants to produce human therapeutic proteins is receiving worldwide interest. In addition to a low risk for human pathogen contamination, plants have the potential for providing both cost effectiveness and scalability. Validation of plants as powerful production factories for pharmaceutical proteins has been provided by: (i) the approval of a plant-made recombinant enzyme to treat a lysosomal storage disease, (ii) the encouraging story of a plant-produced antibody cocktail as a potentially life-saving drug during the recent Ebola outbreak and (iii) the plant-derived vaccine candidates against various influenza strains, to name a few. Proteins expressed in a cell frequently undergo glycosylation, a structural modification that entails the covalent addition of carbohydrate moieties to specific amino acids. There is mounting evidence that protein pharmaceuticals need to be properly glycosylated to exhibit optimum therapeutic efficacy. Production of recombinant proteins in plants leads to non-mammalian-like glycosylation, which can be overcome by glyco-engineering approaches resulting in tailored glycosylation. The adverse activity of plant glycosidases trimming N-glycan sugar chains limits the use of plants as production system for recombinant glycoproteins. A lack of fundamental knowledge about this large group of plant enzymes prevents the design of effective strategies for glyco-engineering. The present project aims to identify a specific glycosidase that cleaves off terminal galactose residues from glycoproteins. Candidates for this -galactosidase have been identified in the secreted proteome of N. benthamiana plants. The molecular and biological function of the -galactosidases will be investigated using biochemical, cell and molecular biological methods. Once fully characterized, their contribution to galactose trimming from recombinant glycoproteins will be investigated in N. benthamiana plants devoid of the relevant enzyme activity. The results expected from the current proposal will make relevant contributions to plant biotechnology and plant physiology as many -galactosidases play a specific role during the remodeling of the plant cell wall.

The use of plants to produce human therapeutic proteins known as Plant Molecular farming (PMF) is receiving worldwide interest. In addition to a low risk for human pathogen contamination, plants have the potential for providing both cost effectiveness and scalability. The majority of therapeutic proteins undergo glycosylation, a structural modification that entails the addition of sugar chains (glycans) to specific amino acids. There is mounting evidence that protein pharmaceuticals need to be properly glycosylated to exhibit optimum therapeutic efficacy. When produced in plants, these proteins have non-mammalian-like glycosylation, but this can be overcome by glyco-engineering resulting in tailored glycosylation. One important sugar on protein glycans is galactose. However, the fact that plant cells have enzymes (galactosidases, BGAL) that trim galactose from glycans, limits the use of plants as production system for recombinant glycoproteins. We have identified a BGAL and showed that this enzyme is responsible for removing terminal galactose sugars from proteins expressed in Nicotiana benthamiana plants. We have then used (i) RNA interference and (ii) genome editing to abolish the BGAL activity and showed that these two strategies are efficient to increase the amounts of fully galactosylated glycans on plant- glycoproteins. The results are highly encouraging for a future establishment of yet another plant-based expression platform, depleted of -galactosidase activity, and thus enabling human-galactosylation on a diverse group of glycoproteins. In early 2020 we started a second line of investigation to used plants to fight the COVID-19 pandemic. In collaboration with scientists from "Medicines for Future (M4F)" initiative our goal was "to produce efficient and affordable drugs for people around the globe by innovating drug discovery and scalable manufacturing of drugs in plants". Among the various options to block SARS-CoV-2 infections by drugs, the inhibition of the binding of the virus to its receptor (ACE2) on human cells is one of the most promising approaches for COVID-19 therapy. This is mainly because emerging new virus variants are characterized by increased binding of their spike protein to the ACE2 receptor. In contrast to other antivirals such as antibodies, ACE2 decoys show increased binding to virus variants, making the decoy more active in neutralizing the virus. We have developed a decoy molecule that consists of an ACE2 fused to the Fc domain of an antibody (ACE2-Fc). When produced in plants, ACE2-Fc is decorated with simplified glycans that seem to improve its binding to SARS-CoV-2 and enhanced virus-neutralizing activity, compared to conventional biotechnological production systems.