Expression of Schistosome-derived Omega-1 with diantennary N-glycans carrying Lewis X motifs in Nicotiana benthamiana plants

Omega-1 is a T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and is a key factor for the induction of Th2 cell differentiation. Induction of Th2 responses by omega-1 is dependent on its RNase activity as well as the N-glycan mediated internalisation by antigen presenting cells. Omega-1 carries two core-difucosylated diantennary N-glycans containing terminal Lewis X motifs. Lewis X is known to be an immunomodulatory glycan due to its interaction with DC-SIGN. However, the main receptor required for omega-1 induced Th2 polarisation was shown to be the mannose receptor. The exact role of N-glycans on the immunomodulatory properties of helminth secreted glycoproteins remains to be elucidated. As the purification of a single glycoprotein from Schistosome egg extracts is relatively inefficient, a platform is required that enables high expression of these helminth glycoproteins with their native N¬-glycan structures. Here we show that Schistosome-derived omega-1 can be efficiently produced in Nicotiana benthamiana plants by means of agroinfiltration. Omega-1 was purified from the intercellular space (apoplast) of Nicotiana benthamiana leafs and was shown to have RNase activity. Omega-1 produced in wild-type plants carried diantennary N-glycans containing typical plant ß1,2-xylose and core a1,3-fucose, but lacked terminal GlcNAc's. By the controlled co-expression of two glycosyltransferases, omega-1 could be engineered to carry terminal Lewis X motifs. Furthermore, both wild-type omega-1 and omega-1 carrying Lewis X motifs were able to induce Th2 cell polarisation. All-in-all our results demonstrate that plants are a promising platform for the expression of helminth glycoproteins carrying engineered N-glycans, which opens up a new field of research.