Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants

Ruud H P Wilbers,Lotte B Westerhof,Aska Goverse,Kim Van Noort,Nicole N Driessen,Jaap Bakker,Katja Obieglo,Gabriele Schramm,Geert Smant,Bart Everts,Sonja I Gringhuis,Maria Yazdanbakhsh,Hermelijn H Smits,Cornelis H Hokke,Arjen Schots

doi:10.1038/srep45910

Abstract

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

Highlights

N-glycosylation machinery was engineered by introducing glycosyltransferases that allow the synthesis of LeX or LDN-F motifs
Helminth glycoproteins are efficiently produced in N. benthamiana
These differences in N-glycan composition most likely arise from different intrinsic protein characteristics, which in the case of omega-1 and kappa-5 could be a difference in sensitivity towards endogenous β-hexosaminidase activity

Summary

Introduction

N-glycosylation machinery was engineered by introducing (hybrid) glycosyltransferases that allow the synthesis of LeX or LDN-F motifs. In the last two decades, plants have emerged as a versatile expression platform for the production of recombinant proteins[12] This holds for the expression of glycoproteins, as plants are highly compliant to engineered adaptations of their endogenous N-glycosylation machinery[13]. Some of the typical characteristics of the limited plant glycome match those of helminths, including the non-mammalian N-glycan core modifications and the lack of sialylation. All these characteristics together inspired us to investigate if plants can be used to produce biologically active helminth glycoproteins with a defined and tailored N-glycan composition. The immunomodulatory functionalities of plant-produced omega-1 with and without the LeX motif were assessed in vitro and in vivo to confirm the bioactivity of the glycoprotein as well as the critical contribution of properly engineered glycosylation

Methods

Results

Conclusion