Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants

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.

Figure 1. Plant-based production of helminth glycoproteins with tailored N-glycans.

(a-b) SDS-PAGE and Coomassie blue staining of crude extracts (CE) and apoplast fluids (AF) from omega-1 (ω1), kappa-5 (κ5) or empty vector (EV) infiltrated plants reveals efficient secretion of both omega-1 (a) and kappa-5 (b) into the leaf extracellular space (apoplast). (c-d) Efficient secretion enables single-step purification from the leaf apoplast fluid by cation exchange chromatography (CEX) for omega-1 (c) or Ni-NTA chromatography for kappa-5 (d). (e) A schematic overview of the successive N-glycan modifying steps in the plant Golgi-system. MNSI: Golgi-α-mannosidase I; GnTI: N-acetyl-glucosaminyltransferase I; GMII: Golgi- α-mannosidase II; GnTII: N-acetyl-glucosaminyltransferase II; XYLT: β1,2-xylosyltransferase; FUT11/12: core α1,3-fucosyltransferase. The plant N-glycosylation machinery was engineered by introducing (hybrid) glycosyltransferases that allow the synthesis of LeX or LDN-F motifs.

Figure 2. Engineering of LDN carrying N-glycans in plants.

Co-expression of different combinations of native GalNAcT, UDP-GalNAc transporters (sqv-7, nstp-4 and nstp-5) and/or C4 epimerase was performed to determine which of these genes are required for in planta engineering of LDN carrying N-glycans. (a) Soybean agglutinin (SBA) binding assay on total soluble proteins from apoplast fluids reveals that expression of native GalNAcT from C. elegans is sufficient for the synthesis of LDN carrying N-glycans. (b) MALDI-TOF MS N-glycan profile for kappa-5 upon co-expression of native GalNAcT reveals the synthesis of LDN motifs. (c) MALDI-TOF MS N-glycan profile for kappa-5 upon co-expression of trans-Golgi-targeted sialFucT and native GalNAcT reveals the synthesis of LDN-F motifs. Sugar residues are placed between brackets when a MS peak represents multiple N-glycan structures of identical mass.

Figure 3. Controlled expression of sialGalT enables synthesis of non-hybrid LeX glycan motifs.

Engineering of LeX on omega-1 was attempted with different expression strategies for sialGalT. N-glycan composition was analyzed by MALDI-TOF-MS. (a) N-glycan profile upon co-expression of d35S:sialFucT and d35S:sialGalT both in the pBIN vector. (b) N-glycan profile upon co-expression of d35S:sialFucT and Gpa2:sialGalT both in the pBIN vector. The weaker constitutive Gpa2 promoter was chosen to reduce sialGalT expression. (c) N-glycan profile upon co-expression of d35S:sialFucT and Gpa2:sialGalT, but the latter being expressed using the pHYG vector. The pHYG vector was chosen as it generally yields more protein compared to pBIN.

Figure 4. Th2 priming capacity of plant-produced omega-1.

(a) Human monocyte-derived dendritic cells (DCs) were pulsed with indicated reagents for 48 hours, after which they were co-cultured with naïve CD4⁺ T cells to evaluate their T cell-polarizing capacity. Th2/Th1 polarization was determined based on percentage of T cells staining positive for IL-4/IFN-γ by intracellular staining 10 days later. Data are normalized to DCs pulsed with LPS and represent mean+/−S.E.M. of 4 independent experiments. (b) DCs were stimulated with indicated reagents for 6 hours in the presence of neutralizing antibody against DC-SIGN or control antibody after which IL-10 mRNA expression was determined. Expression values are normalized to LPS-stimulated DCs and GAPDH was used as reference gene. Data represent mean+/− S.E.M. of 3 independent experiments. SEA, S. mansoni soluble egg antigens; n-ω1, native omega-1 (2 and 0.5 ug/ml); p-ω1, plant-derived omega-1 (8, 2 and 0.5 ug/ml) that carries WT plant N-glycans; p-ω1^H58F, p-ω1 with H58F mutation in the catalytic site that abrogates RNase activity; p-ω1^LeX, plant-derived omega-1 that carries the LeX motif.

Figure 5. The LeX glycan motif on omega-1 contributes to Th2 polarisation in vivo.

(a) Plant-derived omega-1 glycoforms and mouse serum albumin (MSA) were injected into mouse footpads and 7 days later cytokine responses were determined in draining lymph nodes (LNs) following antigen-specific (Ag) or polyclonal restimulation (ConA) of LN cells ex vivo. Cytokine levels were determined in (a) supernatants of 3 day restimulated LN cells or (b) intracellularly in CD4⁺ T cells following PMA/Ionomycin restimulation. One representative of 2 experiments is shown. Data represent mean+/− S.E.M. of 3 to 4 mice per group (*P < 0.05). p-ω1, plant-derived omega-1 that carries WT plant N-glycans; p-ω1^LeX, plant-derived omega-1 that carries the LeX motif.