Limits...
Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5385521&req=5

f1: 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.

Mentions: To achieve high expression levels of recombinant omega-1 and kappa-5, we applied an in-house codon optimization strategy to match the codon use of these helminth genes to highly expressed genes in plants. Expression was achieved by agroinfiltration of Nicotiana benthamiana plants. Accumulation of omega-1 (~30 kDa) and kappa-5 (~41 kDa) in crude extracts and apoplast fluids (leaf extracellular space) was analyzed by SDS-PAGE (Fig. 1a,b). The majority of both glycoproteins (~90%) was recovered from the apoplast, which indicates that both omega-1 and kappa-5 were secreted with remarkable efficiency. Only few endogenous plant proteins were present in these apoplast fluids, which facilitated single-step purification of >0.5 mg of omega-1 or kappa-5 per plant (3–4 gram fresh leaf material) by cation-exchange and Ni-NTA chromatography, respectively (Fig. 1c,d). The N-glycan composition of purified omega-1 and kappa-5 was assessed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis of released N-glycans. The predominant N-glycans on both proteins carry typical plant β1,2-xylose and core α1,3-fucose residues (Supplemental Fig. 1). However, omega-1 mainly displays terminal mannose residues on paucimannosidic glycans (Supplemental Fig. 1a), whereas the kappa-5 N-glycan trimannosyl core is substituted with GlcNAc residues (Supplemental Fig. 1b). So, even though both omega-1 and kappa-5 are isolated and purified from the apoplast, their N-glycan composition is strikingly different. Previously, we also observed the presence of high proportions (~50%) of Lewis A on the N-glycans of recombinant human IL-22 when isolated and purified from the apoplast of N. benthamiana plants14. 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.


Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385521&req=5

f1: 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.
Mentions: To achieve high expression levels of recombinant omega-1 and kappa-5, we applied an in-house codon optimization strategy to match the codon use of these helminth genes to highly expressed genes in plants. Expression was achieved by agroinfiltration of Nicotiana benthamiana plants. Accumulation of omega-1 (~30 kDa) and kappa-5 (~41 kDa) in crude extracts and apoplast fluids (leaf extracellular space) was analyzed by SDS-PAGE (Fig. 1a,b). The majority of both glycoproteins (~90%) was recovered from the apoplast, which indicates that both omega-1 and kappa-5 were secreted with remarkable efficiency. Only few endogenous plant proteins were present in these apoplast fluids, which facilitated single-step purification of >0.5 mg of omega-1 or kappa-5 per plant (3–4 gram fresh leaf material) by cation-exchange and Ni-NTA chromatography, respectively (Fig. 1c,d). The N-glycan composition of purified omega-1 and kappa-5 was assessed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis of released N-glycans. The predominant N-glycans on both proteins carry typical plant β1,2-xylose and core α1,3-fucose residues (Supplemental Fig. 1). However, omega-1 mainly displays terminal mannose residues on paucimannosidic glycans (Supplemental Fig. 1a), whereas the kappa-5 N-glycan trimannosyl core is substituted with GlcNAc residues (Supplemental Fig. 1b). So, even though both omega-1 and kappa-5 are isolated and purified from the apoplast, their N-glycan composition is strikingly different. Previously, we also observed the presence of high proportions (~50%) of Lewis A on the N-glycans of recombinant human IL-22 when isolated and purified from the apoplast of N. benthamiana plants14. 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.

View Article: PubMed Central - PubMed

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.

No MeSH data available.