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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.


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-ω1H58F, p-ω1 with H58F mutation in the catalytic site that abrogates RNase activity; p-ω1LeX, plant-derived omega-1 that carries the LeX motif.
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f4: 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-ω1H58F, p-ω1 with H58F mutation in the catalytic site that abrogates RNase activity; p-ω1LeX, plant-derived omega-1 that carries the LeX motif.

Mentions: To evaluate the functionality of plant-produced helminth glycoproteins with different engineered glycans we investigated the immunomodulatory capacity of recombinant omega-1 in a number of functional assays. First we established that omega-1 produced in unmodified N. benthamiana (p-ω1) is a functional RNase capable of degrading liver RNA at a protein concentration of 0.5 μg/ml (Fig. S5). This is comparable to native and HEK-cell produced omega-1 under similar assay conditions as described by Everts and co-workers4. Next, a strongly increased percentage of IL-4 positive T cells was observed in a co-culture of human T cells with dendritic cells (DCs) that were pre-conditioned with p-ω1 or with LeX-modified recombinant omega-1 from plants (p-ω1LeX) in the presence of LPS (Fig. 4a). This Th2 polarizing effect was similar to that of purified native schistosomal omega-1 (n-ω1). In accordance to previous observations using HEK-cell produced omega-14, the Th2 inducing capacity of p-ω1 is dependent on its RNase activity, as p-ω1 with a H58F mutation in the active site of the T2 RNase domain is not capable of Th2 polarisation (Fig. 4a). Previously, the in vitro Th2 inducing capacity of omega-1 has also been shown to be dependent on binding to the mannose receptor (MR) on DCs4. Binding of omega-1 by MR is facilitated by the plant wild-type paucimannosidic glycans on p-ω1 as well as by the LeX substituted glycans on omega-1 from LeX-engineered plants (p-ω1LeX) (Supplemental Fig. 6).


Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants
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-ω1H58F, p-ω1 with H58F mutation in the catalytic site that abrogates RNase activity; p-ω1LeX, plant-derived omega-1 that carries the LeX motif.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: 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-ω1H58F, p-ω1 with H58F mutation in the catalytic site that abrogates RNase activity; p-ω1LeX, plant-derived omega-1 that carries the LeX motif.
Mentions: To evaluate the functionality of plant-produced helminth glycoproteins with different engineered glycans we investigated the immunomodulatory capacity of recombinant omega-1 in a number of functional assays. First we established that omega-1 produced in unmodified N. benthamiana (p-ω1) is a functional RNase capable of degrading liver RNA at a protein concentration of 0.5 μg/ml (Fig. S5). This is comparable to native and HEK-cell produced omega-1 under similar assay conditions as described by Everts and co-workers4. Next, a strongly increased percentage of IL-4 positive T cells was observed in a co-culture of human T cells with dendritic cells (DCs) that were pre-conditioned with p-ω1 or with LeX-modified recombinant omega-1 from plants (p-ω1LeX) in the presence of LPS (Fig. 4a). This Th2 polarizing effect was similar to that of purified native schistosomal omega-1 (n-ω1). In accordance to previous observations using HEK-cell produced omega-14, the Th2 inducing capacity of p-ω1 is dependent on its RNase activity, as p-ω1 with a H58F mutation in the active site of the T2 RNase domain is not capable of Th2 polarisation (Fig. 4a). Previously, the in vitro Th2 inducing capacity of omega-1 has also been shown to be dependent on binding to the mannose receptor (MR) on DCs4. Binding of omega-1 by MR is facilitated by the plant wild-type paucimannosidic glycans on p-ω1 as well as by the LeX substituted glycans on omega-1 from LeX-engineered plants (p-ω1LeX) (Supplemental Fig. 6).

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.