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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.


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.
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f2: 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.

Mentions: Soybean agglutinin (SBA) binding assays were used to screen for the presence of LDN carrying N-glycans on total apoplast proteins and showed that co-expression of native GalNAcT is sufficient for LDN synthesis in plants (Fig. 2a). Co-expression of the C4 epimerase increased the binding of SBA to total apoplast proteins, however increased binding to purified kappa-5 was not observed (data not shown). Strikingly, nstp4 almost completely blocks the synthesis of LDN. This could indicate that nstp-4 re-directs the UDP-GalNAc substrate to a Golgi compartment where LDN synthesis does not take place. MALDI-TOF MS analysis confirmed the presence of LDN motifs on the N-glycans released from purified kappa-5 (Fig. 2b). Approximately 30% of the isolated N-glycans from kappa-5 carried a single LDN motif as was shown by enzymatic digestion with β-N-acetylhexosaminidase and β-N-acetylglucosaminidase and subsequent MALDI-TOF MS product analysis (Supplemental Fig. 2).


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

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

f2: 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.
Mentions: Soybean agglutinin (SBA) binding assays were used to screen for the presence of LDN carrying N-glycans on total apoplast proteins and showed that co-expression of native GalNAcT is sufficient for LDN synthesis in plants (Fig. 2a). Co-expression of the C4 epimerase increased the binding of SBA to total apoplast proteins, however increased binding to purified kappa-5 was not observed (data not shown). Strikingly, nstp4 almost completely blocks the synthesis of LDN. This could indicate that nstp-4 re-directs the UDP-GalNAc substrate to a Golgi compartment where LDN synthesis does not take place. MALDI-TOF MS analysis confirmed the presence of LDN motifs on the N-glycans released from purified kappa-5 (Fig. 2b). Approximately 30% of the isolated N-glycans from kappa-5 carried a single LDN motif as was shown by enzymatic digestion with β-N-acetylhexosaminidase and β-N-acetylglucosaminidase and subsequent MALDI-TOF MS product analysis (Supplemental Fig. 2).

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.