N-glycosylation of intrinsic and engineered N-X-S/T motifs by Pichia pastoris can be exploited to ligate the mannose receptor but reveals no gain in immunogenicity per se
N-glycosylation of intrinsic and engineered N-X-S/T motifs by Pichia pastoris can be exploited to ligate the mannose receptor but reveals no gain in immunogenicity per se
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DissertationPrüfungsdatum
07.10.2016Datum der Veröffentlichung
22.12.2016Erstgutachter
Burgdorf, SvenZweitgutachter
Thiele, ChristophMetadaten
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Inhalt
Vaccination has the power to eradicate viral diseases and is a promising approach to cure cancer. However, modern vaccination strategies repeatedly fail in inducing a robust cytotoxic CD8+ T cell response, which is needed to eradicate virus-infected or malignantly transformed cells. One way to improve this induction is the targeting of C-type lectin receptors on dendritic cells that lead to the presentation of antigens on MHC class I molecules to CD8+ T cells in a process termed cross-presentation. In addition, certain C-type lectin receptors have the capacity to induce DC maturation, which provides the second crucial signal to induce cytotoxic T cell activation. Yeasts such as Pichia pastoris produce N-glycans that are able to ligate such C-type lectin receptors and are thus generally considered to be immunostimulatory. N-glycosylation by P. pastoris might hence be exploited in vaccine strategies to promote both MHC I-restricted antigen presentation and DC maturation. However, the benefit of such a vaccination approach remains elusive since the particular effect of P. pastoris-derived N-glycans on a cellular and humoral response in vivo has not been investigated so far.
Here we tested whether it is possible to introduce N-glycans on proteins that are not glycosylated in their native state by recombinant expression in P. pastoris in order to target C-type lectin receptors and increase antigen cross-presentation.
For this purpose we expressed β-galactosidase (β-gal), a cytosolic Escherichia coli protein bearing several potential glycosylation sites, and a GFP-derivative with an engineered glycosylation site (NST-GFP) in P. pastoris. We show that both intrinsic and artificially designed N-glycosylation motifs are readily glycosylated after secretion by P. pastoris. We demonstrate that the attached N-glycans ligate the calcium-dependet carbohydrate recognition domains of the mannose receptor (MR), a C-type lectin receptor that mediates cross-presentation of the model antigen ovalbumin (OVA). Antigens internalized by bone marrow-derived dendritic cells (BM-DCs) were consistently routed to OVA-positive compartments related to cross-presentation. However, subsequent in vitro analysis revealed that P. pastoris-derived N-glycans had no immunostimulatory capacity on BM-DCs per se.
To elucidate the impact of such enforced N-glycosylation in vivo we subcutaneously immunized mice with N-glycosylated or enzymatically deglycosylated β-gal or NST-GFP. Surprisingly, the effect of N-glycosylation on in vivo cross-presentation proved to be dependent on the nature of the antigen. The presence of N-glycans increased the in vivo cytotoxicity against β-gal, whereas a decrease was observed against NST-GFP fused to an immunodominant OVA-epitope. Importain vivo ntly, for both antigens tested we observed a reduction of the humoral immune response in the presence of N-glycans as indicated by decreased serum IgG in comparison to the group immunized with deglycosylated proteins.
Our data demonstrate that P. pastoris can be used to tag non-glycoproteins with N-glycans that target the MR. However, a beneficial effect of such N-glycans on in vivo cross-presentation was dependent on the antigen, and the presence of N-glycans consistently decreased a humoral response. These findings have important implications on recombinant vaccines using Pichia pastoris as an expression system.
Here we tested whether it is possible to introduce N-glycans on proteins that are not glycosylated in their native state by recombinant expression in P. pastoris in order to target C-type lectin receptors and increase antigen cross-presentation.
For this purpose we expressed β-galactosidase (β-gal), a cytosolic Escherichia coli protein bearing several potential glycosylation sites, and a GFP-derivative with an engineered glycosylation site (NST-GFP) in P. pastoris. We show that both intrinsic and artificially designed N-glycosylation motifs are readily glycosylated after secretion by P. pastoris. We demonstrate that the attached N-glycans ligate the calcium-dependet carbohydrate recognition domains of the mannose receptor (MR), a C-type lectin receptor that mediates cross-presentation of the model antigen ovalbumin (OVA). Antigens internalized by bone marrow-derived dendritic cells (BM-DCs) were consistently routed to OVA-positive compartments related to cross-presentation. However, subsequent in vitro analysis revealed that P. pastoris-derived N-glycans had no immunostimulatory capacity on BM-DCs per se.
To elucidate the impact of such enforced N-glycosylation in vivo we subcutaneously immunized mice with N-glycosylated or enzymatically deglycosylated β-gal or NST-GFP. Surprisingly, the effect of N-glycosylation on in vivo cross-presentation proved to be dependent on the nature of the antigen. The presence of N-glycans increased the in vivo cytotoxicity against β-gal, whereas a decrease was observed against NST-GFP fused to an immunodominant OVA-epitope. Importain vivo ntly, for both antigens tested we observed a reduction of the humoral immune response in the presence of N-glycans as indicated by decreased serum IgG in comparison to the group immunized with deglycosylated proteins.
Our data demonstrate that P. pastoris can be used to tag non-glycoproteins with N-glycans that target the MR. However, a beneficial effect of such N-glycans on in vivo cross-presentation was dependent on the antigen, and the presence of N-glycans consistently decreased a humoral response. These findings have important implications on recombinant vaccines using Pichia pastoris as an expression system.
Schlagwörter
Pichia pastoris, Mannose Rezeptor, N-Glykosylierung
Klassifikation (DDC)
570 Biowissenschaften, Biologie 610 Medizin, GesundheitKreer, Christoph: N-glycosylation of intrinsic and engineered N-X-S/T motifs by Pichia pastoris can be exploited to ligate the mannose receptor but reveals no gain in immunogenicity per se. - Bonn, 2016. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-45465
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-45465
@phdthesis{handle:20.500.11811/6926,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-45465,
author = {{Christoph Kreer}},
title = {N-glycosylation of intrinsic and engineered N-X-S/T motifs by Pichia pastoris can be exploited to ligate the mannose receptor but reveals no gain in immunogenicity per se},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2016,
month = dec,
note = {Vaccination has the power to eradicate viral diseases and is a promising approach to cure cancer. However, modern vaccination strategies repeatedly fail in inducing a robust cytotoxic CD8+ T cell response, which is needed to eradicate virus-infected or malignantly transformed cells. One way to improve this induction is the targeting of C-type lectin receptors on dendritic cells that lead to the presentation of antigens on MHC class I molecules to CD8+ T cells in a process termed cross-presentation. In addition, certain C-type lectin receptors have the capacity to induce DC maturation, which provides the second crucial signal to induce cytotoxic T cell activation. Yeasts such as Pichia pastoris produce N-glycans that are able to ligate such C-type lectin receptors and are thus generally considered to be immunostimulatory. N-glycosylation by P. pastoris might hence be exploited in vaccine strategies to promote both MHC I-restricted antigen presentation and DC maturation. However, the benefit of such a vaccination approach remains elusive since the particular effect of P. pastoris-derived N-glycans on a cellular and humoral response in vivo has not been investigated so far.
Here we tested whether it is possible to introduce N-glycans on proteins that are not glycosylated in their native state by recombinant expression in P. pastoris in order to target C-type lectin receptors and increase antigen cross-presentation.
For this purpose we expressed β-galactosidase (β-gal), a cytosolic Escherichia coli protein bearing several potential glycosylation sites, and a GFP-derivative with an engineered glycosylation site (NST-GFP) in P. pastoris. We show that both intrinsic and artificially designed N-glycosylation motifs are readily glycosylated after secretion by P. pastoris. We demonstrate that the attached N-glycans ligate the calcium-dependet carbohydrate recognition domains of the mannose receptor (MR), a C-type lectin receptor that mediates cross-presentation of the model antigen ovalbumin (OVA). Antigens internalized by bone marrow-derived dendritic cells (BM-DCs) were consistently routed to OVA-positive compartments related to cross-presentation. However, subsequent in vitro analysis revealed that P. pastoris-derived N-glycans had no immunostimulatory capacity on BM-DCs per se.
To elucidate the impact of such enforced N-glycosylation in vivo we subcutaneously immunized mice with N-glycosylated or enzymatically deglycosylated β-gal or NST-GFP. Surprisingly, the effect of N-glycosylation on in vivo cross-presentation proved to be dependent on the nature of the antigen. The presence of N-glycans increased the in vivo cytotoxicity against β-gal, whereas a decrease was observed against NST-GFP fused to an immunodominant OVA-epitope. Importain vivo ntly, for both antigens tested we observed a reduction of the humoral immune response in the presence of N-glycans as indicated by decreased serum IgG in comparison to the group immunized with deglycosylated proteins.
Our data demonstrate that P. pastoris can be used to tag non-glycoproteins with N-glycans that target the MR. However, a beneficial effect of such N-glycans on in vivo cross-presentation was dependent on the antigen, and the presence of N-glycans consistently decreased a humoral response. These findings have important implications on recombinant vaccines using Pichia pastoris as an expression system.},
url = {https://hdl.handle.net/20.500.11811/6926}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-45465,
author = {{Christoph Kreer}},
title = {N-glycosylation of intrinsic and engineered N-X-S/T motifs by Pichia pastoris can be exploited to ligate the mannose receptor but reveals no gain in immunogenicity per se},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2016,
month = dec,
note = {Vaccination has the power to eradicate viral diseases and is a promising approach to cure cancer. However, modern vaccination strategies repeatedly fail in inducing a robust cytotoxic CD8+ T cell response, which is needed to eradicate virus-infected or malignantly transformed cells. One way to improve this induction is the targeting of C-type lectin receptors on dendritic cells that lead to the presentation of antigens on MHC class I molecules to CD8+ T cells in a process termed cross-presentation. In addition, certain C-type lectin receptors have the capacity to induce DC maturation, which provides the second crucial signal to induce cytotoxic T cell activation. Yeasts such as Pichia pastoris produce N-glycans that are able to ligate such C-type lectin receptors and are thus generally considered to be immunostimulatory. N-glycosylation by P. pastoris might hence be exploited in vaccine strategies to promote both MHC I-restricted antigen presentation and DC maturation. However, the benefit of such a vaccination approach remains elusive since the particular effect of P. pastoris-derived N-glycans on a cellular and humoral response in vivo has not been investigated so far.
Here we tested whether it is possible to introduce N-glycans on proteins that are not glycosylated in their native state by recombinant expression in P. pastoris in order to target C-type lectin receptors and increase antigen cross-presentation.
For this purpose we expressed β-galactosidase (β-gal), a cytosolic Escherichia coli protein bearing several potential glycosylation sites, and a GFP-derivative with an engineered glycosylation site (NST-GFP) in P. pastoris. We show that both intrinsic and artificially designed N-glycosylation motifs are readily glycosylated after secretion by P. pastoris. We demonstrate that the attached N-glycans ligate the calcium-dependet carbohydrate recognition domains of the mannose receptor (MR), a C-type lectin receptor that mediates cross-presentation of the model antigen ovalbumin (OVA). Antigens internalized by bone marrow-derived dendritic cells (BM-DCs) were consistently routed to OVA-positive compartments related to cross-presentation. However, subsequent in vitro analysis revealed that P. pastoris-derived N-glycans had no immunostimulatory capacity on BM-DCs per se.
To elucidate the impact of such enforced N-glycosylation in vivo we subcutaneously immunized mice with N-glycosylated or enzymatically deglycosylated β-gal or NST-GFP. Surprisingly, the effect of N-glycosylation on in vivo cross-presentation proved to be dependent on the nature of the antigen. The presence of N-glycans increased the in vivo cytotoxicity against β-gal, whereas a decrease was observed against NST-GFP fused to an immunodominant OVA-epitope. Importain vivo ntly, for both antigens tested we observed a reduction of the humoral immune response in the presence of N-glycans as indicated by decreased serum IgG in comparison to the group immunized with deglycosylated proteins.
Our data demonstrate that P. pastoris can be used to tag non-glycoproteins with N-glycans that target the MR. However, a beneficial effect of such N-glycans on in vivo cross-presentation was dependent on the antigen, and the presence of N-glycans consistently decreased a humoral response. These findings have important implications on recombinant vaccines using Pichia pastoris as an expression system.},
url = {https://hdl.handle.net/20.500.11811/6926}
}
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