title: N-glycosylation converts non-glycoproteins into mannose receptor ligands and reveals antigen-specific T cell responses in vivo.
abstract: N-glycosylation is generally accepted to enhance the immunogenicity of antigens because of two main reasons. First, the attachment of glycans enables recognition by endocytic receptors like the mannose receptor (MR) and hence increased uptake by dendritic cells (DCs). Second, foreign glycans are postulated to be immunostimulatory and their recognition could induce DC activation. However, a direct comparison between the immunogenicity of N-glycosylated vs. de-glycosylated proteins in vivo and a direct effect of N-glycosylated antigens on the intrinsic capacity of DCs to activate T cells have not been assessed so far.To analyze whether enforced N-glycosylation is a suited strategy to enhance the immunogenicity of non-glycosylated antigens for vaccination studies, we targeted non-glycoproteins towards the MR by introduction of artificial N-glycosylation using the methylotrophic yeast Komagataella phaffii (previously termed Pichia pastoris). We could demonstrate that the introduction of a single N-X-S/T motif was sufficient for efficient MR-binding and internalization. However, addition of N-glycosylated proteins neither influenced DC maturation nor their general capacity to activate T cells, pointing out that enforced N-glycosylation does not increase the immunogenicity of the antigen per se. Additionally, increased antigen-specific cytotoxic T cell responses in vivo after injection of N-glycosylated compared to de-glycosylated proteins were observed but this effect strongly depended on the epitope tested. A beneficial effect of N-glycosylation on antibody production could not be detected, which might be due to MR-cross-linking on DCs and to concomitant differences in IL-6 production by CD4+ T cells.These observations point out that the effect of N-glycosylation on antigen immunogenicity can vary between different antigens and therefore might have important implications for the development of vaccines using K. phaffii.
authors.affiliation: Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Kreer
authors.affiliation: Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, 53105 Bonn, Germany.
authors.forename: Janina M
authors.initials: JM
authors.lastname: Kuepper
authors.affiliation: Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Zehner
authors.affiliation: Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Quast
authors.affiliation: Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Kolanus
authors.affiliation: Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, 53105 Bonn, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Schumak
authors.affiliation: Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Burgdorf
mesh_terms: D000818:Animals; D002450:Cell Communication; D049109:Cell Proliferation; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D000071497:Immunogenicity, Vaccine; D037181:Lectins, C-Type / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008213:Lymphocyte Activation; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010047:Ovalbumin / Q000276:immunology / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014614:Vaccines, Synthetic / Q000276:immunology / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D016207:Cytokines; D000939:Epitopes; D037181:Lectins, C-Type; D008024:Ligands; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011956:Receptors, Cell Surface; D014614:Vaccines, Synthetic; D010047:Ovalbumin; D001616:beta-Galactosidase
keywords: Komagataella phaffii; T cell activation; antigen presentation; cross-presentation; receptor-mediated endocytosis