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.

Distinct Subcellular Compartments of Dendritic Cells Used for Cross-Presentation

“…The fact that MR-deficient BMDCs showed poor CP ability for glycosylated proteins, supports this hypothesis [145]. The role of sugar chains in CP are now under investigation, although evidence suggests that optimum glycosylation might function as a useful adjuvant for CP [147] (Figure 2).…”

The Mannose Receptor: From Endocytic Receptor and Biomarker to Regulator of (Meta)Inflammation

“…The di-aromatic YF motif is responsible for its intracellular trafficking into early endosomes (27). Specific intracellular routing of MR-internalized antigens into early endosomes (28)(29)(30)(31)(32)(33) was shown to have pronounced consequences for its role in antigen presentation. In fact, MRinternalized antigens are targeted towards a distinct nondegradative early endosome population, where they are rescued from lysosomal degradation and concomitant presentation on MHC II molecules (28).…”

Soluble mannose receptor induces pro-inflammatory macrophage activation and metaflammation

“…The MR as a member of the C-type lectin family has mainly been described as an endocytic receptor recognizing glycosylated antigens and mediating antigen processing and presentation 34 . However, the extracellular region of the MR (sMR) can be shed by metalloproteases and released as a soluble protein in the extracellular space.…”