1992;267:1719C1726. to MR-cross-linking on DCs and to concomitant variations 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 as right designation for this strain [10, 11]. Because of its potential to target C-type lectin receptors, fungal glycosylation is considered to be immunogenic [12, 9, 13], although direct AMAS evidence for this hypothesis remains elusive. Moreover, in vaccination studies, and assessed the immunogenic properties of N-glycosylated proteins depended on the nature AMAS of the antigen. RESULTS AMAS Glycan-dependent binding of the MR to non-glycoproteins after manifestation by K. phaffii Since the model antigen OVA is definitely internalized very efficiently from the MR [21], we first analyzed whether binding of OVA to the MR was mediated by glycosylation. Consequently, we performed a binding assay using chimeric proteins consisting of the Fc region of a human being IgG1 attached to the CTLD4-7 of the MR (MR-CTLD) or to the N-terminal region of the MR consisting of the CR, FN II region and CTLD1-2 (MR-Nterm) [22]. We could demonstrate that acknowledgement of OVA is definitely specifically mediated by MR-CTLD (Number ?(Figure1A),1A), whereas MR-Nterm acknowledged collagen as previously described [22]. Such binding was confirmed by far western blot using MR-CTLD (Number ?(Figure1B).1B). Since CTLD4-7 are responsible for acknowledgement of mannosylated glycans [4, 23], we treated OVA with the glycosidase PNGase F and shown that acknowledgement of OVA by MR-CTLD indeed was mediated by glycans (Number ?(Number1A1A and ?and1B).1B). Additionally, MR-mediated uptake of OVA by bone marrow-derived DCs (BM-DCs) was inhibited when OVA was de-glycosylated, confirming glycan-dependent binding of the MR to OVA (Number ?(Number1C1C). Open in a separate window Number 1 Glycan-dependent binding of the MR to -gal indicated in K. phaffiiA. Binding of MR-CTLD, MR-Nterm or the AMAS Fc part IkBKA of a human being IgG isotype control to OVA, PNGase-treated OVA, collagen or BSA was determined by ELISA. B. OVA and PNGase F-treated OVA were separated by SDS-PAGE and subjected to coomassie staining or far-western blot analysis using 50 g/ml MR-CTLD. C. BM-DCs were incubated with fluorochrome-labeled OVA or PNGase F-treated OVA and AMAS analyzed by circulation cytometry. D. Plan of -gal indicated by by affinity chromatography. F. Binding of MR-CTLD, MR-Nterm or isotype control to -gal, OVA, collagen or BSA was determined by ELISA. G. as with F) using glycosylated or de-glycosylated -gal. H. Far-western blot analysis using MR-CTLD on untreated, PNGase F-treated Endo H-treated -gal from and and purified the protein from your supernatant by affinity chromatography (Number ?(Figure1E).1E). Binding assays shown that purified -gal indeed was identified by MR-CTLD (Number ?(Figure1F).1F). Such binding was prevented in the presence of mannan, a competitive inhibitor of MR-mediated endocytosis, or in the absence of calcium (Supplementary Number 1), pointing out that binding to -gal indeed depended on the C-type lectin activity of the MR. Consistently, MR binding was abrogated after treatment of -gal with PNGase F or Endo H (Number 1G, 1H). Furthermore, -gal indicated in and purified from was not identified by MR-CTLD (Number ?(Number1H).1H). These data demonstrate that indeed glycosylates putative N-glycosylation sites in proteins that are normally non-glycosylated and therefore creates ligands of the MR. MR-mediated uptake of mannosylated -gal and focusing on in early endosomes Subsequently, we monitored whether acknowledgement of glycosylated -gal from the MR resulted in.