Effect of differential N-linked and O-linked mannosylation on recognition of fungal antigens by dendritic cells.
BACKGROUND
An experimental approach for improving vaccine efficacy involves targeting antigens to mannose receptors (MRs) on dendritic cells (DCs) and other professional antigen presenting cells. Previously, we demonstrated that mannosylated Pichia pastoris-derived recombinant proteins exhibited increased immunogenicity compared to proteins lacking mannosylation. In order to gain insight into the mechanisms responsible for this observation, the present study examined the cellular uptake of the mannosylated and deglycosylated recombinant proteins.


RESULTS
Utilizing transfected cell lines, roles for the macrophage mannose receptor (MMR, CD206) and DC-SIGN (CD209) in the recognition of the mannosylated, but not deglycosylated, antigens were demonstrated. The uptake of mannosylated antigens into murine bone marrow-derived DCs (BMDCs) was inhibited by yeast mannans (YMs), suggesting a mannose-specific C-type lectin receptor-dependent process, while the uptake of deglycosylated antigens remained unaffected. In particular, antigens with both N-linked and extensive O-linked mannosylation showed the highest binding and uptake by BMDCs. Finally, confocal microscopy studies revealed that both mannosylated and deglycosylated P. pastoris-derived recombinant proteins localized in MHC class II+ compartments within BMDCs.


CONCLUSIONS
Taken together with our previous results, these data suggest that increased uptake by mannose-specific C-type lectin receptors is the major mechanism responsible for the enhanced antigenicity seen with mannosylated proteins. These findings have important implications for vaccine design and contribute to our understanding of how glycosylation affects the immune response to eukaryotic pathogens.
Lam
Jennifer S
JS
Department of Medicine and the Immunology Training Program, Boston University School of Medicine, Boston, Massachusetts, United States of America.
Huang
Haibin
H
Levitz
Stuart M
SM
D000818:Animals; D017951:Antigen Presentation; D000946:Antigens, Fungal / Q000737:chemistry*; D001854:Bone Marrow Cells / Q000378:metabolism; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D037181:Lectins, C-Type / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008358:Mannose / Q000737:chemistry*; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D010843:Pichia / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D000946:Antigens, Fungal; D015815:Cell Adhesion Molecules; D037181:Lectins, C-Type; D008358:Mannose; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin