Fig 2: Glycosylation acts as a buffer for CMG2 ectodomain mutations. A) Graphic showing the disulfide bridge C39-C218 (blue) present in CMG2 WT B) Fibroblast cells were treated or not with tunicamycin for 16h and TCE were analyzed by SDS-PAGE and Western Blot. Representative Western Blot with control fibroblasts and patient fibroblasts. Calnexin serves as a loading control. C) Quantification of total protein levels. CMG2 levels are normalized to WT protein level without tunicamycin treatment, which was set to 100%. Statistics were calculated using an unpaired t-test. Errors represent standard deviation. n = 3. * p=0.05

Fig 3: Localization of TEM8 and CMG2 glycosylation mutants. A) Immunofluorescence of transiently transfected HeLa cells. Cells were transfected for 48h with the respective cDNAs. Cells were fixed, permeabilized and stained for TEM8-HA, endogenous BiP and Hoechst. Scalebars represent 10 µm. B) Immunofluorescence of transiently transfected HeLa cells. Cells were transfected for 48h with the respective cDNAs. Cells were fixed, permeabilized and stained for CMG2-V5, endogenous BiP and Hoechst. Scalebars represent 10 µm.

Fig 4: Loss of glycosylation affects binding of Anthrax toxin to TEM8 but not to CMG2. A and C) HeLa cells were transfected for 48h with the respective cDNAs. Cells were treated for 1h at 4°C with 500 ng/ml PA83 and shifted to 37°C for 10 min to induce cleavage and heptamerization. Immunoprecipitates against TEM8-HA/CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against PA and TEM8-HA/CMG2-V5. Control cells are non-transfected. D50A is a binding deficient mutant that serves as a negative control. B and D) HeLa cells were transfected for 48h with the respective cDNAs. Cells were lysed and incubated for 1h at 4°C with 1 µg/ml PA83. Immunoprecipitates against TEM8-HA/CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against PA and TEM8-HA/CMG2-V5. Control cells are non-transfected. D50A is a binding deficient mutant that serves as a negative control.

Fig 5: Number and localization of glycan sidechains determine trafficking efficiency of TEM8 and CMG2. A) Endoglycosidase H (EndoH) treatment on TEM8 and CMG2 single mutants. HeLa cells were transfected for 48h with the respective cDNAs. 40 µg of cell extracts were treated or not with EndoH as described before. Samples were analyzed by SDS-PAGE and Western Blotting. B) Quantification of surface biotinylation experiments to determine amount of TEM8 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n = 3. * p=0.05, ** p=0.01, *** p=0.001 C) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against TEM8-HA. D) Quantification of surface biotinylation experiments to determine amount of CMG2 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n = 3. * p=0.05, ** p=0.01, *** p=0.001 E) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against CMG2-V5.