Incorporation of desmocollin‐2 into the plasma membrane requires N‐glycosylation at multiple sites

Figures

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  Figure 1. Structural overview about the ECD of DSC2. (A) Molecular structures of the EC1 (5J5J) and EC2‐5 fragments of DSC2 (5ERP, https://www.rcsb.org) 10. The sugar residues of the N‐glycosylation sites are shown in dark blue, and the mannose residues of the O‐mannosylation sites are shown in light blue. The protein backbone is colored in green, Ca2+ ions are shown in orange, and cysteine residues forming disulfide bridges are labeled yellow. (B‐E) Schematic overviews about the generated DSC2 constructs. N‐glycosylation sites are shown in red, and O‐mannosylation sites are labeled in blue. Disulfide bridges are indicated by yellow and red lines. (B) DSC2 wild‐type construct. (C) N‐glycosylation‐ deficient DSC2 constructs. (D) O‐mannosylation‐ deficient constructs. (E) Disulfide bridge‐deficient DSC2 constructs.

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  Figure 2. Cellular localization of N‐glycosylation‐ deficient DSC2 mutants. (A) Representative fluorescence images and corresponding magnifications of transfected HT1080 cells expressing wild‐type DSC2‐eYFP or N‐glycosylation‐ deficient mutants are shown (green). Endogenous DSG2 was stained using anti‐DSG2 antibodies and is shown in red. Scale bars represent 50 μm. (B) Quantitative analysis of transfected cells with DSC2 membrane localization. Nonparametric Kruskal–Wallis test was used for statistical analysis. ***P < 0.001; n = 6. Error bars indicate mean ± SD. (C) Representative fluorescence images of transfected HL‐1 cells expressing wild‐type DSC2‐eYFP or N‐glycosylation‐ deficient mutants are shown (green). Endogenous F‐actin was stained using phalloidin conjugated to Texas Red. Scale bars represent 10 μm. White arrows indicate localization within the Golgi apparatus, and gray arrowheads indicate plasma membrane localization.

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  Figure 3. Cellular localization of O‐mannosylation‐ deficient DSC2 mutants. (A) Representative fluorescence images of transfected HT1080 cells expressing wild‐type DSC2‐eYFP or O‐mannosylation‐ deficient mutants are shown (green). Endogenous desmoglein‐2 is shown in red. Scale bars represent 50 μm. (B) Quantification of transfected HT1080 cells with DSC2 membrane localization revealed no significant differences between wild‐type and disulfide bridge‐deficient DSC2 mutants. Nonparametric Kruskal–Wallis test was used for statistical analysis. n = 3. Error bars indicate mean ± SD. (C) Representative fluorescence images of transfected HL‐1 cells expressing wild‐type DSC2‐eYFP (green) and disulfide bridge‐deficient mutants are shown. Endogenous F‐actin is labeled with phalloidin conjugated with Texas Red (red). Scale bars represent 10 μm.

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  Figure 4. Cellular localization of disulfide bridge‐deficient DSC2 mutants. (A) Representative fluorescence images of transfected HT1080 cells expressing wild‐type DSC2‐eYFP or disulfide bridge‐deficient mutants are shown (green). Endogenous desmoglein‐2 labeled with antibodies is shown in red. Scale bars represent 50 μm. (B) Quantification of transfected HT1080 cells with DSC2 membrane localization revealed no significant differences between wild‐type and disulfide bridge‐deficient DSC2 mutants. Nonparametric Kruskal–Wallis test was used for statistical analysis. n = 3. Error bars indicate mean ± SD. (C) Representative fluorescence images of transfected HL‐1 cells expressing wild‐type DSC2‐eYFP or disulfide bridge‐deficient mutants are shown (green). Endogenous F‐actin is labeled with phalloidin conjugated with Texas Red (red). Scale bars represent 10 μm.

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  Figure 5. Colocalization analysis of compartment trackers and the N‐glycosylation‐ deficient DSC2 mutants. HT1080 cells were transfected with (A) wild‐type and (B) mutant DSC2‐p.N166Q‐p.N392Q‐p.N546Q‐p.N629Q‐p.T338V‐p.T340‐p.T558V‐p.T560V constructs. Cell organelles were stained using compartment trackers. Colocalization of DSC2‐eYFP and the compartment trackers were evaluated using Leica Application Suite X software. Pearson's correlation of DSC2 mutant: 0.83; overlap coefficient: 0.88; colocalization rate: 85.4%. Scale bars represent 10 μm. (C) Schematic overview. ER, endoplasmic reticulum; G, Golgi apparatus; N, nucleus; PM, plasma membrane. (Images for ER and G are licenced from shutterstock.de)