Figure 1.
Lysosomal HEX-catalyzed paucimannose is exposed on the cell surface via lysosomal exocytosis. (A) Paucimannosidic proteins identified in HeLa, THP-1, and HEK293 cell lines. Paucimannosidic proteins are plotted on the x-axis, and the detected number of paucimannosidic peptides is plotted on the y-axis. Proteins of known lysosomal origin are indicated in red. Insert: Gene ontology (GO) enrichment analysis (cell component, CC) based on the paucimannosidic proteins detected in HeLa, THP-1, and HEK293 cells. See also Tables S1 and S2 for data. (B) Representative MS2 spectra of paucimannosidic peptides derived from LAMP1 (UniProt ID: P11279) and PSAP (UniProt ID: P07602) in HeLa cells. (C) Quantitative glycomics analysis of the relative abundance and the ratio of the paucimannose precursor Man3GlcNAc4Fuc1 and the paucimannosidic Man3GlcNAc2Fuc1 structure in WT, HEXA-, and HEXB-KO cells. Bar plots: ratio metric assessment of the relative abundance of relevant glycan structures in WT versus KO cells. (n = 3, independently analyzed samples, mean ± SD, unpaired one-tailed t test; *P ≤ 0.05, **P ≤ 0.01). (D) Representative confocal laser scanning microscopy (CLSM) images of WT HeLa cells stained by Mannitou Ab under unpermeabilized conditions with or without pitstop2 treatment. Bar, 10 µm. (E) Quantitative analysis of cell surface paucimannose level with or without pitstop2 treatment using Mannitou Ab. RFI, relative fluorescent intensity. The mean ± SD fluorescent intensity of mock treatment was set to unity (n = 15 images; N = 3 biological replicates; unpaired two-tailed t test; ***P ≤ 0.001). See also Fig. S1 A. (F) Representative CLSM images of WT HeLa cells stained by Mannitou Ab under unpermeabilized conditions with or without brefeldin A or vacuolin-1 treatment. Bar, 10 µm. (G) Quantitative analysis of cell surface paucimannose level with or without brefeldin A or vacuolin-1 treatment. The mean ± SD fluorescent intensity of mock treatment was set to unity (n = 15 images; N = 3 biological replicates; One-way ANOVA with Dunnett correction; ns, P > 0.05; **P ≤ 0.01). (H) Representative CLSM images of WT HeLa cells stained by Mannitou Ab under unpermeabilized conditions with or without ionomycin treatment. Bar, 10 µm. (I) Quantitative analysis of cell surface paucimannose level with or without ionomycin treatment. The mean ± SD fluorescent intensity of ionomycin treatment was set to unity (n = 15 images; N = 3 biological replicates; unpaired two-tailed t test; ***P ≤ 0.001). (J) Immunoblotting of biotinylated plasma membrane proteins. Cells were incubated in Hanks’ balanced salt solution (HBSS) containing Mg2+ and Ca2+ with or without ionomycin for 5 min at 37°C before biotinylation. After the isolation of biotinylated cell surface proteins by NeutrAvidin beads, CD44, LAMP1, and paucimannosidic proteins were detected by antibodies. Source data are available for this figure: SourceData F1.

Lysosomal HEX-catalyzed paucimannose is exposed on the cell surface via lysosomal exocytosis. (A) Paucimannosidic proteins identified in HeLa, THP-1, and HEK293 cell lines. Paucimannosidic proteins are plotted on the x-axis, and the detected number of paucimannosidic peptides is plotted on the y-axis. Proteins of known lysosomal origin are indicated in red. Insert: Gene ontology (GO) enrichment analysis (cell component, CC) based on the paucimannosidic proteins detected in HeLa, THP-1, and HEK293 cells. See also Tables S1 and S2 for data. (B) Representative MS2 spectra of paucimannosidic peptides derived from LAMP1 (UniProt ID: P11279) and PSAP (UniProt ID: P07602) in HeLa cells. (C) Quantitative glycomics analysis of the relative abundance and the ratio of the paucimannose precursor Man3GlcNAc4Fuc1 and the paucimannosidic Man3GlcNAc2Fuc1 structure in WT, HEXA-, and HEXB-KO cells. Bar plots: ratio metric assessment of the relative abundance of relevant glycan structures in WT versus KO cells. (n = 3, independently analyzed samples, mean ± SD, unpaired one-tailed t test; *P ≤ 0.05, **P ≤ 0.01). (D) Representative confocal laser scanning microscopy (CLSM) images of WT HeLa cells stained by Mannitou Ab under unpermeabilized conditions with or without pitstop2 treatment. Bar, 10 µm. (E) Quantitative analysis of cell surface paucimannose level with or without pitstop2 treatment using Mannitou Ab. RFI, relative fluorescent intensity. The mean ± SD fluorescent intensity of mock treatment was set to unity (n = 15 images; N = 3 biological replicates; unpaired two-tailed t test; ***P ≤ 0.001). See also Fig. S1 A. (F) Representative CLSM images of WT HeLa cells stained by Mannitou Ab under unpermeabilized conditions with or without brefeldin A or vacuolin-1 treatment. Bar, 10 µm. (G) Quantitative analysis of cell surface paucimannose level with or without brefeldin A or vacuolin-1 treatment. The mean ± SD fluorescent intensity of mock treatment was set to unity (n = 15 images; N = 3 biological replicates; One-way ANOVA with Dunnett correction; ns, P > 0.05; **P ≤ 0.01). (H) Representative CLSM images of WT HeLa cells stained by Mannitou Ab under unpermeabilized conditions with or without ionomycin treatment. Bar, 10 µm. (I) Quantitative analysis of cell surface paucimannose level with or without ionomycin treatment. The mean ± SD fluorescent intensity of ionomycin treatment was set to unity (n = 15 images; N = 3 biological replicates; unpaired two-tailed t test; ***P ≤ 0.001). (J) Immunoblotting of biotinylated plasma membrane proteins. Cells were incubated in Hanks’ balanced salt solution (HBSS) containing Mg2+ and Ca2+ with or without ionomycin for 5 min at 37°C before biotinylation. After the isolation of biotinylated cell surface proteins by NeutrAvidin beads, CD44, LAMP1, and paucimannosidic proteins were detected by antibodies. Source data are available for this figure: SourceData F1.

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