Fig. 2: WT IDS is subjected to proteolytic cleavage in the Golgi apparatus.

a Schema of IDS tagged with Flag (N-terminus) and V5 (C-terminus). Arrows indicate the cleavage sites. ss: signal sequence. b Western blotting analysis in HeLa cells expressing Flag-IDS-V5 (N = 3). Anti-Flag antibodies could not detect N-terminal fragments (5 kDa) generated by the cleavage (left panel). The C-terminal fragments (11 kDa) were also not detected by anti-V5 antibodies (right panel). Asterisk: nonspecific bands. c Immunofluorescence staining analysis using HeLa cells expressing Flag-IDS-V5. Immunoreactivities of precursor IDS were found in the calnexin-positive ER and GM130-positive cis-Golgi apparatus but not in the LAMP1-positive lysosome. Bars: 10 µm. d Immunofluorescence staining analysis using HeLa cells expressing Flag-IDS-V5. Cells were treated with 100 nM bafilomycin A1 (lysosomal protease inhibitor) for 12 h. Precursor IDS detected by anti-Flag and -V5 antibodies were not observed in lysosomes. Bar: 10 µm. e Western blotting analysis in HeLa cells expressing WT IDS (N = 3). The processed mature forms increased by treatment of cells with 1 ng/mL brefeldin A (BFA) for 8 h. f Quantification of relative protein levels of mature forms of IDS in (e) (mean ± SD, N = 3, Student’s t-test, ***P < 0.001). g Putative model of glycosylation and proteolytic cleavage of IDS. Precursor IDS is modified by glycosylation in the ER and Golgi apparatus. The glycosylated IDS is subjected to proteolytic cleavage in the Golgi apparatus and subsequently moves into the lysosome as mature forms to degrade GAGs. In contrast, IDS mutants are modified by glycosylation in the ER but accumulate in the ER. GAGs glycosaminoglycans