The missense variant Pro311Leu affects expression, lysosomal targeting, processing, and enzymatic activity of HGSNAT. (A) Pro311Leu HGSNAT mutant lacks enzymatic activity. The N-acetyltransferase activity was measured in homogenates of primary cultured skin fibroblasts of healthy control donor (Control) or fibroblasts transduced with LV vectors encoding for the GFP-tagged WT HGSNAT (LV-HGSNAT) or the Pro311Leu mutant (LV-P311L-HGSNAT). The graph shows individual values, means, and SD of three independent experiments. P values were calculated by one-way ANOVA followed by Tukey post hoc test; ****, P < 0.0001. (B) The 75-kD (with EGFP tag) nonglycosylated precursor is the main HGSNAT form detected in the homogenates of cells transduced with the mutant virus, while the fully glycosylated 83-kD precursor and the cleaved 29-kD α-subunit are detected in cells expressing the WT enzyme. The 50-kD band represents a nonspecific cross-reacting protein also present in nontransduced cells. The panel shows a representative blot from three independent experiments yielding similar results. (C and D) The Pro311Leu HGSNAT mutant protein is not targeted to lysosomes. Representative confocal images show fibroblast cells transduced with LV vectors encoding for the GFP-tagged WT HGSNAT (LV-HGSNAT) and or the Pro311Leu mutant (LV-P311L-HGSNAT). (E) Cells grown on glass slides were labeled with Lysotracker Red for 1 h before fixation (C) or stained for the ER (anti-Calreticulin antibodies; D) or Golgi (anti-P115 antibodies; E; red). Scale bar equals 10 μm. Panels show typical images of triplicate experiments.