Figure 2. Recombinant enzyme replacement in cultured human fibroblasts derived from patients with GM2 gangliosidoses.

(A) Intracellular MUGS–degrading activity in SD fibroblasts after treatment with modB or mod2B. Before enzyme treatment, fibroblasts were treated (+) or not treated (–) with M6P. Error bars show mean ±SEM (n = 3). ANOVA with a Tukey post-hoc test; ***P < 0.001. (B) Vital staining of the β-Hex activity with HMDER-βGlcNAc. SD fibroblasts were treated with modB or mod2B, followed by incubation with HMDER-βGlcNAc. Blue, nuclei; yellow, HMDER. Scale bar: 100 μm. (C) Hex delivery imaging. AFO-modB, AFO-mod2B, or AFO-NHS was added to SD fibroblasts. Living cells were viewed with a confocal fluorescent microscope. Blue, nuclei; red, AFO. Scale bars: 20 μm. (D and E) TSD fibroblasts were treated with modB or mod2B and immunostained for GM2 (green) and Hex proteins (red), and relative contents were examined by confocal laser scanning microscopy. Scale bars: 20 μm. Error bars show mean ±SEM (n = 6–7). ANOVA with a Tukey post-hoc test; *P < 0.05. (F) GM2-ELISA. TSD fibroblasts were treated with modB or mod2B to evaluate the intracellular GM2-degrading activity. Error bars show mean ±SEM (n = 7). ANOVA with a Tukey post-hoc test; *P < 0.05, ***P < 0.001. (G) The protease sensitivities of modB and mod2B incorporated in SD fibroblasts. Immunoblotting of cell extracts with anti-NAG(A) after enzyme replacement. β-Actin blot is from duplicate samples run on a parallel gel. Each lane contained 7.5 μg of protein.