Figure 6.

rhGAA and modified rhGAAs are differentially processed after internalization in fibroblasts of patients with Pompe disease. Fibroblasts of patients with Pompe disease were treated with 500 nM unmodified rhGAA, oxidized rhGAA, or rhGAA–AOAA for 16 h at 37 °C. Uptake media were replaced by growth media after 16 h, and cells were harvested at the indicated time points, lysed, and analyzed by Western blotting. A, Western blot analysis of unmodified rhGAA, oxidized rhGAA, and rhGAA–AOAA processed in cellulo over a 24-h time course compared with precursor enzyme (lane 1 of each blot) probed with a primary antibody against GAA (top) or actin (bottom). Precursor and processed bands are highlighted by arrows, and time points of harvest are indicated above each lane. B, to differentiate between proteolytic and glycan processing, a PNGase F digest was performed on lysates harvested at 24 h, and Western blots were probed with a primary antibody against GAA (top) or actin (bottom). PNGase F digested (right) and undigested lysates (middle) were compared with unprocessed enzymes (left). AOAA, aminooxyacetic acid; GAA, acid alpha-glucosidase; PNGase F, peptide:N-glycanase F; rhGAA, recombinant human GAA.