The fibroblasts were obtained from Coriell Institute for Medical Research (Camden, NJ) or established in our laboratory after informed consent was obtained. Western Blot Analysis Protein samples for Western blot were prepared as previously described (Shimada et al. which show different response to PC, were overexpressed in HEK293T cells, bortezomib improved the activity of M519V, S529V, and C647W in them (1.3C5.9-fold). These results indicate that bortezomib enhances the activity of some PC-unresponsive GAA mutants as well as PC-responsive mutants. Introduction Pompe disease (OMIM HNPCC2 232300) is an autosomal recessive lysosomal storage disorder arising from a deficiency of acid -glucosidase (GAA), leading to progressive accumulation of glycogen in multiple tissues including skeletal and cardiac muscles (Hers 1963). The clinical presentation of Pompe disease spans a broad spectrum of severity, ranging from a rapid infantile-onset form to a slow late-onset form. The infantile-onset form displays a near complete loss of GAA activity and is characterized by generalized muscle weakness and severe cardiomyopathy, leading to premature death in the first year of life (van den Hout et al. 2003). The late-onset form typically displays some residual GAA activity and manifests as slowly progressive respiratory failure and muscle weakness without cardiac involvement (van der Harpagide Ploeg and Reuser 2008). In 2006, enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) was approved for the treatment of Pompe disease. ERT extends life expectancy, improves cardiac muscle pathology and walking distance, and stabilizes pulmonary function in patients with Pompe disease (Kishnani et al. 2007; van der Ploeg et al. 2010), but the clearance of accumulated glycogen in skeletal muscle remains Harpagide an ongoing challenge. ERT also facilitates the formation of anti-rhGAA antibodies that can reduce the efficacy of treatment (de Vries et al. 2010; Kishnani et al. 2010; Banugaria et al. 2011). Recently, pharmacological chaperone (PC) therapy has emerged as a promising treatment for several lysosomal storage disorders including Pompe disease. PCs are small molecules that stabilize misfolded enzymes and prevent their degradation, thereby rescuing their intracellular trafficking and activity (Fan 2008). In Pompe disease, imino sugars such as deoxynojirimycin (DNJ) and em N /em -butyl-DNJ (NB-DNJ) have shown to improve the function of several mutant GAAs (Okumiya et al. 2007; Parenti et al. 2007; Flanagan et al. 2009). We previously reported that the proteasome inhibitor bortezomib as well as imino sugars can exert a positive effect on mutant GAA in fibroblasts from a patient with Pompe disease carrying the c.546G T Harpagide mutation (Shimada et al. 2011). In addition, bortezomib, which is a therapeutic drug for multiple myeloma, has recently shown to be a safe treatment to induce the effective reduction of anti-rhGAA antibody titers in patients with infantile Pompe disease who received ERT (Banugaria et al. 2013). Thus, bortezomib is attracting interest as a novel treatment for Pompe disease. However, it has not been fully characterized as an enzyme enhancement molecule that is effective for multiple GAA mutations. In this study, we investigated the effect of bortezomib treatment on mutant GAAs in patient fibroblasts and transiently expressed cells. Materials and Harpagide Methods Chemicals and Antibodies Bortezomib was purchased from Toronto Research Chemicals Inc. (North York, Canada). Protease inhibitor cocktail (PIC) was obtained from Roche Diagnostics (Indianapolis, IN) Anti-GAA antibody was a gift from Genzyme Corporation (Cambridge, MA). Anti-LAMP2 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–actin antibody was purchased from Cell Signaling Technologies (Beverly, MA), and 4-methylumbelliferyl -d-glucopyranoside and all other chemicals were obtained from Sigma (St. Louis, MO). Cell Cultures HEK293T cells and skin fibroblasts were maintained at subconfluent densities in Dulbeccos Modified Eagles Medium supplemented with 10% fetal bovine serum at 37C and 5% CO2. The fibroblasts were obtained from Coriell Institute for Medical Research (Camden, NJ) or established in our laboratory after informed consent was obtained. Western Blot Analysis Protein samples for Western blot were prepared as previously described (Shimada et al. 2011). Briefly, fibroblasts were extracted with 50?mM TrisCHCl, pH?7.5 containing 2% SDS, and PIC. Cell lysates were resolved by SDSCPAGE on 4C20% acrylamide gradient gels and transferred onto a nitrocellulose membrane. The membranes were blocked with 50?mM TrisCHCl, pH?7.5 containing 150?mM NaCl, 0.1% gelatin, 0.1% casein, and 0.05% Tween 20, and then incubated with each primary antibody. After a brief washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibody (Nichirei Corp., Tokyo, Japan) and detected using Immunostar LD (Wako Pure Chemicals, Tokyo, Japan). Immunocytochemistry Immunofluorescence staining against both GAA and LAMP2 was performed according to a procedure previously described (Shimada et al. 2011). Fibroblasts were incubated with the primary antibodies and visualized by using Alexa 488-conjugated anti-rabbit IgG and Alexa 594-conjugated anti-mouse IgG secondary antibodies (Invitrogen, Carlsbad, CA) followed by counterstaining with DAPI (Vector Laboratories). Site-Directed Mutagenesis and Expression of Mutated GAA in HEK293T Cells Human GAA cDNA was purchased from OriGene Technologies (Rockville, MD). Site-directed mutagenesis was performed to introduce mutations into.