Pharmacological chaperones facilitate the post-ER transport of recombinant N370S mutant β-glucocerebrosidase in plant cells: Evidence that N370S is a folding mutant

• Published in: Molecular genetics and metabolism 2012-07, Vol.106 (3), p.323-329
• Main Authors: Babajani, Gholamreza, Tropak, Michael B., Mahuran, Don J., Kermode, Allison R.
• Format: Article
• Language: English
• Subjects: Biological Transport; Catalytic Domain; Cells, Cultured; Endoplasmic Reticulum - metabolism; Gaucher Disease - enzymology; Gaucher Disease - genetics; Glucosylceramidase - genetics; Glucosylceramidase - metabolism; Humans; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Mutation; N370S mutation; Pharmacological chaperones; Plant Cells - metabolism; Plants, Genetically Modified; Protein Folding; Protoplasts; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Secretion; Tobacco BY2 cells; β-glucocerebrosidase
• ISSN: 1096-7192; 1096-7206
• DOI: 10.1016/j.ymgme.2012.04.018

Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a N370S missense mutation in the GCase protein. As part of a larger endeavor to study the fate of mutant human proteins expressed in plant cells, the N370S mutant protein along with the wild-type- (WT)-GCase, both equipped with a signal peptide, were synthesized in transgenic tobacco BY2 cells, which do not possess lysosomes. The enzymatic activity of plant-recombinant N370S GCase lines was significantly lower (by 81–95%) than that of the WT-GCase lines. In contrast to the WT-GCase protein, which was efficiently secreted from tobacco BY2 cells, and detected in large amounts in the culture medium, only a small proportion of the N370S GCase was secreted. Pharmacological chaperones such as N-(n-nonyl) deoxynojirimycin and ambroxol increased the steady-state mutant protein levels both inside the plant cells and in the culture medium. These findings contradict the assertion that small molecule chaperones increase N370S GCase activity (as assayed in treated patient cell lysates) by stabilizing the enzyme in the lysosome, and suggest that the mutant protein is impaired in its ability to obtain its functional folded conformation, which is a requirement for exiting the lumen of the ER. ► A novel plant system is used to study mutant glucocerebrosidase (GCase). ► Recombinant N370S GCase shows impaired ER exit and reduced catalytic activity. ► Pharmacological chaperones (PCs) promote post-ER transport of N370S GCase. ► Our findings contradict PC-mediated stabilization of N370S GCase in lysosomes. ► Ambroxol is an effective active-site inhibitor of N370S GCase at neutral (ER) pH.