Isolation and characterisation of a recombinant, precursor form of lysosomal acid alpha-glucosidase

Glycogenosis type II (GSD II, Pompe disease) is an autosomal recessive lysosomal storage disease that results from a deficiency of acid alpha-glucosidase (GAA). Patients with this disorder are unable to break down lysosomal glycogen, which consequently accumulates in the lysosome. To evaluate enzyme replacement therapy for GSD II patients, we have expressed human GAA cDNA in Chinese hamster ovary-K1 cells utilising a vector that places the cDNA under the transcriptional control of the human polypeptide chain elongation factor la gene promoter. A clonal cell line that secreted precursor recombinant GAA at approximately 18 mg . l(-1). day(-1) was identified. The precursor recombinant GAA was purified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters similar to those of GAA purified from human tissues. The partial N-terminal amino acid sequence of recombinant GAA conformed to that derived from the nucleotide sequence of the cloned cDNA. The recombinant enzyme was taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocytosed GAA was localised to the lysosome and showed evidence of intracellular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were cultured in the presence of mannose 6-phosphate.