ISOLATION AND CHARACTERIZATION 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 alph a-glucosidase (GAA). Patients with this disorder are unable to break d own 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 c ell line that secreted precursor recombinant GAA at approximately 18 m g . l(-1). day(-1) was identified. The precursor recombinant GAA was p urified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters simi lar to those of GAA purified from human tissues. The partial N-termina l amino acid sequence of recombinant GAA conformed to that derived fro m the nucleotide sequence of the cloned cDNA. The recombinant enzyme w as taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocyto sed GAA was localised to the lysosome and showed evidence of intracell ular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were culture d in the presence of mannose 6-phosphate.