Molecular cloning of a gene encoding acid alpha-glucosidase from Tetrahymena pyriformis

Lysosomal acid alpha-glucosidase is essential for the degradation of glycogen to glucose in lysosomes. The ciliated protozoan Tetrahymena pyriformis secretes acid alpha-glucosidase into its culture medium. We have earlier reported the purification and characterization of acid alpha-glucosidase from T. pyriformis. The exact molecular mechanism of secretion of this enzyme has not yet been clarified. In the present study we have isolated a full length cDNA clone encoding acid alpha-glucosidase from T. pyriformis. The isolated clone (3019 bp) contained an open reading frame encoding 923 amino acids, and has an estimated molecular mass of 104 kDa. Northern blot analysis revealed that the isolated cDNA hybridized to a 2.8-kb mRNA transcript. N-terminal amino acids after the first methionine fulfilled the requirement of a signal peptide. The deduced amino acid sequence contains the amino acid sequences determined of several peptides derived from the purified enzyme, and was found to have 34% identity and 45% similarity with that of human lysosomal enzyme, with 75% identity in the 16 amino acids at the proposed active site.