MOLECULAR-CLONING OF A GENE ENCODING ACID ALPHA-GLUCOSIDASE FROM TETRAHYMENA-PYRIFORMIS

Lysosomal acid alpha-glucosidase is essential for the degradation of g lycogen to glucose in lysosomes. The ciliated protozoan Tetrahymena py riformis secretes acid alpha-glucosidase into its culture medium. We h ave earlier reported the purification and characterization of acid alp ha-glucosidase from T. pyriformis. The exact molecular mechanism of se cretion of this enzyme has not yet been clarified. In the present stud y we have isolated a full length cDNA clone encoding acid alpha-glucos idase from T. pyriformis. The isolated clone (3019 bp) contained an op en reading frame encoding 923 amino acids, and has an estimated molecu lar mass of 104 kDa. Northern blot analysis revealed that the isolated cDNA hybridized to a 2.8-kb mRNA transcript. N-terminal amino acids a fter the first methionine fulfilled the requirement of a signal peptid e. The deduced amino acid sequence contains the amino acid sequences d etermined of several peptides derived from the purified enzyme, and wa s found to have 34% identity and 45% similarity with that of human lys osomal enzyme, with 75% identity in the 16 amino acids at the proposed active site.