Identification of the base-pair substitution responsible for a human acid alpha glucosidase allele with lower "affinity" for glycogen (GAA 2) and transient gene expression in deficient cells.

The lysosomal enzyme termed acid alpha glucosidase (GAA), or acid maltase, is genetically polymorphic, with three alleles segregating in the normal population. The rarer GAA 2 allozyme has a lower affinity for glycogen and starch but not for lower-molecular-weight substrates. The GAA 2 allozyme can be detected by "affinity" electrophoresis in starch gel, since the lower affinity for the starch matrix results in a more rapid migration to the anode. Previously, we have isolated and sequenced the cDNA for GAA and transiently expressed the cDNA in deficient fibroblasts. In order to determine the molecular basis for the GAA 2 allozyme, we constructed a cDNA and a genomic DNA library from a GAA 2 cell line and determined the nucleotide sequence of the coding region. Only a single base-pair substitution of an A for a G at base-pair 271 was found, resulting in substitution of asparagine for aspartic acid at codon 91. This amino acid substitution is consistent with the more basic pI of the GAA 2 enzyme. The base-pair substitution also abolishes a Taq-I site, predicting the generation of a larger DNA fragment. This larger Taq-I fragment was also seen in two other individuals expressing the GAA 2 allozyme. A 5' fragment containing the base-pair substitution was ligated to the remaining 3' cDNA from a GAA 1 allele and cloned into an expression vector, and the hybrid cDNA was transiently expressed in SV40-transformed GAA-deficient fibroblasts. The enzyme activity exhibited the altered mobility of the GAA 2 allozyme, as demonstrated by electrophoresis in starch gel.(ABSTRACT TRUNCATED AT 250 WORDS)