Signal sequence and DNA-mediated expression of human lysosomal α-galactosidase A

Twelve complementary DNA clones for human lysosomal α- galactosidase A were isolated from an Okayama-Berg library constructed from SV40-transformed human fibroblasts. The identity of these clones was confirmed by complete colinearity of the nucleotide-deduced amino acid sequence with that determined by direct chemical sequencing of human placental α-galactosidase A. Hybridization of the α-galactosidase A cDNA to genomic DNA from individuals with varying numbers of X chromosomes as well as from interspecies somatic-cell hybrids showed only a single locus in the genoma at Xq 13.1 - Xq 22. One cDNA clone (pcD-AG210) contained the complete coding sequence for both the signal peptide and mature α-galactosidase A. The signal peptide of 31 amino acids contains the expected hydrophobic domains consisting of Leu-Gly-Cys-Ala-Leu-Ala-Leu and Phe-Leu-Ala-Leu-Val and has Ala at the signal peptidase cleavage site. Twelve out of 15 G residues flanking the 5' end of the cDNA in pcD-AG210 were removed and the truncated fragment was ligated into the original vector. This construct, pcD-AG502, encoded enzymatically active human α-galactosidase A in monkey COS cells.