Signal sequence and DNA-mediated expression of human lysosomal alpha-galactosidase A.
Twelve complementary DNA clones for human lysosomal alpha-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 alpha-galactosidase A. Hybridization of the alpha-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 genome at Xq 13.1-Xq 22. One cDNA clone (pcD-AG210) contained the complete coding sequence for both the signal peptide and mature alpha-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 fifteen 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 alpha-galactosidase A in monkey COS cells.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D005696:Galactosidases / Q000235:genetics*; D005815:Genetic Code; D006801:Humans; D006822:Hybrid Cells; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D009693:Nucleic Acid Hybridization; D021382:Protein Sorting Signals / Q000235:genetics*; D000519:alpha-Galactosidase / Q000235:genetics*
D021382:Protein Sorting Signals; D004247:DNA; D005696:Galactosidases; D000519:alpha-Galactosidase