Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

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Abstract
Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, .alpha.-galactosidase A (.alpha.-Gal A; .alpha.-D-galactosidase galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of .alpha.-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human .alpha.-Gal A was isolated from a .lambda.gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an .alpha.-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 .times. 107 plaques. Of these, only one clone (designated .lambda.AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5'' end of the 1250-base-pair EcoRI insert of clone .lambda.AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone .lambda.AG18 appears to contain the full-length coding region of the processed, enzymtically active .alpha.-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.