Molecular Biology of the Alpha-L-Fucosidase Gene and Fucosidosis

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Abstract
Human α-L-fucosidase, a lysosomal enzyme, hydrolyzes α-L-fucose from glycolipids and glycoproteins. Its activity is deficient in human fucosidosis an autosomal recessive disease. In order to understand the molecular basis of this lysosomal storage disorder we have cloned several cDNAs coding for human α-L-fucosidase from a human hepatoma and a human liver cDNA library constructed in Agtl 1. Compiling the cDNA sequences of these clones we have identified 1,829 base pairs (bp) encoding human a-L-fucosidase. This includes an open reading frame of 1,172 bp, a consensus polyadenylation signal AAT AAA and a poly (A)^+ tail. The sequence is incomplete at the 5'-end, and clones encoding the amino terminus of the native protein, the propeptide and leader signal have not yet been isolated. The open reading frame encodes for 390 amino acids with a calculated Mr of 45,557. This represents 78-95% of the mature processed α-L-fucosidase. The availability of these cDNA clones has enabled us to map the structural gene for α-L-fucosidase to chromosome lp34.1- lp36.1 by Southern blot analysis of DNA from human-rodent somatic cell hybrids and by in situ hybridization. Furthermore, a Pvu II restriction fragment length polymorphism (RFLP) has been identified at the human α-L-fucosidase gene locus. Analysis of mRNA by Northern blotting gives a major species of 2.25 kb. In 4 patients with fucosidosis no mRNA signal was detected and Western blots gave no immunoreactive enzyme. Southern blotting after Eco RI digestion in two fucosidosis families revealed a banding abnormality (extra 6-kb band). In one family 2 affected sibs were heterozygous for this abnormality; in another the patient was homozygous for the same abnormality. Using two probes which flanked the single Eco RI site in the open reading frame, the abnormality was pinpointed to this site. Studies are underway to determine whether this abnormality is responsible for fucosidosis in humans.

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