Karl F. Johnson

Molecular defect in processing α-fucosidase in fucosidosis

Abstract In normal human skin fibroblasts, an enzymatically active 53,000-dalton form of α-fucosidase is processed to a 50,000-dalton mature form. Endoglycosidase-H treatment of [ 35 S]methionine pulse-chase labelled material immunoprecipated with a polyclonal antibody to α-L-fucosidase (Andrews-Smith & Alhadeff, Biochim. Biophys. Acta 715 : 90–96 (1982)) indicated the removal of a single N -linked oligosaccharide unit from both precursor and nature form of α-L-fucosidase. Tunicamycin pretreatment of normal fibroblasts indicated that no other N -linked oligosaccharide units were present. Studies on fibroblasts from patients with less than 5% of normal α-L-fucosidase activity (fucosidosis) showed 8 of 11 patients synthesized no detectable α-fucosidase protein whereas 2 synthesized normal amounts of 53,000 dalton precursor, none of the mature 50,000 dalton form was detectable and one contained small amounts of cross-reacting material. This is the first evidence for processing of α-L-fucosidase in cells and the first precise evidence of a molecular defect in fucosidosis.

Synthesis and processing of lysosomal α-fucosidase in cultured human fibroblasts

Abstract The lysosomal enzyme α- l -fucosidase from human skin fibroblasts is synthesized as a 53 kDa glycosylated precursor which is then proteolytically processed to a 50 kDa mature form. This was confirmed by pulse-chase labeling studies with chase times up to 72 h. In fibroblasts treated with 1-deoxymannojirimycin to prevent trimming of high mannose oligosaccharides, endoglycosidase H (endo H) treatment completely deglycosylated and reduced the size of immunoprecipitated α-fucosidase by 4–5 kDa, suggesting the presence of two oligosaccharide units. Endoglycosidase H and endo F studies on untreated α-fucosidase suggested the presence of one complex-type and one high mannose-type unit, and that the final processing from 53 to 50 kDa did not involve the removal of carbohydrate. Processing was inhibited by the thiol proteinase inhibitor Ep-459, but not by Ep-475 or leupeptin. Since Ep-459 treatment increased both α-fucosidase activity (3-fold) and the amount of immunoprecipitable α-fucosidase protein in normal human skin fibroblasts, this suggests a role for cysteine-like proteinases either directly or indirectly in lysosomal hydrolase processing and turnover. Subcellular fractionation studies revealed that the proteolytic processing of the 53 kDa precursor to the 50 kDa mature form occurred in the lysosome, or some other dense organelle.

Molecular heterogeneity in lysosomal storage diseases

The availability of specific antibodies and cDNA probes for lysosomal hydrolases has revealed unexpected heterogeneity among the human inherited lysosomal storage diseases. Using alpha-fucosidase and N-acetyl-beta-D-hexosaminidase deficiency variants as examples, it has been determined that a lysosomal hydrolase deficiency can result from DNA deletion mutations, failure to synthesize mRNA because of defective splicing, posttranslational defects in assembly, and synthesis of a precursor enzyme that is prematurely proteolytically degraded through lack of a protective protein. In some cases (fucosidosis), the different genotypes cannot be distinguished phenotypically, whereas in others (beta-hexosaminidoses) the phenotypes can range from infantile neurodegeneration through juvenile motor neuron disease to adult neurodysfunction. Biochemical studies on both diseases have revealed several distinct genotypes. We show that some forms of fucosidosis result from unstable enzyme that can be stabilized by protease inhibitors, whereas partial beta-hexosaminidase deficiencies cannot be corrected by these protease inhibitors.