Abstract
The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD). This inherited disorder is characterized by the accumulation of acidic monosaccharides in lysosomal compartments and a concomitant decrease of their buoyant density. We demonstrate that C-terminal trimming of the lysosomal cysteine proteinase cathepsin B is inhibited in SASD fibroblasts. This late event in the biosynthesis of cathepsin B normally takes place in mature lysosomes, suggesting an impaired biogenesis of these organelles in SASD cells. When normal fibroblasts are loaded with sucrose, which inhibits transport from late endosomes to lysosomes, C-terminal cathepsin B processing is prevented to the same extent. Further characterization of the terminal endocytic compartments of SASD cells revealed properties usually associated with late endosomes/prelysosomes. In addition to a decreased buoyant density, SASD "lysosomes" show a reduced acidification capacity and appear smaller than their normal counterparts. We conclude that the accumulation of small non-diffusible compounds within endocytic compartments interferes with the formation of mature lysosomes and that the acidic environment of the latter organelles is a prerequisite for C-terminal processing of lysosomal hydrolases.
Highlights
The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD)
We have addressed the impact of an altered endocytic environment on the function and the biogenesis of these organelles in fibroblasts from patients suffering from sialic acid storage disease (SASD)
Partial co-localization of FITC-dextran with the SASD is characterized by the aberrant accumulation of sialic acid in late endocytic compartments and a concomitant decrease of their buoyant density
Summary
Materials—The human skin fibroblast lines GM 5520 (ISSD-1), GM 8496 (Salla-1), GM 5521, and GM 5522 (from the unaffected parents of GM 5520, controls 1 and 2, respectively) were obtained from the Human Genetic Mutant Cell Repository (Camden, NJ). The ratio between the two fluorescence intensities obtained was used to estimate the pH of the terminal endocytic compartment by comparison with a standard curve generated with FITC-dextran-loaded microsomes at defined pH To this end, microsomes were labeled by loading of fibroblasts with FITC-dextran as described above. Determination of the Acidification Capacity of Endocytic Compartments in Vitro—SASD and normal fibroblasts were labeled with FITCdextran, disrupted, and fractionated by differential centrifugation as described above. Digital Image Analysis of Terminal Endocytic Compartments—To determine the size of the terminal endocytic compartments, fibroblasts were labeled with FITC-dextran as described above, fixed with 3% paraformaldehyde in phosphate-buffered saline, and examined by fluorescence microscopy. The membranes were probed with affinity purified rabbit antibodies to human cathepsin B or its C-terminal extension and incubated with horseradish peroxidaseconjugated goat anti-rabbit IgG immunoglobulins as reported [30]. Total protein was determined using bovine serum albumin as a standard [38]
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