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Apparently normal extracellular acidic alpha-mannosidase in fibroblast cultures from patients with mannosidosis
Abstract:
Fibroblasts from patients with mannosidosis, cultured in medium supplemented with fetal calf serum from which acidic alpha-mannosidase (alpha-D-mannoside mannohydrolase, E.C.3.2.1.24) has been removed, secreted a normal amount of apparently unaffected acidic alpha-mannosidase into fetal calf serum-free medium. Both the intracellular and extracellular acidic alpha-mannosidase activities were completely precipitated by antiserum to placenta alpha-mannosidase B. In contrast to the heat-lability of the intracellular acidic alpha-mannosidase and its low affinity for artificial mannoside substrate, the extracellular enzyme exhibited both normal thermostability and normal kinetics. Mixing experiments with the intercellular enzymes suggested that the decreased activity in the patients' fibroblasts is not the effect of an inhibitor or absence of an activator. However, incubation of the mannosidosis extracellular enzyme with either normal or patient cell lysate resulted in a partial loss of activity, whereas an additive value was observed with the normal extracellular enzyme. In contrast to normal culture medium, the medium from mannosidosis cell culture was unable to enhance the rate of reduction of intracellular radioactivity in mucolipidosis type II fibroblasts precultured in the presence of radiolabeled mannose. These findings suggest that the defect in mannosidosis is expressed only after the enzyme has been delivered to lysosomes and presumably undergone some form of processing there.
Insights
Mannosidosis patients secrete normal amounts of alpha-mannosidase, but it becomes defective after lysosomal processing. This suggests the mannosidosis defect occurs post-secretion within the cell.
Area of Science:
- Biochemistry
- Genetics
- Cell Biology
Background:
- Mannosidosis is a lysosomal storage disorder caused by deficient activity of the enzyme alpha-mannosidase.
- Understanding the precise defect in alpha-mannosidase is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the characteristics of alpha-mannosidase secreted by fibroblasts from mannosidosis patients.
- To determine if the enzyme defect in mannosidosis arises intracellularly or extracellularly.
Main Methods:
- Culturing fibroblasts from mannosidosis patients in specialized media.
- Assessing intracellular and extracellular alpha-mannosidase activity, thermostability, and kinetics.
- Performing enzyme mixing experiments and evaluating effects on mucolipidosis type II fibroblasts.
Main Results:
- Mannosidosis fibroblasts secreted normal quantities of alpha-mannosidase with seemingly unaffected activity.
- The secreted enzyme exhibited normal thermostability and kinetics, unlike the intracellular enzyme.
- The mannosidosis enzyme's activity decreased upon incubation with cell lysates, and its medium failed to correct mucolipidosis type II fibroblasts.
Conclusions:
- The alpha-mannosidase defect in mannosidosis is likely expressed after the enzyme enters the lysosomes and undergoes processing.
- The study differentiates the defect from other lysosomal storage disorders, such as mucolipidosis type II.