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Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages
Abstract:
beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.
Insights
Fibroblasts secrete beta-glucuronidase, which is rapidly taken up by macrophages and fibroblasts via distinct cellular receptors. This finding has implications for treating mucopolysaccharidoses with fibroblast transplants.
Area of Science:
- Cell Biology
- Biochemistry
- Enzyme kinetics
Background:
- Lysosomal enzymes are crucial for cellular function.
- Defects in lysosomal enzymes lead to diseases like mucopolysaccharidoses.
- Understanding enzyme uptake mechanisms is key for therapeutic strategies.
Purpose of the Study:
- To investigate the uptake mechanisms of secreted beta-glucuronidase by different cell types.
- To identify the specific receptors involved in this endocytosis process.
- To explore the implications for fibroblast transplantation therapies.
Main Methods:
- In vitro uptake assays using radiolabeled beta-glucuronidase.
- Competition assays with mannose-containing compounds and specific phosphorylated sugars.
- Enzyme treatment with alkaline phosphatase prior to uptake studies.
- Comparative analysis of uptake in mouse 3T3 fibroblasts and human fibroblasts, and mouse peritoneal macrophages.
Main Results:
- Beta-glucuronidase uptake was rapid and saturable in both fibroblasts and macrophages.
- Mannose compounds inhibited uptake in macrophages but not fibroblasts.
- Mannose-6-phosphate significantly inhibited uptake in both cell types, with complete abolition in fibroblasts.
- Alkaline phosphatase pre-treatment abolished fibroblast uptake but not macrophage uptake.
- Differential inhibition patterns suggest distinct receptor-mediated endocytosis pathways.
Conclusions:
- Fibroblasts secrete beta-glucuronidase recognized by distinct receptors on fibroblasts and macrophages.
- Endocytosis of this lysosomal enzyme involves different receptor systems in these cell types.
- Findings support the potential of fibroblast transplants for treating mucopolysaccharidoses.