Macrophage endosomes contain proteases which degrade endocytosed protein ligands.
Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D002846:Chromatography, Affinity; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D008024:Ligands / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology* / Q000201:enzymology; D008358:Mannose / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D010436:Pepstatins / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011650:Pulmonary Alveoli / Q000166:cytology; D011817:Rabbits; D012709:Serum Albumin; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013997:Time Factors
D008024:Ligands; D010436:Pepstatins; D011480:Protease Inhibitors; D012709:Serum Albumin; C023840:mannose-bovine serum albumin conjugate; D000643:Ammonium Chloride; C014195:Streptomyces pepsin inhibitor; D012710:Serum Albumin, Bovine; D008985:Monensin; D010447:Peptide Hydrolases; D002402:Cathepsin D; D008358:Mannose; D009550:Nigericin; C031375:pepstatin