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The fate of peptides pinocytosed by macrophages in vitro
Abstract:
A series of small peptides, such as might arise in the course of intralysosomal protein digestion, were screened for the ability to escape, intact, from mouse peritoneal macrophage lysosomes. Inability to penetrate lysosomal membranes was inferred from a peptide's induction of lysosomal swelling, or vacuolization, in cultured macrophages. Two of the peptides tested, (D-Glu)(2) and (D-Ala)(3), induced vacuolization. Neither peptide was susceptible to hydrolysis by enzymes in macrophages or in the serum-containing culture medium. Their morphological effect was inhibited by parafluorophenylalanine, an inhibitor of pinocytosis. Once formed by either peptide, the vacuoles persisted for several hours in peptide-free medium. Quantitative studies of radioactively labeled (D-Glu)(2) confirmed the morphological evidence that (D-Glu)(2) is taken up by pinocytosis and stored, intact, in macrophage lysosomes. The majority of the peptides which failed to induce vacuolization-(L-Ala)(2), L-Ser.L-Ala, L-Val.L-Ala, L-Ala.L-Thr, Gly.D, L-Phe, L-Ala.D-His, (L-Ala)(3), (L-Glu)(2), and D-Leu.L-Tyr-were found to be susceptible to hydrolysis by cellular or serum peptidases. Their failure to induce vacuolization was attributed to their hydrolysis to subunits capable of penetrating lysosomal membranes. Some of the peptides which had failed to induce vacuolization-(D-Ala)(2), D-Ser.D-Ala, D-Val.D-Ala, Gly-D-Asn, D-Ala.D-Thr, and D-Arg.D-Val-were found to be indigestible. Except for the cytotoxic peptide D-Arg.D-Val, peptides in this category all had lower molecular weights and volumes than (Glu)(2) or (Ala)(3). It is inferred that these peptides are small enough to escape from macrophage lysosomes, while (Glu)(2) and (Ala)(3) are too large to escape intact. The implications of this inference for the mechanism of intracellular digestion of pinocytosed proteins are discussed.
Insights
Small peptides were tested for their ability to escape macrophage lysosomes. Larger peptides like (D-Glu)(2) and (D-Ala)(3) caused swelling and were retained, indicating they cannot penetrate lysosomal membranes.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Lysosomes are key organelles for intracellular digestion of proteins.
- Understanding how proteins or peptides interact with and cross lysosomal membranes is crucial for comprehending cellular processing.
- Macrophage lysosomes play a significant role in processing internalized material via pinocytosis.
Purpose of the Study:
- To investigate the ability of small peptides, mimicking products of protein digestion, to escape intact from mouse peritoneal macrophage lysosomes.
- To determine the factors influencing peptide permeability across lysosomal membranes.
- To elucidate the mechanism of intracellular digestion of pinocytosed proteins.
Main Methods:
- Screening of various small peptides for their ability to penetrate lysosomal membranes in cultured mouse peritoneal macrophages.
- Induction of lysosomal swelling (vacuolization) as an indicator of membrane impermeability.
- Assessment of peptide susceptibility to hydrolysis by cellular and serum peptidases.
- Quantitative studies using radioactively labeled peptides to track uptake and storage.
Main Results:
- Two peptides, (D-Glu)(2) and (D-Ala)(3), induced lysosomal vacuolization, indicating they are retained within lysosomes.
- These vacuolizing peptides were resistant to hydrolysis and were taken up by pinocytosis, storing intact in lysosomes.
- Most other tested peptides, being susceptible to hydrolysis, were broken down into smaller subunits capable of crossing lysosomal membranes.
- Indigestible peptides, smaller in molecular weight and volume than (D-Glu)(2) and (D-Ala)(3), were inferred to escape lysosomes.
Conclusions:
- Lysosomal membrane permeability is size-dependent, with larger peptides like (D-Glu)(2) and (D-Ala)(3) being too large to escape intact.
- Hydrolysis by peptidases is a critical factor allowing smaller peptide fragments to penetrate lysosomal membranes and exit.
- The findings provide insights into the mechanisms governing the intracellular digestion of pinocytosed proteins within macrophages.