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A pulse-chase study tracking the conversion of macrophage-endocytosed serum amyloid A into extracellular amyloid
Barbara Kluve-Beckerman1, John J Manaloor, Juris J Liepnieks
1Dpartment of Pathology and laboratoty Medicine, Indiana University School of Medicine, Indianapolis, 46202, USA. bkluvebe@iupui.edu
Objective:
To determine whether serum amyloid A (SAA) is internalized by and processed in macrophages en route to deposition as extracellular amyloid.
Methods:
SAA was tracked in cultures of peritoneal macrophages, using a pulse-chase protocol. Macrophages were pulsed with either fluorescently (with Texas Red) tagged SAA (TxR-SAA) or iodinated SAA ((125)I-SAA). Cells were then rinsed and shifted to chase medium containing unlabeled SAA and amyloid-enhancing factor (AEF) to induce amyloid formation. At selected times, TxR-SAA in living cells was observed by confocal scanning microscopy. (125)I-SAA was visualized and quantified in cell lysates and medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorimaging. The presence of amyloid was confirmed by Congo red staining.
Results:
Confocal microscopy immediately after the pulse revealed TxR-SAA in endosomal vesicles, with no extracellular or cell surface accumulation. After 24 hours and 72 hours of chase, virtually all TxR-SAA remained intracellular. By 10 days, extracellular fluorescence was very strong, indicating that SAA had moved out of cells. Congo red staining revealed amyloid colocalized with areas of extracellular fluorescence. Experiments using (125)I-SAA showed that while 90-95% of internalized (125)I-SAA was degraded within 24 hours, 5-10% persisted as intact SAA or SAA peptides. Immediately after the pulse, SAA was full-length, but within 24 hours, discrete (125)I-SAA peptides were seen. Each peptide had an intact SAA amino-terminus, as expected for AA protein. Amyloid was detected in cultures as early as 24 hours after initiation of treatment with SAA and AEF and appeared to be intracellular.
Conclusion:
The results of this study provide direct evidence that SAA internalized by and processed in macrophages forms extracellular amyloid. Based on the presence of (125)I-AA protein in macrophage lysates prior to the appearance of extracellular TxR-labeled amyloid, it was concluded that cleavage of SAA to AA occurs intracellularly.
Insights
Serum amyloid A (SAA) is taken up by macrophages and processed intracellularly, leading to the formation of extracellular amyloid deposits. This study demonstrates the macrophage
Area of Science:
- Immunology and Inflammation Research
- Protein Misfolding Diseases
- Cell Biology
Background:
- Serum amyloid A (SAA) is a precursor protein implicated in amyloidosis.
- The cellular mechanisms of SAA deposition as extracellular amyloid remain incompletely understood.
Purpose of the Study:
- To investigate the cellular uptake and processing of SAA by macrophages.
- To determine the role of macrophages in the formation of extracellular amyloid from SAA.
Main Methods:
- Pulse-chase experiments using fluorescently (Texas Red) and radioactively (iodine-125) labeled SAA in peritoneal macrophage cultures.
- Confocal scanning microscopy to track intracellular SAA localization.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorimaging to quantify SAA degradation and peptide formation.
- Congo red staining to confirm amyloid presence.
Main Results:
- Fluorescently tagged SAA was rapidly internalized into endosomal vesicles within macrophages.
- The majority of internalized SAA was degraded within 24 hours, with a small fraction persisting as intact SAA or peptides.
- Extracellular amyloid deposits, confirmed by Congo red staining, appeared by 10 days and colocalized with extracellular fluorescence, indicating SAA egress from cells.
- Amyloid formation was detected intracellularly as early as 24 hours.
Conclusions:
- Macrophages internalize, process, and subsequently release SAA, which then forms extracellular amyloid.
- Cleavage of SAA into AA protein, the amyloidogenic form, occurs intracellularly within macrophages prior to extracellular deposition.