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Purification and characterization of the D-mannose receptor from J774 mouse macrophage cells
1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract:
Macrophages display on their cell surface a D-mannose-specific receptor which facilitates the scavenging of certain pathogens and deleterious macromolecules from the extracellular fluid as part of the host defense mechanism. The mouse D-mannose receptor was purified from J774 E macrophages and an antiserum was generated against the receptor protein. In mouse macrophages, the newly synthesized receptor has an Mr of 157,000 Da and rapidly matures to a protein with an Mr of 172,000 Da. Both forms of the receptor protein are tightly associated with cell membranes. The receptor is found in a number of mouse macrophage cell types but is not present in mouse fibroblasts. An assay was developed to characterize D-mannose receptor-ligand binding based on immunoprecipitation of the detergent-solubilized receptor protein. The dissociation constant, determined for receptor and the neoglycoprotein D-mannose-BSA, was 1.67nM. Receptor-ligand binding was calcium and pH dependent. Monosaccharides, such as D-mannose and L-fucose, partially inhibited receptor binding to the ligand D-mannose-BSA.
Insights
Researchers purified the mouse D-mannose receptor from macrophages, revealing its role in host defense. This D-mannose receptor binds ligands, with binding dependent on calcium and pH levels.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophages possess a D-mannose-specific receptor on their surface.
- This receptor is crucial for host defense by scavenging pathogens and macromolecules.
- Understanding the D-mannose receptor's properties is key to comprehending macrophage function.
Purpose of the Study:
- To purify the mouse D-mannose receptor from J774 E macrophages.
- To generate an antiserum against the purified receptor protein.
- To characterize the binding properties and molecular characteristics of the D-mannose receptor.
Main Methods:
- Purification of the D-mannose receptor from mouse macrophage cell line J774 E.
- Generation of specific antiserum against the receptor protein.
- Development of an immunoprecipitation assay for receptor-ligand binding characterization.
- Analysis of receptor molecular weight and cell membrane association.
Main Results:
- The mouse D-mannose receptor was successfully purified, and an antiserum was generated.
- Newly synthesized receptor (157,000 Da) matures to a 172,000 Da form, both membrane-associated.
- The receptor is present in various mouse macrophage types but absent in fibroblasts.
- A dissociation constant of 1.67 nM was determined for receptor-neoglycoprotein (D-mannose-BSA) binding.
- Receptor-ligand binding is dependent on calcium and pH, and inhibited by D-mannose and L-fucose.
Conclusions:
- The study successfully characterized the mouse D-mannose receptor, including its molecular forms and membrane association.
- The developed assay allows for the study of D-mannose receptor-ligand interactions.
- The findings provide insights into the molecular basis of D-mannose receptor function in host defense mechanisms.