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Isolation and characterization of a mannose receptor from human pigment epithelium
V L Shepherd1, B I Tarnowski, B J McLaughlin
1Department of Veterans Affairs, Vanderbilt University, Nashville, Tennessee.
Abstract:
Recent work demonstrated that a mannose receptor is involved in the phagocytosis of rod outer segments by the rat retinal pigment epithelium (RPE). In this study the binding of soluble mannose-containing ligands by human RPE explants is described. In addition, the authors report the isolation of a mannose receptor from human RPE and describe its relationship to the macrophage mannose receptor. Epithelial explants bound the soluble ligand 125I-mannose bovine serum albumin (BSA) by a mannose-specific process. The protein involved in mannose recognition was extracted from human tissue and purified using ligand-affinity chromatography. The protein that bound to the affinity column had a molecular weight of 175 kD by sodium dodecyl sulfate gel electrophoresis and migrated with the same mobility as the human macrophage mannose receptor. Antibodies directed against the macrophage receptor crossreacted with the mannose receptor from human RPE by immunoblot analysis. Binding specificity studies demonstrated that mannose and mannan inhibited ligand binding to the purified receptor by 65% and 90%, respectively; galactose had no effect. Using immunogold labeling of human RPE cells in explant culture, antimacrophage mannose receptor was localized at the apical plasma membrane. These results suggest that human RPE expresses a mannose receptor on its apical surface (as does the rat RPE) and that this receptor is similar to the human macrophage mannose receptor.
Insights
Human retinal pigment epithelium (RPE) expresses a mannose receptor similar to the macrophage mannose receptor. This receptor is involved in binding mannose-containing ligands and is located on the apical surface of RPE cells.
Area of Science:
- Ophthalmology
- Cell Biology
- Immunology
Background:
- Previous research identified a mannose receptor in rat retinal pigment epithelium (RPE) involved in phagocytosis.
- The function and characteristics of mannose receptors in human RPE remain less understood.
Purpose of the Study:
- To investigate the binding of mannose-containing ligands by human RPE explants.
- To isolate and characterize the mannose receptor from human RPE.
- To determine the relationship between the human RPE mannose receptor and the macrophage mannose receptor.
Main Methods:
- Human RPE explants were used to study the binding of 125I-mannose bovine serum albumin (BSA).
- A mannose receptor was isolated from human RPE using ligand-affinity chromatography.
- Sodium dodecyl sulfate gel electrophoresis and immunoblot analysis were performed.
- Immunogold labeling was used to localize the receptor on human RPE cells.
Main Results:
- Human RPE explants demonstrated mannose-specific binding of soluble mannose-BSA ligands.
- A 175 kD protein, identified as a mannose receptor, was isolated from human RPE.
- This receptor showed similar molecular weight and mobility to the human macrophage mannose receptor.
- Antibodies against the macrophage mannose receptor cross-reacted with the human RPE receptor.
- Mannose and mannan significantly inhibited ligand binding, while galactose had no effect.
- Immunogold labeling localized the receptor to the apical plasma membrane of human RPE cells.
Conclusions:
- Human RPE expresses a mannose receptor on its apical surface, analogous to rat RPE.
- The human RPE mannose receptor is similar in characteristics and localization to the human macrophage mannose receptor.
- This suggests a conserved role for mannose receptors in RPE function across species.