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Characterization and isolation of an intracellular D-mannose-specific receptor from human promyelocytic HL60 cells
V Pimpaneau1, P Midoux, M Monsigny
1Département de Biochimie des Glycoconjugués et Lectines Endogènes, CNRS, INSERM et Université, Orléans, France.
Abstract:
Most mammalian macrophages express D-mannose-specific receptor (membrane lectin, Mr 175,000) allowing endocytosis of their ligands, but cells of the monocytic lineage (HL60, U937, monocyte) lack this receptor. However, after permeabilization, promyelocytic, promonocytic cells and monocytes bound fluoresceinylated D-mannose-terminated neoglycoproteins as evidenced by flow cytometry. Under these conditions, confocal analysis confirmed the intracellular membrane localization of the labeling and the absence of nuclear binding. An intracellular D-mannose-specific receptor was isolated from the human promyelocytic cell line HL60, by affinity chromatography on 4-isothiocyanatophenyl alpha-D-mannopyranoside-substituted Affi-gel as a 60,000-Mr membrane protein requiring divalent cations for the ligand binding. Under the same conditions, mouse macrophages were shown to express a 175,000-Mr D-mannose-specific receptor but not the 60,000-Mr receptor.
Insights
Monocytes and related cells lack the typical D-mannose receptor found on macrophages. However, these cells possess an intracellular D-mannose receptor, distinct from the macrophage receptor, which binds mannose-terminated neoglycoproteins.
Area of Science:
- Immunology
- Cell Biology
- Glycobiology
Background:
- Mammalian macrophages typically express a D-mannose-specific receptor (175,000 Mr) involved in ligand endocytosis.
- Cells of the monocytic lineage, including HL60, U937, and monocytes, generally lack this surface D-mannose receptor.
Purpose of the Study:
- To investigate the presence and characteristics of D-mannose-specific receptors in monocytic cells.
- To identify and isolate the D-mannose-binding protein in human promyelocytic cell line HL60.
Main Methods:
- Flow cytometry was used to detect D-mannose-neoglycoprotein binding to permeabilized monocytic cells.
- Confocal microscopy confirmed intracellular localization of the bound ligands.
- Affinity chromatography was employed to isolate the D-mannose-specific receptor from HL60 cells.
Main Results:
- Permeabilized monocytic cells (HL60, U937, monocytes) bound D-mannose-neoglycoproteins, indicating an intracellular receptor.
- Confocal analysis revealed intracellular membrane localization, excluding nuclear binding.
- A 60,000 Mr intracellular D-mannose-specific receptor, requiring divalent cations, was isolated from HL60 cells.
- Mouse macrophages expressed the 175,000 Mr receptor but not the 60,000 Mr receptor.
Conclusions:
- Human monocytic cells possess an intracellular D-mannose-specific receptor distinct from the 175,000 Mr macrophage receptor.
- This intracellular receptor plays a role in the recognition of D-mannose-terminated ligands within monocytic cells.