Identification of the Macrophage Mannose Receptor as a 175-kDa Membrane Protein

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 83; no. 8; pp. 2501 - 2505
• Main Authors: Wileman, Thomas E., Lennartz, Michelle R., Stahl, Philip D.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.04.1986; National Acad Sciences
• Subjects: Affinity Labels; Alveolar macrophages; Animals; Biological and medical sciences; Cell Membrane - analysis; Cell Membrane - metabolism; Cell membranes; Cell physiology; Endocytosis; Enzymes; Fundamental and applied biological sciences. Psychology; Glycoproteins - metabolism; Iodine Radioisotopes; Lactoperoxidase; Lectins, C-Type; Ligands; Lungs; Macrophages; Macrophages - analysis; Macrophages - metabolism; Mannans; Mannose-Binding Lectins; Membrane Glycoproteins; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Molecular and cellular biology; Molecular Weight; Oxidases; Rabbits; Receptors; Receptors, Cell Surface; Receptors, Immunologic - isolation & purification; Receptors, Immunologic - metabolism; Yeasts; Characterization; Vertebrata; Endocytosis; Membrane protein; Mammalia; Internalization; Glycoproteins; Identification; Mannose; Macrophage; Biological receptor
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.83.8.2501

Mannose-lactoperoxidase, a neoglycoprotein prepared by reaction of lactoperoxidase with cyanomethyl 1-thiomannoside, bound to alveolar macrophages at 4 degrees C (Kd = 5.8 × 10-8 M) and was rapidly internalized at 37 degrees C (Kuptake = 2 × 10-8 M). Mannose-lactoperoxidase binding and uptake were blocked by yeast mannan, and mannose-lactoperoxidase inhibited uptake of 125I-labeled mannose-BSA (bovine serum albumin). Radioiodination of cells with surface-bound mannose-lactoperoxidase was carried out in the presence of glucose and glucose oxidase. A major polypeptide (175 kDa) was radioiodinated by this procedure. Iodination of the 175-kDa polypeptide appeared to be receptor-mediated, since it was blocked by the presence of yeast mannan. Specific iodination was absent from receptor-negative cells. To demonstrate that the 175-kDa species is a ligand-binding protein, cells were iodinated by the standard lactoperoxidase method. Washed cells were then allowed to bind mannose-BSA. Receptor-ligand complexes, prepared by detergent extraction, were passed over anti-BSA IgG affinity columns. Mannose, but not mannose 6-phosphate or galactose, eluted a radioactive protein from the column that migrated with an apparent molecular mass of 175 kDa on NaDodSO4/PAGE. Detergent extracts of crude membranes prepared from macrophage-enriched whole rabbit lung were adsorbed to mannose-Sepharose; the fraction obtained by elution with mannose contained two protein components of 175 and 55 kDa. Subsequent chromatography on N-acetylglucosamine-agarose yielded a single protein of 175 kDa. The 175-kDa polypeptide was shown to bind 125I-labeled mannose-BSA in a precipitation assay. This binding could be blocked with mannan or mannose-BSA. The results indicate that the cell-surface mannose receptor is a 175-kDa protein.