Identification of the macrophage mannose receptor as a 175-kDa membrane protein.
Mannose-lactoperoxidase, a neoglycoprotein prepared by reaction of lactoperoxidase with cyanomethyl 1-thiomannoside, bound to alveolar macrophages at 4 degrees C (Kd = 5.8 X 10(-8) M) and was rapidly internalized at 37 degrees C (K uptake = 2 X 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.
Wileman
T E
TE
Lennartz
M R
MR
Stahl
P D
PD
D000345:Affinity Labels; D000818:Animals; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D004705:Endocytosis; D006023:Glycoproteins / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007784:Lactoperoxidase; D037181:Lectins, C-Type; D008264:Macrophages / Q000032:analysis / Q000378:metabolism*; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011817:Rabbits; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D006023:Glycoproteins; D007457:Iodine Radioisotopes; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C025363:mannoproteins; D007784:Lactoperoxidase