Isolation and characterization of a mannose-specific endocytosis receptor from rabbit alveolar macrophages.
Rabbit alveolar macrophages express a plasma-membrane receptor that recognizes glycoprotein ligands bearing terminal mannose, fucose or N-acetylglucosamine residues. Macrophage membranes were washed extensively with buffers containing high salt and mannose or EDTA to remove endogenously bound ligand, before Triton X-100 extraction. The extracts were chromatographed on mannose-Sepharose. Elution with mannose, followed by dialysis and a second mannose-Sepharose step with EDTA elution, produced a preparation that migrated as single protein band of Mr 175,000 on SDS/polyacrylamide-gel electrophoresis. The purified protein binds mannose-BSA (bovine serum albumin) with a dissociation constant of 1.9 X 10(-8) M. Ligand binding is Ca2+ and pH-dependent, with maximal binding at neutral pH and low binding below pH 6.0. The binding of 125I-mannose-BSA is inhibited by ligands bearing high-mannose oligosaccharides, such as mannan or beta-glucuronidase, as well as the monosaccharides mannose, fucose and N-acetylglucosamine. Galactose, galactosylated BSA, glucose and mannose 6-phosphate are non-inhibitory. Amino acid compositional analyses indicate that the receptor contains high concentrations of aspartate/asparagine and glutamate/glutamine, and low amounts of methionine. The carbohydrate composition was studied by lectin overlays of electrophoretically transferred receptor, and the results indicate the presence of N-linked complex and O-linked sialylated oligosaccharides. A protein of Mr 175,000 was immunoprecipitated from radio-iodinated macrophage membranes with an antibody generated against purified rabbit lung mannose receptor.
Lennartz
M R
MR
Department of Cell Biology and Physiology, Washington University, St. Louis, MO 63110.
Wileman
T E
TE
Stahl
P D
PD
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D004705:Endocytosis; D037181:Lectins, C-Type; D008024:Ligands; D008264:Macrophages / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011817:Rabbits; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D012709:Serum Albumin; D012710:Serum Albumin, Bovine / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D037181:Lectins, C-Type; D008024:Ligands; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D012709:Serum Albumin; C023840:mannose-bovine serum albumin conjugate; D012710:Serum Albumin, Bovine; D008358:Mannose; D002118:Calcium