Purification and characterization of a cation-dependent mannose 6-phosphate receptor from murine P388D1 macrophages and bovine liver.
A new binding protein which recognizes the phosphomannosyl recognition marker of lysosomal enzymes has been isolated from P388D1 macrophages which lack the 215-kDa Man-6-P receptor. This receptor was detergent-solubilized from total membranes and purified by chromatography on a lysosomal enzyme affinity column. An identical protein was purified from bovine liver using a two-step procedure which is based on the difference in ion requirement and ligand specificity of the new Man-6-P binding protein and the previously characterized 215-kDa Man-6-P receptor. After detergent solubilization of membranes, both receptors were co-purified on a phosphomannan-Sepharose affinity column run in the presence of MnCl2 and then were separated using a Dictyostelium discoideum lysosomal enzyme-Affi-Gel column which, in the absence of MnCl2, retains only the 215-kDa Man-6-P receptor. The new Man-6-P receptor binds efficiently to phosphomannosyl monoester-containing ligands when MnCl2 is present. This binding is specifically inhibited by Man-6-P or by low pH. The cation-dependent Man-6-P receptor has a subunit molecular size of 46 kDa and appears to be an oligomer composed of three subunits. The receptor contains both high mannose (or hybrid)- and complex-type oligosaccharide units on the basis of sensitivity to digestion with endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F. Radioimmunoassays carried out with two different antibodies demonstrate that the 46-kDa cation-dependent Man-6-P receptor and the 215-kDa cation-independent Man-6-P receptor not only differ in their properties but are also immunologically distinct.
Hoflack
B
B
Kornfeld
S
S
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002412:Cations; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D006600:Hexosephosphates / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D011863:Radioimmunoassay; D017527:Receptor, IGF Type 2
D002352:Carrier Proteins; D002412:Cations; D006600:Hexosephosphates; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2