Purification and characterization of an osteoclast membrane glycoprotein with homology to manganese superoxide dismutase.
The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the inorganic and organic components of bone matrix. Isolated avian osteoclasts have been used to immunize mice and generate an osteoclast-directed monoclonal antibody library (J. Cell Biology, 100:1592). A subset of these monoclonal antibodies recognizes antigens which are expressed on osteoclasts and which are absent or nearly so on multinucleated giant cells formed in vitro from monocyte or marrow mononuclear cells. One of these antibodies, designated 121F, has been used to identify and purify an osteoclast plasma membrane-associated glycoprotein. Western blot analysis on disulfide bond-reduced extracts from osteoclasts or multinucleated giant cells formed in vitro demonstrates that the 121F antibody recognizes a 150 kDa protein detectable only in osteoclasts. This high molecular weight protein has been purified by a combination of immunoaffinity and gel filtration chromatography procedures, in conjunction with electroelution of a single band from SDS-polyacrylamide gels. Silver staining of the purified antigen on SDS-polyacrylamide gels has revealed a single protein species larger than 200 kDa in its unreduced form and 150 kDa when disulfides are reduced. Isoelectric focusing of the purified antigen reveals a single species, having a neutral pl point of 6.95. Whereas endoglycosidase treatment and lectin affinity chromatographic analyses demonstrate that the antigen recognized by the 121F antibody possesses complex N-linked sugars, trifluoromethanesulfonic acid treatment indicates there are no additional O-linked carbohydrate components. Periodate oxidation and monosaccharide hapten inhibition studies provide no evidence for the antigenic epitope bound by the 121F antibody being carbohydrate in nature. Although the native antigen is blocked at its N-terminus, amino acid analysis of a hydroxylamine generated peptide disclosed a striking relationship between the osteoclast antigen recognized by the 121F monoclonal antibody and manganese and iron superoxide dismutase. Therefore, in addition to serving as a distinguishing cell type-specific marker for osteoclasts, this cell surface glycoprotein may function directly in osteoclast-mediated bone resorption.
Oursler
M J
MJ
Department of Cell Biology, Washington University School of Dental Medicine, St. Louis, Missouri.
Li
L
L
Osdoby
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000276:immunology; D015153:Blotting, Western; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D007525:Isoelectric Focusing; D008345:Manganese / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000737:chemistry*; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D002241:Carbohydrates; D000939:Epitopes; D008562:Membrane Glycoproteins; D008345:Manganese; D013482:Superoxide Dismutase