Tissue distribution, structural characterization, and biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity

Abstract

Mac-3 is a mouse macrophage differentiation antigen defined by a rat anti-mouse monoclonal antibody (MAb),M3/84. The structure, biosynthesis, quantitative surface expression, and distribution of Mac-3 have been studied by radiolabeling and isolation with MAb-Sepharose, saturation binding, absorption, and immunofluorescence flow cytometry. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mac-3 migrates as a diffuse band with average Mr = 110,000. Labeling of intact cells with 125I and accessibility to MAb show it is present at least in part on the cell surface. Saturation labeling with 125I-MAb shows 4.2 X 10(4) cell surface sites on thioglycollate medium-elicited peritoneal macrophages. [35S]Methionine and [3H]glucosamine incorporation into Mac-3 by purified macrophages show it is a glycoprotein synthesized by these cells. Absorption shows Mac-3 is strongest in macrophages, present in lower quantities in lung, liver, bone marrow, and spleen, and undetectable in thymus, lymph node, brain, and heart. Immunofluorescent flow cytometry shows surface expression on thioglycollate-elicited macrophages but not bone marrow, spleen, lymph node, or thymus cell suspensions. Similar amounts of Mac-3 are immunoprecipitated from resident macrophages or macrophages elicited by sterile inflammatory agents, intracellular parasites, or immunomodulators, but the average Mr of Mac-3 varies from 92,000 to 110,000. Mac-3 is synthesized from precursor(s) of Mr = 74,000 and 79,000, identical in the different macrophages. Processing into the mature molecule, which migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a more diffuse band and varies in Mr among macrophage elicited by different agents and to a lesser degree between different preparations of the same type of macrophage, occurs in 15 to 30 min.