title: Purification, biochemical composition, and biosynthesis of the Mo3 activation antigen expressed on the plasma membrane of human mononuclear phagocytes.
abstract: Mo3 is an activation Ag expressed on the surface of human mononuclear phagocytes stimulated in vitro or in vivo by various activating factors. Mo3 is obtained by immunoprecipitation with anti-Mo3 mAb from lysates of PMA-stimulated U-937 cells. The Ag is a heterogeneous glycoprotein with a molecular mass range of 42 to 66 kDa (nonreducing conditions) containing N-linked carbohydrate chains. When the cells are treated with phosphatidylinositol-specific phospholipase C, greater than 60% of total precipitable gp42-66 Ag is released in the supernatant. This phosphatidylinositol-specific phospholipase C-sensitive linkage to the plasma membrane has provided a means for the one-step purification of Mo3 by immunoaffinity chromatography. The eluted soluble Mo3 (sMo3) was greater than 90% pure as documented by the appearance of a single major protein peak on reverse phase HPLC and SDS-PAGE. The average yield was 12.1 micrograms/10(8) cells. Sufficient quantities of sMo3 have been purified to permit the determination of amino acid and carbohydrate composition. Complex N-linked carbohydrates make up nearly 50% of the glycoprotein content and contribute to its heterogeneity. An anti-Mo3 polyclonal antiserum generated from sMo3 was used to immunoprecipitate Mo3 and its precursor from biosynthetically labeled, PMA-stimulated U-937 cells or LPS-stimulated monocytes. These 35S-methionine "pulse-chase" experiments demonstrated the existence of a 40- to 42-kDa endo-beta-N-acetylglucosaminidase-sensitive precursor, which over a period of 4 to 5 h gave rise to an endo-beta-N-acetylglucosaminidase-resistant, but N-glycanase-sensitive 42- to 66-kDa mature form.
authors.affiliation: Simpson Memorial Institute, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109.
authors.forename: I F
authors.initials: IF
authors.lastname: Mizukami
authors.forename: S D
authors.initials: SD
authors.lastname: Vinjamuri
authors.forename: F
authors.initials: F
authors.lastname: Perini
authors.forename: D Y
authors.initials: DY
authors.lastname: Liu
authors.forename: R F
authors.initials: RF
authors.lastname: Todd
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008562:Membrane Glycoproteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D010586:Phagocytes / Q000276:immunology*; D011233:Precipitin Tests; D011498:Protein Precursors / Q000032:analysis; D011817:Rabbits
chemical_list: D000596:Amino Acids; D000954:Antigens, Surface; D002241:Carbohydrates; D007106:Immune Sera; D008562:Membrane Glycoproteins; D011498:Protein Precursors