Purification, biochemical composition, and biosynthesis of the Mo3 activation antigen expressed on the plasma membrane of human mononuclear phagocytes.
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.
Mizukami
I F
IF
Simpson Memorial Institute, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109.
Vinjamuri
S D
SD
Perini
F
F
Liu
D Y
DY
Todd
R F
RF
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
D000596:Amino Acids; D000954:Antigens, Surface; D002241:Carbohydrates; D007106:Immune Sera; D008562:Membrane Glycoproteins; D011498:Protein Precursors