Mizukami I F, Vinjamuri S D, Perini F, Liu D Y, Todd R F
Simpson Memorial Institute, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109.
J Immunol. 1991 Aug 15;147(4):1331-7.
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.
Mo3是一种激活抗原，在体外或体内受到各种激活因子刺激的人单核吞噬细胞表面表达。Mo3是通过用抗Mo3单克隆抗体对佛波酯（PMA）刺激的U-937细胞裂解物进行免疫沉淀获得的。该抗原是一种异质性糖蛋白，分子量范围为42至66 kDa（非还原条件下），含有N-连接的碳水化合物链。当细胞用磷脂酰肌醇特异性磷脂酶C处理时，超过60%的可沉淀gp42 - 66抗原释放到上清液中。这种与质膜的磷脂酰肌醇特异性磷脂酶C敏感连接为通过免疫亲和层析一步纯化Mo3提供了一种方法。通过反相高效液相色谱（HPLC）和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）上出现的单个主要蛋白峰证明，洗脱的可溶性Mo3（sMo3）纯度大于90%。平均产量为12.1微克/10⁸个细胞。已纯化出足够量的sMo3以确定其氨基酸和碳水化合物组成。复杂的N-连接碳水化合物占糖蛋白含量的近50%，并导致其异质性。从sMo3产生的抗Mo3多克隆抗血清用于从生物合成标记的、PMA刺激的U-937细胞或脂多糖（LPS）刺激的单核细胞中免疫沉淀Mo3及其前体。这些³⁵S-甲硫氨酸“脉冲追踪”实验证明存在一种40至42 kDa的内切β-N-乙酰氨基葡萄糖苷酶敏感前体，该前体在4至5小时内产生一种内切β-N-乙酰氨基葡萄糖苷酶抗性但N-糖苷酶敏感的42至66 kDa成熟形式。
