Pitt A, Mayorga L S, Stahl P D, Schwartz A L
Edward Mallinkrodt Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Clin Invest. 1992 Nov;90(5):1978-83. doi: 10.1172/JCI116077.
We investigated the protein composition of J774-E clone macrophage phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D,beta-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing phagosome were the FcII receptor, the mannose receptor, and alpha-adaptin. These results indicate that although the macrophage phagosome's total protein composition changes little during phagolysosome formation, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins.
我们研究了在吞噬溶酶体生物发生的不同阶段分离得到的J774-E克隆巨噬细胞吞噬体的蛋白质组成。通过密度梯度离心法分离内化抗体包被的金黄色葡萄球菌3分钟后再分别追踪0、4、9或15分钟所形成的吞噬体。通过放射性标记配体回收、酶标记物和电子显微镜对吞噬体制剂的富集和纯度进行定量分析。对分离得到的吞噬体进行一维SDS-PAGE分析显示其蛋白质组成几乎相同。然而，使用针对沿内吞途径已知行程的选定蛋白质的抗体进行蛋白质印迹分析表明，吞噬体的蛋白质组成存在明显差异。在成熟的吞噬体内积累的有液泡质子泵的31-kD亚基、组织蛋白酶D、β-葡萄糖醛酸酶、阳离子依赖性甘露糖6-磷酸受体和LAMP-1。在成熟的吞噬体内减少的有FcII受体、甘露糖受体和α-衔接蛋白。这些结果表明，虽然巨噬细胞吞噬体的总蛋白质组成在吞噬溶酶体形成过程中变化不大，但成熟的吞噬体通过蛋白质循环既接收又消除特定的膜蛋白和隔离蛋白。
