Nath J, Ohno Y, Gallin J I, Wright D G
Department of Hematology, Walter Reed Army Institute of Research, Washington, DC 20307.
J Immunol. 1992 Nov 15;149(10):3360-71.
Activation of human neutrophils by PMA causes a post-translational incorporation of 14C-labeled tyrosine into multiple neutrophil (PMN) proteins, that is distinctly different from the enzymatic tyrosinolation of tubulin in FMLP-stimulated PMN. Post-translational incorporation of other radiolabeled amino acids, including the structurally similar amino acid phenylalanine, does not occur under identical conditions of neutrophil activation, suggesting an involvement of the phenolic hydroxyl group of tyrosine in the PMA-mediated reaction. Similar to the stimulation of PMN tubulin tyrosinolation by FMLP, the PMA-induced incorporation of tyrosine into multiple PMN proteins is closely associated with activation of the NADPH oxidase-mediated respiratory burst in stimulated PMN and can be inhibited by a variety of reducing agents, inhibitors of peroxidase-mediated reactions, and intracellular scavengers of oxygen radicals. Moreover, the PMA-induced post-translational incorporation of tyrosine does not occur in PMN from patients with chronic granulomatous disease and is significantly reduced (50%) in PMN of an individual with myeloperoxidase deficiency. A similar stimulus-induced incorporation of tyrosine into multiple PMN proteins is also observed in PMN exposed to various phagocytic stimuli, and the incorporated radioactivity in cells undergoing phagocytosis is substantially enriched (40- to 50-fold) in isolated PMN phagolysosomes. Consistent with this latter observation, HPLC fractionation of stimulated PMN proteins and analysis of the incorporated radioactivity reveal that the 14C label is primarily associated with PMN membrane proteins. Furthermore, this post-translational incorporation of tyrosine, like that associated with PMA stimulation, is associated with production of oxygen radicals and the generation of protein carbonyl derivatives, which are indicative of oxidative protein modifications via mixed function oxidases. Our findings indicate that tyrosine incorporation into membrane proteins of stimulated PMN is functionally relevant to the physiologic host-defense responses of human neutrophils undergoing phagocytosis.
佛波醇酯（PMA）激活人中性粒细胞会导致14C标记的酪氨酸在翻译后掺入多种中性粒细胞（PMN）蛋白中，这与在N-甲酰甲硫氨酸-亮氨酸-苯丙氨酸（FMLP）刺激的PMN中微管蛋白的酶促酪氨酸化明显不同。在相同的中性粒细胞激活条件下，其他放射性标记氨基酸（包括结构相似的苯丙氨酸）的翻译后掺入不会发生，这表明酪氨酸的酚羟基参与了PMA介导的反应。与FMLP刺激PMN微管蛋白酪氨酸化类似，PMA诱导的酪氨酸掺入多种PMN蛋白与刺激的PMN中烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶介导的呼吸爆发激活密切相关，并且可以被多种还原剂、过氧化物酶介导反应的抑制剂和细胞内氧自由基清除剂抑制。此外，PMA诱导的酪氨酸翻译后掺入在慢性肉芽肿病患者的PMN中不会发生，并在髓过氧化物酶缺乏个体的PMN中显著减少（50%）。在暴露于各种吞噬刺激的PMN中也观察到类似的刺激诱导的酪氨酸掺入多种PMN蛋白，并且在吞噬细胞的分离的PMN吞噬溶酶体中，吞噬细胞中掺入的放射性显著富集（40至50倍）。与后一观察结果一致，对刺激的PMN蛋白进行高效液相色谱（HPLC）分级分离并分析掺入的放射性表明，14C标记主要与PMN膜蛋白相关。此外，这种酪氨酸的翻译后掺入，与PMA刺激相关的情况一样，与氧自由基的产生和蛋白质羰基衍生物的生成有关，这表明通过混合功能氧化酶进行了氧化蛋白质修饰。我们的研究结果表明，酪氨酸掺入受刺激的PMN膜蛋白在功能上与进行吞噬作用的人中性粒细胞的生理宿主防御反应相关。
