Lara F A, Lins U, Bechara G H, Oliveira P L
Departamento de Bioquímica Médica, ICB, Universidade Federal do Rio de Janeiro, Brazil.
J Exp Biol. 2005 Aug;208(Pt 16):3093-101. doi: 10.1242/jeb.01749.
Heme is present in all cells, acting as a cofactor in essential metabolic pathways such as respiration and photosynthesis. Moreover, both heme and its degradation products, CO, iron and biliverdin, have been ascribed important signaling roles. However, limited knowledge is available on the intracellular pathways involved in the flux of heme between different cell compartments. The cattle tick Boophilus microplus ingests 100 times its own mass in blood. The digest cells of the midgut endocytose blood components and huge amounts of heme are released during hemoglobin digestion. Most of this heme is detoxified by accumulation into a specialized organelle, the hemosome. We followed the fate of hemoglobin and albumin in primary cultures of digest cells by incubation with hemoglobin and albumin labeled with rhodamine. Uptake of hemoglobin by digest cells was inhibited by unlabeled globin, suggesting the presence of receptor-mediated endocytosis. After endocytosis, hemoglobin was observed inside large digestive vesicles. Albumin was exclusively associated with a population of small acidic vesicles, and an excess of unlabeled albumin did not inhibit its uptake. The intracellular pathway of the heme moiety of hemoglobin was specifically monitored using Palladium-mesoporphyrin IX (Pd-mP) as a fluorescent heme analog. When pulse and chase experiments were performed using digest cells incubated with Pd-mP bound to globin (Pd-mP-globin), strong yellow fluorescence was found in large digestive vesicles 4 h after the pulse. By 8 h, the emission of Pd-mP was red-shifted and more evident in the cytoplasm, and at 12 h most of the fluorescence was concentrated inside the hemosomes and had turned green. After 48 h, the Pd-mP signal was exclusively found in hemosomes. In methanol, Pd-mP showed maximal emission at 550 nm, exhibiting a red-shift to 665 nm when bound to proteins in vitro. The red emission in the cytosol and at the boundary of hemosomes suggests the presence of heme-binding proteins, probably involved in transport of heme to the hemosome. The existence of an intracellular heme shuttle from the digestive vesicle to the hemosome acting as a detoxification mechanism should be regarded as a major adaptation of ticks to a blood-feeding way of life. To our knowledge, this is the first direct observation of intracellular transport of heme in a living eukaryotic cell. A similar approach, using Pd-mP fluorescence, could be applied to study heme intracellular metabolism in other cell types.
血红素存在于所有细胞中，在呼吸和光合作用等基本代谢途径中作为辅因子发挥作用。此外，血红素及其降解产物一氧化碳、铁和胆绿素都被认为具有重要的信号传导作用。然而，对于不同细胞区室之间血红素通量所涉及的细胞内途径，我们了解得还很有限。微小牛蜱摄取的血液重量是其自身重量的100倍。中肠的消化细胞通过胞吞作用摄取血液成分，在血红蛋白消化过程中会释放出大量血红素。大部分血红素通过积累到一种特殊的细胞器——血色素体中而被解毒。我们通过用罗丹明标记的血红蛋白和白蛋白孵育，追踪了消化细胞原代培养物中血红蛋白和白蛋白的去向。未标记的球蛋白可抑制消化细胞对血红蛋白的摄取，这表明存在受体介导的内吞作用。内吞作用后，在大型消化泡内观察到了血红蛋白。白蛋白仅与一群小的酸性囊泡相关，过量的未标记白蛋白并不抑制其摄取。使用钯-中卟啉IX（Pd-mP）作为荧光血红素类似物，专门监测了血红蛋白血红素部分的细胞内途径。当用与球蛋白结合的Pd-mP（Pd-mP-球蛋白）孵育消化细胞进行脉冲追踪实验时，脉冲后4小时在大型消化泡中发现强烈的黄色荧光。到8小时时，Pd-mP的发射发生红移，在细胞质中更明显，12小时时大部分荧光集中在血色素体内并变成绿色。48小时后，Pd-mP信号仅在血色素体中发现。在甲醇中，Pd-mP在550nm处显示最大发射峰，在体外与蛋白质结合时发射峰红移至665nm。细胞质中和血色素体边界处的红色发射表明存在血红素结合蛋白，可能参与血红素向血色素体的转运。从消化泡到血色素体存在一种细胞内血红素穿梭机制作为解毒机制，这应被视为蜱对吸血生活方式的一种主要适应性。据我们所知，这是首次在活的真核细胞中直接观察到血红素的细胞内运输。一种类似的方法，利用Pd-mP荧光，可用于研究其他细胞类型中的血红素细胞内代谢。
