Smith A, Hunt R C
Department of Biochemistry and Molecular Biology, LSU Medical Center, New Orleans.
Eur J Cell Biol. 1990 Dec;53(2):234-45.
Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.
在体内和体外，血红素结合蛋白通过受体介导的方式转运血红素，结果导致血红素分解代谢，但蛋白质不受影响，这表明完整的脱辅基血红素结合蛋白可从细胞中循环利用。然而，迄今为止，受体介导的内吞作用对血红素结合蛋白的细胞内转运情况仍有待确定。对培养的人肝癌细胞系HepG2和小鼠肝癌细胞Hepa进行的生化研究表明，血红素结合蛋白被转运至细胞内，内吞作用后又完整地回到培养基中。在37℃孵育期间，血红素结合蛋白在细胞内积聚约15分钟后达到平台期，而表面结合在5分钟时达到饱和。在4℃孵育期间，没有配体发生内化。这些及其他数据表明，血红素结合蛋白受体可循环利用，此外，在37℃短期孵育期间，莫能菌素孵育显著抑制了细胞相关的血红素-[125I]血红素结合蛋白的量，这与受体循环受阻一致。氯化铵和甲胺的抑制作用较小。对血红素-[125I]血红素结合蛋白进行电子显微镜放射自显影显示，在人肝癌细胞系HepG2中，血红素结合蛋白存在于经典内吞途径的囊泡中，证实了血红素结合蛋白的内化。胶体金偶联的血红素结合蛋白和电子显微镜显示，4℃时与受体结合的血红素结合蛋白最初分布在整个细胞表面，包括微绒毛和被膜小窝。在37℃孵育后，血红素结合蛋白-金位于细胞内的被膜小泡中，然后位于小的内体和多泡体中。血红素结合蛋白和转铁蛋白在细胞内的共定位通过两种方式得以显示。使用二氨基联苯胺诱导的密度转移测定法，观察到放射性碘化血红素结合蛋白与转铁蛋白的辣根过氧化物酶偶联物存在于同一亚细胞区室中。此外，血红素-血红素结合蛋白和二价铁转铁蛋白的胶体金衍生物在被膜小窝、被膜小泡、内体和多泡体中共同存在。因此，血红素结合蛋白和转铁蛋白通过类似的受体介导机制发挥作用，即转运蛋白在内吞作用后从细胞中循环利用，以进行进一步的细胞内转运循环。
