Hidaka H, Hidaka E, Tozuka M, Nakayama J, Katsuyama T, Fidge N
Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan.
J Lipid Res. 1999 Jun;40(6):1131-9.
We previously reported the identity and purification of two HDL3-binding proteins in rat liver plasma membranes. As these proteins are candidate high density lipoprotein (HDL) receptors and probably multifunctional, including a role in HDL metabolism, we have considerable interest in identifying corresponding proteins that are present in human tissue. This report describes the identification of HDL3-binding sites on human monocytes with the use of fluorescence microscopy and flow cytometry assay. After the incubation of mononuclear cells from human blood with fluorescein isothiocyanate (FITC)-labeled human HDL3, fluorescence micrographs showed dense signals of fluorescent grains on monocytes, but not lymphocytes. A significant increase in FITC intensity on monocytes, but not lymphocytes, was observed by flow cytometry analysis, and the interaction between FITC-HDL3 and human monocytes was concentration-dependent. Although very low density (VLDL) and low density lipoprotein (LDL) were ineffective competitors and HDL2 only partially competed for binding, a 50-fold concentration of HDL3 did compete effectively for binding of FITC-HDL3 to human monocytes. Trypsin treatment reduced the FITC intensity of monocytes, showing that a portion of cell-associated FITC-HDL3 remained bound to the cell surface. Two major HDL-binding proteins were identified in CHAPS-solubilized human mononuclear cells by ligand blotting, using HDL3 as the ligand. Both showed similar binding parameters, specificity, and molecular weight identical to HB1 and HB2 from rat liver plasma membrane. We conclude that corresponding candidate HDL receptors or a similar receptor complex also exist on human blood monocytes.
我们之前报道了大鼠肝细胞膜中两种高密度脂蛋白3（HDL3）结合蛋白的鉴定及纯化。由于这些蛋白是候选的高密度脂蛋白（HDL）受体，可能具有多种功能，包括在HDL代谢中发挥作用，因此我们对鉴定人类组织中存在的相应蛋白非常感兴趣。本报告描述了利用荧光显微镜和流式细胞术检测法鉴定人类单核细胞上HDL3结合位点的过程。用人血单核细胞与异硫氰酸荧光素（FITC）标记的人HDL3孵育后，荧光显微镜照片显示单核细胞上有密集的荧光颗粒信号，而淋巴细胞上没有。通过流式细胞术分析观察到单核细胞上FITC强度显著增加，而淋巴细胞上没有，并且FITC-HDL3与人单核细胞之间的相互作用呈浓度依赖性。尽管极低密度脂蛋白（VLDL）和低密度脂蛋白（LDL）作为竞争者无效，HDL2仅部分竞争结合，但HDL3浓度提高50倍时确实能有效竞争FITC-HDL3与人单核细胞的结合。胰蛋白酶处理降低了单核细胞的FITC强度，表明一部分与细胞结合的FITC-HDL3仍与细胞表面结合。通过配体印迹法，以HDL3作为配体，在CHAPS溶解的人单核细胞中鉴定出两种主要的HDL结合蛋白。两者显示出相似的结合参数、特异性，分子量与大鼠肝细胞膜中的HB1和HB2相同。我们得出结论，人类血液单核细胞上也存在相应的候选HDL受体或类似的受体复合物。
