Membrane Biology and Biochemistry Unit, Life and Medical Sciences Institute (LIMES), Bonn, Germany.
J Lipid Res. 2010 Jul;51(7):1747-60. doi: 10.1194/jlr.M003822. Epub 2010 Feb 23.
We examined the effect of Niemann-Pick disease type 2 (NPC2) protein and some late endosomal lipids [sphingomyelin, ceramide and bis(monoacylglycero)phosphate (BMP)] on cholesterol transfer and membrane fusion. Of all lipid-binding proteins tested, only NPC2 transferred cholesterol at a substantial rate, with no transfer of ceramide, GM3, galactosylceramide, sulfatide, phosphatidylethanolamine, or phosphatidylserine. Cholesterol transfer was greatly stimulated by BMP, little by ceramide, and strongly inhibited by sphingomyelin. Cholesterol and ceramide were also significantly transferred in the absence of protein. This spontaneous transfer of cholesterol was greatly enhanced by ceramide, slightly by BMP, and strongly inhibited by sphingomyelin. In our transfer assay, biotinylated donor liposomes were separated from fluorescent acceptor liposomes by streptavidin-coated magnetic beads. Thus, the loss of fluorescence indicated membrane fusion. Ceramide induced spontaneous fusion of lipid vesicles even at very low concentrations, while BMP and sphingomyelin did so at about 20 mol% and 10 mol% concentrations, respectively. In addition to transfer of cholesterol, NPC2 induced membrane fusion, although less than saposin-C. In this process, BMP and ceramide had a strong and mild stimulating effect, and sphingomyelin an inhibiting effect, respectively. Note that the effects of the lipids on cholesterol transfer mediated by NPC2 were similar to their effect on membrane fusion induced by NPC2 and saposin-C.
我们研究了尼曼-匹克病 2 型（NPC2）蛋白和一些晚期内体脂质[神经鞘磷脂、神经酰胺和双（单酰基甘油）磷酸酯（BMP）]对胆固醇转移和膜融合的影响。在所有测试的脂质结合蛋白中，只有 NPC2 以相当高的速率转移胆固醇，而没有转移神经酰胺、GM3、半乳糖脑苷脂、硫酸脑苷脂、磷脂酰乙醇胺或磷脂酰丝氨酸。BMP 极大地刺激了胆固醇的转移，神经酰胺的刺激作用很小，神经鞘磷脂的抑制作用很强。在没有蛋白质的情况下，胆固醇和神经酰胺也被显著转移。这种胆固醇的自发转移被神经酰胺大大增强，被 BMP 稍微增强，被神经鞘磷脂强烈抑制。在我们的转移测定中，生物素化供体脂质体通过链霉亲和素包被的磁性珠与荧光受体脂质体分离。因此，荧光的损失表明发生了膜融合。神经酰胺甚至在非常低的浓度下诱导脂质囊泡的自发融合，而 BMP 和神经鞘磷脂分别在约 20 mol%和 10 mol%的浓度下诱导融合。除了胆固醇的转移，NPC2 还诱导了膜融合，尽管不如脑苷脂激活蛋白 C（saposin-C）。在这个过程中，BMP 和神经酰胺具有很强和温和的刺激作用，而神经鞘磷脂则具有抑制作用。需要注意的是，这些脂质对 NPC2 介导的胆固醇转移的影响与它们对 NPC2 和脑苷脂激活蛋白 C 诱导的膜融合的影响相似。
