Setälä Niko L, Holopainen Juha M, Metso Jari, Wiedmer Susanne K, Yohannes Gebrenegus, Kinnunen Paavo K J, Ehnholm Christian, Jauhiainen Matti
Department of Ophthalmology, University of Helsinki, Helsinki, Finland.
Biochemistry. 2007 Feb 6;46(5):1312-9. doi: 10.1021/bi0621866.
In circulation the phospholipid transfer protein (PLTP) facilitates the transfer of phospholipid-rich surface components from postlipolytic chylomicrons and very low density lipoproteins (VLDL) to HDL and thereby regulates plasma HDL levels. To study the molecular mechanisms involved in PLTP-mediated lipid transfer, we studied the interfacial properties of PLTP using Langmuir phospholipid monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) to follow the transfer of 14C-labeled phospholipids and [35S]PLTP between lipid vesicles and HDL particles. The AsFlFFF method was also used to determine the sizes of spherical and discoidal HDL particles and small unilamellar lipid vesicles. In Langmuir monolayer studies high-activity (HA) and low-activity (LA) forms of PLTP associated with fluid phosphatidylcholine monolayers spread at the air/buffer interphase. Both forms also mediated desorption of [14C]dipalmitoylphosphatidylcholine (DPPC) from the phospholipid monolayer into the buffer phase, even when it contained no physiological acceptor such as HDL. After the addition of HDL3 to the buffer, HA-PLTP caused enhanced lipid transfer to them. The particle diameter of HA-PLTP was approximately 6 nm and that of HDL3 approximately 8 nm as determined by AsFlFFF analysis. Using this method, it could be demonstrated that in the presence of HA-PLTP, but not LA-PLTP, [14C]DPPC was transferred from small unilamellar vesicles (SUV) to acceptor HDL3 molecules. Concomitantly, [35S]-HA-PLTP was transferred from the donor to acceptor, and this transfer was not observed for its low-activity counterpart. These observations suggest that HA-PLTP is capable of transferring lipids by a shuttle mechanism and that formation of a ternary complex between PLTP, acceptor, and donor particles is not necessary for phospholipid transfer.
在血液循环中，磷脂转运蛋白（PLTP）促进富含磷脂的表面成分从脂解后的乳糜微粒和极低密度脂蛋白（VLDL）转移至高密度脂蛋白（HDL），从而调节血浆HDL水平。为研究PLTP介导的脂质转移所涉及的分子机制，我们利用Langmuir磷脂单层膜和不对称流场流分级法（AsFlFFF）研究了PLTP的界面特性，以追踪14C标记的磷脂和[35S]PLTP在脂质囊泡与HDL颗粒之间的转移。AsFlFFF方法还用于测定球形和盘状HDL颗粒以及小单层脂质囊泡的大小。在Langmuir单层膜研究中，高活性（HA）和低活性（LA）形式的PLTP与在空气/缓冲液界面铺展的流体磷脂酰胆碱单层膜相关联。即使缓冲液中不含有诸如HDL等生理受体，这两种形式也都介导了[14C]二棕榈酰磷脂酰胆碱（DPPC）从磷脂单层膜解吸至缓冲液相。向缓冲液中添加HDL3后，HA-PLTP导致脂质向其转移增强。通过AsFlFFF分析测定，HA-PLTP的颗粒直径约为6 nm，HDL3的颗粒直径约为8 nm。使用该方法可以证明，在存在HA-PLTP而非LA-PLTP的情况下，[14C]DPPC从小单层囊泡（SUV）转移至受体HDL3分子。同时，[35S]-HA-PLTP从供体转移至受体，而其低活性对应物未观察到这种转移。这些观察结果表明，HA-PLTP能够通过穿梭机制转移脂质，并且PLTP、受体和供体颗粒之间形成三元复合物对于磷脂转移并非必要。
