Ségui Bruno, Allen-Baume Victoria, Cockcroft Shamshad
Department of Physiology, University College London, London WC1E 6JJ, U.K.
Biochem J. 2002 Aug 15;366(Pt 1):23-34. doi: 10.1042/BJ20020317.
Mammalian phosphatidylinositol transfer proteins (PITPs) alpha and beta, which share 77% identity, have been shown to exhibit distinct lipid-transfer activities. In addition to transferring phosphatidylinositol (PI) and phosphatidylcholine (PC), PITPbeta has been shown to transfer sphingomyelin (SM), and this has led to the suggestion that PITPbeta is important for the regulation of SM metabolism. In the present study, we have analysed the ability of human PITPbeta to transfer and regulate the metabolism of cellular SM. We report that, in vitro, the two PITP isoforms were comparable in mediating PI, PC or SM transfer. Using permeabilized HL-60 cells as the donor compartment, both PITP isoforms efficiently transferred PI and PC, and were slightly active towards SM, with the activity of PITPbeta being slightly greater. To identify which cellular lipids were selected by PITPs, PITPalpha and PITPbeta were exposed to permeabilized HL-60 cells, and subsequently repurified and analysed for their bound lipids. Both PITPs were able to select only PI and PC, but not SM. SM synthesis takes place at the Golgi, and PITPbeta was shown to localize in that compartment. To examine the role of PITPbeta in SM biosynthesis, Golgi membranes were used. Purified Golgi membranes had lost their endogenous PITPbeta, but were able to recruit PITPbeta when added exogenously. However, PITPbeta did not enhance the activities of either SM synthase or glucosylceramide synthase. Further analysis in COS-7 cells overexpressing PITPbeta showed no effects on (a) SM and glucosylceramide biosynthesis, (b) diacylglycerol or ceramide levels, (c) SM transport from the Golgi to the plasma membrane, or (d) resynthesis of SM after exogenous sphingomyelinase treatment. Altogether, these observations do not support the suggestion that PITPbeta participates in the transfer of SM, the regulation of SM biosynthesis or its intracellular trafficking.
哺乳动物的磷脂酰肌醇转移蛋白（PITPs）α和β，它们的同源性为77%，已被证明具有不同的脂质转移活性。除了转移磷脂酰肌醇（PI）和磷脂酰胆碱（PC）外，PITPβ还被证明能转移鞘磷脂（SM），这表明PITPβ对SM代谢的调节很重要。在本研究中，我们分析了人PITPβ转移和调节细胞SM代谢的能力。我们报告，在体外，这两种PITP异构体在介导PI、PC或SM转移方面相当。以通透化的HL-60细胞作为供体区室，两种PITP异构体都能有效地转移PI和PC，对SM的活性较弱，其中PITPβ的活性略高。为了确定PITPs选择了哪些细胞脂质，将PITPα和PITPβ暴露于通透化的HL-60细胞，随后重新纯化并分析其结合的脂质。两种PITPs都只能选择PI和PC，而不能选择SM。SM合成发生在高尔基体，PITPβ被证明定位于该区室。为了研究PITPβ在SM生物合成中的作用，使用了高尔基体膜。纯化的高尔基体膜已失去其内源性PITPβ，但在外源添加时能够募集PITPβ。然而，PITPβ并没有增强SM合酶或葡萄糖神经酰胺合酶的活性。在过表达PITPβ的COS-7细胞中进行的进一步分析表明，对（a）SM和葡萄糖神经酰胺生物合成、（b）二酰甘油或神经酰胺水平、（c）SM从高尔基体到质膜的转运或（d）外源性鞘磷脂酶处理后SM的再合成均无影响。总之，这些观察结果不支持PITPβ参与SM转移、SM生物合成调节或其细胞内运输的观点。
