Prion rods contain small amounts of two host sphingolipids as revealed by thin-layer chromatography and mass spectrometry
Prion rods contain small amounts of two host sphingolipids as revealed by thin-layer chromatography and mass spectrometry
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DOI：
10.1515/bchm.1998.379.6.655
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发表时间：
1998-06-01
影响因子：
3.7
通讯作者：
Riesner, D
中科院分区：
文献类型：
--
作者：
Klein, TR;Kirsch, D;Riesner, D
在朊病毒（传染性海绵状脑病的病原体）中检测到鞘脂。对高度纯化的感染性朊病毒棒进行分析，所述朊病毒棒主要由N-末端截短的朊病毒蛋白（所谓的PrP 27-30）的不溶性聚集体组成。通过高效薄层色谱法定量脂质类，其中每种脂质类的检测限为25-50 ng。基质辅助激光解吸/电离质谱首次应用于复杂生物样品中的脂质分析。一种新开发的制备技术将灵敏度提高到1-20 pg/分子种类。在朊病毒棒的氯仿/甲醇（2：1 v/v）提取物中，仅始终观察到鞘脂、半乳糖神经酰胺和鞘磷脂。PrP与鞘脂的摩尔比在2：1和40：1之间，这取决于朊病毒制剂的纯度。相同的脂质也存在于朊病毒杆在0.2%SDS中超声处理后的梯度离心的低密度级分中。从两种替代方案来看，鞘脂是朊病毒功能所需的，或者是依赖于PrP在小窝中的细胞位置，第二种替代方案似乎更合理，因为最高特异性感染性的制剂含有最低量的鞘脂。
Sphingolipids were detected in prions, the agents of transmissible spongiform encephalopathies. The analysis was carried out on highly purified, infectious prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein, so-called PrP 27-30, Lipid classes were quantified by high performance thin-layer chromatography with a detection limit of 25-50 ng per lipid class. Matrix-assisted laser desorption/ionization mass spectrometry was applied for the first time to lipid analysis in complex biological samples. A newly developed preparation technique improved the sensitivity to 1-20 pg per molecular species, Only the sphingolipids, galactosylceramide and sphingomyelin, were consistently observed in chloroform/methanol (2:1 v/v) extracts of prion rods. The molar ratio of PrP to the sphingolipids was between 2:1 and 40:1, depending on the purity of the prion preparation. The same lipids were also present in the low density fraction of a gradient centrifugation of prion-rods after sonication in 0.2% SDS, From the two alternatives, that the sphingolipids are either required for prion function or are relies from the cellular location of PrP in caveolae, the second alternative appears more plausible since the preparation of highest specific infectivity contained the lowest amount of sphingolipids.