Atshaves Barbara P, Jefferson John R, McIntosh Avery L, Gallegos Adalberto, McCann Bonnie M, Landrock Kerstin K, Kier Ann B, Schroeder Friedhelm
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, USA.
Lipids. 2007 Oct;42(10):871-84. doi: 10.1007/s11745-007-3091-z. Epub 2007 Aug 7.
Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.
尽管鞘脂是富含于脂筏/小窝中的非常重要的信号分子，但对于可能调节鞘脂向活细胞脂筏/小窝分布的因素，如鞘脂结合蛋白等，人们了解得相对较少。由于早期研究表明固醇载体蛋白2（SCP-2）在体外可增强鞘糖脂从膜上的转移，因此研究了SCP-2表达对活细胞中鞘脂向脂筏/小窝分布的影响。使用非去污剂亲和色谱法从纯化的L细胞膜中分离脂筏/小窝和非脂筏，结果表明，与非脂筏结构域相比，脂筏/小窝中富含多种鞘脂，包括神经酰胺、酸性鞘糖脂（神经节苷脂GM1）、中性鞘糖脂（单己糖、二己糖、球苷脂）和鞘磷脂。SCP-2的过表达进一步增加了脂筏/小窝结构域中总鞘脂和特定鞘脂种类的含量。荧光结合和置换数据分析表明，纯化的人重组SCP-2对所有测试的鞘脂类别均表现出高结合亲和力（纳摩尔范围）。结合亲和力按以下顺序降低：神经酰胺>酸性鞘糖脂（神经节苷脂GM1）>中性鞘糖脂（单己糖、己糖、球苷脂）>鞘磷脂。在表达SCP-2的质膜中，各个鞘脂类别在脂筏/小窝与非脂筏中的富集情况与那些与SCP-2结合最紧密的类别（神经酰胺、GM1>中性鞘糖脂（单己糖、二己糖和球苷脂）>鞘磷脂）密切相关。综合这些数据表明，SCP-2在活细胞中起着选择性调节鞘脂向脂筏/小窝分布的作用。
