Milis Daniel G, Moore Messiah K, Atshaves Barbara P, Schroeder Friedhelm, Jefferson John R
Department of Chemistry, Luther College, Decorah, Iowa, 52101-1045, USA.
Mol Cell Biochem. 2006 Feb;283(1-2):57-66. doi: 10.1007/s11010-006-2270-1.
The influence of sterol carrier protein-2 (SCP-2) on the cellular metabolism of sphingolipids was examined in control mouse L-cells and stably transfected clones expressing the protein SCP-2. Three approaches were used to examine for differences; (1) compositional analysis of endogenous sphingolipid classes, (2) metabolism of NBD-ceramide, and (3) live cell labelling via endocytic uptake of BODIPY-sphingomyelin. SCP-2 over expression significantly altered the endogenous levels of both neutral and acidic sphingolipid classes. Among the neutral sphingolipids, expression of SCP-2 induced a 1.7-fold increase in the level of lactosylceramide (LacCer, p < 0.05) and a similar fold decrease in the level of the higher-order neutral glycosylceramides (p < 0.05). Among the acidic sphingolipids, SCP-2 resulted in a 5.2-fold decrease in the endogenous plasma membrane level of ganglioside GM1 (p < 0.03). Incubation of both control and transfected cell lines with NBD-ceramide resulted in the rapid establishment of a steady-state distribution of NBD-labelled sphingomyelin (NBD-SM) and glucosylceramide (NBD-GlcCer). In the SCP-2 expressing clones the conversion of NBD-Cer to NBD-GlcCer was 30% lower during incubation periods between 5 and 30 min (p < 0.025). Inspection of the cells by fluorescence microscopy after incubation with BODIPY labelled sphingomyelin (BODIPY-SM) revealed similar punctuated patterns with no distinguishable differences between the cell types. These results imply that SCP-2 plays a role in modulating enzymatic steps involved in metabolism of sphingolipid homeostasis.
在对照小鼠L细胞和稳定转染表达固醇载体蛋白-2（SCP-2）的克隆中，研究了SCP-2对鞘脂细胞代谢的影响。采用了三种方法来检测差异：（1）内源性鞘脂类别的组成分析，（2）NBD-神经酰胺的代谢，以及（3）通过内吞摄取BODIPY-鞘磷脂进行活细胞标记。SCP-2的过表达显著改变了中性和酸性鞘脂类别的内源性水平。在中性鞘脂中，SCP-2的表达导致乳糖基神经酰胺（LacCer）水平增加1.7倍（p<0.05），而高阶中性糖基神经酰胺水平则有类似倍数的下降（p<0.05）。在酸性鞘脂中，SCP-2导致神经节苷脂GM1的内源性质膜水平下降5.2倍（p<0.03）。用NBD-神经酰胺孵育对照细胞系和转染细胞系，均导致NBD标记的鞘磷脂（NBD-SM）和葡萄糖基神经酰胺（NBD-GlcCer）迅速建立稳态分布。在表达SCP-2的克隆中，在5至30分钟的孵育期内，NBD-神经酰胺向NBD-葡萄糖基神经酰胺的转化降低了30%（p<0.025）。用BODIPY标记的鞘磷脂（BODIPY-SM）孵育后，通过荧光显微镜检查细胞，发现细胞类型之间没有明显差异的类似点状模式。这些结果表明，SCP-2在调节参与鞘脂稳态代谢的酶促步骤中发挥作用。
