Hanada K, Nishijima M, Akamatsu Y, Pagano R E
Department of Biochemistry and Cell Biology, National Institute of Health, Tokyo, Japan.
J Biol Chem. 1995 Mar 17;270(11):6254-60. doi: 10.1074/jbc.270.11.6254.
SPB-1, a Chinese hamster ovary cell variant defective in serine palmitoyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol deprivation on cellular functions and membrane properties. To investigate whether there was an interaction among sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (PLAP) in SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubility of PLAP in Triton X-100. Although the PLAP solubility of the membranes isolated from the control cells in Triton X-100 was only 10%, deprivation of sphingolipid and cholesterol further enhanced the solubility, which reached 50% when both sphingolipids and cholesterol were deprived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes changed independently, eliminating the possibility that sphingolipid deprivation induced a reduction in cellular cholesterol and enhanced PLAP solubility and vice versa. It was also unlikely that the enhanced solubility was due to structural changes in PLAP molecules since, regardless of sphingolipid and cholesterol deprivations, almost all PLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected by sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the PLAP insolubility and suggested that these lipids coordinately played a role in formation of Triton X-100-resistant complexes.
SPB - 1是一种中国仓鼠卵巢细胞变体，其在用于鞘脂合成的丝氨酸棕榈酰转移酶活性方面存在缺陷，为研究鞘脂和/或胆固醇缺乏对细胞功能和膜特性的影响提供了一个有用的系统。为了研究生物膜中鞘脂、胆固醇和糖基磷脂酰肌醇（GPI）锚定蛋白之间是否存在相互作用，我们通过稳定转染将人胎盘碱性磷酸酶（PLAP）引入SPB - 1细胞和野生型细胞中，并研究了鞘脂和/或胆固醇缺乏对PLAP在Triton X - 100中的溶解度的影响。虽然从对照细胞分离的膜在Triton X - 100中的PLAP溶解度仅为10%，但鞘脂和胆固醇的缺乏进一步提高了溶解度，当鞘脂和胆固醇都缺乏时，溶解度达到50%。通过外源性鞘氨醇和胆固醇的代谢互补，增强的溶解度被抑制到对照水平。分离膜中鞘脂和胆固醇的含量独立变化，排除了鞘脂缺乏导致细胞胆固醇减少并增强PLAP溶解度，反之亦然的可能性。增强的溶解度也不太可能是由于PLAP分子的结构变化，因为无论鞘脂和胆固醇是否缺乏，几乎所有的PLAP都具有GPI锚定部分，并且在去污剂处理膜的上清液和沉淀部分中蛋白质的表观分子量没有差异。此外，分离膜中窖蛋白的表达水平不受鞘脂和/或胆固醇消耗的显著影响。这些结果表明鞘脂和胆固醇都与PLAP的不溶性有关，并表明这些脂质在形成抗Triton X - 100复合物中协同发挥作用。
