Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin - Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase
Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin - Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase
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DOI：
10.1074/jbc.273.50.33787
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发表时间：
1998-12-11
影响因子：
4.8
通讯作者：
Nishijima, M
中科院分区：
文献类型：
--
作者：
Hanada, K;Hara, T;Nishijima, M
Lysenin是一种来源于蚯蚓的溶血蛋白，对鞘磷脂有很高的亲和力。中国仓鼠卵巢(CHO)细胞对Lysenin表现出高度的细胞溶解敏感性，但鞘磷脂酶处理使细胞对Lysenin产生抗性。鞘磷脂合成缺陷的温度敏感型CHO突变细胞对Lysenin产生抗性，这种Lysenin抗性被鞘脂的代谢互补所抑制。从诱变的CHO细胞中筛选出两种类型的鞘磷脂缺失突变体，这两种突变体都显示出比野生型细胞更弱的赖氨酸结合能力。另一株突变株(命名为LY-B)不能从头合成任何一种鞘磷脂，也不具有丝氨酸棕榈酰基转移酶(SPT；EC 2.3.1.50)催化鞘磷脂合成第一步的活性。LY-B细胞缺乏SPT的一种成分LCB1蛋白，而将仓鼠LCB1基因导入LY-B细胞，可以恢复细胞的SPT活性和鞘磷脂的合成。亲和肽标记的LCB1蛋白在LY-B细胞中的表达导致内源性LCB2蛋白吸附到标记亲和基质上。此外，抗仓鼠LCB2蛋白抗体将SPT活性和野生型LCB1蛋白与LCB2蛋白进行了免疫共沉淀。因此，细胞表面鞘磷脂是Lysenin诱导的细胞溶解所必需的，并且Lysenin是分离鞘磷脂缺失突变体的有用工具，此外，这些结果表明SPT酶包括LCB1和LCB2蛋白。
Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, has a high affinity for sphingomyelin. Chinese hamster ovary (CHO) cells exhibited a high cytolytic sensitivity to lysenin, but treatment with sphingomyelinase rendered the cells resistant to lysenin. Temperature-sensitive CHO mutant cells defective in sphingolipid synthesis were resistant to lysenin, and this lysenin resistance was suppressed by metabolic complementation of sphingolipids. Selection of lysenin-resistant variants from mutagenized CHO cells yielded two types of sphingomyelin-deficient mutants, both of which showed less lysenin binding capability than wildtype cells. One mutant strain was severely defective in sphingomyelin synthesis but not glycosphingolipid synthesis, and another strain (designated LY-B) was incapable of de novo synthesis of any sphingolipid species and had no activity of serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzing the first step of sphingolipid biosynthesis. LY-B cells lacked the LCB1 protein, a component of SPT, and transfection of LY-B cells with the hamster LCB1 cDNA restored both SPT activity and sphingolipid synthesis to the cells. Expression of an affinity peptide-tagged LCB1 protein in LY-B cells caused the endogenous LCB2 protein to adsorb to a tag affinity matrix. In addition, an anti-hamster LCB2 protein antibody co-immunoprecipitated both SPT activity and the wild-type LCB1 protein with the LCB2 protein. Thus, cell surface sphingomyelin ia; essential for lysenin-induced cytolysis, and lysenin is a useful tool for isolation of sphingomyelin-deficient mutants, Moreover, these results demonstrate that the SPT enzyme comprises both the LCB1 and LCB2 proteins.