The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, while downregulation of all 3 ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome editing strategy, we established stable single ORMDL3 knockout (ORMDL3-KO) and ORMDL1/2/3 triple knockout (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. Additionally, triple ORMDL1/2/3 knockout but not ORMDL3 single knockout dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.

中文翻译：

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ORMDLs 的 CRISPR/Cas9 缺失揭示了鞘脂代谢的复杂性。

丝氨酸棕榈酰转移酶 (SPT) 复合物催化神经酰胺（鞘脂的前体）从头生物合成中的限速步骤。哺乳动物 ORMDL 亚型 (ORMDL1-3) 是 SPT 的负调节剂。然而，单个 ORMDL 异构体的作用尚不清楚。使用针对单个 ORMDL 的 siRNA，只有单个 siORMDL3 对二氢神经酰胺和神经酰胺水平有适度的影响，而所有 3 个 ORMDL 的下调诱导更明显的增加。借助基于 CRISPR/Cas9 的基因组编辑策略，我们建立了稳定的单 ORMDL3 敲除 (ORMDL3-KO) 和 ORMDL1/2/3 三重敲除 (ORMDL-TKO) 细胞系，以进一步了解 ORMDL 蛋白在鞘脂生物合成中的作用。虽然 ORMDL3-KO 适度增加了二氢神经酰胺和神经酰胺的水平，ORMDL-TKO 细胞在这些鞘脂前体的积累中显着增加。SPT 活性仅在 ORMDL-TKO 细胞中增加。此外，三重 ORMDL1/2/3 敲除而不是 ORMDL3 单敲除显着增加了半乳糖神经酰胺、葡萄糖神经酰胺和乳糖神经酰胺的水平，其升高的 N-酰基链分布与神经酰胺种类的增加广泛相关。令人惊讶的是，虽然 C16:0 是主要的鞘磷脂种类，但它仅在 ORMDL3-KO 中增加，而所有其他 N-酰基链鞘磷脂种类在 ORMDL-TKO 细胞中显着增加。鞘氨醇碱基分析表明，虽然 ORMDL-TKO 细胞中的鞘氨醇仅增加了 2 倍，但二氢鞘氨醇、二氢鞘氨醇-1-磷酸、ORMDL-TKO 细胞和 ORMDL3-KO 细胞中 1-磷酸鞘氨醇显着增加，而不是在 ORMDL3-KO 细胞中。因此，ORMDL 蛋白可能在细胞鞘脂的生物合成和调节中具有复杂的、多方面的作用。