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标题：CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism
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作者：Christopher D. Green ; Cynthia Weigel ; Clement Oyeniran 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2021
卷号：62
页码：100082
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：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, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (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. In addition, ORMDL-TKO but not ORMDL3-KO 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.