CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism

Christopher D. Green,Clement Oyeniran,Jason Newton,Usha Mahawar,Sarah Spiegel,Briana N. James,Deanna Davis,Cynthia Weigel,Michael Maceyka,Binks W. Wattenberg

doi:10.1016/j.jlr.2021.100082

Christopher D. Green, Clement Oyeniran + Show 8 more

Open Access

https://doi.org/10.1016/j.jlr.2021.100082

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Abstract

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.

Highlights

The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids
Effects of ORMDL knockdown on ceramide levels Given previous results with ORMDL3 regulation of ceramide levels in epithelial cells [21] together with the recent observation in mice that ORMDL3 controls brain sphingolipids [18], we sought to compare the effect of downregulating individual ORMDLs with that of ORMDL1/2/3 triple knockdown in A549 cells, which express all three isoforms, on dihydroceramide, an intermediate formed solely in the de novo sphingolipid biosynthetic pathway, and on ceramide that can be generated both by de novo and recycling/salvage pathways
Immunoblotting with an antibody that recognizes all three ORMDL isoforms showed that the ORMDL3-KO1 cells had modestly reduced levels of ORMDL proteins, suggesting that ORMDL3 was absent and that ORMDL1 and ORMDL2 were not upregulated at the protein level (Fig. 2A)

Summary

Introduction

The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. With the CRISPR/Cas9-based genomeediting strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 tripleKO (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. 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. ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids. Infection and Immunity, The Ohio State University, Columbus, OH, USA. Current address for Deanna Davis: University of Louisville, Diabetes and Obesity Center, Louisville, KY, USA

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