ORMDLs results in elevated sphingolipid synthesis under control conditions and reverses the homeostatic inhibition of sphingolipid synthesis resulting from elevated cellular sphingolipid

The ORM1 ( Saccharomyces cerevisiae )-like proteins (ORMDLs) and their yeast orthologs, the ORMs, regulate the initiating and rate-limiting enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT) ( 1–4 ). The three ORMDL isoforms (ORMDL1–3) are small (17.5 kDa) hydrophobic membrane proteins that are situated in the endoplasmic reticulum along with SPT. The three isoforms are highly homologous and their functions appear to be redundant at the cellular level ( 3, 4 ). The ORMDLs are negative regulators of SPT and mediate the homeostatic regulation of SPT in response to cellular sphingolipid levels. siRNA depletion of the ORMDLs results in elevated sphingolipid synthesis under control conditions and reverses the homeostatic inhibition of sphingolipid synthesis resulting from elevated cellular sphingolipid content ( 1, 3, 4 ). The Orms and ORMDLs form stable physical complexes with SPT ( 1 ), even under conditions in which the ORMDLs are minimally inhibiting SPT. In yeast, the Orm regulatory activity is controlled by Orm phosphorylation. This may not be the case for the mammalian ORMDLs, which lack the sequences that are phosphorylated in the yeast Orms. Clinical interest in ORMDL function has been sparked by the observation that SNPs adjacent to the ORMDL3 gene are highly correlated with Abstract The ORM1 ( Saccharomyces cerevisiae )-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex. —Siow, D., M. Sunkara, T. M. Dunn, A. J. Morris, and B. Wattenberg. ORMDL/ serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis. J. Lipid Res. 2015. 56: 898–908.