ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis

Abstract

Very long-chain fatty acids (VLCFAs) exert a variety of cellular functions and are associated with numerous diseases. However, the precise pathway behind their elongation has remained elusive. Moreover, few regulatory mechanisms for VLCFAs synthesis have been identified. Elongases catalyze the first of four steps in the VLCFA elongation cycle; mammals have seven elongases (ELOVL1-7). In the present study, we determined the precise substrate specificities of all the ELOVLs by in vitro analyses. Particularly notable was the high activity exhibited by ELOVL1 toward saturated and monounsaturated C20- and C22-CoAs, and that it was essential for the production of C24 sphingolipids, which are unique in their capacity to interdigitate within the membrane as a result of their long chain length. We further established that ELOVL1 activity is regulated with the ceramide synthase CERS2, an enzyme essential for C24 sphingolipid synthesis. This regulation may ensure that the production of C24-CoA by elongation is coordinated with its utilization. Finally, knockdown of ELOVL1 caused a reduction in the activity of the Src kinase LYN, confirming that C24-sphingolipids are particularly important in membrane microdomain function.

Fig. 1.

Each ELOVL protein exhibits a characteristic substrate specificity. HEK 293T cells were transfected with a vector (pCE-puro 3xFLAG-1) or a plasmid encoding the indicated 3xFLAG-tagged human ELOVL protein. (A) Total membrane proteins (0.1 μg protein) prepared from the transfected cells were treated with Endo H, separated by SDS-PAGE, and detected by immunoblotting with anti-FLAG antibodies. (B) Total membrane proteins (20 μg protein) were incubated with the indicated acyl-CoA (50 μM) and 0.075 μCi [¹⁴C]malonyl-CoA for 30 min at 37 °C. After termination of the reactions, lipids were saponified, acidified, extracted, and separated by normal-phase TLC, followed by detection and quantification by a bioimaging analyzer BAS-2500. Values presented are FA levels and represent the mean ± SD from three independent experiments. Statistically significant differences compared to vector transfected cells are indicated (*p < 0.05, **p < 0.01; t test). (C) Total membrane proteins (20 μg protein) were incubated with C18:0- or C20:0-CoA (20 μM) and 100 μM (0.075 μCi) [¹⁴C]malonyl-CoA for 30 min at 37 °C. After termination of the reactions, lipids were subjected to methanolysis, extraction, separation by reverse-phase TLC, and detection by BAS-2500. (D) VLCFA elongation pathways found in mammals and the ELOVLs proposed to be involved in each pathway are illustrated. The numbers located on the left side of the arrows indicate the ELOVL protein (ELOVL1–7). Parentheses exhibit weak activities of those enzymes in the indicated reactions. SFA, saturated FA; MUFA, monounsaturated FA.

Fig. 2.

Tissue-specific expression patterns of human ELOVL mRNAs. ELOVL and GAPDH cDNAs were amplified by PCR from human tissue cDNAs or from the pCE-puro 3xFLAG-ELOVLx plasmid using specific primers. Amplified fragments were separated by agarose gel electrophoresis and stained with ethidium bromide.

Fig. 3.

ELOVL1 is important for C24 sphingolipid production. HeLa cells were transfected with 16 nM control or ELOVL1 siRNA 4 d prior to experiments. (A) Total membrane proteins (40 μg) were incubated with the indicated acyl-CoA (50 μM) and 0.025 μCi [¹⁴C]malonyl-CoA for 1 h at 37 °C. After termination of the reactions, lipids were saponified, acidified, extracted, and separated by TLC, followed by detection and quantification by a bioimaging analyzer BAS-2500. Values represent the mean ± SD from three independent experiments. Statistically significant differences are indicated (**p < 0.01; t test). (B) Total RNA was prepared from the transfected cells and subjected to RT-PCR using primers specific for ELOVL1 or GAPDH. (C) Cells were labeled with 2 μCi [³H]sphingosine for 2 h at 37 °C. Lipids were extracted, separated by reverse-phase TLC, and detected by autoradiography (Right). The left panel shows C16:0 Cer, C20:0 Cer, C24:0 Cer, and C24:1 Cer standards, each at 10 nmol, separated by reverse-phase TLC, and stained with cupric acetate/phosphoric acid solution. (D) Lipids were extracted from the transfected cells and subjected to LC/ESI-MS/MS analysis. SM ions, determined as those losing the 60 Da neutral ion, were quantified. Values represent the relative amounts of certain SM species compared to total SM levels, and are the mean ± SD from three independent experiments. Note that ionization efficiencies varied among molecular species of SMs. Statistically significant differences are indicated (**p < 0.01; t test). E1, ELOVL1; cont., control.

Fig. 4.

CERS2 regulates ELOVL1 activity. (A) Total membrane proteins (40 μg) prepared from HEK 293T cells were incubated with the indicated acyl-CoA (40 μM) and 0.025 μCi [¹⁴C]malonyl-CoA in the presence or absence of 20 μM FB₁ for 1 h at 37 °C. After termination of the reactions, lipids were saponified, acidified, extracted, and separated by TLC, followed by detection and quantification by a bioimaging analyzer BAS-2500. Values represent the mean ± SD from three independent experiments. Statistically significant differences are indicated (*p < 0.05, **p < 0.01; t test). (B) HeLa cells were transfected with the indicated siRNAs (6 nM) 4 d prior to experiments. Total RNA was prepared then subjected to RT-PCR using primers specific for CERS2, CERS4, CERS5, or GAPDH. (C) Total membrane proteins (40 μg) prepared from HeLa cells transfected with the indicated siRNAs were subjected to an in vitro elongase assay as in A using the indicated acyl-CoA (50 μM) and 0.04 μCi [¹⁴C]malonyl-CoA. Values represent the mean ± SD from three independent experiments. Statistically significant differences are indicated (*p < 0.05; t test). (D) HEK 293T cells were transfected with a pcDNA3 HA-1 control vector, or a pcDNA3 HA-CERS2 or pcDNA3 HA-CERS4 plasmid, and with a pCE-puro 3xFLAG-ELOVL1 plasmid or pCE-puro 3xFLAG-1 control vector. Total cell lysates were prepared from the transfected cells and solubilized with 1% Triton X-100. Total lysates (Left) or proteins immunoprecipitated with anti-FLAG M2 agarose (Right) were subjected to immunoblotting with anti-FLAG or anti-HA antibodies. IP, immunoprecipitation; IB, immunoblotting; Total, total cell lysates.

Fig. 5.

Knockdown of ELOVL1 mRNA causes a reduction in LYN activity. HeLa cells were transfected with control, ELOVL1, ELOVL3, CERS2, or CERS4 siRNAs (15 nM) 4 d prior to experiments, and with the pCE-puro LYN-3xFLAG plasmid 2 d prior to experiments. Total lysates were prepared from the cells, solubilized with Triton X-100, and immunoprecipitated with anti-FLAG M2 antibodies. Immunoprecipitates were incubated with 5 μCi [γ-³²P]ATP for 5 min at 37 °C, then separated by SDS-PAGE, and detected by autoradiography (Upper) or by immunoblotting with anti-FLAG antibodies (Lower). p-LYN, phosphorylated LYN.