Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids

Abstract

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification.

Keywords: acyl-coenzyme A; arachidonic acid; cytochrome P450; fatty acid/metabolism; phospholipids.

Fig. 1.

FLAG column purified F-ACSL isoforms overexpressed in E. coli were resolved by PAGE and immunoblotted with anti-FLAG antibody.

Fig. 2.

Neutral loss of phosphoadenosine diphosphate (507.1 Da) scan of the [M+H]⁺ ions from F-ACSL4 no FA blank control (A), authentic AA-CoA positive control (Avanti Polar Lipids) (B), reaction product from F-ACSL4 and AA (C), reaction product from F-ACLS4 and 8,9-EET (D), reaction product from F-ACSL4 and 11,12-EET (E), and reaction product from F-ACSL4 and 14,15-EET (F). AA-CoA 1,054.3 Da and EET-CoAs 1,070.4 Da.

Fig. 3.

Purified F-ACSLs can activate either EETs (A) or HETEs (B) as substrates. ACS activity with different substrates at 5 μM (final concentration). Error bars reflect SEM from three separate experiments. S.A., specific activity.

Fig. 4.

Overexpressed ACSL1 and ACSL4 in COS7 cells. Immunoblots of membranes (50 μg) from COS7 cells overexpressing ACSL1 (A) and ACSL4 (B). GAPDH served as loading control. EV, empty vector.

Fig. 5.

Neutral loss of phosphoadenosine diphosphate (507.1 Da) scan of the [M+H]⁺ ions from COS7-ACSL4 and no FA control (A), reaction product from COS7-ACSL4 and AA (B), reaction product from COS7-ACSL4 and 8,9-EET (C), reaction product from COS7-ACSL4 and 11,12-EET (D), and reaction product from COS7-ACSL4 and 14,15-EET (E). AA-CoA 1,054.3 Da and EET-CoAs 1,070.4 Da.

Fig. 6.

ACSL1 and ACSL4 overexpressed in COS7 cells use both EETs and HETEs as substrates. ACS activity with 5 μM final substrate concentrations of AA, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 12-HETE, and 15-HETE. Error bars reflect SEM from three separate experiments. S.A., specific activity.

Fig. 7.

Michaelis-Menten kinetic enzyme activity curves from purified F-ACSL isoforms assayed spectrophotometrically with different FAs. F-ACSL1 (A), F-ACSL3 (B), F-ACSL4 (C), F-ACSL5 (D), and F-ACSL6 (E). Data points represent the mean of determinations from proteins obtained in three independent experiments.

Fig. 8.

Michaelis-Menten enzyme activity curves from membranes of COS7 cells overexpressing ACSL1 (A) or ACSL4 (B) with different FAs. Data points represent the mean of determinations from three independent experiments.

Fig. 9.

Scheme of the synthesis of EETs from unesterified AA catalyzed by CYP450 monooxygenase (Cyp450 MOase). EETs can then be activated by ACSL to produce EET-CoA. Once activated, the EET-CoA can be esterified by a phospholipid acyltransferase such as MBOAT5/LPCAT3, specific to lysophosphatidylcholine and lysophosphatidylethanolamine, or MBOAT7/LPIAT1, specific to lysophosphatidylinositol, to produce oxidized phospholipids.