Characterization of Recombinant Long-Chain Rat Acyl-CoA Synthetase Isoforms 3 and 6:  Identification of a Novel Variant of Isoform 6

Abstract

The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 microM. DHA was also strongly preferred by ACSL6_v2. The apparent Km value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.

Acyl-CoA Synthetase Activity Links Wild-Type but Not Mutant α-Synuclein to Brain Arachidonate Metabolism

“…On the other hand, we demonstrated a 32% reduction in brain microsomal acyl-CoA synthetase activity (Vmax) in preparations from α-synuclein deficient mouse brains ( Figure 5 and Table 3). In addition, the Vmax/Km ratio was also significantly depressed in the gene-ablated mice (Table 3), demonstrating that under physiological conditions (75) there would be an effect by α-synuclein on Acsl activity. While we demonstrated the presence of α-synuclen in snca +/+-microsomes (Figure 6), others have also noted that α-synuclein is particularly enriched in microsomal fractions containing primarily ER (23) as well as colocalized with ER in synaptosomal preparations (21,80,89), but not found associated with synaptic vesicles (80).…”

The implications of alternative splicing in the ENCODE protein complement

“…Kinetics assays show that this isoform has conspicuously different ATP-binding affinities (29). A further locus for which there is evidence of distinct functions is XX-FW83563B9.3 (TAZ), where it has been shown that alternative isoform 002, which has a 31-residue deletion from skipping the fifth exon, may in fact be the principal isoform, because it is the only isoform to have full cardiolipin metabolic activity (30).…”

Suppression of Long Chain Acyl-CoA Synthetase 3 Decreases Hepatic de Novo Fatty Acid Synthesis through Decreased Transcriptional Activity

“…Based upon these data, it has been predicted that ACSL3 has an active role in phospholipid or eicosanoid metabolism (36). ACSL3 has a broad range of substrate preferences toward C 16 -C 24 fatty acids (35,37). Although few studies have focused on the physiological function of ACSL3 (16,35), its localization on lipid droplets or endoplasmic reticulum membranes (31,32) suggests that this enzyme is likely involved in lipid synthesis.…”