Mammalian long-chain acyl-CoA synthetases

Abstract

Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo- and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.

Figure 1

Representation of the spliced events affecting initiation of translation (A) and the fatty acid Gate domains (B). (A) Cartoon showing the exon-intron organization of the 5′-end of the ACSL genes, corresponding spliced variants (mRNA) and their products. The long isoforms start at the first AUG codon (AUG1, Met1) and the short isoform at the second AUG codon (AUG2, Met2). Note that two isoforms of ACSL3 are generated from the same mRNA. No variation has been reported for ACSL1. (B) Representation of the exclusive-exon pair encoding the two versions of the fatty acid Gate-domains (F- and Y-Gate) downstream of the exon encoding the ATP-binding site (P-loop). ACSL3, ACSL4, and ACSL5 genes lack the F-exon and encoded only the Y-Gate-domain version.

Figure 2

Residue alignments of the amino-terminus (A) and fatty acid Gate-domains (B) of all known human and mouse ACSL isoforms. The spliced variant annotation is shown on the left and the GenBank accession numbers are indicated in panel B. A color version of this figure is available in the online journal.

Figure 3

In silico analysis of the amino-terminus of the human ACSL isoforms. Prediction analysis of signal sequence was performed with SignalP on the long (left) and short (right) isoforms. The plots represent the probability of each residue to not be a part of the mature product (display as the signal peptide score, green curve), the probably of the presence of a site for signal peptidase (cleavage site score, red bar) and the predicted start of the mature product based on these two parameters (predicted, blue curve). ACSL1 encoded three isoforms that are identical in their N-terminus and are not predicted to carry a signal peptide. The residue alignments of the two isoforms are shown above the graphs with the putative signal peptide indicated in a box. A bar indicates the signal peptide position on the graphs.

Figure 4

Residue alignment of the amino-terminus of the ACSL1 forms of various species. The alignment was performed with CLUSTALW and shows the presence of an extra residue (Threonine), indicated with an arrow, in the rodent forms. The peptide used to raise a rat ACSL1 antibody is shown underlined (21). GenBank accession numbers were: human, NP_001986.2; Pan troglodytes, XP_517555.1; Sus scrofa, AAT79534; Gallus gallus, NP_001012596; Danio rerio, NP_001027007.1; mouse, NP_032007.2; rat, NP_036952.1. A color version of this figure is available in the online journal.

Figure 5

Spliced variants of human (A), mouse (B), and rat (C) ACSL4. Spliced sites occurring between the two codon initiators of translation, boxed ATG, are indicated by an arrow. Note that only a partial sequence of the exon is shown, indicated with a dashed line. Genbank accession numbers are given in Table 1. A color version of this figure is available in the online journal.

Figure 6

Sequence alignment of human ACSL6 gene and cloned variant 1. Alignment showing the missing bases and mismatches in the first encoding exon (exon 2) of the original cDNA of ACSL6, isolated from K562 cells (13). Missing bases are indicated with dashes (–) and matching bases with dots (.). This exon was interpreted as a UTR and the cloned cDNA was annotated as encoding a short isoform initiated at the ATG present in exon 3. Note that only a partial sequence of exons is shown. The predicted translated product of the full-length isoform is shown above the exon sequence with the two in-frame codon initiators boxed (ATG) and the product of the cloned variant is indicated below the sequence of the isolated cDNA.

Figure 7

Identification of the full-length rat ACSL6 isoform. (A) Residue alignment of the long products of human and rodent ACSL6 isoforms. Rat ACSL6 lacks the first encoding exon (AUG1 exon). (B) Identification of the first encoding exon of rat ACSL6. The alignment shows the nucleotide sequence of rat chromosome 10, position q22 39718637 to 39718787, compared with the sequence of an isolated cDNA obtained by reverse transcription-PCR of total RNA isolated from rat brain. The sequence was deposited at GenBank under GenBank accession number EF490998. (C) Residue alignment showing conservation of the first encoding exon in mammalian ACSL6 orthologues. A color version of this figure is available in the online journal.

Figure 8

Erroneous translation (A), update (B), and annotation (C) of mouse Acsl6 isoform 1. (A) Sequence of cDNA and product of version 0.1. The first 25 codons present in the annotated transcript (NM_144823.1) were mistakenly not translated and the annotated product (NP_659072.1) was initiated at the second ATG. (B) Residue alignment of the fatty acid Gate-domain of version 0.1 and the updated version, 0.3, of the Acsl6 product, NP_659072. Version 0.1 (NP_659072.1) lacked the first 25 residues (697 aa in length, residues shown +295 to +335) and contained the Y-Gate domain, whereas version 0.3 (NP_659072.3) was initiated at the first ATG (722 aa in length, residues shown +320 to +360) but contained the F-Gate domain version. (C) Residue alignment of the fatty acid Gate-domains of the two isoforms before and after the update. The original isoform 1 (NP_659072.1) and isoform 2 (AAO38689.1) represent the two versions of the Gate-domain, Y-Gate and F-Gate, respectively, which was mistakenly switched to the F-Gate (NP_659072.3) and Y-Gate (NP_001028769.1) during the update. RefSeq mistakes occurred during the correction of another mistake of the RefSeq annotated product of NM_144823.1. The first annotated product NP_659072.1 (AUG2, Y-exon) was erroneously translated from the second AUG and lacked the first 25 residues predicted by the nucleotide sequence of NM_144823 version 0.1 (AUG1, Y-exon) (panel A). Instead of correcting the wrongly translated product of NM_144823 version 0.1, NP_659072.1, and updating the product to version 0.2 (AUG1, Y-exon), RefSeq chose to update the cDNA sequence to version 0.2. It did so by truncating the AUG1-exon present in NM_144823.1, using an EST lacking the first AUG, and whose translated product would match the wrong product NP_659072.1. Then, version 0.2 of the cDNA and 0.1 of the product were both updated to version 0.3 and 0.2, respectively, using a third different EST. This EST contains AUG1, as in version 0.1, but also contains the F-exon instead of the Y-exon. Hence, version 0.2 of the product represented a different isoform (AUG1, F-exon). Moreover, the third cDNA also contained 5 base mismatches (two residues), hence a fourth EST was used to perform another update of both the cDNA and the product sequence, to version 0.4 and 0.3 respectively (panel B). Genbank accession numbers are given in Table 1. A color version of this figure is available in the online journal.