Activity of the acyl-CoA synthetase ACSL6 isoforms: role of the fatty acid Gate-domains
© Soupene et al; licensee BioMed Central Ltd. 2010
Received: 20 November 2009
Accepted: 29 April 2010
Published: 29 April 2010
Activation of fatty acids by acyl-CoA synthetase enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6.
Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms.
The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.
In mammals, long-chain acyl-CoA synthetases (ACSLs) are necessary for fatty acid degradation, phospholipid remodeling, and the production of long acyl-CoA esters that regulate many physiological processes . These membrane-bound enzymes act on non-polar hydrophobic substrates, fatty acids, generating acyl-CoAs, important activated intermediates in lipid synthesis pathways, that are water-soluble as well as powerful detergents [2–7]. In the first step of the two-step reaction catalyzed by these enzymes, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with co-enzyme A to produce the activated acyl-CoA. The five long-chain acyl-CoA synthetase genes can give rise to multiple spliced transcript variants . 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 [1, 2, 8–10]. In particular, the bacterial form acts as a dimer, and a conserved short motif, called the fatty acid Gate domain, is implicated in controlling access of the fatty acid substrate to the catalytic site of each of the two sub-units .
These membrane proteins can not be purified in the absence of detergent and can form aggregates when over-expressed in E. coli . Defining the enzymatic characteristics of the mammalian forms in vivo as well as in vitro has proven a challenge and has resulted in rather contradictory results . Isoforms of the human ACSL6 members, predominately found in brain tissue and erythrocytes, can be expressed as active enzymes in the membrane of E. coli [8, 11–13]. We determined the activity of several isoforms representing alternative spliced variants which differ at their amino-terminus and have different short motifs of 26 residues in close proximity of the ATP-binding site . These alternative motifs, specific for the mammalian forms, were predicted to contain different versions of the bacterial homologue fatty acid Gate-domain [2, 8, 14, 15]. We now show that alternative splicing of these motifs generates isoforms of ACSL6 with strikingly different activity, and we identified the aromatic residues representing the Gating residue for fatty acid entry for each version of the Gate-domains of human ACSL6.
Results and Discussion
Activity of the Tyrosine-Gate, Phenylalanine-Gate and no-Gate domain isoforms
Identification of the Gating residues
These results indicate that the splicing event affecting the two alternative fatty acid Gate-domains of the human and rodent ACSL6 forms produces enzyme species with different Gating residues and enzymatic properties. In addition, these results establish that, for a Y-Gate enzyme, the presence of a tyrosine residue at position 319 was essential for activity, whereas any aromatic residue could be tolerated in the F-Gate domain. Finally, the difference of activity of the Y-to-F mutant compared to a normal F-Gate enzyme, and of a F-to-Y mutant compared a normal Y-Gate enzyme indicate that other residues are involved in defining the specific activity of isoform 1 and 2.
Specific Gate-domain residues essential for activity
ATP requirement and inhibition of activity
ATP is needed for the initial formation of the acyl-AMP intermediate in the two-step reaction catalyzed by ACSL. CoA-SH is subsequently exchanged to form the final acyl-CoA product. To determine if excess ATP could inhibit the CoA-exchange reaction and limit the formation of the product by the active enzymes, all 14C-labeled compounds present in the reaction mixture were separated, identified and quantified by thin-layer chromatography (see Methods section). The distribution of the 14C-labeled compounds was similar at 1 and 20 mM ATP. Under these conditions, 70-80% of the substrate was converted and the relative ratio of the two acyl-AMP and acyl-CoA species was similar, despite a 20-fold difference in ATP concentration (Figure 5, panel C). However, for reactions performed at 50 mM ATP, the acyl-AMP and acyl-CoA species were not formed, and most of the 14C-label was found in the unreacted acyl substrate (acyl left), as was the case for the reaction performed without addition of ATP (0 mM column). Hence, the inhibitory effect of ATP was also observed on the formation of the intermediary acyl-AMP species and accounted for the decrease in production of the final acyl-CoA product. As this effect was not observed with an enzyme lacking strong activity, these results may indicate that the released pyrophosphate at the first-step and/or the released AMP during the second step can inhibit the acyl-AMP reaction. The reported higher Km value for the lesser active rat ACSL6 isoform 1 may have been erroneously determined because of the lack of inhibition and of the slightly increased activity obtained at high ATP concentration, as observed with human isoform 1.
Inhibition of acyl-CoA formation by Triton X-100
Alteration of the Carboxy-terminal domain affects activity
Removal of the Amino-terminus stimulates activity
The activities of the truncated forms (ΔN constructs) were assayed with oleic acid (C18:1-OH) and compared to that of their respective full-length versions (isoform 1 and 2) (Figure 1 and 2). Surprisingly, removal of the N-terminus resulted in enzymes with far greater activity (Figure 2). These differences were most noticeable in the 10-fold increase in velocity value of the otherwise poorly active Y-Gate form. For the more active F-Gate form, removal of the N-terminus resulted in an increase of the apparent affinity rather than in the Vmax value. Similarly, large increases in activity rates were observed with palmitic acid (C16:0-OH) and arachidonic acid (C20:4-OH) (not shown). As anticipated, truncation of the N-terminus did not rescue the activity of the inactive form truncated of its Gate-domain (ΔGate and ΔN-ΔGate) (Figure 1, right column).
Amino-terminal domain is not essential for oligomer formation
On denaturing polyacrylamide gels, the migration pattern of the truncated F-Gate form was noticeably different compare to the full-length protein. As reported previously , when proteins were solubilized from membranes under mild condition (1% SDS at 37°C) rather than boiled before separation on gels containing 0.1% SDS, human ACSL6 isoforms were detected as two bands with apparent molecular mass of 70 kDa and 140 kDa (Figure 8 and Additional file 1). The calculated mass of a monomeric form was 80 kDa (see legend), but as commonly observed for hydrophobic proteins, human ACSL6 migrates faster than the molecular weight standard of the corresponding mass. The species of higher molecular mass was twice the apparent mass of a monomeric protein and since the two bacterial homologues (FadD and ttACS) are dimeric enzymes [1, 2, 4], it likely represents the dimeric form of human ACSL6. The same pattern was obtained with rat ACSL6 isoform 1 and isoform 2 expressed in E. coli (data not shown). The two bands (70 and 140 kDa) were also recognized by an antibody raised against the epitope tag present at the N-terminus (Additional file 1) and by an affinity-purified antibody raised against human ACSL6 . Both bands were also detected in protein extracts of mammalian cells expressing ACSL6 . Although, these two species were detected with all forms and recombinant constructs, their relative amounts were significantly different for the ΔN-(F-Gate) truncated construct (Figure 8. Panel A, lane 3 vs. lane 5; Panel B, lane 4 vs. 6). As seen on a gel, the ratio of high to low molecular mass species was greater for the truncated ΔN-(F-Gate) protein than for its full-length version (Figure 8, panels B and C). Hence, the N-terminal domain is not essential for formation of the dimeric complexes but its removal increased dimer formation and/or stability in the presence of SDS. Based on the findings that the bacterial forms act as dimers, these complexes probably represent the active species of human ACSL6. Their relative increased in abundance could account for the higher activity observed with the truncated F-Gate construct (Figure 2). The ΔN-(Y-Gate) protein was also more active than its full-length version, but even under the mild denaturing conditions we used, we could not detect a difference in the dimer to monomer ratio. Repeated attempts to resolve and quantify these oligomeric forms under non-denaturing conditions [21, 22] or by gel filtration chromatography were not successful. Differences in stability of the two full-length forms (isoform 1 versus isoform 2) might also account for their unexpected differences of activity (Figure 2).
Detection of interaction between isoforms
Activity of oligomeric complexes
Addition of a tag at the C-terminus of the active isoform 2 resulted in an enzyme with very low activity. This tagged version of isoform 2 provided an additional test to detect the formation of oligomeric complexes. Activity assays were performed with membrane fractions obtained from cells carrying a single vector that allowed co-expression of two differently tagged proteins from the same promoter. This provided an assay for the measurement of the activity of different isoforms associated in oligomeric complexes. For example, ACSL activity of cells co-expressing each of the two tagged versions of isoform 2, the active Nter-2 with the inactive Cter-2, were compared to cells that co-express the active Nter-2 with the inactive Cter-3. In addition, different combinations were made to generate reference and control cells (Figure 7). To detect the putative effects of the inactive sub-unit (Cter-2 or Cter-3) on activity of the enzyme complex (e.g. Nter-2/Cter-2 or Nter-2/Cter-3), the activity rates were normalized to the amount of active sub-units present in the complexes, i.e. Nter-2 (see methods section and Figure 7, panel A). When the two different tagged versions of isoform 2 (Figure 7, 4th bar) were co-expressed, the rate of formation of acyl-CoA by membranes obtained from these cells was far lower than the rate of synthesis obtained with the Nter-2 enzyme expressed alone (1st bar). Even in the absence of normalization for the relative amount of the active species (Figure 7, panel B), activity rate of the Nter-2/Cter-2 enzyme was nearly as low as the value obtained with the Cter-2 enzyme expressed alone (2nd bar) or with co-expression of the two tagged versions of the inactive isoform 3 (Nter-3/Cter-3) (Figure 7, last bar). This result strongly suggests that the inactivated isoform 2 had a dominant effect on the activity of the active unit, Nter-isoform 2. This effect of one protein on the activity of another confirmed that oligomeric complexes were formed as established above. The effect of the inactivated Cter-isoform 2 on an active form was compared to the effect of the naturally occurring inactive isoform 3 (the truncated form that misses the essential Gate-domain) on that active form. This test may mimic the in vivo circumstances in which various spliced isoforms with different activity rates are expressed in the same cells [1, 8]. Intriguingly, interaction with isoform 3 did not result in inhibition of the isoform 2. The species Nter-2/Cter-3 (Figure 7, 5th bar) was as active as the homodimer Nter-2 (1st bar).
The C-terminus hammer domain of ACSL is connected to the anvil domain, carrying the ATP-binding site and the Gate-domain, by a flexible linker . The hammer and anvil form the catalytic site and the correct positioning of the C-terminus is essential to prevent escape as well as entry of acyl-AMP until the second step of the reaction with CoA-SH has occurred, and acyl-CoA is formed. The defect of the extended C-terminally tagged enzyme could render the second active sub-unit of the dimeric complex structurally defective resulting in a slower acyl-CoA synthetase activity. Isoform 3 lacks the motif carrying the fatty acid Gate-domain. This domain is essential for the entry and access of the fatty acid to the catalytic site. The removal of the short region connecting the fatty acid tunnel to the ATP-binding site might not preclude processing of fatty acids once they have found their way to the site. The apparent activity of the isoform 3 in the complex formed with isoform 2 could indicate that entry of the fatty acids through one of the sub-units provides substrate to the other one through an internal channel event. Efforts to solve the structure of these homo- and heterodimeric complexes are underway.
The alternative fatty acid Gate-domain motifs play an essential role in the activity of the human ACSL6 enzymes. The aromatic residues representing the Gating residues of the two versions of the Gate-domain were identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2. A tyrosine and tryptophan could be substituted to the phenylalanine residue of isoform 2 without affecting activity whereas the tyrosine residue was essential for the activity of isoform 1. We also identified another residue, a leucine, present in the F-containing Gate domain (F-Gate), that is essential for the activity of that form. The three isoforms can form dimeric complexes with identical units and with different isoforms. As previously proposed, the diversity of acyl-CoA synthetase activities of the many ACSL6 enzyme species might represent an adaptation mechanism of an enucleated cell, the red blood cell, to the absolute necessity to repair damaged lipids and to maintain the lipid composition of the membrane [1, 23].
The activation of fatty acids to acyl-CoA is required for many fatty acid utilizing pathways in physiology. All plasma membranes need to maintain a defined lipid composition, both in relative amount of the different classes and in molecular species within each class. Acylation of lysophospholipids with different acyl-CoA species is an essential step, and ACSL enzymes play a central role in this process . In the red blood cell membrane, ACSL6 is responsible of maintaining the phospholipid composition of this enucleated cell lacking de novo lipid synthesis. For the iron-containing oxygen-carrying red blood cell, the activities of ACSL6 isoforms are especially important for the repair of phospholipids damaged by oxidation. ACSL6 is one of the major forms expressed in neuronal cells, and as in other cells that rapidly turn-over their plasma membrane lipids, activities of the different ACSL6 complexes might be essential to their plasma membrane plasticity . An understanding of how the different isoforms of ACSL6 act, and interact, is essential to determine the mechanisms sustaining the formation of the various acyl-CoA species needed to maintain plasma membrane composition. Additional enzymatic studies combined with structural information are needed to better understand the function of these enzymes in physiologic processes.
List of oligonucleotides used in this study.
sequence 5' to 3'
Site-directed mutagenesis experiments were performed with the QuikChange Multi Site-directed Mutagenesis kit (Stratagene) according to the manufacturer instruction. Primers were designed with the QuikChange® Primer Design Program (Stratagene). The presence of the intended nucleotide change(s) and the absence of unwarranted mutations were verified by full-length sequencing of the constructs.
Detection of protein-protein interactions was performed in E. coli using the BacterioMatch II Two-hybrid system (Stratagene). As indicated in Figure 9 and Additional file 2, the different isoforms were cloned by PCR at the Not I/BamH I restriction sites of the bait vector (pBT) and of the target vector (pTRG). Primers were designed to introduce an Eag I and a Xho I site at the 5' and 3' end, respectively, of the ACSL6 amplicons. Plasmids were propagated into the E. coli strain XL1-Blue MRF' Kan (Stratagene). All constructs were verified by sequencing and expression of the different fusion proteins was verified by immuno-detection with an affinity-purified anti-ACSL6 antibodies . Results are presented in Additional file 2, panel C. Expression of the fused proteins was induced with a concentration of IPTG of 30 μM. Interaction analysis was performed in E. coli BacterioMatch II Validation reporter cells, carrying a his B mutation and the HIS3 reporter cassette, according to the manufacturer instructions (Stratagene).
Co-expression of isoforms fused at their amino-terminus with an hexahistidine-tag, or at their carboxy-terminus with a S-tag, were performed in E. coli cells transformed with various construct made in the pCOLA-DUET vector of Novagen. With this vector, two constructs can be cloned under the control of an identical promoter and expressed simultaneously upon addition of IPTG at a concentration of 100 μM in the culture medium. N-terminally His-tagged constructs were obtained by PCR cloning at the BamH I and Hind III restriction sites, whereas C-terminally S-tagged constructs were cloned at the Nae I and Xho I restriction sites. The two primer sets were designed to introduce BamH I and Hind III sites and Pml I and Xho I sites, respectively. The two primer sets were Cola-BamH with Cola-Hind, and Cola-Pml with Cola-Xho. Correct fusion of the different constructs was verified by sequencing, and expression of the fusion proteins was confirmed by immuno(affinity)-detection with an anti-Histidine antibody (Pierce) and S-protein (Novagen), respectively (see below).
Expression and immunodetection of ACSL6
ACSL6 isoforms cloned into pET28a and pCOLA-DUET vectors were transformed into E. coli BL21(DE3) cells (Novagen). Expression and detection with an HRP-conjugated anti-Histidine or anti-ACSL6 antibody were performed as previously described . Cells were disrupted in breakage buffer containing Tris-HCl 50 mM, pH 8.0, EDTA 5 mM, NaCl 0.3 M, DTT 5 mM and PMSF 0.1 mM, by two passages in a French press cell at 12,000 psi. for The lysate was cleared by centrifugation at 8,000 g for 20 min at 4°C. To prepare the membrane and soluble fractions, the total extract was subjected to centrifugation at 100,000 g for 60 min at 10°C. The supernatant represented the soluble fraction. The pellet represents the membrane fraction and was suspended in breakage buffer supplemented with 10% glycerol and, stored frozen at -80°C. Detection of the S-tagged constructs with a HRP-conjugated-(S-protein) (Novagen) was performed under the same condition used with the anti-His antibody. Expression of isoforms cloned in the pBait and pTRG vectors was performed in M9 salt synthetic medium with 0.2% glucose supplemented with 1 mM MgS04, 1 mM thiamine-HCl, 0.2 mM adenine, with 30 μM IPTG (induced condition), and with 5 mM 3-amino-1,2,4-triazole, freshly made in DMSO at 1 M, and when appropriate, supplemented with 25 μg/ml kanamycin, 25 μg/ml chloramphenicol, and 10 μg/ml tetracycline, according to the manufacturer instructions (Stratagene). Cells were grown at 37°C for 24 to 48 hrs or at 30°C for 48 to 72 hrs. Production of the different fusion proteins was analyzed by immuno-detection of protein extracts of cells grown in presence of IPTG with an affinity-purified anti-ACSL6 antibody which recognized all the isoforms and the ΔN constructs fused to either the lambda-cI protein (Bait vector set) or the amino-terminal domain of the RNA polymerase alpha subunit (Additional file 2). The analysis was done with cells transformed with only one of each of the constructs so as to confirm that each construct was correctly fused and produced at level comparable to each other. Predicted molecular mass (in kDa) of the fusions were: cI-isoform 1, 103; cI-isoform 2, 103; cI-isoform 3, 95; cI-ΔN-isoform2, 99; RNAPa-isoform 1, 105; RNAPa-isoform 2, 105; RNAPa-isoform 3, 97.
Acyl-CoA synthetase assay
All measurements were performed with membrane fractions obtained from E. coli BL21(DE3) cells expressing the different isoforms and constructs. Prior to measurements, control experiments were performed with the different enzyme preparations to determine the correct amount of proteins that will not process more than 20% of the radio-labeled fatty acids under the reaction condition and the period of the incubation. The particular reaction conditions of each assay are given in the legend of the figures. In most experiments, 0.25 to 10 μg of proteins were used and the reactions were performed at 30°C for an incubation period ranging from 0 to 8 min. Unless indicated otherwise, reactions were performed in a final volume of 150 μl and initiated by the addition of 130 μl of a mixture containing the enzyme to the fatty acid dissolved in 20 μl. The fatty acid was usually first dispatched in glass tubes and pre-warmed at 30°C before addition of the pre-warmed enzyme mixture. When appropriate, protein diluted in 10 μl was first dispatched in the tubes and reactions were initiated by addition of 140 μl of the reaction mixture containing the fatty acids mixture. 14C-labeled fatty acid suspensions were usually provided at a concentration of 4 μM (unless otherwise indicated in legend of figures) with a specific activity of 50-54 mCi/mmol. Dried fatty acids were suspended in 20 μl of 13.5 mM NaHCO3 and 0.55 mM Triton X-100 and were diluted to 1.8 mM NaHCO3 and 74 μM Triton X-100 in the final reaction volume. The 130 μl enzyme mixture was diluted with the fatty acid buffer to a final concentration of 0.1 M Tris.HCl pH 8.0, 1 mM DTT, 20 mM MgCl2, 20 mM ATP and 0.5 mM CoA. The reaction was stopped by addition of 2.25 ml of isopropanol/heptane/2 M H2SO4 (40:10:1, v/v) and vigorous vortexing. Newly synthesized 14C-acyl-CoA was separated from un-esterified 14C-fatty acid by successive addition of 1 ml of heptane and 1 ml of H2O, and vigorous vortexing. The two phases were separated by centrifugation at 500 g for 3 min. The upper organic phase was carefully removed and collected in a scintillation vial. The lower aqueous phase was then extracted twice with 2 ml heptane saturated with 4 mg/ml palmitic acid, and once with 2 ml heptane. Each of the upper phases were collected and pooled with the first one. The amounts of newly synthesized 14C-acyl-CoA and of[14C-fatty acid were measured by scintillation counting in the lower and upper phases, respectively. For each reaction, the total amount of 14C present was calculated by addition of the dpm values of the upper and lower phases. Control reactions were performed with membranes obtained from cells carrying the vector pET28a and the dpm values obtained were subtracted to those obtained with the enzyme preparations. For calculation of the kinetic parameters, data were displayed as a scatter plot of activity rates in function of the concentration of substrate (Figure 2) using Prism graphing software version 5 (GraphPad Software, Inc.), and the kinetic values were obtained by non-linear regression of the data computed by the software.
Thin-layer chromatography detection of the acyl-species was performed as described in . Reactions were performed in 15 μl with 10 μM 14C-oleic acid and 1 μg of membrane proteins. After 10 min incubation at 30°C, the reaction was stopped with 1.6 μl of acetic acid 50% and spotted on a silica plate. Along with standards, species were separated at 4°C for 4-5 hrs in a solvent system made of 1-butanol/acetic acid glacial/water (80/25/40; v/v). The plates were dried and analyzed with a PhosphoImager Storm Scanner (Molecular Dynamics, GE Healthcare).
This work was supported in part by grant from the National Institutes of Health to F.A.K (HL070583).
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