Substrate Specificity of Human Long-Chain Acyl-CoA Synthetase ACSL6 Variants

Abstract

Long-chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their active form, acyl-CoAs. Recent knock-out mouse studies revealed that among ACSL isoenzymes, ACSL6 plays an important role in the maintenance of docosahexaenoic acid (DHA)-containing glycerophospholipids. Several transcript variants of the human ACSL6 gene have been found; the two major ACSL6 variants, ACSL6V1 and V2, encode slightly different short motifs that both contain a conserved structural domain, the fatty acid Gate domain. In the present study, we expressed recombinant human ACSL6V1 and V2 in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system, and then, using our novel ACSL assay system with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined the substrate specificities of the recombinant human ACSL6V1 and V2 proteins. The results showed that both ACSL6V1 and V2 could convert various kinds of long-chain fatty acids into their acyl-CoAs. Oleic acid was a good common substrate and eicosapolyenoic acids were poor common substrates for both variants. However, ACSL6V1 and V2 differed considerably in their preferences for octadecapolyenoic acids, such as linoleic acid, and docosapolyenoic acids, such as DHA and docosapentaenoic acid (DPA): ACSL6V1 preferred octadecapolyenoic acids, whereas V2 strongly preferred docosapolyenoic acids. Moreover, our kinetic studies revealed that ACSL6V2 had a much higher affinity for DHA than ACSL6V1. Our results suggested that ACSL6V1 and V2 might exert different physiological functions and indicated that ACSL6V2 might be critical for the maintenance of membrane phospholipids bearing docosapolyenoic acids such as DHA.

INTRODUCTION

Long-chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their active form, acyl-CoAs, for both the synthesis of cellular lipids and degradation via β-oxidation.^1,2) In humans and rodents, five ACSL isoforms have been identified: ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6.²⁾ Each ACSL isoform has a distinct substrate preference, subcellular localization, and tissue distribution, and each has been suggested to be involved in the modulation of various pathophysiological events via the generation of long-chain acyl-CoA. Among these ACSL isoforms, we and others found that ACSL4 has a preference for arachidonic acid (AA) as well as other highly unsaturated fatty acids (HUFAs) and plays an important role in the maintenance of HUFA-containing glycerophospholipids and arachidonate metabolism.^3–7)

Like ACSL4, ACSL6 has also been shown to play an important role in the maintenance of HUFA-containing glycerophospholipids. Overexpression of ACSL6 in PC12 cells increased docosahexaenoic acid (DHA) internalization, stimulated phospholipid synthesis, and then enhanced neurite outgrowth.⁸⁾ In ACSL6-deficient mice, the levels of DHA- or docosapentaenoic acid (DPA)-containing glycerophospholipids were decreased in tissues highly abundant with ACSL6, such as brain and testis.^9–11) Moreover, ACSL6 deficiency disrupted normal brain function and spermatogenesis. ACSL6-deficient mice exhibited motor dysfunction and increased astrogliosis.⁹⁾ ACSL6 was also shown to be required for normal spermatogenesis, and ACSL6-deficient male mice were severely subfertile.^10,11)

ACSL6 was first identified in rat brain.¹²⁾ Thereafter, several transcript variants of the ACSL6 gene have been reported.^13–15) The two major variants, ACSL6V1 and ASCL6V2, arise from an alternative splicing of exons 14 or 13, respectively, in the human ACSL6 gene^2,15) (Fig. 1A). These two variants encode slightly different short motifs containing a conserved structural domain called the fatty acid Gate domain, which is shown to form a fatty acid-binding tunnel and which is an essential determinant of ACSL activity.^16,17) The amino acid sequence of the Gate domains in each ACSL6 variant is highly conserved among species (Fig. 1B). Van Horn et al. showed that in a direct competition assay with palmitic acid, ACSL6V2 preferred palmitic acid slightly more than AA and strongly preferred DHA.¹³⁾ Those authors also showed that ACSL6V1 strongly preferred linoleic and linolenic acids over palmitic acid. However, the precise substrate specificities of ACSL6V1 and V2 have not been fully elucidated.

Fig. 1. Human ACSL6V1 and V2 and Amino Acid Sequences of Their Gate Domains

A: Schematic representation of human ACSL6V1 and V2. Exons are represented by shaded boxes and alternative exons by black boxes. Translation initiation site AUG is also indicated. B: Alignment of the Gate domain motifs of human, mouse, and rat ACSL6V1 and V2.¹⁾ Conserved amino acid residues are shaded.

Very recently, we established an in vitro ACSL assay system using liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is highly sensitive and applicable to various kinds of fatty acids.^5,6) In the present study, we cloned the cDNAs of human ACSL6V1 and V2, expressed these recombinant enzymes in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system, and then, using our ACSL assay system, examined the substrate specificity of the recombinant human ACSL6V1 and V2 proteins.

MATERIALS AND METHODS

Materials

Human brain total RNA and Ex Taq were purchased from TaKaRa Bio Inc. (Shiga, Japan). Human liver total RNA and restriction endonucleases were from BioChain Institute Inc. (Newark, CA, U.S.A.). KOD plus DNA polymerase Ver.2 was from Toyobo (Osaka, Japan). The pTAKN-2 vector was from BioDynamics Laboratory Inc. (Tokyo, Japan). SuperScript III reverse transcriptase, oligo(dT)_12–18 primer, Bac-to-Bac™ Baculovirus Expression System, Cellfectin reagent, pFASTBac1 vector, and goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) were from Invitrogen (Carlsbad, CA, U.S.A.). Max Efficiency DH10Bac Competent Cells were from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Anti-ACSL6 rabbit antibody, palmitic acid (16 : 0), γ-linolenic acid (18 : 3), dihomo-γ-linolenic acid (20 : 3), docosatetraenoic acid (22 : 4), DPA (n-3) (20 : 5), and various acyl-CoA standards were from Sigma-Aldrich (St. Louis, MO, U.S.A.). Linoleic acid (18 : 2), α-linolenic acid (18 : 3), AA (20 : 4), eicosapentaenoic acid (20 : 5), DPA (n-6) (22 : 5), and DHA (22 : 6) were from Cayman Chemical Company (Ann Arbor, MI, U.S.A.). Oleic acid (18 : 1) was from Nacalai Tesque Inc. (Kyoto, Japan). Heptadecanoyl-CoA (17 : 0-CoA) was from Avanti Polar Lipids Inc. (Birmingham, AL, U.S.A.).

Expression of Recombinant Human ACSL6 Variants

Human brain and liver cDNAs were synthesized from human brain total RNA and liver total RNA using oligo(dT)_12–18 primers with SuperScript III reverse transcriptase, respectively. The cDNAs encoding human ACSL6V1 and V2 were prepared by two rounds of PCR reactions from these cDNAs. The first PCR used the sense primer (5′-CGG GCC CCG CTG AC-3′) and antisense primer (5′-GCT CAG TCA TAA AAT CAG ATG CTT ATT C-3′), both of whose sequences are common to ACSL6V1 and V2 and contain a few nucleotide sequences in the untranslated region just outside the translated sequence of ACSL6. With each primer and high-fidelity KOD plus DNA polymerase Ver.2, cDNA products were amplified from the human brain and liver cDNA by PCR (10 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2.5 min). In the second PCR, cDNA products were amplified from the first PCR product by PCR (35 cycles of 95 °C for 30 s and 55 °C for 30 s, followed by 72 °C for 2.5 min) using ExTaq and each primer for ACSL6: the sense primer (5′-CAC CATGCTG ACC TTC TTC CTC GT-3′) and the antisense primer (5′-TTA ATG GTG ATG GTG ATG ATG CAT GGA GAT TGA GTA AAG CTC TTC-3′). These were then subcloned into the pTAKN-2 vector, and the DNA sequences were determined. By outsourcing the plasmid DNA sequencing analysis to FASMAC Co., Ltd. (Kanagawa, Japan), we confirmed that the amino acid sequences from the plasmid DNA are consistent with those of ACSL6 V1 and V2 registered in GenBank. We used BamHI/XhoI to digest the plasmids carrying ACSL6V1 cDNA or ACSL6V2 cDNA and ligated the resulting inserts into a pFASTBac1 vector at the BamHI/XhoI sites. The Bac-to-Bac Baculovirus Expression System was used to produce recombinant baculoviruses. The obtained plasmid was transfected into DH10Bac-competent Escherichia (E.) coli, and recombinant bacmid DNA was prepared. The recombinant baculovirus carrying ACSL6 was produced by transfecting the purified bacmid into insect cell Sf9 using Cellfectin reagent. The virus was amplified by adding an aliquot of the initial virus pool to fresh, sub-confluent Sf9 cells in 150-mm dishes. After 5 d of incubation at 27 °C, all infected cells were centrifuged to pelleted. The pellet was washed with phosphate-buffered saline (PBS) and resuspended in 20 mM phosphate buffer pH 7.0 containing 500 mM NaCl (buffer A). The cell suspensions were sonicated by a sonicator (Branson Ultrasonics, Brookfield, CT, U.S.A.) at 25% output (1 mM phenylmethylsulfonyl fluoride (PMSF) was added before sonication), and centrifuged at 10000 × g for 10 min. The supernatant at 10000 × g was used as the ACSL6 V1 or V2 in the ACSL enzyme assay.

Measurement of ACSL Activity

Enzyme preparations (1 µg) were incubated with 0.01–100 nmol of the indicated fatty acids in 100 µL assay buffer (50 mM phosphate buffer pH 7.0, 1% Triton-X 100, 5 mM dithiothreitol, 15 mM MgCl₂, 1 mM CoA, 10 mM ATP) for 20 min at 37 °C. After incubation, 1 mL of methanol containing an internal standard (5 pmol heptadecanoyl-CoA, 17 : 0-CoA) was added to terminate the reaction followed by centrifugation at 20000 × g for 5 min. The supernatant was dried in a centrifugal evaporator, suspended in 400 µL of methanol, and centrifuged at 20000 × g for 5 min. The resulting supernatant was further diluted 25-fold and then analyzed by LC-MS/MS to quantify the acyl-CoA species. All mass spectrometry was performed according to a previously reported method.^5,6) In the present study, we used a linear ion trap quadrupole mass spectrometer (QTRAP6500plus; SCIEX, Framingham, MA, U.S.A.).

RESULTS

Expression of Recombinant Human ACSL6V1 and V2

We first cloned the cDNA for two major human ACSL6 variants, ACSL6V1 and V2, from human liver and brain cDNAs, respectively. Plasmids containing the cDNAs for ACSL6V1 and V2 were used for bacmid preparation; then recombinant ACSL6 variants with a C-terminal His-tag were expressed in Sf9 cells. Expression of recombinant human ACSL6V1 and V2 in 10000 × g supernatants of Sf9 cells was confirmed by Coomassie Brilliant Blue (CBB) staining and immunoblotting with anti-ACSL6 antibody (Fig. 2). Although ACSL6 is predicted to carry one transmembrane spanning segment at its N-terminus, it is still unclear if ACSL6 is inserted into the membrane or if it is only associated to membranes.¹⁾ ACSL6 may be loosely bound to the membrane. Since our immunoblot analysis revealed that both ACSL6V1 and V2 were detected in 10000 × g supernatants of baculovirus-infected Sf9 cells expressing ACSL6 variants, we here used the 10000 × g supernatant as the enzyme sources for the ACSL enzyme assay.

Fig. 2. Expression of Recombinant ACSL6V1 and V2 in Sf9 Cells

SDS-PAGE was performed using 10000 × g supernatant of Sf9 cells infected with baculovirus carrying ACSL6V1 or ACSL6V2 for 5 d or without baculovirus (control), and the expression levels of recombinant ACSL6 proteins were confirmed by CBB staining (upper panel) and Western blotting with anti-ACSL6 antibody (lower panel). The following samples were applied to lanes 1–3: 10000 × g supernatant (25 µg protein) of uninfected Sf9 cells (lane 1) and Sf9 cells infected with baculovirus carrying ACSL6V1 or V2 (lanes 2 and 3, respectively). For Western blotting, anti-ACSL6 rabbit antibody and goat anti-rabbit IgG-HRP were used as the primary and secondary antibodies, respectively. In lanes 2 and 3, the bands of ACSL6V1 and V2, respectively, were detected around approximately 75 kDa.

Substrate Preference of Recombinant Human ACSL6V1 and V2

We recently established an ACSL assay system using LC-MS/MS that is applicable to various kinds of fatty acids.^5,6) With this system, the acyl-CoA species were identified using multiple reaction monitoring (MRM) transition and retention times, and the acyl-CoA species were quantified on the basis of the analyte peak area of the MRM transition. We could not generate a calibration curve for quantitative analysis of the kinetics of ACSL6V1 and V2 on the long-chain fatty acids whose acyl-CoA products were not commercially available. Therefore, we first evaluated the substrate specificity of the recombinant human ACSL6V1 and V2 proteins on the basis of the ratio of the peak area of the analyte to the internal standard (17 : 0-CoA).

As a result, we found that both ACSL6V1 and V2 could convert various kinds of long-chain fatty acids into their respective acyl-CoAs (Fig. 3). The enzymatic activities on oleic acid (18 : 1) were almost the same for both variants, but their preferences for octadecapolyenoic acids, such as linoleic acid (18 : 2) and linolenic acid (18 : 3), and for docosapolyenoic acids, such as DHA (22 : 6) and DPA (22 : 5), differed considerably. Whereas ACSL6V1 preferred octadecapolyenoic acids, ACSL6V2 strongly preferred DHA (22 : 6) and other docosapolyenoic acids over the other long-chain fatty acids. On the other hand, AA (20 : 4) and other eicosapolyenoic acids were poor substrates for both ACSL6V1 and V2.

Fig. 3. Fatty Acid Preferences of Recombinant Human ACSL6V1 and V2 Expressed in Sf9 Cells

Ten thousand × g supernatants of Sf9 cells (1 µg protein) infected with baculovirus carrying ACSL6V1 or V2 (ACSL6V1 or ACSL6V2, respectively) or without baculovirus (control) were incubated with fatty acids (100 µM) having different chain lengths and different degrees of unsaturation at 37 °C for 20 min. The acyl-CoA values generated in these reactions were measured on the basis of the ratio of the peak area of the analyte to the internal standard (17 : 0-CoA) by LC-MS/MS (mean + standard error of the mean (S.E.M,) n = 3). Each data point represents the mean of three measurements obtained in each experiment. S.E.Ms. are indicated by error bars.

Kinetic Studies of Recombinant Human ACSL6V1 and V2

Since 18 : 1-CoA, 18 : 2-CoA, 20 : 4-CoA and 22 : 6-CoA are commercially available, their use as standards enabled us to perform quantitative analyses of the kinetics of ACSL6V1 and V2 on oleic acid (18 : 1), linoleic acid (18 : 2), AA (20 : 4), and DHA (22 : 6). The substrate concentrations of these long-chain unsaturated fatty acids were varied (1–100 µM), and then Michaelis–Menten plots on these four fatty acids were generated for ACSL6V1 and V2 (Fig. 4). As shown in Table 1, ACSL6V1 and V2 had similar K_m values and V_max for oleic acid (18 : 1) and AA (20 : 4), but these two variants had very different K_m values for linoleic acid (18 : 2) and DHA (22 : 6). Whereas the K_m value for linoleic acid (18 : 2) of ACSL6V2 was 5-fold higher than that of ACSL6V1, the K_m value for DHA (22 : 6) of ACSL6V2 was 7-fold lower than that of ACSL6V1. In addition, V_max for DHA (22 : 6) of ACSL6V2 was 6-fold higher than that of ACSL6V1.

Fig. 4. Michaelis–Menten Plots from Recombinant Human ACSL6V1 and V2 Expressed in Sf9 Cells Assayed with Different Fatty Acids

With indicated concentrations (0.1–1000 µM) of oleic acid (18 : 1), linoleic acid (18 : 2), AA (20 : 4), and DHA (22 : 6)), ACSL6 assay was performed using 10000 × g supernatants of Sf9 cells (1 µg) infected with baculovirus carrying ACSL6V1 or V2 (ACSL6V1 or ACSL6V2, respectively) at 37 °C for 20 min. The specific activity levels of the ACSL6 variants were measured by LC-MS/MS (means ± S.E.M., n = 3). Each data point represents the mean of three measurements obtained in each experiment. SEMs are indicated by error bars.

Table 1. Kinetic Constants for Recombinant Human ACSL6V1 and V2 with Oleic Acid (18 : 1), Linoleic Acid (18 : 2), AA (20 : 4), and DHA (22 : 6)

	C18:1	C18:2	C20:4	C22:6
ACSL6V1
Km (µM)	97.0	16.8	67.8	109.0
Vmax (nmol/min/mg)	75.7	35.6	12.4	53.5
Vmax/Km (mL/min/mg)	0.78	2.12	0.18	0.49
ACSL6V2
Km (µM)	116.9	86.6	53.2	14.7
Vmax (nmol/min/mg)	96.7	21.3	14.2	340.0
Vmax/Km (mL/min/mg)	0.83	0.25	0.27	23.19

K_m and V_max values were calculated by Michaelis–Menten plots as shown in Fig. 3.

V_max/K_m is the rate constant for the capture of a substrate by an enzyme into a productive complex.¹⁸⁾ In Table 1, the V_max/K_m data indicate that ACSL6V2 interacts with DHA (22 : 6) at a much higher catalytic efficiency than with the other three long-chain fatty acids. The V_max/K_m values of ACSL6V2 were in the order of DHA (22 : 6) >> oleic acid (18 : 1) > AA (20 : 4) > linoleic acid (18 : 2), although those of ACSL6V1 were in the order of linoleic acid (18 : 2) > oleic acid (18 : 1) > DHA (22 : 6) > AA (20 : 4).

DISCUSSION

In this study, we prepared recombinant human ACSL6V1 and V2 proteins using the baculovirus expression system and then examined those enzymes’ substrate specificities by using our optimized ACSL assay system with LC-MS/MS. This revealed distinctive differences in substrate preference between ACSL6V1 and V2. Whereas ACSL6V2 strongly preferred docosapolyenoic acids, ACSL6V1 preferred octadecapolyenoic acids. Especially, ACSL6V2 exhibited a remarkably high affinity and catalytic efficiency for DHA. The V_max/K_m value of ACSL6V2 for DHA was about 50-fold higher than that of ACSL6V1. It was noteworthy that these two variants encode identical amino acid sequences except for the fatty acid Gate domain (Fig. 1A). These results suggested that the Gate domain of ACSL6V2 determines its substrate specificity for docosapolyenoic acids, although structural analyses of ACSL6V1 and V2 are needed to uncover the precise molecular mechanism(s) by which they recognize specific fatty acids as their substrates.

In the brain and testis of ACSL6-deficient mice, the levels of DHA-containing glycerophospholipids were decreased while those of AA-containing ones were increased.^9–11) It was also shown that the levels of DPA-containing glycerophospholipids were decreased in ACSL6-deficient testis.¹¹⁾ These phenotypes in ACSL6-deficient mice corresponded to the substrate preference of ACSL6V2 in this study. Mouse ACSL6V2 has an identical Gate domain to human ACSL6V2 (Fig. 1B). Among ACSL6 variants, ACSL6V2 appears to be critical for the maintenance of docosapolyenoic acid-containing glycerophospholipids in these tissues. Several DHA-selective lysophospholipid acyltransferase isozymes, such as LPAAT3 and LPAAT4, have been identified.^19–21) Since both LPAAT3-deficient mice and ACSL6-deficient mice also exhibited impaired spermatogenesis,^10,11,20) ACSL6V2 might be coupled with these DHA-selective acyltransferase isozymes.

Finally, we here found that ACSL6V1 preferred octadecapolyenoic acids as its substrate. Our PCR analysis using commercial human tissue cDNAs revealed that both ACSL6V1 and V2 were highly expressed in human brain and testis (data not shown). ACSL6V1 and V2 might have different functions in brain and testis development, and ACSL6 gene splicing might be involved in the development of these tissues. Further studies of the molecular mechanism by which the splicing of ACSL6 variants is regulated are needed in order to clarify the physiological roles of ACSL6 variants.

Acknowledgments

This work was supported in part by a Grants-in-Aid for Scientific Research (B) (No. 16H05108 and 19H03375 to S.H.) and for Scientific Research (C) (No. 19K07087 to H.K.) from the Japan Society for the Promotion of Science, and by the Private University Research Branding Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Soupene E, Kuypers FA. Mammalian long-chain acyl-CoA synthetases. Exp. Biol. Med. (Maywood), 233, 507–521 (2008).
• 2) Mashek DG, Bornfeldt KE, Coleman RA, Berger J, Bernlohr DA, Black P, DiRusso CC, Farber SA, Guo W, Hashimoto N, Khodiyar V, Kuypers FA, Maltais LJ, Nebert DW, Renieri A, Schaffer JE, Stahl A, Watkins PA, Vasiliou V, Yamamoto TT. Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family. J. Lipid Res., 45, 1958–1961 (2004).
• 3) Kang MJ, Fujino T, Sasano H, Minekura H, Yabuki N, Nagura H, Iijima H, Yamamoto TT. A novel arachidonate-preferring acyl-CoA synthetase is present in steroidogenic cells of the rat adrenal, ovary, and testis. Proc. Natl. Acad. Sci. U.S.A., 94, 2880–2884 (1997).
• 4) Klett EL, Chen S, Yechoor A, Lih FB, Coleman RA. Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids. J. Lipid Res., 58, 884–894 (2017).
• 5) Shimbara-Matsubayashi S, Kuwata H, Tanaka N, Kato M, Hara S. Analysis on the substrate specificity of recombinant human acyl-CoA synthetase ACSL4 variants. Biol. Pharm. Bull., 42, 850–855 (2019).
• 6) Kuwata H, Nakatani E, Shimbara-Matsubayashi S, Ishikawa F, Shibanuma M, Sasaki Y, Yoda E, Nakatani Y, Hara S. Long-chain acyl-CoA synthetase 4 participates in the formation of highly unsaturated fatty acid-containing phospholipids in murine macrophages. Biochim. Biophys. Acta Mol. Cell Biol. Lipids, 1864, 1606–1618 (2019).
• 7) Kuwata H, Hara S. Role of acyl-CoA synthetase ACSL4 in arachidonic acid metabolism. Prostaglandins Other Lipid Mediat., 144, 106363 (2019).
• 8) Marszalek JR, Kitidis C, Dirusso CC, Lodish HF. Long-chain acyl-CoA synthetase 6 preferentially promotes DHA metabolism. J. Biol. Chem., 280, 10817–10826 (2005).
• 9) Fernandez RF, Kim SQ, Zhao Y, Foguth RM, Weera MM, Counihan JL, Nomura DK, Chester JA, Cannon JR, Ellis JM. Acyl-CoA synthetase 6 enriches the neuroprotective omega-3 fatty acid DHA in the brain. Proc. Natl. Acad. Sci. U.S.A., 115, 12525–12530 (2018).
• 10) Hale BJ, Fernandez RF, Kim SQ, Diaz VD, Jackson SN, Liu L, Brenna JT, Hermann BP, Geyer CB, Ellis JM. Acyl-CoA synthetase 6 enriches seminiferous tubules with the ω-3 fatty acid docosahexaenoic acid and is required for male fertility in the mouse. J. Biol. Chem., 294, 14394–14405 (2019).
• 11) Shishikura K, Kuroha S, Matsueda S, Iseki H, Matsui T, Inoue A, Arita M. Acyl-CoA synthetase 6 regulates long-chain polyunsaturated fatty acid composition of membrane phospholipids in spermatids and supports normal spermatogenic processes in mice. FASEB J., 33, 14194–14203 (2019).
• 12) Fujino T, Yamamoto T. Cloning and functional expression of a novel long-chain acyl-CoA synthetase expressed in brain. J. Biochem., 111, 197–203 (1992).
• 13) Van Horn CG, Caviglia JM, Li LO, Wang S, Granger DA, Coleman RA. Characterization of recombinant long-chain rat acyl-CoA synthetase isoforms 3 and 6: identification of a novel variant of isoform 6. Biochemistry, 44, 1635–1642 (2005).
• 14) Lee EJ, Kim HC, Cho YY, Byun SJ, Lim JM, Ryoo ZY. Alternative promotion of the mouse acyl-CoA synthetase 6 (mAcsl6) gene mediates the expression of multiple transcripts with 5′-end heterogeneity: genetic organization of mAcsl6 variants. Biochem. Biophys. Res. Commun., 327, 84–93 (2005).
• 15) Soupene E, Kuypers FA. Multiple erythroid isoforms of human long-chain acyl-CoA synthetases are produced by switch of the fatty acid gate domains. BMC Mol. Biol., 7, 21 (2006).
• 16) Hisanaga Y, Ago H, Nakagawa N, Hamada K, Ida K, Yamamoto M, Hori T, Arii Y, Sugahara M, Kuramitsu S, Yokoyama S, Miyano M. Structural basis of the substrate-specific two-step catalysis of long chain fatty acyl-CoA synthetase dimer. J. Biol. Chem., 279, 31717–31726 (2004).
• 17) Soupene E, Dinh NP, Siliakus M, Kuypers FA. Activity of the acyl-CoA synthetase ACSL6 isoforms: role of the fatty acid Gate-domains. BMC Biochem., 11, 18 (2010).
• 18) Northrop DB. On the meaning of K_m and V/K in enzyme kinetics. J. Chem. Educ., 75, 1153–1157 (1998).
• 19) Eto M, Shindou H, Shimizu T. A novel lysophosphatidic acid acyltransferase enzyme (LPAAT4) with a possible role for incorporating docosahexaenoic acid into brain glycerophospholipids. Biochem. Biophys. Res. Commun., 443, 718–724 (2014).
• 20) Iizuka-Hishikawa Y, Hishikawa D, Sasaki J, Takubo K, Goto M, Nagata K, Nakanishi H, Shindou H, Okamura T, Ito C, Toshimori K, Sasaki T, Shimizu T. Lysophosphatidic acid acyltransferase 3 tunes the membrane status of germ cells by incorporating docosahexaenoic acid during spermatogenesis. J. Biol. Chem., 292, 12065–12076 (2017).
• 21) Eto M, Shindou H, Yamamoto S, Tamura-Nakano M, Shimizu T. Lysophosphatidylethanolamine acyltransferase 2 (LPEAT2) incorporates DHA into phospholipids and has possible functions for fatty acid-induced cell death. Biochem. Biophys. Res. Commun., 526, 246–252 (2020).