A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters

Abstract

Acyl-CoA-dependent O-acyltransferases catalyze reactions in which fatty acyl-CoAs are joined to acyl acceptors containing free hydroxyl groups to produce neutral lipids. In this report, we characterize a human multifunctional O-acyltransferase (designated MFAT) that belongs to the acyl-CoA:diacylglycerol acyltransferase 2/acyl-CoA:monoacylglycerol acyltransferase (MGAT) gene family and is highly expressed in the skin. Membranes of insect cells and homogenates of mammalian cells overexpressing MFAT exhibited significantly increased MGAT, acyl-CoA:fatty acyl alcohol acyltransferase (wax synthase), and acyl-CoA:retinol acyltransferase (ARAT) activities, which catalyze the synthesis of diacylglycerols, wax monoesters, and retinyl esters, respectively. Furthermore, when provided with the appropriate substrates, intact mammalian cells overexpressing MFAT accumulated more waxes and retinyl esters than control cells. We conclude that MFAT is a multifunctional acyltransferase that likely plays an important role in lipid metabolism in human skin.

Fig. 1

Human multifunctional acyltransferase (MFAT), a member of the acyl-CoA:diacylglycerol acyltransferase 2/acyl-CoA: monoacylglycerol acyltransferase (DGAT2/MGAT) gene family. A: Dendrogram of the human DGAT2/MGAT gene family. Chromosome locations were from the National Center for Biotechnology Information LocusLink database (http://www.ncbi.nlm.nih.gov/Locus-Link/list.cgi). MGAT1 was designated as DC2, MGAT2 as DC5, and MFAT as DC4 by Cases et al. (20). B: Hydrophobicity plots of DGAT2/MGAT gene family members as assessed by Kyte-Doolittle analysis (35). Thick horizontal lines indicate predicted transmembrane domains (http://www.cbs.dtu.dk/services/TMHMM/). The X axis represents the positions of amino acids, numbering from the N terminus to the C terminus.

Fig. 2

Tissue expression pattern of human MFAT. A: Northern blot analysis. Human MFAT mRNA expression was examined with 20 μg of total RNA from the indicated human tissues. Sk., skeletal; Sm., small. B: Quantitative PCR. Relative mRNA levels in the indicated human tissues (compared with white adipose tissue) were assessed with real-time PCR. Cyclophilin mRNA levels served as internal controls.

Fig. 3

Expression of MFAT in Sf9 insect cells. A: Immunoblots of insect cell membranes. Expression of MFAT and control proteins was verified by immunoblotting with an anti-FLAG antibody. Membrane proteins (5 μg) were analyzed from Sf9 cells infected with wild-type virus or FLAG-tagged versions of mouse DGAT1, mouse DGAT2, mouse MGAT1, human MGAT2, or human MFAT recombinant baculoviruses. B–F: Expression of MFAT confers in vitro acyltransferase activities. MGAT (B), DGAT (C), wax monoester synthase (D), wax diester synthase (E), and acyl-CoA:retinol acyltransferase (ARAT; F) activities were detected by incorporation of [¹⁴C]palmitoyl-CoA (25 μM) into diacylglycerol (DAG), triacylglycerol (TAG), wax monoester (ME), wax diester (DE), or retinyl ester (RE) in the presence of 100 μM added sn-2-monooleoylglycerol, sn-1,2-dioleoylglycerol, 1-hexadecanol, 1,2-hexadecandiol, or all-trans-retinol, respectively. Neutral lipids were extracted, separated by TLC, and exposed to X-ray film. Arrows indicate the putative products of each acyltransferase activity. In E, the prominent acylation product when 1,2-hexadecandiol was provided is most likely the wax monoester 2-hydroxylhecadecyl hexadecanoate (lower arrow). Results are representative of three independent experiments.

Fig. 4

Demonstration of multiple acyl-CoA acyltransferase activities for MFAT by labeled activity-specific acyl acceptors and by dependence on acyl-CoA as an acyl donor. A: MGAT activity assessed by the amount of [³H]sn-2-monooleoylglycerol (~3,000 cpm/nmol) incorporated into both diacylglycerol (black bar) and triacylglycerol (gray bar). B: Wax monoester synthase (WS) activity assessed by the amount of [¹⁴C]1-hexadecanol (~8,000 cpm/nmol) incorporated into wax monoester. C: ARAT activity assessed by the amount of [³H]all-trans-retinol (~320,000 cpm/nmol) incorporated into retinol ester. For all panels, each acyl acceptor was tested without (−CoA) and with (+CoA) added palmitoyl-CoA. Values shown are means of duplicate measurements. The experiment was repeated twice with similar results.

Fig. 5

Dependence of acyltransferase activities in membranes expressing MFAT on acyl acceptor concentrations. A: Acylglycerol acyltransferase (both DGAT and MGAT) activities assessed by the amount of [¹⁴C]palmitoyl-CoA incorporated into both diacylglycerol and triacylglycerol in the presence of different concentrations of sn-2-monooleoylglycerol. The inset shows the amount of [¹⁴C]palmitoyl-CoA incorporated into diacylglycerol only. B: DGAT activity assessed by the amount of [¹⁴C]palmitoyl-CoA incorporated into triacylglycerol in the presence of different concentrations of sn-1,2-dioleoylglycerol. C: Wax monoester synthase activity assessed by the amount of [¹⁴C]palmitoyl-CoA incorporated into wax monoester in the presence of different concentrations of 1-hexadecanol. D: ARAT activity assessed by the amount of [¹⁴C]palmitoyl-CoA incorporated into retinyl ester in the presence of different concentrations of all-trans-retinol. Values are means of duplicate measurements. The experiment was repeated twice with similar results.

Fig. 6

In vitro MGAT, wax synthase, and ARAT activities in mammalian cells overexpressing MFAT. A: Immunoblotting of FLAG-tagged proteins demonstrating the expression of FLAG-tagged MFAT and control proteins. Cells were transfected with expression vectors containing cDNAs for β-galactosidase (LacZ) or FLAG-tagged versions of DGAT1, DGAT2, MGAT1, MGAT2, or MFAT. B–F: In vitro acyltransferase activities conferred by MFAT expression. MGAT (B), DGAT (C), wax monoester synthase (D), wax diester synthase (E), and ARAT (F) activities were detected by the incorporation of [¹⁴C]palmitoyl-CoA (25 μM) into diacylglycerol (DAG), triacylglycerol (TAG), wax monoester (ME), wax diester (DE), or retinyl ester (RE) in the presence of 100 μM added sn-2-monooleoylglycerol, sn-1,2-dioleoylglycerol, 1-hexadecanol, 1,2-hexadecandiol, or all-trans-retinol, respectively. Neutral lipids were extracted, separated by TLC, and exposed to X-ray film. Arrows indicate the putative products of each acyltransferase activity. In E, the prominent acylation product when 1,2-hexadecandiol was provided is most likely the wax monoester 2-hydroxylhecadecyl hexadecanoate (lower arrow). Results are representative of two experiments.

Fig. 7

Esterification of monoacylglycerol, fatty acyl alcohol, and retinol in intact cells overexpressing MFAT. A: Immunoblotting of FLAG-tagged proteins demonstrating the expression of FLAG-tagged DGAT1 and control proteins (LacZ). B–D: Esterification of monoacylglycerol, fatty acyl alcohol, and retinol in COS-7 cells overexpressing MFAT as assessed by the accumulation of prospective products. Cells were plated in six-well dishes (10⁵/well) for 24 h and transfected with vectors containing cDNAs for MFAT or control enzymes. Twenty-four hours later, cells were incubated in medium containing 25 μM [³H]sn-2-monooleoylglycerol (specific activity, ~15,000 cpm/nmol) for 18 h (B), 10 μM [¹⁴C]1-hexadecanol (~47,000 cpm/nmol) for 6 h (C), or 2.5 μM [³H]retinol (~200,000 cpm/nmol) for 18 h (D). Neutral lipids were separated by TLC, visualized with a Bioscan imaging scanner, and scraped to assess the incorporation of radioactivity into diacylglycerol (black bar) and triacylglycerol (gray bar) (B), wax esters (C), and retinol esters (D). Values represent results from pooled six-well dishes for each group. Similar results were found in two experiments.