A, expression cloning of wax synthase cDNAs from mouse preputial gland. A cDNA library was prepared from male preputial gland mRNA and divided into pools of ∼200 cDNAs each. Plasmid DNA from individual pools was combined with pVA1 plasmid (to increase expression from the expression plasmids (15)) and introduced via FuGENE 6 reagent into HEK 293 cells. [1-¹⁴C]Hexadecanol was added to the medium 16–20 h later, and after a further incubation of 6–18 h, the cells were harvested by scraping into the medium. Lipid metabolites were extracted into chloroform:methanol (2:1, v/v) and resolved by TLC in solvent system 2. An autoradiogram prepared by exposure of the TLC plate to Kodak BioMax MR film for 72 h is shown. Lane 1, radiolabeled lipids from mock transfected cells; lane 2, lipids synthesized in cells transfected with a 200-member cDNA pool encoding wax synthase activity; lane 3, lipids synthesized from a subpool of 20 cDNAs derived from the pool analyzed in lane 2; lane 4, lipids from cells transfected with a single positive cDNA derived from the subpool of lane 3. The positions to which cholesteryl esters and a wax monoester standard (cetylpalmitate) migrated to on the plate are shown on the right. Only the portion of the autoradiogram containing radiolabeled cholesteryl and wax esters is shown. B, expression of mouse and human wax synthase enzyme activities in HEK 293 cells. Expression vectors encoding nothing (pCMV, lane 1), the human wax synthase enzyme (hWS, lane 2), or the mouse wax synthase enzyme (mWS, lane 3) were introduced into HEK 293 cells by transfection. Subsequent assay for wax ester synthesis was carried out as described in A.

A, the deduced amino acid sequences of the human (hWS) and mouse (mWS) wax synthase proteins are aligned with identities between the two enzymes indicated by black boxes. Amino acids are numbered on the right. Arrowheads above the alignment indicate the positions of introns in the encoding genes. Dots indicate every 10th amino acid residue. The GenBank™/EBI Data Bank accession number for the human wax synthase cDNA sequence is AY605053 and for the mouse are AY611031 and AY611032. B, structures and chromosomal locations of the human and mouse wax synthase genes. Exons are indicated by boxes and introns by connecting lines. Amino acids encoded at the beginning and end of the protein are indicated above the gene schematic. The structures of the gene and their chromosomal locations were deduced by comparing the sequences of the cloned cDNAs with those of genomic DNAs (GenBank™/EBI Data Bank accession number HsX_11826_33.52 for human gene and MmX_78942_32 for mouse gene). C, amino acid sequence identities between mouse wax synthase and other acyltransferase enzymes. The percent sequence identities were deduced by pairwise comparisons using the BLASTP 2.2.6 program.

Aliquots (15–25 μg) of HEK 293 membrane protein prepared from cells expressing the mouse wax synthase enzyme were incubated with the indicated radiolabeled and unlabeled lipids at 37 °C for 25–30 min. Reactions were stopped by acidification and lipids extracted for TLC analysis in solvent system 2 as described under “Experimental Procedures.” The radiolabeled lipids used in these reactions are incorporated into multiple products by endogenous enzymes, and for this reason only the portion of the autoradiogram containing wax esters is shown. A, fatty alcohol substrates. Individual fatty alcohols tested are designated by chain length and number of unsaturated bonds (e.g. 10:0 is decanol, which has 10 carbons and 0 double bonds; 18:3 is 9,12,15 linolenyl alcohol, which has 18 carbons and 3 double bonds). B, fatty acyl-CoA substrates. Fatty acyl groups are designated by chain length and number of unsaturated bonds. C, isoprenol substrates. C₁₀, farnesol, C₁₅, geraniol, C₂₀, geranylgeraniol, 16:0, hexadecanol control. The results shown in A—C are typical of those obtained in at least two separate experiments carried out on different days using recombinant mouse and human wax synthases.

A, HEK 293 cells were transfected with expression plasmids encoding the indicated acyltransferase enzymes modified to contain FLAG epitopes and then assayed for the synthesis of wax esters after the addition of [¹⁴C]hexadecanol to the culture medium (top panel). The positions to which cholesteryl and wax esters migrated on the TLC plate developed in solvent system 2 are indicated on the right of the autoradiogram. To determine relative expression levels of the acyltransferase enzymes, cell membranes were prepared from duplicate dishes and subjected to immunoblotting using a monoclonal antibody directed against the FLAG epitope (bottom panel). The positions to which protein standards of known molecular mass migrated on the SDS-polyacrylamide gel are indicated on the right of the lumigram. B, insect Sf9 cells were infected with baculovirus vectors encoding the indicated acyltransferase or control enzyme (steroid 5β-reductase, 5β-Red) modified to contain a His₆ epitope, and then assayed for the synthesis of wax esters as described in A (top panel). Endogenous levels of ACAT enzyme activity are low in Sf9 cells, and thus the formation of cholesteryl esters in the infected cells was not detected in the exposure shown. Cell lysates from duplicate dishes were subjected to immunoblotting using a monoclonal antibody directed against the His₆ epitope (bottom panel). The results shown are representative of at least two experiments performed on different days.

Insect Sf9 cells were infected with baculovirus vectors encoding the mouse wax synthase (WS), indicated acyltransferase, or control enzyme (steroid 5β-reductase, 5β-Red), each modified to contain a His₆ epitope, and then assayed for the synthesis of diacylglycerols and triacylglycerols after the addition of BSA-conjugated [¹⁴C]palmitate to the culture medium (top panel). TLC was performed in solvent system 3. Only a product of unknown structure was detected in uninfected cells (Cells), whereas those infected with the control steroid 5β-reductase virus synthesized this product and an enhanced level of triacylglycerols. Cells infected with the MGAT2-expressing virus produced elevated levels of diacylglycerols as well as triacylglycerols, and those infected with the DGAT2-expressing virus produced elevated levels of triacylglycerols. No increase in acyltransferase activity was detected in cells infected with the wax synthase-expressing virus. To determine relative expression levels of the various enzymes, cell lysates from duplicate dishes were subjected to immunoblotting using a monoclonal antibody directed against the His₆ epitope (bottom panel). The MGAT2 and wax synthase enzymes were expressed at higher levels than the steroid 5β-reductase and DGAT2 enzymes. The results shown in both panels are typical of those obtained in three independent experiments.

Chinese hamster ovary-K1 cells were transfected with the indicated expression vectors encoding nothing (pCMV) or a FLAG epitope-tagged wax synthase (Wax Synthase) enzyme. After transient expression, cells were fixed, permeabilized, and incubated with a rabbit polyclonal antiserum directed against the endoplasmic reticulum protein calnexin (Anti-Calnexin) and a mouse monoclonal antibody directed against the FLAG epitope (Anti-FLAG). A and D, detection of rabbit polyclonal antibodies directed against calnexin with rhodamine-conjugated goat anti-rabbit antiserum. B and E, detection of FLAG epitope with fluoresceinconjugated goat anti-mouse antiserum. C and F, merged images of rhodamine and fluorescein signals. Double indirect immunofluorescence microscopy was performed on a Zeiss 510 Laser Scanning Confocal microscope using a 63 × 1.3 NA PlanApo objective. Both the calnexin and FLAG-wax synthase enzymes are detected in endoplasmic reticulum membranes of Chinese hamster ovary cells. These data are representative of two separate experiments.

The relative levels of wax synthase mRNA were determined by real time PCR in the tissues and cell types indicated on the left of the figure using cyclophilin mRNA levels as a reference standard. The data were normalized to the threshold values (C_T) determined in the liver (C_T = 29.2) and then expressed on a log₁₀ scale. This experiment was repeated twice using the same preparations of tissue RNAs isolated from pools of organs or dishes of cells (macrophages).

The indicated plasmid DNAs were introduced into HEK 293 cells by transfection and allowed to express for 26 h. Cell lysates were prepared, and the conversion of BSA-conjugated [¹⁴C]palmitate into lipids was determined. TLC was performed in solvent system 2. Lane 1, mock transfected cells synthesized an unknown product and triacylglycerols. Lane 2, cells expressing FAR1 produced these same two products and hexadecanol. Lane 3, cells expressing wax synthase (WS) produced only the two background lipids, indicating the near absence of FAR activity in HEK 293 cells. Lane 4, cells transfected with the FAR1 and wax synthase cDNAs produced the two background lipids, hexadecanol, wax esters, and an additional lipid of unknown structure. Lane 5, the mixing of equal amounts of lysate derived from cells transfected individually with the FAR1 and wax synthase cDNAs resulted in the production of the background lipids plus hexadecanol and wax esters. Lane 6, positive control showing synthesis of hexadecanol and wax esters by membranes from mouse preputial glands. This experiment was performed in various guises many times.