Fig 1.
Comparison of WS and DGAT enzyme reaction.
Wax synthases (WSs) or acyl-CoA:wax alcohol acyltransferases catalyze the condensation of a fatty alcohol with an acyl-CoA, thereby forming wax esters (WEs). In contrast, acyl-CoA:diacylglycerol O-acyltransferases (DGATs) catalyze the condensation of an acyl-CoA molecule with diacylglycerol (DAG) to yield triacylglycerols (TAGs).
Fig 2.
A. Topological model of AWAT2 in accordance to data generated by the services of Phobius [18], TMHMM [16] and SOSUI [17]. According to the model, AWAT2 is predicted to contain an N-terminal cytoplasmic tail that is linked to two TM domains, which are connected by a short linker. The remaining C-terminal sequence forms again a cytosolic domain, which contains the catalytic HPHG motif of the enzyme. A hydrophobic patch that may form a membrane contact is located in the middle of this domain. B. Predicted domain structures of mouse AWAT2 and mouse DGAT2. The conserved TM domains (dark grey/white boxes), the hydrophobic patch (hatched boxes), the active site motif HPHG and the putative neutral lipid binding motif “FLXLXXX” of DGAT2 are indicated. C. Domain swap variants of mouse AWAT2 and mouse DGAT2.
Again, the conserved TM domains (dark grey/white boxes) and the hydrophobic patch (hatched boxes) are indicated. The sequences of mouse AWAT2 and mouse DGAT2 were used to construct seven domain swap variants (V1-V7), in which different parts of mouse AWAT2 were exchanged for the respective parts of mouse DGAT2. Mouse DGAT2 derived parts are shown in black (non-TM domains) and in white (TM-domains), respectively.
Fig 3.
Separation of neutral lipids derived from yeast cultures expressing different mouse AWAT2 variants, an empty vector control, AWAT2 wt or DGAT2 wt.
pYES2/NT empty vector control (left side), mouse AWAT2 and DGAT2 wild type enzymes (middle) as well as domain swap variants derived from those two enzymes (right) were expressed in S. cerevisiae H1246 and the cultures were supplemented with 18:1-OH. The left part of the figure, which shows the empty vector control, originates from a different TLC plate than the rest of the shown samples do. Similar results were obtained when cultures were supplemented with 16:0-OH (not shown). VLC fatty acyl-containing WEs are indicated by arrows. COOH = free fatty acids, OH = fatty alcohols, TAG = triacylglycerols, WE = wax esters, VLC WE = very long chain fatty acyl-containing wax esters, SE = sterol esters. Data are representative for at least three independent biological replicates.
Fig 4.
Comparison of WEs derived from yeast cultures expressing AWAT2 or V2.
A. GC analysis of WEs derived from a culture expressing mouse AWAT2 either fed with 16:0-OH (left chromatogram) or 18:1-OH (right chromatogram). B. GC analysis of WEs derived from a culture expressing V2 either fed with 16:0-OH (left chromatogram) or 18:1-OH (right chromatogram). In comparison to AWAT2, V2 expressing cultures synthesize four additional WE upon feeding 16:0-OH (left chromatogram) and a single additional WE upon feeding 18:1-OH (right chromatogram). The results shown here for cultures expressing V2 are also representative for cultures expressing V5, mouse AWAT2 N36R as well as AWAT2 A25F N36R (not shown). Data are representative for at least three independent biological replicates.
Fig 5.
Mass spectra of VLC fatty acyl-harboring WEs produced by mouse cultures expressing V2 fed with 16:0-OH.
A. GC analysis of WEs derived from a culture expressing V2 (same chromatogram as in Fig 4B left hand side). B. Mass spectrum of peak (B) identified it as hexadecanoyl-eicosanoate (16:0–20:0), C. mass spectrum of peak (C) identified it as hexadecanoyl-docosanoate (16:0–22:0), mass spectrum of peak (D) identified it as hexadecanoyl-tetracosanoate (16:0–24:0) and mass spectrum of peak (E) identified it as hexadecanoyl-hexacosanoate (16:0–26:0). The results shown here for cultures expressing V2 are also representative for cultures expressing V5, mouse AWAT2 N36R as well as AWAT2 A25F N36R (not shown).
Table 1.
Acyl chain composition of WEs synthesized by cultures expressing mouse AWAT2 and mouse AWAT2-derived domain swap variants in comparison with the acyl CoA-pool composition upon feeding of 16:0-OH.
Table 2.
Acyl chain composition of WEs synthesized by cultures expressing mouse AWAT2 and AWAT2-derived domain swap variants in comparison with the acyl CoA-pool composition upon feeding of 18:1-OH.
Fig 6.
HPLC analysis of acyl-CoA species derived from a culture expressing pYES2/NT (empty vector control) either fed with 16:0-OH (left chromatogram) or 18:1-OH (right chromatogram).
un = unidentified). Data are representative for at least three independent biological replicates.
Fig 7.
Alignment of the N-terminal part of vertebrate-derived AWAT2 and DGAT2 sequences.
Vertebrate-derived sequences were obtained from the UniProt database [37] and aligned using the Clustal Omega tool [38]. The membrane topology was analyzed by using the SOSUI tool [17]. Predicted TM domains are highlighted in gray, whereas parts of the sequence corresponding to the putative neutral lipid binding domain “FLXLXXX” in mouse DGAT2 are highlighted in black. The conserved motifs pGGRR and YFP are underlined. Mouse DGAT2 and AWAT2, which were used in this study, are printed in bold. In case of mouse DGAT2, the indicated TM structure represents the actual topology determined by Stone et al. 2006 [19]. Besides an abbreviation for each enzyme, also the respective UniProt-identifier is listed. A set of 10 conserved amino acid positions were identified, that were different between DGAT2 and AWAT2 sequences. 9 of these positions are in the shown region and marked by arrow heads.
Table 3.
Acyl chain composition of WEs synthesized by cultures expressing mouse AWAT2 and AWAT2-derived single amino acid exchange variants in comparison with the acyl CoA-pool composition upon feeding of 16:0-OH.
Table 4.
Acyl chain composition of WEs synthesized by cultures expressing mouse AWAT2 and AWAT2-derived single amino acid exchange variants in comparison with the acyl CoA-pool composition upon feeding of 18:1-OH.
Fig 8.
Separation of neutral lipids derived from yeast cultures expressing different mouse AWAT2 variants.
All variants were expressed in S. cerevisiae H1246 and supplemented with 18:1-OH. Similar results were obtained when cultures were supplemented with 16:0-OH (not shown). VLC fatty acyl-containing WEs are indicated by arrows. COOH = free fatty acids, OH = fatty alcohols, TAG = triacylglycerols, WE = wax esters, VLC WE = very long chain fatty acyl-containing wax esters, SE = sterol esters. Data are representative for at least three independent biological replicates.