Identification of avian wax synthases

Abstract

Background: Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms.

Results: Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands.

Conclusions: We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities.

Figure 1

Phylogenetic analysis of putative avian wax synthases in comparison to different acyltransferases from human, plants, yeast, Tetrahymena and bacteria. The phylogram shows the relation of the analyzed avian proteins with acyltransferases from different organisms. The tree is based on the alignment of the following sequences (NCBI accession numbers are given): HsAWAT1 [Homo sapiens, NP_001013597.1], HsAWAT2 [H. sapiens, NP_001002254.1], HsMOGAT3 [H. sapiens, NP_835470], HsMOGAT2 [H. sapiens, NP_079374], HsMOGAT1 [H. sapiens, NP_477513.2], HsACAT1 [H. sapiens, NP_003092.4], HsACAT2 [H. sapiens, NP_003569.1], HsDGAT2 [H. sapiens, NP_115953.2], HsDGAT1 [H. sapiens, NP_036211.2], SchWS [Simmondsia chinensis, AF149919_1], EgWS [Euglena gracilis, ADI60058.1], PxhWS [Petunia x hybrida, AAZ08051.1], AcWS/DGAT [Acinetobacter sp. ADP1, YP_045555.1], GgWS1 [Gallus gallus, XP_424082.2], GgWS2 [G. gallus, JQ031643], GgWS4 [G. gallus, XP_419207.1], GgWS5 [G. gallus, NP_001026192.1], AdWS5 [Anser domesticus, JQ031647], TaWS5 [Tyto alba, JQ031646], AdWS4 [A. domesticus, JQ031643], TaWS4 [T. alba, JQ031645], HsTMEM68 [H. sapiens, Q96MH6.2], GgDGAT1 [G. gallus, JQ031642], Tetrahymena WS [Tetrahymena thermophila, XP_001027910, XP_001026090, XP_001008104, XP_001019739], MhWS1 [Marinobacter hydrocarbonoclasticus, ABO21021.1], Arabidopsis WSD [Arabidopsis thaliana, NP_568547.1, NP_177356.1, NP_850307.1, NP_200151.2] MrDGAT2B [Umbelopsis ramanniana, AAK84180.1], MrDGAT2A [U. ramanniana,AAK84179.1], ScDGA1p [Saccharomyces cerevisiae, NP_014888.1], ScARE1p [S. cerevisiae, CAA42296.1], ScARE2p [S. cerevisiae, CAA96298.1], Arabidopsis WS [A. thaliana, NP_200345.1, XP_002866091.1, NP_200349.1, NP_200346.1]. The scale corresponds to amino acid substitutions per site in the alignment of 41 sequences with a total of 188 positions. Numbers at the branches are bootstrap values indicating the probability of this relationship in %. Values above 95 can be regarded as correct. The dendrogram was created with ClustalX2 and MEGA5 software.

Figure 2

Alignment of typical motifs of DGAT1 (a) and DGAT2 (b) families. Figure 2a shows the alignment of the FYxDWWN motif which is a potential acyl-CoA binding motif in proteins of the DGAT1 family [44]. Figure 2b represents the partially modified HPHG motif representing a potential part of the active site in DGAT2 family proteins [45]. NCBI accession numbers of reference proteins: HsDGAT1 [Homo sapiens, NP_036211.2], HsACAT1 [H. sapiens, NP_003092.4], HsACAT2 [H. sapiens, NP_003569.1], HsDGAT2 [H. sapiens, NP_115953.2], HsAWAT1 [H. sapiens, NP_001013597.1], HsAWAT2 [H. sapiens, NP_001002254.1].

Figure 3

Expression profiles of avian WS sequences in different tissues of chicken. RNA was converted to cDNA with gene specific primers and used as template for partial transcript amplification. Reactions were performed with or without (+ and - marks) reverse transcriptase (C, positive control with plasmid DNA).

Figure 4

Production of lipids in transgenic yeast cultures. Cultures expressing one of the respective WS sequences were supplemented with a mixture of 125 μM 10:0-OH, 12:0-OH, 14:0-OH, 16:0-OH and 18:0-OH and induced for 48 hours. Lipids of the harvested cells were extracted and analyzed. Mean values and standard deviations of two independent experiments are given (a). TLC analysis of lipid extracts from yeast cells expressing GgFAR1 or co-expressing GgFAR1 and GgWS4 from cultures supplemented with 14:0-fatty acid (b). (FA: fatty acids, FOH: fatty alcohols, TAG: triacylglycerols, WE: wax esters)

Figure 5

Acyl-CoA specificities of avian enzymes. Relative WS (a and c) and DGAT (b) activities of isolated yeast membranes from yeast cells expressing an avian WS sequence. Assays were conducted with 10:0-OH and the given straight-chain (a and b) or branched-chain (c) acyl-CoA thioesters under standard assay conditions (a and b) or increased protein amount (10 μg) and incubation time (2 h) (c). The relative activities of each protein are given, 100% corresponds to the activities with 10:0-OH and 16:0-CoA under identical conditions. Values are mean values from at least two independent assay series. (GgWS1: 116 pmol*min^-1*mg^-1; GgWS2: 105 pmol*min^-1*mg^-1; GgWS4: 149 pmol*min^-1*mg^-1; AdWS4: 126 pmol*min^-1*mg^-1; TaWS4: 149 pmol*min^-1*mg^-1).

Figure 6

Acyl-acceptor specificities of avian proteins. Relative WS activities of membrane fractions of yeast cells harboring an avian enzyme with 16:0-CoA and the given acyl-acceptors under otherwise standard conditions. 100% corresponds to the activity with 16:0-CoA and 10:0-OH (GgWS1: 116 pmol*min^-1*mg^-1; GgWS2: 105 pmol*min^-1*mg^-1; GgWS4: 149 pmol*min^-1*mg^-1; AdWS4: 126 pmol*min^-1*mg^-1; TaWS4: 149 pmol*min^-1*mg^-1), values are mean values from at least two independent assay series.

Figure 7

Properties of WS activities of preen gland membranes of chicken, goose and barn owl. Relative WS activities of preen gland membranes with 12:0-OH and different acyl-CoA thioesters (a) or 16:0-CoA and different alcohols (b). 100% corresponds to the turnover of 12:0-OH with 16:0-CoA. Standard assays were used apart of higher protein amounts (50 μg) and incubation times (2 h).