Comparative genomics and proteomics of vertebrate diacylglycerol acyltransferase (DGAT), acyl CoA wax alcohol acyltransferase (AWAT) and monoacylglycerol acyltransferase (MGAT)

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Abstract

BLAT (BLAST-Like Alignment Tool) analyses of the opossum (Monodelphis domestica) and zebrafish (Danio rerio) genomes were undertaken using amino acid sequences of the acylglycerol acyltransferase (AGAT) superfamily. Evidence is reported for 8 opossum monoacylglycerol acyltransferase-like (MGAT) (E.C. 2.3.1.22) and diacylglycerol acyltransferase-like (DGAT) (E.C. 2.3.1.20) genes and proteins, including DGAT1, DGAT2, DGAT2L6 (DGAT2-like protein 6), AWAT1 (acyl CoA wax alcohol acyltransferase 1), AWAT2, MGAT1, MGAT2 and MGAT3. Three of these genes (AWAT1, AWAT2 and DGAT2L6) are closely localized on the opossum X chromosome. Evidence is also reported for six zebrafish MGAT- and DGAT-like genes, including two DGAT1-like genes, as well as DGAT2-, MGAT1-, MGAT2- and MGAT3-like genes and proteins. Predicted primary, secondary and transmembrane structures for the opossum and zebrafish MGAT-, AWAT- and DGAT-like subunits and the intron–exon boundaries for genes encoding these enzymes showed a high degree of similarity with other members of the AGAT superfamily, which play major roles in triacylglyceride (DGAT), diacylglyceride (MGAT) and wax ester (AWAT) biosynthesis. Alignments of predicted opossum, zebrafish and other vertebrate DGAT1, DGAT2, other DGAT2-like and MGAT-like amino acid sequences with known human and mouse enzymes demonstrated conservation of residues which are likely to play key roles in catalysis, lipid binding or in maintaining structure. Phylogeny studies of the human, mouse, opossum, zebrafish and pufferfish MGAT- and DGAT-like enzymes indicated that the common ancestors for these genes predated the appearance of bony fish during vertebrate evolution whereas the AWAT- and DGAT2L6-like genes may have appeared more recently prior to the appearance of marsupial and eutherian mammals.

Introduction

Acylglycerol acyltransferases (AGATs) are predominantly responsible for triglyceride synthesis in the body, via two major pathways: the glycerol phosphate (GP) pathway (Kennedy, 1957) and the monoacylglycerol (MG) pathway (see Coleman and Lee, 2004, Yen et al., 2008). The final step of both pathways involves diacylglycerol and fatty acyl CoA being catalytically converted into triglyceride via two distinct DGAT (diacylglycerol O-acyltransferase, E.C.2.3.1.20) families, DGAT1 and DGAT2 (Cases et al., 1998, Oelkers et al., 1998). The two pathways differ in the mode of synthesis of diacylglycerols prior to catalysis by DGAT1 and/or DGAT2, with the GP pathway involving de novo synthesis from glycerol-3-phosphate and fatty acyl CoA, whereas the MG pathway uses partially hydrolyzed monoacylglycerols and fatty acyl CoA. This penultimate step in the MG pathway is catalyzed by enzymes encoded by the monoacylglycerol acyltransferase (MGAT; 2-acylglycerol O-acyltransferase; EC 2.3.1.22) gene sub-family, for which three MGAT (also designated as MGOT and MOGAT) genes have been reported in humans (Yen et al., 2002, Yen and Farese, 2003, Cheng et al., 2003). The human DGAT2-like gene family comprises at least seven members, including DGAT2 (Cases et al., 2001), MGAT1 (Yen et al., 2002), MGAT2 (Yen and Farese, 2003), MGAT3 (Cheng et al., 2003), and three genes located together on the X chromosome (DGAT2L6, AWAT1 and AWAT2) (Turkish et al., 2005). The latter two genes encode enzymes with acyl CoA wax alcohol acyltransferase (AWAT) activity, and are specifically expressed in sebocytes (skin), and play a role in preventing surface desiccation by the formation of wax esters.

The triglyceride forming pathways are differentially distributed in the body with the GP pathway being widely distributed and responsible for triglyceride synthesis in most tissues of the body. The MG pathway however is predominantly localized in specific cell types, including enterocytes (intestine), hepatocytes (liver) and adipocytes (adipose tissue), where large amounts of triglycerides are synthesized or stored. In particular, MG is the major pathway of the small intestine, where partially hydrolyzed fats are used to synthesize triglycerides following lipid ingestion (see reviews in Coleman and Lee, 2004, Yen et al., 2008).

This study describes the predicted sequences, structures and phylogeny of diacylglycerol acyltransferase-like (DGAT) and monoacylglycerol acyltransferase-like (MGAT) genes and enzymes from eutherian (human and mouse) and marsupial (opossum) mammals and from a bony fish (zebrafish) species. Computational methods were used to predict the primary, secondary and transmembrane structures for these enzymes, as well as gene locations, exonic structures and sequences for MGAT- and DGAT-like genes, using published data from genome sequences. Predictions of MGAT- and DGAT-like enzyme sequences from a wider range of vertebrates were also used to examine the phylogeny and evolution of these genes and to identify conserved amino acid residues, including likely candidates for the active sites and substrate binding regions for these enzymes.

Section snippets

DGAT and MGAT gene and enzyme identification

BLAT (BLAST-Like Alignment Tool) studies were undertaken using the UC Santa Cruz web browser [http://genome.ucsc.edu/cgi-bin/hgBlat] (Altschul et al., 1990, Kent et al., 2002) with the default settings. UniProtKB/Swiss-Prot database derived amino acid sequences for human, mouse and zebrafish DGAT-like and MGAT-like enzymes [http://au.expasy.org], Genbank derived amino acid sequences for zebra fish DGAT-like and MGAT-like enzymes [http://www.ncbi.nlm.nih.gov/Genbank/], and predicted NCBI

Alignments of opossum and zebrafish DGAT1 amino acid sequences with human and mouse DGAT1: evidence for two zebrafish DGAT1-like genes

The deduced amino acid sequences of a predicted opossum DGAT1 subunit and two predicted forms of zebrafish DGAT1 subunits (designated as DGAT1A and DGAT1B) are shown in Fig. 1 together with the previously reported sequences for human and mouse DGAT1 (Oelkers et al., 1998, Buhman et al., 2002) (see Table 1). The enzymes contained 488–505 amino acid residues in sequence and exhibited predicted MWs of ~ 55–58 kDa and high theoretical isoelectric points (pI values of 9.4–9.6) (Table 1). The

Conclusions

BLAT analyses of the opossum and zebrafish genomes using the amino acid sequences reported for human and mouse DGAT1 and DGAT2-like protein subunits were undertaken to interrogate these genomes. Evidence is reported for an opossum DGAT1 gene and six DGAT2-like genes, including DGAT2, AWAT1, AWAT2, DGAT2L6, MGAT1, MGAT2 and MGAT3 genes. Evidence for two zebrafish and pufferfish DGAT1-like genes and four DGAT2-like genes (DGAT2, MGAT1, MGAT2 and MGAT3) is also described. Three of the opossum

Acknowledgements

I am grateful to Dr Laura Cox and Dr John VandeBerg of the Southwest Foundation for Biomedical Research in San Antonio Texas USA for helpful discussions and advice.

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