In this study, we report the discovery, within the BayGenomics gene-trapping resource, of Agpat6
, a new member of the glycerolipid acyltransferase family. AGPAT6 is 48 kDa in size (with the V5-His tag) and is found exclusively within the ER. Agpat6
is predominantly expressed in brown adipose tissue and mammary epithelium. The milk from Agpat6−/−
mice is depleted in diacylglycerols and triacylglycerols, and the mammary epithelium from Agpat6−/−
mice is underdeveloped and depleted in intracellular fat droplets. In an accompanying article (25
), we show that the triacylglycerol content of brown and white adipose tissue in Agpat6−/−
mice is also significantly reduced. These experimental findings, together with sequence similarities between AGPAT6 and other glycerolipid acyltransferases (), suggest that AGPAT6 is a bona fide
acyltransferase with an important role in the synthesis of triacylglycerols.
Proposed glycerolipid acyltransferase subgroups as assessed by alignment of amino acid sequences of putative orthologues from human, mouse, C. elegans, and D. melanogaster.
The mammary gland abnormalities in Agpat6−/−
mice are reminiscent of those in mice lacking acylCoA:diacylglycerol acyltransferase 1 (DGAT1), an enzyme that adds an acyl group to diacylglycerol to generate triacylglycerols. Dgat1−/−
nursing mothers have underdeveloped mammary glands and lack the ability to produce triacylglycerol-rich milk droplets (26
). The fact that the mammary glands from Agpat6
knockout females appeared underdeveloped suggests that defective lipid biosynthetic pathways interfere, directly or indirectly, with mammary gland development.
This identification of Agpat6
within BayGenomics illustrates the utility of gene trapping for inactivating a broad spectrum of genes, including novel genes that had escaped scientific scrutiny. Once the sequence tag for the Agpat6
ES cell line was in hand, we were able to classify Agpat6
as being a member of the glycerolipid acyltransferase family, and then move on to examine the mouse and human genomes for additional Agpat6
-like sequences. By searching the DNA sequence databases, we quickly identified a novel, never-previously-reported gene resembling Agpat6
, which we have provisionally designated Agpat8
(). Both AGPAT6 and AGPAT8 contain the classic sequence motifs (I–IV) characteristic of glycerolipid acyltransferases (5
). When the sequences spanning motifs I–IV were analyzed for relatedness, it was apparent that AGPAT6 and AGPAT8 were most related to each other () (66% identical at the amino acid level, but only ~2–15% identical with the other family members). More importantly, we found that both AGPAT6 and AGPAT8 contain sequences within motifs I–IV that distinguish them from other members of the family (). Domain III is strikingly conserved between AGPAT6 and AGPAT8, representing a signature sequence for these two proteins (). Also, the arginine in the VPEGTR consensus sequence within motif III is changed to a cysteine in AGPAT6 and AGPAT8, a feature shared only by AGPAT7 and the unknown protein at locus 270084. Remarkably, orthologues for Agpat6
appear to exist in the genomes of plants, worms, and flies (), suggesting that, together, AGPAT6 and AGPAT8 probably play a unique, fundamental, and conserved function in lipid biosynthesis.
Fig. 9 Dendrogram illustrating the amino acid sequence relatedness of mouse glycerolipid acyltransferases within the region of the proteins spanning functional domains I–IV (5–7). Alignments were performed with the Clustal W algorithm (http://www.ebi.ac.uk/clustalw/ (more ...)
By analyzing conserved sequences (motifs I–IV) from multiple glycerolipid acyltransferases in four species (human, mouse, D. melanogaster
, and C. elegans
), we divided the family into eight subgroups (separated by double horizontal lines in ). AGPAT1 and AGPAT2, which have been proven to carry out the AGPAT reaction (conversion of lysophosphatidic acid to phosphatidic acid) (2
), belong to the same subgroup. AGPAT3, AGPAT4, and AGPAT5, which have been reported to have very weak AGPAT activities (18
), constitute two distinct subgroups. GPAM and GNPAT, two enzymes that add acyl groups to the sn-1
), are more closely related to each other than to any other member of the family. Lysocardiolipin acyltransferase (LYCAT) (15
) falls into a fifth subgroup. Interestingly, we identified three putative C. elegans
orthologues for mammalian LYCAT (15
). Tafazzin falls into a sixth subgroup; tafazzin is involved in cardiolipin remodeling but its precise biochemical role remains to be established (17
). AGPAT7 (19
) and a closely related novel protein (locus 270084) form a seventh subgroup; their biological importance and biochemical function remain to be determined. AGPAT6 and AGPAT8 form the eighth subgroup.
For the entire glycerolipid acyltransferase family, we hypothesize that sequence relatedness within domains I–IV will ultimately be shown to correlate with biochemical function. For example, in the case of AGPAT6 and AGPAT8, we hypothesize that these two enzymes will ultimately be shown to have acyl acceptor and/or donor preferences that are similar to each other and distinct from those of other AGPATs, GPAMs, GNPATs, or LYCATs.
The identification of enzymatic activities for putative lipid biosynthetic enzymes can be straightforward (2
), but in some cases has been very challenging. For example, despite sequence motifs suggesting an acyltransferase activity (32
) and despite years of biochemical studies by multiple groups, the biochemical role for tafazzin in cardiolipin remodeling has not yet been identified with certainty. This has certainly been the case for several of the AGPATs.
We expressed AGPAT2, GPAM, and AGPAT6 in insect cells, with sequence-verified plasmids, and then tested the membrane fractions for GPAM or AGPAT activities under a variety of reaction conditions. While the biochemical activities of our experimental controls, GPAM and AGPAT2, were invariably extremely robust (>5–10×background), we have not observed any AGPAT or GPAM activities in insect cell membranes overexpressing AGPAT6 (at least no activity above background) (A. Beigneux, S. G. Young, unpublished results). Similarly, we have not identified AGPAT or GPAM activities in E. coli membranes overexpressing AGPAT6.
One way to explain these results, of course, is to postulate that none of the reaction conditions were appropriate for AGPAT6, although they were perfectly suitable for the AGPAT2 and GPAM controls. This is definitely possible. For example, DGAT1 and DGAT2 carry out the same reaction but have different requirements for magnesium (31
), so it would be a mistake to assume that AGPAT2 and AGPAT6 would share identical in vitro
reaction conditions. Given the phenotypes of the mice, an AGPAT or GPAM activity for AGPAT6 would make a lot of sense. A reduced capacity for synthesizing lysophosphatidic acid or phosphatidic acid would probably explain reduced amounts of diacylglycerols and triacylglycerols in the milk and in the brown fat (25
) of Agpat6−/−
On the other hand, it is possible that AGPAT6 catalyzes a distinct biochemical activity. One reason for suspecting a different activity is that we have been unable, thus far, to detect even a small increase in AGPAT or GPAM activity in AGPAT6-enriched membranes, under a variety of assay conditions with various sn
-1-acylglycerols, and acyl-CoA species as substrates. A second reason for suspecting a different enzymatic function is that it would be astonishing if mammals (and lower organisms such as worms and flies) truly require seven or more distinct AGPAT enzymes. Enzymes for crucial steps in lipid biosynthesis are frequently redundant (6
), but the need for seven or more different AGPATs would be quite remarkable, particularly since the fatty acyl chain specificities of AGPAT1 and AGPAT2 are broad (2
) and since the loss of AGPAT2
causes such striking disease phenotypes (33
In the end, biochemical studies, as well as the characterization of knockout mouse for each of the glycerolipid acyltransferases, will be critical for understanding lipid synthesis and lipid storage in different tissues, and for understanding the potential relevance of this family of enzymes to health and disease.