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Journal of Lipid Research, Vol. 47, 734-744, April 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Agpat6—a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium

Anne P. Beigneux^1,*, Laurent Vergnes, Xin Qiao^*, Steven Quatela, Ryan Davis^**, Steven M. Watkins^**, Rosalind A. Coleman, Rosemary L. Walzem^**, Mark Philips, Karen Reue and Stephen G. Young^*

^* Division of Cardiology, Department of Internal Medicine, University of California, Los Angeles, CA 90095
Departments of Medicine and Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA 90073
Department of Medicine, Cell Biology, and Pharmacology, New York University School of Medicine, New York, NY 10016
^** Lipomics Technologies, West Sacramento, CA 95691
Department of Nutrition, University of North Carolina, Chapel Hill, NC 27599

Published, JLR Papers in Press, January 31, 2006.

¹ To whom correspondence should be addressed. e-mail: abeigneux{at}mednet.ucla.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource), we identified a novel gene, 1-acylglycerol-3-phosphate O-acyltransferase (Agpat6), with sequence similarities to previously characterized glycerolipid acyltransferases. Agpat6's closest family member is another novel gene that we have provisionally designated Agpat8. Both Agpat6 and Agpat8 are conserved from plants, nematodes, and flies to mammals. AGPAT6, which is predicted to contain multiple membrane-spanning helices, is found exclusively within the endoplasmic reticulum (ER) in mammalian cells. To gain insights into the in vivo importance of Agpat6, we used the Agpat6 ES cell line from BayGenomics to create Agpat6-deficient (Agpat6^–/–) mice. Agpat6^–/– mice lacked full-length Agpat6 transcripts, as judged by northern blots. One of the most striking phenotypes of Agpat6^–/– mice was a defect in lactation. Pups nursed by Agpat6^–/– mothers die perinatally. Normally, Agpat6 is expressed at high levels in the mammary epithelium of breast tissue, but not in the surrounding adipose tissue. Histological studies revealed that the aveoli and ducts of Agpat6^–/– lactating mammary glands were underdeveloped, and there was a dramatic decrease in the size and number of lipid droplets within mammary epithelial cells and ducts. Also, the milk from Agpat6^–/– mice was markedly depleted in diacylglycerols and triacylglycerols. Thus, we identified a novel glycerolipid acyltransferase of the ER, AGPAT6, which is crucial for the production of milk fat by the mammary gland.

Supplementary key words acyltransferase • transacylase • milk fat

Abbreviations: AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase; DGAT, diacylglycerol acyltransferase; ECFP, enhanced cyan fluorescent protein; ER, endoplasmic reticulum; ES, embryonic stem; GNPAT, glyceronephosphate O-acyltransferase; GPAM, glycerol-3-phosphate acyltransferase; LYCAT, lysocardiolipin acyltransferase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
BayGenomics is a genomics program that uses gene-trapping vectors to produce mutant lines of mouse embryonic stem (ES) cells (http://baygenomics.ucsf.edu/) (1). The gene inactivated by the insertion of the gene-trapping vector can easily be identified with a unique DNA sequence tag. To date, BayGenomics has inactivated >3,000 unique genes in ES cells and has distributed >2,500 different cell lines to the research community for the purpose of creating knockout mice. Aside from producing mutant ES cell clones, BayGenomics also produces a few knockout mice from the gene-trap ES cell lines, with the goal of identifying genes relevant to lipid metabolism and cardiopulmonary disease.

While analyzing the sequence tags for BayGenomics ES cell clones, we encountered a novel gene with sequence similarities to 1-acylglycerol-3-phosphate O-acyltransferases [AGPATs, which convert lysophosphatidic acid to phosphatidic acid (2, 3)] and other members of the glycerolipid acyltransferase family. Because of amino acid sequence similarities to AGPAT1, AGPAT2, and other putative AGPATs (AGPAT3, AGPAT4, and AGPAT5), the novel gene was provisionally designated Agpat6. Although this provisional name was based on similarities to other AGPATs, it should be noted that AGPAT6 also has sequence similarities to a variety of "non-AGPAT" glycerolipid acyltransferases. At about the same time, another report drew attention to the existence of AGPAT6 in the human cDNA databases (4); however, there have been no published data on the intracellular localization of the enzyme or its in vivo importance.

The glycerolipid acyltransferase protein family, which has been defined largely on the basis of amino acid sequence similarities, includes glycerol-3-phosphate acyltransferase (GPAM), glyceronephosphate O-acyltransferase (GNPAT), tafazzin, lysocardiolipin acyltransferase (LYCAT), 2-acylglycerophosphoethanolamine acyltransferase, the AGPATs, and a number of novel enzymes of unknown function. Amino acid sequence alignments of GPAM, AGPAT1, AGPAT2, and GNPAT have defined four regions of homology (motifs I–IV) that represent signatures for glycerolipid acyltransferases (5–7). Site-directed mutagenesis, followed by expression studies in Escherichia coli, have indicated that motifs I and IV are involved in catalysis, whereas motifs II and III are required for glycerol-3-phosphate binding (5, 8, 9). Recent studies of naturally occurring mutations in AGPAT2 (10, 11) have revealed a fifth important sequence motif (V), but its function has not yet been identified.

The biochemical properties, subcellular localization, and biological relevance of GPAM, GNPAT, tafazzin, LYCAT, AGPAT1, and AGPAT2 have been at least partially characterized either in humans or mice (2, 3, 12–15). GPAM is located in mitochondria and catalyzes the acylation of glycerol-3-phosphate at the sn-1 position to generate lysophosphatidic acid, and studies with Gpam-deficient mice have revealed that this enzyme plays a major role in triacylglycerol synthesis (12). GNPAT is located in peroxisomes and catalyzes the acylation of glyceronephosphate to generate 1-acyl-glycerone-3-phosphate, a precursor in plasmalogen synthesis (16). Mutations in GNPAT cause rhizomelic chondrodysplasia punctata type 2, characterized by shortening of the upper extremities, mental retardation, and cataracts (13). The activity of tafazzin, which is located in mitochondria, is not known with certainty, but it could be involved in transferring acyl groups from phosphatidylcholine or phosphatidylethanolamine to monolysocardiolipin (17). Mutations in tafazzin cause Barth syndrome, an X-linked disease associated with dilated cardiomyopathy, skeletal myopathy, neutropenia, and growth retardation (14). Another cardiolipin biosynthetic enzyme, LYCAT, was identified recently (15).

The biochemical roles for AGPAT1 and AGPAT2 in generating phosphatidic acid are well documented (2, 3). Mutations in AGPAT2, which is expressed largely in adipose tissue, cause congenital generalized lipodystrophy (10), a disease characterized by a striking absence of subcutaneous and abdominal fat, hypertriglyceridemia, and severe insulin resistance. The other putative AGPATs (AGPAT3, AGPAT4, AGPAT5, and AGPAT7) (18, 19) were identified by sequence homology, and little is known about their biochemical properties or physiologic importance. AGPAT activity was attributed to AGPAT3, AGPAT4, and AGPAT5 in one study (18), but the activity levels were very low and in no way comparable to that observed for AGPAT2 (18). The physiological relevance of the novel BayGenomics gene, Agpat6, had never been investigated.

We sought to determine the intracellular localization of AGPAT6 and to define its relatedness, in terms of amino acid sequence, to known glycerolipid acyltransferases. In addition, we sought to ascertain the biological importance of AGPAT6 by examining the phenotypes of Agpat6-deficient (Agpat6^–/–) mice. Here, we report that AGPAT6 is located exclusively in the endoplasmic reticulum (ER) and that its absence leads to underdeveloped mammary epithelium and the production of milk depleted in diacylglycerols and triacylglycerols. In addition, we report the identification of another novel gene, provisionally designated Agpat8, which is the closest homolog of Agpat6 within the glycerolipid acyltransferase family. Remarkably, AGPAT6 and AGPAT8 are conserved from plants, flies, and worms to mammals.

	MATERIALS AND METHODS

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Agpat6^–/– mice
A mouse ES cell line (DTM030, strain 129/OlaHsd) containing an insertional mutation in Agpat6 was identified by BayGenomics, a gene-trapping resource (1). The gene-trap vector used (pGT1dTMpfs) contains a splice-acceptor sequence upstream of the reporter gene ßgeo (a fusion of ß-galactosidase and neomycin phosphotransferase II) (1). As judged by 5' rapid amplification of cDNA ends (20), the insertional mutation in DTM030 was located in the second intron of Agpat6. Thus, the mutation results in the production of an in-frame fusion transcript consisting of exons 1 and 2 from Agpat6 and ßgeo. Another BayGenomics ES cell line, RRF360, was used to create Agpat4 knockout mice.

We determined the exact site of insertion of the vector within intron 2, which allowed us to design a PCR strategy to genotype the mice. The following primers were used for genotyping: primer 1, 5'-ACAGGCTTTTGTGGTTTGGTTTGCT-3'; primer 2, 5'-AGAAATCCTCCCCAACAGTGGGACT-3'; and primer 3 (from vector sequences), 5'-CGTGTCCTACAACACACACTCCAACC-3'. The wild-type allele was detected with primers 1 and 2 (located in sequences flanking the insertion, yielding a 458 bp product), whereas the mutant allele was detected with primers 1 and 3, yielding a 378 bp fragment.

ES cell line DTM030 was injected into C57BL/6 blastocysts to generate chimeric mice, which were bred to establish Agpat6 knockout mice. All mice had a mixed genetic background (C57BL/6 and 129/OlaHsd). The mice were weaned at 21 days of age, housed in a barrier facility with a 12 h light/12 h dark cycle, and fed a chow diet containing 4.5% fat (Ralston Purina, St. Louis, MO).

Northern blots
Total RNA was isolated from 50–150 mg of mouse tissue with Tri-Reagent (Sigma, St. Louis, MO). Total RNA (5 µg) was separated by electrophoresis on 1% agarose/formaldehyde gels and transferred to a Nytran SuPerCharge membrane (Schleicher and Schuell, Keene, NH). A mouse multiple-tissue poly(A)⁺ RNA blot and a mouse embryo poly(A)⁺ RNA blot (Clontech, Palo Alto, CA) were used to determine the tissue pattern of Agpat6 expression in adult mice and to examine the temporal expression of Agpat6 during embryogenesis. Bands on Northern blots were visualized by autoradiography (Fuji Super RX films; Fujifilm, Tokyo, Japan) and quantified by densitometry (Molecular Imager FX; Bio-Rad, Hercules, CA). A cDNA probe comprising sequences within the 3' untranslated region of Agpat6 (a region not conserved in Agpat8) was amplified by RT-PCR with 5'-GTGGCAGGACAAGGTCAGAGCTACA-3' and 5'-TCCCTCCTGACTCACCAGTTCTTCC-3'. The lacZ probe was prepared as described previously (21). ß-Actin and 18S cDNA probes were used as controls for RNA integrity and loading. [³²P]dCTP-labeled cDNA probes were prepared with All-in-One random prime labeling mixture (Sigma). Standard prehybridization, hybridization, and washing procedures were used (21).

Western blots
The complete open reading frame of Agpat6 was amplified from a mouse embryo cDNA library by RT-PCR with primers 5'-ACCATGTTCCTGTTGCTACCT-3' and 5'-GGACCGGCTGCGGTCCTCATGGTTTCC-3'. For expression in mammalian cells, the Agpat6 coding sequence was cloned in-frame with a C-terminal V5-His tag into pcDNA3.1/V5-His TOPO TA expression vector (Invitrogen, Carlsbad, CA). For expression in insect cells, Agpat6 was amplified by PCR from pcDNA3.1/V5-His TOPO with the C-terminal V5-His tag with primers 5'-ATCGGAATTCATGTTCCTGTTGCTACCT-3' and 5'-CGATGAATTCTCAATGGTGATGGTGATGATGACC-3', subcloned into pCR2.1 (Invitrogen), and then subsequently cloned into the EcoRI site into pBacPAK8 (Clontech). The integrity of the Agpat6 sequence within each construct was verified by sequencing. High-Five insect cells infected with C-terminal V5-tagged Agpat6, Agpat2, or Gpam-recombinant baculovirus, or COS-7 cells transfected with the Agpat6-pcDNA3.1/V5-His construct, were collected in 1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS containing a mixture of protease inhibitors (Complete Mini EDTA-free; Roche Applied Science, Indianapolis, IN). Samples were sonicated on ice and clarified by centrifugation at 500 g for 10 min at 4°C. The protein content of the supernatant fluid (whole-cell extract) was determined with a Bradford assay (Bio-Rad). Denatured proteins (5 ng for insect cell extracts, 1 µg for COS-7 cell extracts) were size-fractionated on 4–12% Bis-Tris NuPAGE gels (Invitrogen). After electrotransfer to a sheet of nitrocellulose membrane (Invitrogen), the blots were blocked with phosphate-buffered saline containing 0.2% Tween and 5% nonfat powdered milk overnight at 4°C, then incubated for 1 h at room temperature with a horseradish peroxidase-conjugated mouse monoclonal antibody against the V5 tag (Invitrogen) (1:5,000). Antibody binding was detected with the ECL Plus Western blotting kit (Amersham Biosciences, Piscataway, NJ) and Fuji Super RX X-ray film (Fujifilm).

Histology and immunohistochemistry
Mice were anesthetized intraperitoneally with a ketamine/xylazine cocktail and perfusion-fixed with normal saline followed by 4% paraformaldehyde. For ß-galactosidase staining, tissues were fixed and stained as described previously (21). For hematoxylin/eosin staining, mammary glands were fixed in 10% buffered formalin for 24 h, dehydrated in ethanol, transitioned into xylene, embedded in paraffin, sectioned (1 µm thick), and stained.

For fat staining, mice were deeply anesthetized intraperitoneally with a ketamine/xylazine cocktail and perfusion-fixed with 0.1 M cacodylate followed by 2.5% glutaraldehyde in 0.1 M cacodylate. Mammary glands were dissected out and cleaved into thin slices to ensure thorough fixation. After fixation, tissues were stained in 1% osmium tetroxide, dehydrated in ethanol, transitioned into acetonitrile, and then embedded in Epon resin.

For some immunohistochemistry studies, the Agpat6-pcDNA3.1/V5-His construct described above was transfected into HeLa cells. AGPAT6 subcellular localization was compared with that of protein disulfide isomerase (an ER marker) and of manganese superoxide dismutase (a mitochondrial marker). The following antibodies were used: affinity-purified rabbit anti-6xHistine (1:6,000; Immunology Consultants Laboratory, Newberg, OR); Alexa Fluor 568-labeled goat anti-rabbit IgG (1:800; Molecular Probes, Eugene, OR); mouse monoclonal anti-protein disulfide isomerase (1:700; Abcam, Cambridge, MA); Alexa Fluor 488-labeled goat anti-mouse IgG (1:800; Molecular Probes); FITC-conjugated mouse anti-V5 tag (1:1,500; Invitrogen); rabbit polyclonal anti-manganese superoxide dismutase (1 µg/ml; Stressgen, Victoria, Canada); Cy3-labeled goat anti-rabbit IgG (1:800; Abcam).

In addition, the complete coding sequences for Agpat2, Agpat6, and Gpam were inserted in-frame with a C-terminal enhanced cyan fluorescent protein marker in pECFP-N1 (Clontech). The ECFP-tagged constructs were expressed in COS-1 cells along with M1-YFP, a yellow fluorescent protein-tagged ER marker (22). To assess mitochondrial localization, ECFP-tagged constructs were expressed in COS-1 cells labeled with a MitoTracker dye (Molecular Probes). Cells were imaged alive with an inverted Zeiss 510 laser scanning confocal microscope (63x lens).

Analysis of lipids in milk
Three hours after removing newborn pups, lactating females were given two successive intraperitoneal injections of oxytocin (5 µl/g body weight of a 20 U/ml solution; injections separated by 20 min). At the time of the second oxytocin injection, mice were deeply anesthetized with a ketamine/xylazine cocktail, and milk was collected from the mammary glands and stored at –80°C for analysis. For all experiments, the milk was collected 24 h postpartum, when pups were still alive and suckling.

Milk lipids were extracted in the presence of authentic internal standards by the method of Folch, Lees, and Sloane Stanley (23) with chloroform-methanol (2:1, v/v). Twenty microliters of milk was used for each analysis. In some experiments, neutral lipids were separated by thin-layer chromatography in hexane-ethyl ether-acetic acid (80:20:2), subsequently visualized with iodine vapor, and identified by comigration with lipid standards. In other experiments, individual lipid classes within each extract were separated by preparative HPLC. Each isolated lipid class fraction was transesterified in 3 N methanolic-HCl in a sealed vial under nitrogen at 100°C for 45 min. The fatty acid methyl esters were extracted from the mixture with hexane containing 0.05% butylated hydroxytoluene and prepared for gas chromatography by sealing the hexane extracts under nitrogen. Fatty acid methyl esters were then separated and quantified by capillary gas chromatography with a gas chromatograph (model 6890; Hewlett-Packard, Wilmington, DE) equipped with a 30-m DB-225MS capillary column (J&W Scientific, Folsom, CA) and a flame-ionization detector, as described previously (24). Lipid metabolome data were expressed as nanomoles per gram and were assembled in tables as means ± SD for each group.

	RESULTS

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Agpat6 knockout mice
The BayGenomics library of mutant ES cells contained a mouse ES cell line with an insertional mutation in Agpat6, which was used to generate Agpat6^–/– mice. The site of insertion was identified within intron 2, making it possible to design a PCR strategy to distinguish heterozygous (Agpat6^+/–) from homozygous (Agpat6^–/–) mice (Fig. 1A ). As expected, full-length Agpat6 transcripts were absent in Agpat6^–/– embryos (Fig. 1B).

View larger version (23K): [in this window] [in a new window]   Fig. 1. An insertional mutation in 1-acylglycerol-3-phosphate O-acyltransferase (Agpat6). A: Scheme of the insertion event, and detection of the insertional mutation by PCR with genomic DNA from Agpat6+/+, Agpat6+/–, and Agpat6–/– mice. Numbers indicate exons. Primer orientation and location are indicated with arrows. SA, splice acceptor; SV40pA, poly(A) tail. B: Northern blot with total RNA showing the expression of Agpat6 in Agpat6+/+, Agpat6+/–, and Agpat6–/– embryos. An 18S cDNA was used for normalization.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Structural features of AGPAT6. A: Kyte-Doolittle hydrophobicity profiles for mouse AGPAT6, AGPAT1, and AGPAT2. Numbers on the x axis refer to amino acid residues. AGPAT6 (456 amino acids) is larger than AGPAT1 (285 amino acids) and AGPAT2 (278 amino acids), mainly because AGPAT6 contains 140 extra amino acid residues upstream of the region containing the signature glycerolipid acyltransferase sequence motifs (5–9) (highlighted in gray). Bold horizontal lines indicate predicted transmembrane domains (as judged by http://www.cbs.dtu.dk/services/TMHMM-2.0/, http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0E.html, and http://smart.embl-heidelberg.de/). The question mark in the AGPAT6 profile indicates a potential transmembrane domain that is predicted by one of the three sequence analysis programs (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0E.html). B: Western blot, with an anti-V5 antibody, showing that the molecular mass of the tagged version of AGPAT6 is 48 kDa, both in extracts from COS-7 cells transfected with V5-tagged Agpat6 (+) and in High-Five cells infected with a V5-tagged Agpat6 recombinant baculovirus (+). The 48 kDa band was absent in extracts from nontransfected COS-7 cells (–) and noninfected High-Five cells (–). C: Western blot of total membrane fractions (5 ng each) from High-Five cells infected with V5-tagged AGPAT6 (48 kDa), AGPAT2 (31 kDa), or glycerol-3-phosphate acyltransferase (GPAM; 93.7 kDa) baculoviruses. The molecular mass of AGPAT2 was identical to that predicted for the full-length open reading frame. Membranes from noninfected cells (niC) were included as a control.

View larger version (47K): [in this window] [in a new window]   Fig. 3. AGPAT6 localizes to the endoplasmic reticulum (ER). A: Enhanced cyan fluorescent protein (ECFP)-tagged Agpat2, Agpat6, or Gpam constructs were cotransfected into COS-1 cells with M1-YFP (a yellow fluorescent protein-tagged ER marker) (22). To assess mitochondrial localization, ECFP-tagged constructs were expressed in COS-1 cells labeled with a MitoTracker dye. ECFP-AGPAT6 colocalized with M1-YFP but not with the MitoTracker dye, indicating an ER membrane localization for AGPAT6. Representative enlargements are shown as inserts in the panels for AGPAT6. AGPAT2 and GPAM were used as controls for ER and mitochondrial localization, respectively. B: HeLa cells transfected with an expression vector for a V5-His-tagged Agpat6. The subcellular localization of AGPAT6 was compared with that of an ER marker, protein disulfide isomerase (PDI), and a mitochondrial marker, manganese superoxide dismutase (MnSOD). Merged images show colocalization of AGPAT6 and PDI but not MnSOD.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Agpat6 expression during development. A: ß-Galactosidase staining of an Agpat6 knockout embryo (E15). Arrowheads point to expression in brain (b), dorsal fat pad (df), lung (lg), liver (lv), and mesenchyme (m) of the guts. B: A mouse embryo poly(A)+ RNA blot showing the expression of Agpat6 throughout embryogenesis in wild-type mice. ß-Actin was used for normalization.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Agpat6 expression in adult mice. A: A mouse poly(A)+ RNA blot showing the expression of Agpat6 in adult tissues. ß-Actin cDNA was used for normalization. Sk., skeletal. B: A mouse total RNA blot showing prominent expression of Agpat6 in testis and brown adipose tissue in adult mice. 18S cDNA was used for normalization. BAT, brown adipose tissue. C–G: ß-Galactosidase staining of brown adipose tissue (C), testis (D), cerebellum (E), hippocampus (F), and kidney (G) from a 6 week old Agpat6–/– male. Arrowheads point to expression in spermatids (sp), Sertoli cells (s), cerebellar lobule (cb), hippocampus (h), dentate gyrus (dg), and tubular cells (t).

Agpat6 is expressed in nonlactating mammary gland, and the expression levels are upregulated during lactation, as judged by Northern blot analysis. This upregulation was evident both for the full-length Agpat6 transcript in wild-type mice and for the Agpat6-ßgeo fusion transcript in Agpat6^–/– mice (Fig. 6A ). ß-Galactosidase staining revealed that Agpat6 is expressed predominantly in epithelial cells of the mammary gland; no staining was detected in the surrounding white adipose tissue (Fig. 6B). Hematoxylin and eosin-stained sections revealed that the alveoli and ducts of the mammary glands of Agpat6^–/– lactating females were underdeveloped compared with those of wild-type mice (Fig. 6C). Furthermore, reduced numbers of fat droplets were evident in the epithelial cells of Agpat6^–/– mammary glands compared with wild-type controls (Fig. 6D).

View larger version (50K): [in this window] [in a new window]   Fig. 6. Expression of Agpat6 in mammary gland. A: Northern blot analysis of total RNA from Agpat6+/+ and Agpat6–/– mammary glands. An Agpat6 cDNA was used to detect the full-length Agpat6 mRNA in wild-type tissues, and a lacZ probe was used to detect the fusion transcript in the knockout mice. An 18S cDNA was used for normalization. B: ß-Galactosidase staining of mammary gland from Agpat6–/– and Agpat6+/+ lactating females, revealing a high level of Agpat6 expression in epithelial cells of the mammary gland. C, D: Hematoxylin and eosin staining of Agpat6–/– and Agpat6+/+ mammary glands, showing reduced size and number of alveoli in the mammary glands of lactating Agpat6–/– mice (C) as well as reduced numbers of fat droplets in Agpat6–/– epithelial cells (D). In all experiments, the mammary glands were dissected 24 h postpartum, when pups were still alive and suckling.

View larger version (45K): [in this window] [in a new window]   Fig. 7. Influence of Agpat6 deficiency on the composition of milk. A: Milk streak in a pup nursed by an Agpat6–/– female. B, C: Osmium tetroxide-stained sections of mammary glands from Agpat6–/– (B) and Agpat6+/+ (C) lactating females, showing decreased lipid droplets in the alveoli and ducts of mammary glands from the Agpat6–/– female. D: Reduced triacylglycerol (TG) content of milk from an Agpat6–/– female, compared with heterozygous and wild-type controls, as assessed by thin-layer chromatography. The intensity of the cholesteryl ester (CE) band was not significantly reduced in milk from Agpat6–/– females. E: Reduced diacylglycerol (DAG) and triacylglycerol content of milk from Agpat6–/– females, as assessed by gas chromatography. In all experiments, the milk was collected (or the mammary glands were dissected) 24 h postpartum, when pups were still alive and suckling. Diacylglycerol and triacylglycerol content of milk is expressed as mean ± SD for each group.

View larger version (55K): [in this window] [in a new window]   Fig. 8. Agpat4 expression in mammary gland (A) and testis (B), as judged by ß-galactosidase staining. Agpat4 expression in the mammary gland was undetectable, whereas it was robust in the Sertoli cells of the testis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 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 (Table 1). Both AGPAT6 and AGPAT8 contain the classic sequence motifs (I–IV) characteristic of glycerolipid acyltransferases (5–7). When the sequences spanning motifs I–IV were analyzed for relatedness, it was apparent that AGPAT6 and AGPAT8 were most related to each other (Fig. 9 ) (66% identical at the amino acid level, but only 2–15% identical to 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 (Table 1). Domain III is strikingly conserved between AGPAT6 and AGPAT8, representing a signature sequence for these two proteins (Table 1). 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, orthologs for Agpat6 and Agpat8 appear to exist in the genomes of plants, worms, and flies (Table 1), suggesting that, together, AGPAT6 and AGPAT8 probably play a unique, fundamental, and conserved function in lipid biosynthesis.

View larger version (6K): [in this window] [in a new window]   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/).

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, 3, 30, 31), but in some cases it 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. Although the biochemical activities of our experimental controls, GPAM and AGPAT2, were invariably extremely robust (>5–10x background), we have not observed any AGPAT or GPAM activities in insect cell membranes overexpressing AGPAT6 (at least no activity above background) (A. Beigneux and 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 the reaction conditions were not 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 the reduced amounts of diacylglycerols and triacylglycerols in the milk and in the brown fat (25) of Agpat6^–/– mice.

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, to date, 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 because the fatty acyl chain specificities of AGPAT1 and AGPAT2 are broad (2, 3) and the loss of AGPAT2 causes such striking disease phenotypes (33).

In the end, biochemical studies, as well as the characterization of a 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.

	ACKNOWLEDGMENTS

 
The authors are grateful to Drs. Tal Lewin and Diana Mehedint for biochemical protocols. This work was supported by BayGenomics, a Program for Genomics Applications from the National Heart, Lung, and Blood Institute (UO1 HL-66621).

Manuscript received December 22, 2005 and in revised form January 19, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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