Agpat6--a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium - PubMed (original) (raw)
Agpat6--a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium
Anne P Beigneux et al. J Lipid Res. 2006 Apr.
Abstract
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.
Figures
Fig. 1
An insertional mutation in Agpat6. (A) Schematic of the insertion event, and the 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.
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 to the region containing the signature glycerolipid acyltransferase sequence motifs (–9) (highlighted in grey). 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 sequence 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 weight 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 GPAM (93.7 kDa) baculoviruses. The molecular weight of AGPAT2 was identical to that predicted for the full-length open reading frame. Membranes from noninfected cells (niC) were included as a control.
Fig. 3
AGPAT6 localizes to the ER. (A) ECFP-tagged Agpat2, Agpat6, or Gpam constructs were cotransfected into COS-1 cells with M1-YFP (a YFP-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. A representative enlargement is shown as an insert 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.
Fig. 4
Agpat6 expression during development. (A) β-Galactosidase staining of an Agpat6 knockout embryo (E15). Arrows 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. β-Actin was used for normalization.
Fig. 5
Agpat6 expression in adult mice. (A) A mouse poly(A)+ RNA blot showing the expression of Agpat6 in adult tissues. A β-Actin cDNA was used for normalization. (B) A mouse total RNA blot showing prominent expression of Agpat6 in testis and brown adipose tissue in adult mice. A 18S cDNA was used for normalization. BAT, brown adipose tissue. (C–G) β-Galactosidase staining of brown adipose tissue (C), testis (D), cerebellum (E), hippocampus (F), and kidney (G) from a 6-week-old _Agpat6_−/− male. Arrows point to expression in spermatids (sp), Sertoli cells (s), cerebellar lobule (cb), hippocampus (h), dentate gyrus (dg), and tubular cells (t).
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) β-Galactosidase staining of mammary gland from _Agpat6_−/− and Agpat6+/+ lactating females, revealing a high level of Agpat6 expression in the 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_-deficient epithelial cells (D). In all experiments, the mammary glands were dissected 24 h postpartum, when pups were still alive and suckling.
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 the mammary glands from the _Agpat6_−/− female. (D) Reduced triacylglycerol (TG) content of milk from an _Agpat6_−/− female, compared to heterozygous and wild-type controls, as assessed by thin layer chromatography. The intensity of the cholesterol ester (CE) band was not significantly reduced in milk from _Agpat6_−/− females. (E) Reduced diacylglycerol (DAG) and triacylglycerol (TG) 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.
Fig. 8
Agpat4 expression in mammary gland (A) and testis (B), as judged by β-galactosidase staining. Agpat4 expression in the mammary gland was undetectable, whereas it was robust in the Sertoli cells of the testis.
Fig. 9
Dendrogram illustrating the amino acid sequence relatedness of mouse glycerolipid acyltransferases within the region of the proteins spanning functional domains I–IV (–7). Alignments were performed with the Clustal W algorithm (
http://www.ebi.ac.uk/clustalw/
).
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