Caenorhabditis elegans mboa-7, a member of the MBOAT family, is required for selective incorporation of polyunsaturated fatty acids into phosphatidylinositol - PubMed (original) (raw)

Caenorhabditis elegans mboa-7, a member of the MBOAT family, is required for selective incorporation of polyunsaturated fatty acids into phosphatidylinositol

Hyeon-Cheol Lee et al. Mol Biol Cell. 2008 Mar.

Abstract

Phosphatidylinositol (PI) is a component of membrane phospholipids, and it functions both as a signaling molecule and as a compartment-specific localization signal in the form of polyphosphoinositides. Arachidonic acid (AA) is the predominant fatty acid in the sn-2 position of PI in mammals. LysoPI acyltransferase (LPIAT) is thought to catalyze formation of AA-containing PI; however, the gene that encodes this enzyme has not yet been identified. In this study, we established a screening system to identify genes required for use of exogenous polyunsaturated fatty acids (PUFAs) in Caenorhabditis elegans. In C. elegans, eicosapentaenoic acid (EPA) instead of AA is the predominant fatty acid in PI. We showed that an uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into PI. Incorporation of exogenous PUFA into PI of the living worms and LPIAT activity in the microsomes were greatly reduced in mboa-7 mutants. Furthermore, the membrane fractions of transgenic worms expressing recombinant MBOA-7 and its human homologue exhibited remarkably increased LPIAT activity. mboa-7 encodes a member of the membrane-bound O-acyltransferase family, suggesting that mboa-7 is LPIAT. Finally, mboa-7 mutants had significantly lower EPA levels in PI, and they exhibited larval arrest and egg-laying defects.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

(A) C. elegans PUFA synthetic pathway from bacterial fatty acids. Lipid structures are abbreviated as in 20:4n-6, which has 20 carbons and four double bonds, the first occurring at the n-6 position. fat-1, n-3 desaturase; fat-2, Δ12 desaturase; fat-3, Δ6 desaturase; fat-4, Δ5 desaturase. (B) Rescue of the growth defects of fat-4 fat-1 mutants by dietary supplementation with AA or EPA. Synchronized populations of first-stage larvae were propagated on E. coli OP50 supplemented with AA or EPA, and they were allowed to grow at 15°C. At least 150 worms were scored for each assay and scored as adult if they form vulva. Representative plots of multiple experiments are shown. WT, wild-type. (C) One-dimensional thin-layer chromatographic separation of total lipids from fat-4 fat-1 mutants radiolabeled with [14C]AA (left) and [14C]EPA (right).

Figure 2.

Figure 2.

(A) Knockdown and knockout of mboa-7 inhibit growth rescue by dietary supplementation with AA or EPA. Growth of each strain was scored as described in Figure 1B, except that E. coli HT115 was used as a food source. All experiments were performed at 15°C. (B) Multiple sequence alignment of C. elegans mboa-7 and homologous sequences in human (BB1/LENG4; Fukunaga-Johnson et al., 1996; Wende et al., 2000), mouse, and zebrafish. Residues identical in all four sequences are shaded in black, and residues identical in three proteins are shaded in gray. The numbers on the left indicate amino acid positions. The histidine residue indicated by asterisk is the predicted active site of the MBOAT motif in mboa-7 (Hofmann, 2000; Bosson et al., 2006). Accession numbers for the sequences used were as follows: C. elegans, EU016382; human, EU016381; mouse, EU016380; and zebrafish, NP_609029. (C) Genomic structure of mboa-7 (F14F3.3). The mboa-7 gene is located on chromosome X. Gray boxes indicate coding exons, and white boxes indicate 5′ and 3′ untranslated sequences. The positions of the ATG initiation codon, stop codon (TAG), the trans-spliced leader SL1, and the poly(A) tail are shown. The extent of the deletion in mboa-7(gk399) is indicated by a horizontal line.

Figure 3.

Figure 3.

Incorporation of exogenous [14C]AA and [14C]EPA into phospholipids of mboa-7 mutants. (A) One-dimensional thin layer chromatographic separation of total lipids from fat-4 fat-1 and fat-4 fat-1; mboa-7 mutants radiolabeled with exogenous [14C]AA (left) and [14C] EPA (right). (B) Incorporation of various radiolabeled fatty acids into phospholipids of fat-4 fat-1 and fat-4 fat-1; mboa-7 mutants. The amount of incorporation was expressed as the percentage of radioactivity incorporated into total lipids. All experiments were carried out at 20°C. Note that the radiolabeled fatty acids are not metabolized to AA or EPA in vivo due to the lack of Δ5 and n-3 fatty acid desaturation activities. Each bar represents the mean ± SEM of at least three independent experiments. **p < 0.01.

Figure 4.

Figure 4.

The membrane fraction of mboa-7 mutants lacks LPIAT activity. (A and B) In vitro fatty acid incorporation assay using the membrane fractions of wild-type and mboa-7 mutants and different lysophospholipids as acyl acceptors. [14C] EPA was used as an acyl donor. (A) One-dimensional thin layer chromatographic separation of total lipids of the reaction mixture after incubation. FFA, free fatty acid. (B) Each bar represents the mean ± SEM of at least three independent experiments. **p < 0.01. (C) In vitro fatty acid incorporation into lysoPI with different radiolabeled fatty acids as acyl donors. Each bar represents the mean ± SEM of at least three independent experiments. **p < 0.01. (D) Acyl-CoA:lysophospholipid acyltransferase activity in the membrane fractions of wild-type and mboa-7 mutants. [14C]Arachidonoyl-CoA (12.5 μM) and the indicated concentration of lysoPC, lysoPE, or lysoPS were used. (E) [14C]Arachidonoyl-CoA:lysoPI acyltransferase activity in the membrane fractions of wild-type and mboa-7 mutants. [14C]Arachidonoyl-CoA (12.5 μM) and the indicated concentration of lysoPI were used.

Figure 5.

Figure 5.

Recombinant MBOA-7 and h-MBOA-7 (a human homologue of MBOA-7) expressed in C. elegans show remarkably increased LPIAT activity. [14C]Arachidonoyl-CoA:lysoPI acyltransferase activity in the membrane fractions of the indicated strains were measured. 40 μM lysoPI and 12.5 μM [14C]arachidonoyl-CoA were used as an acyl donor and an acyl acceptor, respectively. The membrane fraction of MBOA-7::GFP-expressing worms showed remarkably increased LPIAT activity both in wild-type and mboa-7 mutants (xhEx1[mboa-7::GFP] and mboa-7; xhEx1[mboa-7::GFP], respectively). Human MBOA-7::GFP also catalyzed LPIAT activity (xhEx4[h-mboa-7::GFP]). MBOA-7H350A::GFP, which is mutated at the predicted active site of the membrane-bound _O_-acyltransferase motif of MBOA-7, had no LPIAT activity (xhEx2[mboa-7 H350A::GFP] and xhEx3[mboa-7 H350A::GFP]).

Figure 6.

Figure 6.

Phenotypes of mboa-7 mutants. (A, B) For comparison of growth, wild-type and mboa-7 embryos were placed onto culture plates, incubated at 20°C, and photographed after 72 h of growth. Arrows indicate mboa-7 mutants that have arrested development at an early larval stage. Bar, 200 μm. (C) Some of the mboa-7 mutants show bags of worms phenotype where embryos hatch within the mother, leaving a cuticle sack containing multiple wriggling larvae (arrowheads). Asterisks indicate the anterior of the mother worm. Bar, 200 μm. (C–G) Expression of MBOA-7::GFP in an early embryo (D and E) and a second-stage larva (F and G). Nomarski micrographs (D and F) and corresponding MBOA-7::GFP expression (E and G). MBOA-7::GFP was ubiquitously expressed throughout development. Asterisks indicate the anterior of the larva (F and G). Bar, 10 μm (D and E) and 200 μm (F and G).

References

    1. Akesson B., Elovson J., Arvidson G. Initial incorporation into rat liver glycerolipids of intraportally injected (3H)glycerol. Biochim. Biophys. Acta. 1970;210:15–27. - PubMed
    1. Arnhold J., Benard S., Kilian U., Reichl S., Schiller J., Arnold K. Modulation of luminol chemiluminescence of fMet-Leu-Phe-stimulated neutrophils by affecting dephosphorylation and the metabolism of phosphatidic acid. Luminescence. 1999;14:129–137. - PubMed
    1. Ashrafi K., Chang F. Y., Watts J. L., Fraser A. G., Kamath R. S., Ahringer J., Ruvkun G. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature. 2003;421:268–272. - PubMed
    1. Baker R. R., Thompson W. Positional distribution and turnover of fatty acids in phosphatidic acid, phosphinositides, phosphatidylcholine and phosphatidylethanolamine in rat brain in vivo. Biochim. Biophys. Acta. 1972;270:489–503. - PubMed
    1. Baker R. R., Thompson W. Selective acylation of 1-acylglycerophosphorylinositol by rat brain microsomes. Comparison with 1-acylglycerophosphorylcholine. J. Biol. Chem. 1973;248:7060–7065. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources