Genetic dissection of polyunsaturated fatty acid synthesis in Caenorhabditis elegans

Abstract

Polyunsaturated fatty acids (PUFAs) are important membrane components and precursors of signaling molecules. To investigate the roles of these fatty acids in growth, development, and neurological function in an animal system, we isolated Caenorhabditis elegans mutants deficient in PUFA synthesis by direct analysis of fatty acid composition. C. elegans possesses all the desaturase and elongase activities to synthesize arachidonic acid and eicosapentaenoic acid from saturated fatty acid precursors. In our screen we identified mutants with defects in each fatty acid desaturation and elongation step of the PUFA biosynthetic pathway. The fatty acid compositions of the mutants reveal the substrate preferences of the desaturase and elongase enzymes and clearly demarcate the steps of this pathway. The mutants show that C. elegans does not require n3 or ⌬5-unsaturated PUFAs for normal development under laboratory conditions. However, mutants with more severe PUFA deficiencies display growth and neurological defects. The mutants provide tools for investigating the roles of PUFAs in membrane biology and cell function in this animal model. P olyunsaturated fatty acid (PUFA) components of phospholipids are necessary to create a fluid membrane environment. In mammals, specific PUFA composition affects many cellular processes including modulation of ion channels (1, 2), endocytosis͞exocytosis (3), and activities of membrane-associated enzymes that are sensitive to the biophysical properties of lipid membranes (4). In addition, certain classes of PUFAs are precursors of powerful eicosanoid effectors such as prostaglandins and leukotrienes (5). Disruptions in proper PUFA intake and metabolism are associated with certain human disease states such as coronary artery disease, hypertension, diabetes, inflammatory disorders, and cancer (6). In most cases the mechanisms through which fatty acid composition affects these conditions are not well understood.Mammals require the essential fatty acids linoleic acid (18:2n6) and linolenic acid (18:3n3) in their diet. Desaturation and elongation of these fatty acids produce the C20 PUFAs that become membrane components and precursors for eicosanoids. Plants produce these essential n6 and n3 PUFAs by desaturating oleic acid (18:1n9) at C12 and C15 positions to produce 18:2n6 and 18:3n3 but rarely possess the enzymes necessary to desaturate and elongate them further into C20 PUFAs. The free-living nematode Caenorhabditis elegans synthesizes a wide range of PUFAs including arachidonic acid (20:4n6) and eicosapentaenoic acid (20:5n3) by using saturated fatty acids obtained from their Escherichia coli diet (7). To this end, C. elegans expresses the full range of desaturase activities found in plants (⌬12 and n3 desaturase) and animals (⌬5 and ⌬6 desaturase) as well as n6 and n3 PUFA elongase activities found in animals. cDNAs corresponding to C. elegans n3, ⌬12, ⌬6, and ⌬5 desaturase genes have been expressed in Arabidopsis and yeast to confirm their desaturase activity (8-11). Another C. elegans ...

Identification of two epoxide hydrolases in Caenorhabditis elegans that metabolize mammalian lipid signaling molecules

“…Several desaturases and elongases in the fatty acid synthetic pathway of C. elegans have mammalian counterparts with parallel activities. [22][23][24][25][26] The cytochrome p450 monooxygenases responsible for production of the EETs and EpOMEs are present in the genome, though few have been characterized. [27][28][29] Finally, C. elegans contain arachidonic acid and linoleic acid, the precursors of these signaling molecules.…”

Caenorhabditis elegans mboa-7, a Member of the MBOAT Family, Is Required for Selective Incorporation of Polyunsaturated Fatty Acids into Phosphatidylinositol

“…fat-4 fat-1 mutants, which do not produce AA(20:4n-6) and n-3 PUFAs such as EPA(20:5n-3), were viable and fertile ( Figure 1A; Watts and Browse, 2002;Kahn-Kirby et al, 2004), but they exhibited slow growth at 15°C ( Figure 1B). Dietary supplementation with 25 M AA or EPA led to incorporation of these PUFAs into the phospholipid fractions of fat-4 fat-1 mutants ( Figure 1C), and it rescued the growth defects of this strain ( Figure 1B).…”

“…Unlike mammals, C. elegans possesses all the required fatty acid biosynthetic enzymes, and it has no dietary requirements for essential fatty acids (Napier and Michaelson, 2001;Wallis et al, 2002). Moreover, a series of mutants have been isolated that are unable to synthesize specific PUFAs due to mutations in genes encoding fatty acid desaturases (fat genes, Watts and Browse, 2002). C. elegans membrane fatty acids can be manipulated by dietary supplementation with fatty acids Watts and Browse, 2006).…”

“…C. elegans membrane fatty acids can be manipulated by dietary supplementation with fatty acids Watts and Browse, 2006). In contrast to mammals in which AA is a major PUFA in membrane phospholipids and eicosapentaenoic acid (EPA) is a minor component, C. elegans possesses abundant EPA but not AA (Satouchi et al, 1993;Wallis et al, 2002;Watts and Browse, 2002). The major PUFA attached to PI in wildtype worms is EPA and not AA.…”

CYP-13A12 of the nematode Caenorhabditis elegans is a PUFA-epoxygenase involved in behavioural response to reoxygenation

“…We cannot exclude the possibility that other EPA-and also AA-derived metabolites share the locomotion-promoting capacity of 17,18-EEQ. In particular, AA-derived metabolites such as 14,15-EET may take over the function of 17,18-EEQ, if EPA is not available as suggested by the normal locomotion behaviour of fat-1 mutants that are unable to synthesise n−3 PUFAs [49]. Moreover, considering that the substrate and reaction specificities of CYP-13A12 and CYP-33E2 largely overlap, further genetic analysis is required to dissect the individual roles of these enzymes in the O2-ON response.…”