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Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.

ABSTRACT: Glycerophospholipids are structural and functional components of cellular membranes as well as precursors of various lipid mediators. Using acyl-CoAs as donors, glycerophospholipids are formed by the de novo pathway (Kennedy pathway) and modified in the remodeling pathway (Lands' cycle). Various acyltransferases, including two lysophosphatidic acid acyltransferases (LPAATs), have been discovered from a 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. Proteins of this family contain putative acyltransferase motifs, but their biochemical properties and physiological roles are not completely understood. Here, we demonstrated that mouse LPAAT3, previously known as mouse AGPAT3, possesses strong LPAAT activity and modest lysophosphatidylinositol acyltransferase activity with a clear preference for arachidonoyl-CoA as a donor. This enzyme is highly expressed in the testis, where CDP-diacylglycerol synthase 1 preferring 1-stearoyl-2-arachidonoyl-phosphatidic acid as a substrate is also highly expressed. Since 1-stearoyl-2-arachidonoyl species are the main components of phosphatidylinositol, mouse LPAAT3 may function in both the de novo and remodeling pathways and contribute to effective biogenesis of 1-stearoyl-2-arachidonoyl-phosphatidylinositol in the testis. Additionally, the expression of this enzyme in the testis increases significantly in an age-dependent manner, and beta-estradiol may be an important regulator of this enzyme's induction. Our findings identify this acyltransferase as an alternative important enzyme to produce phosphatidylinositol in the testis.

SUBMITTER: Yuki K

PROVIDER: S-EPMC2666172 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.

Yuki Koichi K Shindou Hideo H Hishikawa Daisuke D Shimizu Takao T

Journal of lipid research 20081229 5

Glycerophospholipids are structural and functional components of cellular membranes as well as precursors of various lipid mediators. Using acyl-CoAs as donors, glycerophospholipids are formed by the de novo pathway (Kennedy pathway) and modified in the remodeling pathway (Lands' cycle). Various acyltransferases, including two lysophosphatidic acid acyltransferases (LPAATs), have been discovered from a 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. Proteins of this family contain p ...[more]

PMID: 19114731

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Project description:Lipid-modifying enzymes serve crucial roles in cellular processes such as signal transduction (producing lipid-derived second messengers), intracellular membrane transport (facilitating membrane remodeling needed for membrane fusion/fission), and protein clustering (organizing lipid domains as anchoring platforms). The lipid products crucial in these processes can derive from different metabolic pathways, thus it is essential to know the localization, substrate specificity, deriving products (and their function) of all lipid-modifying enzymes. Here we discuss an emerging family of these enzymes, the lysophosphatidic acid acyltransferases (LPAATs), also known as acylglycerophosphate acyltransferases (AGPATs), that produce phosphatidic acid (PA) having as substrates lysophosphatidic acid (LPA) and acyl-CoA. Eleven LPAAT/AGPAT enzymes have been identified in mice and humans based on sequence homologies, and their localization, specific substrates and functions explored. We focus on one member of the family, LPAATδ, a protein expressed mainly in brain and in muscle (though to a lesser extent in other tissues); while at the cellular level it is localized at the trans-Golgi network membranes and at the mitochondrial outer membranes. LPAATδ is a physiologically essential enzyme since mice knocked-out for Lpaatδ show severe dysfunctions including cognitive impairment, impaired force contractility and altered white adipose tissue. The LPAATδ physiological roles are related to the formation of its product PA. PA is a multifunctional lipid involved in cell signaling as well as in membrane remodeling. In particular, the LPAATδ-catalyzed conversion of LPA (inverted-cone-shaped lipid) to PA (cone-shaped lipid) is considered a mechanism of deformation of the bilayer that favors membrane fission. Indeed, LPAATδ is an essential component of the fission-inducing machinery driven by the protein BARS. In this process, a protein-tripartite complex (BARS/14-3-3γ/phosphoinositide kinase PI4KIIIβ) is recruited at the trans-Golgi network, at the sites where membrane fission is to occur; there, LPAATδ directly interacts with BARS and is activated by BARS. The resulting formation of PA is essential for membrane fission occurring at those spots. Also in mitochondria PA formation has been related to fusion/fission events. Since PA is formed by various enzymatic pathways in different cell compartments, the BARS-LPAATδ interaction indicates the relevance of lipid-modifying enzymes acting exactly where their products are needed (i.e., PA at the Golgi membranes).

Project description:Metabolic engineering of the omega-3 (ω3) long chain polyunsaturated fatty acid biosynthesis pathway has generated fish oil-like levels of pharmaceutically and nutritionally important docosahexaenoic acid (DHA) in plant seeds. However, the majority of DHA has been accumulated at the sn-1 and sn-3 positions of triacylglycerol (TAG) in these engineered seeds, leaving only a minor amount (∼10%) at sn-2 position and indicating a strong discrimination (or, a very poor specificity) for DHA by seed lysophosphatidic acid acyltransferases (LPAATs), which mediate the acylation of sn-2 position of glycerol backbone. In order to increase the level of DHA at sn-2 position of TAG and to increase overall DHA level in seeds, we attempted to discover DHA-preferring LPAATs. Several LPAATs for acylation of the sn-2 position of the TAG glycerol backbone were investigated for substrate preference for DHA. In transiently expressing these LPAATs in Nicotiana benthamiana, a Mortierella alpina LPAAT had the highest substrate specificity for accumulating DHA onto oleoyl-lysophosphatidic acid (oleoyl-LPA), while the plant LPAATs tested showed lower preference for DHA. In a competition assay with a pool of four ω3 acyl-Coenzyme A (CoA) substrates involved in the DHA biosynthesis pathway, LPAATs from both M. alpina and Emiliania huxleyi showed a high preference for DHA-CoA acylation onto oleoyl-LPA. When docosahexaenoyl-LPA was used as the acyl receiver, M. alpina LPAAT also showed a high preference for DHA-CoA. Stable overexpression of M. alpina LPAAT in an Arabidopsis line that expressed the DHA biosynthesis pathway significantly increased both the total DHA levels and the distribution of DHA onto the sn-2 position of seed TAG. LC-MS analysis of the seed TAG species also confirmed that overexpression of M. alpina LPAAT increased di-DHA and tri-DHA TAGs, suggesting that the M. alpina LPAAT could enrich DHA at the TAG sn-2 position, leading to a metabolic engineering of oil seed for channeling DHA into the sn-2 position of TAG and to a higher DHA level.

Project description:BackgroundLysophosphatidic acid acyltransferase (LPAAT) is a phospholipid biosynthesis enzyme that introduces a particular set of fatty acids at the sn-2 position of phospholipids. Many bacteria have multiple LPAAT paralogs, and these enzymes are considered to have different fatty acid selectivities and to produce diverse phospholipids with distinct fatty acid compositions. This feature is advantageous for controlling the physicochemical properties of lipid membranes to maintain membrane integrity in response to the environment. However, it remains unclear how LPAAT paralogs are functionally differentiated and biologically significant.ResultsTo better understand the division of roles of the LPAAT paralogs, we analyzed the functions of two LPAAT paralogs, PlsC4 and PlsC5, from the psychrotrophic bacterium Shewanella livingstonensis Ac10. As for their enzymatic function, lipid analysis of plsC4- and plsC5-inactivated mutants revealed that PlsC4 prefers iso-tridecanoic acid (C12-chain length, methyl-branched), whereas PlsC5 prefers palmitoleic acid (C16-chain length, monounsaturated). Regarding the physiological role, we found that plsC4, not plsC5, contributes to tolerance to cold stress. Using bioinformatics analysis, we demonstrated that orthologs of PlsC4/PlsC5 and their close relatives, constituting a new clade of LPAATs, are present in many γ-proteobacteria. We also found that LPAATs of this clade are phylogenetically distant from principal LPAATs, such as PlsC1 of S. livingstonensis Ac10, which are universally conserved among bacteria, suggesting the presence of functionally differentiated LPAATs in these bacteria.ConclusionsPlsC4 and PlsC5, which are LPAAT paralogs of S. livingstonensis Ac10, play different roles in phospholipid production and bacterial physiology. An enzyme belonging to PlsC4/PlsC5 subfamilies and their close relatives are present, in addition to principal LPAATs, in many γ-proteobacteria, suggesting that the division of roles is more common than previously thought. Thus, both principal LPAATs and PlsC4/PlsC5-related enzymes should be considered to decipher the metabolism and physiology of bacterial cell membranes.

Project description:BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels. Nevertheless, products derived from microalgal TAG are not yet economically feasible; increasing TAG content via targeted genetic engineering of genes in TAG biosynthesis pathway are important to achieve economic viability. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with the endogenous enzyme lysophosphatidic acid acyltransferase (NeoLPAAT1) responsible for plastidial TAG biosynthesis RESULTS: NeoLPAAT1 was found to contain all canonical motifs attributed to LPAAT proteins, two hypothetical membrane-spanning domains and a putative chloroplast transit peptide, indicating as a member of plastidial LPAAT type 1 subfamily. The NeoLPAAT1-expression cassette integrated in N. oleoabundans transformant was confirmed by PCR. The neutral lipid content in the transformant detected by Nile red staining was 1.6-fold higher than in wild type. The NeoLPAAT1 transcript was twofold higher in the transformant than wild type. Considerably higher lipid quantity was found in the transformant than wild type: total lipid content increased 1.8- to 1.9-fold up to 78.99 ± 1.75% dry cell weight (DCW) and total lipid productivity increased 1.8- to 2.4-fold up to 16.06 ± 2.68 mg/L/day; while TAG content increased 2.1- to 2.2-fold up to 55.40 ± 5.56% DCW and TAG productivity increased 1.9- to 2.8-fold up to 10.67 ± 2.37 mg/L/day. A slightly altered fatty acid composition was detected in the transformant compared to wild type; polyunsaturated fatty acid (C18:2) increased to 19% from 11%. NeoLPAAT1-overexpression stability was observed in the transformant continuously maintained in solid medium over 150 generations in a period of about 6 years.ConclusionsOur results demonstrate the considerably increased TAG content and productivity in N. oleoabundans by overexpression of plastidial NeoLPAAT1 that are important for products derived from microalgal TAG to achieve economic viability. Plastidial LPAAT1 can be a candidate for target genetic manipulation to increase TAG content in other microalgal species with desired characteristics for production of natural edible oils and biofuels.

Dataset Information

Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.

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Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.

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