Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase

Thai, Thanh-Phuong; Heid, Hans; Rackwitz, Hans-Richard; Hunziker, Andreas; Gorgas, Karin; Just, Wilhelm W.

doi:10.1016/s0014-5793(97)01495-6

Cited by 59 publications

(56 citation statements)

References 35 publications

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“…The human DHAPAT cDNA (14) was removed from the pBluescript SK phagemid vector and inserted into the shuttle vector pBK-CMV (Stratagene, La Jolla, CA) using the Eco RI (5 Ј ) and Xho I (3 Ј ) restriction sites. The construct was used to transform XL1-Blue MRF Ј , which was then grown on kanamycin-containing agar to form colonies.…”

Section: Construction Of An Expression Vector Containing Human Dhapatmentioning

confidence: 99%

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Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids

Liu

Nagan

Just

et al. 2005

Journal of Lipid Research

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Section: Construction Of An Expression Vector Containing Human Dhapatmentioning

confidence: 99%

“…Our lack of knowledge in this area is attributable, in part, to the lack of molecular tools for studying the system. Only recently have any of the enzymes that participate in the biosynthetic pathway for plasmalogens been purified or the genes that code for them cloned (14,15).…”

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Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids

Liu

Nagan

Just

et al. 2005

Journal of Lipid Research

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“…Northern Blot Analysis of DHAPAT mRNA during Cell Differentiation-The human DHAPAT cDNA has been cloned (16,17) and a number of human and mouse EST clones are available. One EST clone (W54198) was used for Northern blot analysis of the DHAPAT mRNA in 3T3-L1 cells during differentiation.…”

Section: Differentiation-induced Increased Activity Of Lipogenic Enzymentioning

confidence: 99%

“…Novikoff and co-workers (4) reported that during differentiation of 3T3-L1 cells there is a large increase in the number of peroxisomes (catalase-containing organelles) and the amount of endoplasmic reticulum (ER), and in an abstract, they also reported that DHAPAT in differentiated 3T3-L1 cells was associated with peroxisomes (14). In light of our progress in purifying specific DHAPAT (15) and the recent cloning of its cDNA (16,17), we examined whether peroxisomal DHAPAT is induced during differentiation of these cells, along with the nonspecific microsomal GPAT, by studying the properties and subcellular distributions of the enzymes. The results indicate that peroxisomal DHAPAT and its mRNA are indeed induced during adipogenesis.…”

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confidence: 99%

Induction of the Peroxisomal Glycerolipid-synthesizing Enzymes during Differentiation of 3T3-L1 Adipocytes

Hajra¹,

Larkins²,

Das³

et al. 2000

Journal of Biological Chemistry

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The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using Preadipocyte cell lines (1) have been widely used to study the induction of a number of enzymes and receptors and also the mechanism of differentiation at the genetic level (2, 3). The fibroblast-like mouse 3T3-L1 cells differentiate in response to high concentrations of insulin and other agents to cells that synthesize large amounts of triglycerides and are morphologically similar to adipose cells (4). A similar cell line (3T3-F442A), on transplantation in mice has been reported to develop into adipose tissue (5). During differentiation, lipogenic enzymes of the triglyceride biosynthetic pathway, as well as the ancillary enzymes needed for the formation of substrates and cofactors are induced (6). As outlined in Fig. 1, enzymes starting from the cytosolic glycerophosphate dehydrogenase (GPD), 1 catalyzing the biosynthesis of sn-glycerol 3-phosphate (GP) followed by the glycerophosphate acyltransferase (GPAT), 1-acyl-GP acyltransferase, and diacylglycerol acyltransferase (DAGAT) are highly induced during differentiation (7-9). Coleman and Bell (10) reported that due to nonspecificity of microsomal GPAT, which catalyzes the acylation of both GP and DHAP, DHAPAT activity is also induced during differentiation of these cells. All animal cells, however, have been shown to contain a specific peroxisomal DHAPAT which does not contain GPAT activity (11,12). This DHAPAT, co-localized in peroxisomes with acyl/alkyl DHAP:NADPH reductase and alkyl-DHAP synthase, is generally believed to be involve...

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“…In most animal tissues, DHAPAT is found in a membrane-bound fraction (15,20) and localized in the luminal side of peroxisomes (15,21). This enzyme was also found to be part of a heterotrimeric complex that includes the 1-alkyl-DHAP synthase (22,23). Alterations in DHAPAT function have been associated with various human diseases such as neonatal adrenoleukodystrophy, infantile Refsum, disease, hyperpipecolic academia, and rhizomelic chondrodyplasia punctata (24 -26).…”

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Leishmania major Expresses a Single Dihydroxyacetone Phosphate Acyltransferase Localized in the Glycosome, Important for Rapid Growth and Survival at High Cell Density and Essential for Virulence

Zufferey

Mamoun

2006

Journal of Biological Chemistry

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Despite major advances in the understanding of pathogenesis of the human protozoan parasite Leishmania major, little is known about the enzymes and the primary precursors involved in the initial steps of synthesis of its major glycerolipids including those involved in virulence. We have previously demonstrated that the initial step of acylation of the precursor glycerol 3-phosphate is not essential for the synthesis of ester and ether phospholipids in this parasite. Here we show that Leishmania expresses a single acyltransferase with high specificity for the precursor dihydroxyacetone phosphate and shows the best activity in the presence of palmitoylCoA. We have identified and characterized the LmDAT gene encoding this activity. LmDAT complements the lethality resulting from the loss of both dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferase activities in yeast. Recombinant LmDAT exhibits biochemical properties similar to those of the native enzyme of the promastigote stage parasites. We show that LmDAT is a glycosomal enzyme and its loss in a ⌬lmdat/⌬lmdat null mutant results in complete abrogation of the parasite dihydroxyacetone phosphate acyltransferase activity. Furthermore, lack of LmDAT causes a major alteration in parasite division during the logarithmic phase of growth, an accelerated cell death during stationary phase, and loss of virulence. Together, our results demonstrate that LmDAT is the only dihydroxyacetone phosphate acyltransferase of the L. major localized in the peroxisome, important for growth and survival and essential for virulence.Worldwide, protozoan parasites of the genus Leishmania cause a large spectrum of important human diseases collectively named leishmaniasis. These parasites develop within the digestive tract of the sand fly vector as flagellated, mobile promastigotes and differentiate into and multiply as non-motile amastigotes within the phagolysosomal compartment of vertebrate host macrophages.Glycerolipids constitute 70% of total lipids in the protozoan parasite Leishmania (1-3). They are classified into ester and ether lipids depending on the substitution at position 1 of the glycerol backbone. Ester lipids harbor an acyl group, whereas ether lipids carry a fatty alcohol moiety. Lipids of Leishmania parasites have been a focus of extensive studies because some of their derivatives, such as lipophosphoglycan and glycosylphosphatidylinositol-anchored protease gp63, were shown to be important for parasite virulence and development (for review, see . Lipids are also essential cell constituents and, therefore, must be constantly synthesized to allow multiplication of the parasite. This suggests that the pathways leading to their synthesis are essential for parasite proliferation and pathogenesis and, thus, offer a reasonable target for rational design of new anti-leishmanial drugs. In fact, a lipidbased drug, miltefosine, is a potent antileishmanial compound that inhibits parasite growth in vitro and in vivo and is currently used for treatment of visceral and muc...

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Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase

Cited by 59 publications

References 35 publications

Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids

Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids

Induction of the Peroxisomal Glycerolipid-synthesizing Enzymes during Differentiation of 3T3-L1 Adipocytes

Leishmania major Expresses a Single Dihydroxyacetone Phosphate Acyltransferase Localized in the Glycosome, Important for Rapid Growth and Survival at High Cell Density and Essential for Virulence

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