The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform

Rosalind A. Coleman - One of the best experts on this subject based on the ideXlab platform.

  • glycerol 3 phosphate Acyltransferase isoform 4 gpat4 limits oxidation of exogenous fatty acids in brown adipocytes
    Journal of Biological Chemistry, 2015
    Co-Authors: Daniel E Cooper, Trisha J. Grevengoed, Eric L. Klett, Rosalind A. Coleman
    Abstract:

    Glycerol-3-Phosphate Acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4(-/-) mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4(-/-) mice fed a 45% fat diet was 12% higher. Core body temperature was 1 oC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4(-/-) mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4(-/-) BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4(-/-) brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.

  • Glycerol-3-Phosphate Acyltransferase-4-deficient mice are protected from diet-induced insulin resistance by the enhanced association of mTOR and rictor
    American journal of physiology. Endocrinology and metabolism, 2014
    Co-Authors: Chongben Zhang, Daniel E Cooper, Trisha J. Grevengoed, Eric L. Klett, James M. Eaton, Thurl E. Harris, Rosalind A. Coleman
    Abstract:

    Glycerol-3-Phosphate Acyltransferase (GPAT) activity is highly induced in obese individuals with insulin resistance, suggesting a correlation between GPAT function, triacylglycerol accumulation, an...

  • Mice deficient in Glycerol-3-Phosphate Acyltransferase-1 have a reduced susceptibility to liver cancer.
    Toxicologic pathology, 2012
    Co-Authors: Jessica M. Ellis, David S. Paul, Michael A. Depetrillo, Bhanu Singh, David E. Malarkey, Rosalind A. Coleman
    Abstract:

    The risk of hepatocellular carcinoma increases with the persistence of non-alcoholic fatty liver disease. Triacylglycerol synthesis is initiated by Glycerol-3-Phosphate Acyltransferase (GPAT). Of four isoforms, GPAT1 contributes 30–50% of total liver GPAT activity, and we hypothesized that it might influence liver susceptibility to tumorigenesis. C57Bl/6 mice deficient in GPAT1 were backcrossed 6 times to C3H mice. After exposure to the carcinogen diethylnitrosamine (DEN) and the tumor promoter phenobarbital, male Gpat1-/- mice, compared with controls (Gpat1+/+), had 93% fewer macroscopically visible nodules per liver at 21 weeks of age and 39% fewer at 34 weeks of age. Microscopically, control mice had increased numbers of foci of altered hepatocytes, particularly the basophilic subtype, as well as more, and malignant, liver neoplasms than did the Gpat1-/- mice. At 21 weeks of age, 50% (4/8) of control mice (50%) had hepatocellular adenomas with an average multiplicity (tumors per tumor-bearing-animal) o...

  • cloning and functional characterization of a novel mitochondrial n ethylmaleimide sensitive glycerol 3 phosphate Acyltransferase gpat2
    Archives of Biochemistry and Biophysics, 2007
    Co-Authors: Shuli Wang, Tal M. Lewin, Nicole M J Schwerbrock, Douglas P Lee, Nan Gong, Douglas G Mashek, Maria R Gonzalezbaro, Cliona Stapleton, Rosalind A. Coleman
    Abstract:

    Abstract Glycerol-3-Phosphate Acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open-reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8 kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 μM; 0.25 μCi) [1-14C]oleate for 6 h increased incorporation of [14C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an ∼89-kDa band in liver and testis from GPAT1 null mice and both 89- and 80-kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.

  • identification of a new glycerol 3 phosphate Acyltransferase isoenzyme mtgpat2 in mitochondria
    Journal of Biological Chemistry, 2004
    Co-Authors: Tal M. Lewin, Nicole M J Schwerbrock, Douglas P Lee, Rosalind A. Coleman
    Abstract:

    Abstract Glycerol-3-Phosphate Acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished, because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT-/- mice, PCR genotyping and Northern analysis showed successful knockout of mtGPAT; however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT-/- liver mitochondria have different isoelectric points. These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT-/- mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtGPAT activity in wild type samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by dihydroxyacetone phosphate and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT-/- mice.

Antoni R. Slabas - One of the best experts on this subject based on the ideXlab platform.

  • Kinetic mechanism and order of substrate binding for sn-Glycerol-3-Phosphate Acyltransferase from squash (Cucurbita moschata).
    FEBS letters, 2002
    Co-Authors: Matthew William Hayman, Tony Fawcett, Antoni R. Slabas
    Abstract:

    sn-Glycerol-3-Phosphate Acyltransferase (G3PAT, EC 2.3.1.15), a component of glycerolipid biosynthesis, is an important enzyme in chilling sensitivity in plants. The three-dimensional structure of the enzyme from squash (Cucurbita moschata), without bound substrate, has been determined [Turnbull et al. (2001) Acta Crystallogr. D 57, 451-453; Turnbull et al. (2001) Structure 9, 347-353]. Here we report the kinetic mechanism of plastidial G3PAT from squash and the order of substrate binding using acyl-acyl carrier protein (acyl-ACP) substrates. The reaction proceeds via a compulsory-ordered ternary complex with acyl-ACP binding before Glycerol-3-Phosphate. We have also determined that the reaction will proceed with C(4:0)-CoA, C(6:0)-CoA and C(12:0)-ACP substrates, allowing a wider choice of acyl groups for future co-crystallisation studies.

  • Engineering Trierucin into Oilseed Rape by the Introduction of a 1-Acyl-sn-Glycerol-3-Phosphate Acyltransferase from Limnanthes Douglasii
    Physiology Biochemistry and Molecular Biology of Plant Lipids, 1997
    Co-Authors: Clare L. Brough, Johan T.m. Kroon, Jane M. Coventry, William W. Christie, Tina L. Barsby, Antoni R. Slabas
    Abstract:

    Currently available cultivars of high erucic acid rape (HEAR) have a theoretical maximum of 66% erucic acid (22:1, Δ13) in their seed oil due to the specificity of the membrane-bound 1-acyl-sn-Glycerol-3-Phosphate Acyltransferase (LPA-AT) enzyme. In HEAR the LPA-AT does not incorporate erucic acid at the s n-2 position of triacylglycerols (TAG) but preferentially incorporates oleic acid (18:1, Δ9), even if this is only a minor component of the total fatty acid pool (1). However, some plant species, e.g Limnanthes, can utilise erucoyl-CoA as a substrate and effectively incorporate erucic acid at the sn-2 position (2, 3).

  • towards the genetic engineering of triacylglycerols of defined fatty acid composition major changes in erucic acid content at the sn 2 position affected by the introduction of a 1 acyl sn glycerol 3 phosphate Acyltransferase from limnanthes douglasii
    Molecular Breeding, 1996
    Co-Authors: Clare L. Brough, Adrian P. Brown, Johan T.m. Kroon, Jane M. Coventry, William W. Christie, Tina L. Barsby, Antoni R. Slabas
    Abstract:

    A cDNA encoding a 1-acyl-sn-Glycerol-3-Phosphate Acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-Glycerol-3-Phosphate Acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.

  • Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-Glycerol-3-Phosphate Acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other Acyltransferases.
    Plant molecular biology, 1994
    Co-Authors: Adrian P. Brown, Jack Coleman, Andrew M. Tommey, Martin D. Watson, Antoni R. Slabas
    Abstract:

    We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-Glycerol-3-Phosphate Acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44°C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-Glycerol-3-Phosphate Acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in Acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42 543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-Glycerol-3-Phosphate Acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.

Tal M. Lewin - One of the best experts on this subject based on the ideXlab platform.

  • cloning and functional characterization of a novel mitochondrial n ethylmaleimide sensitive glycerol 3 phosphate Acyltransferase gpat2
    Archives of Biochemistry and Biophysics, 2007
    Co-Authors: Shuli Wang, Tal M. Lewin, Nicole M J Schwerbrock, Douglas P Lee, Nan Gong, Douglas G Mashek, Maria R Gonzalezbaro, Cliona Stapleton, Rosalind A. Coleman
    Abstract:

    Abstract Glycerol-3-Phosphate Acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open-reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8 kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 μM; 0.25 μCi) [1-14C]oleate for 6 h increased incorporation of [14C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an ∼89-kDa band in liver and testis from GPAT1 null mice and both 89- and 80-kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.

  • identification of a new glycerol 3 phosphate Acyltransferase isoenzyme mtgpat2 in mitochondria
    Journal of Biological Chemistry, 2004
    Co-Authors: Tal M. Lewin, Nicole M J Schwerbrock, Douglas P Lee, Rosalind A. Coleman
    Abstract:

    Abstract Glycerol-3-Phosphate Acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished, because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT-/- mice, PCR genotyping and Northern analysis showed successful knockout of mtGPAT; however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT-/- liver mitochondria have different isoelectric points. These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT-/- mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtGPAT activity in wild type samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by dihydroxyacetone phosphate and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT-/- mice.

  • Rat sn-Glycerol-3-Phosphate Acyltransferase: Molecular cloning and characterization of the cDNA and expressed protein
    Biochimica et biophysica acta, 1999
    Co-Authors: B. Ganesh Bhat, Ping Wang, Ji Hyeon Kim, Tracy M. Black, Tal M. Lewin, Frederick T. Fiedorek, Rosalind A. Coleman
    Abstract:

    Rat mitochondrial Glycerol-3-Phosphate Acyltransferase (GPAT) cDNA was cloned and characterized. We identified a cDNA containing an open reading frame of 828 amino acids that had an 89% homology with the coding region of the previously characterized mouse mitochondrial GPAT and a predicted amino acid sequence that was 96% identical. The rat 5' UTR was only 159 nucleotides, in contrast to the 926 nucleotide 5' UTR of the mouse cDNA and had an internal deletion of 167 nucleotides. GPAT was expressed in Sf21 insect cells, and specific inhibitors strongly suggest that, like the Escherichia coli GPAT, the recombinant mitochondrial GPAT and the mitochondrial GPAT isoform in rat liver contain critical serine, histidine, and arginine residues.

  • analysis of amino acid motifs diagnostic for the sn glycerol 3 phosphate Acyltransferase reaction
    Biochemistry, 1999
    Co-Authors: Tal M. Lewin, Ping Wang, Rosalind A. Coleman
    Abstract:

    Alignment of amino acid sequences from various Acyltransferases [sn-Glycerol-3-Phosphate Acyltransferase (GPAT), lysophosphatidic acid Acyltransferase (LPAAT), acyl-CoA:dihydroxyacetone-phosphate Acyltransferase (DHAPAT), 2-acylglycerophosphatidylethanolamine Acyltransferase (LPEAT)] reveals four regions of strong homology, which we have labeled blocks I−IV. The consensus sequence for each conserved region is as follows:  block I, [NX]-H-[RQ]-S-X-[LYIM]-D; block II, G-X-[IF]-F-I-[RD]-R; block III, F-[PLI]-E-G-[TG]-R-[SX]-[RX]; and block IV, [VI]-[PX]-[IVL]-[IV]-P-[VI]. We hypothesize that blocks I−IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of Acyltransferases. Using Escherichia coli GPAT (PlsB) as a model Acyltransferase, we examined the role of the highly conserved amino acid residues in blocks I−IV in GPAT activity through chemical modification and site-directed mutagenesis experiments. We found that the histidine an...

  • Short sequence-paper Rat sn-Glycerol-3-Phosphate Acyltransferase: molecular cloning and characterization of the cDNA and expressed protein
    1999
    Co-Authors: B. Ganesh Bhat, Ping Wang, Ji Hyeon Kim, Tracy M. Black, Tal M. Lewin, Frederick T. Fiedorek, Rosalind A. Coleman
    Abstract:

    Rat mitochondrial Glycerol-3-Phosphate Acyltransferase (GPAT) cDNA was cloned and characterized. We identified a cDNA containing an open reading frame of 828 amino acids that had an 89% homology with the coding region of the previously characterized mouse mitochondrial GPAT and a predicted amino acid sequence that was 96% identical. The rat 5P UTR was only 159 nucleotides, in contrast to the 926 nucleotide 5P UTR of the mouse cDNA and had an internal deletion of 167 nucleotides. GPAT was expressed in Sf21 insect cells, and specific inhibitors strongly suggest that, like the Escherichia coli GPAT, the recombinant mitochondrial GPAT and the mitochondrial GPAT isoform in rat liver contain critical serine, histidine, and arginine residues. fl 1999 Elsevier Science B.V. All rights reserved.

Choukri Ben Mamoun - One of the best experts on this subject based on the ideXlab platform.

Rachel Zufferey - One of the best experts on this subject based on the ideXlab platform.

  • The Glycerol-3-Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei.
    The Journal of eukaryotic microbiology, 2016
    Co-Authors: Nipul Patel, Karim A. Pirani, Tongtong Zhu, Melanie Cheung-see-kit, Sungsu Lee, Daniel G. Chen, Rachel Zufferey
    Abstract:

    Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the Glycerol-3-Phosphate Acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored Glycerol-3-Phosphate Acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl-CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N-terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked Glycerol-3-Phosphate Acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and GPI-anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial Acyltransferase takes over TbGAT's function in its absence.

  • the initial step of glycerolipid metabolism in leishmania major promastigotes involves a single glycerol 3 phosphate Acyltransferase enzyme important for the synthesis of triacylglycerol but not essential for virulence
    Molecular Microbiology, 2005
    Co-Authors: Rachel Zufferey, Choukri Ben Mamoun
    Abstract:

    : The synthesis of the major phospholipids, including those that play an essential role in Leishmania virulence, initiates with the acylation of Glycerol-3-Phosphate and dihydroxyacetonephosphate at the sn-1 position by Glycerol-3-Phosphate and dihydroxyacetonephosphate Acyltransferases respectively. In this study, we show that Leishmania major promastigotes express a single Glycerol-3-Phosphate Acyltransferase activity important for triacylglycerol synthesis but not essential for virulence. The encoding gene, LmGAT, expressed in yeast results in full complementation of the lethality of a mutant, gat1Deltagat2Delta, lacking Glycerol-3-Phosphate activity. Biochemical analyses revealed that LmGAT is a low-affinity Glycerol-3-Phosphate Acyltransferase and exhibits higher specific activity with unsaturated long fatty acyl-CoA donors. A L. major null mutant, Deltalmgat/Deltalmgat, was created and a thorough analysis of its lipid composition was performed. Deletion of LmGAT resulted in a complete loss of Leishmania Glycerol-3-Phosphate Acyltransferase activity and a major reduction in triacylglycerol synthesis. Consistent with the specificity of LmGAT for Glycerol-3-Phosphate but not dihydroxyacetonephosphate, Deltalmgat/Deltalmgat mutant expressed normal levels of the ether-lipid derivatives and virulence factors, lipophosphoglycan and GPI-anchored proteins, gp63, and its virulence was not affected in mice.

  • The initial step of glycerolipid metabolism in Leishmania major promastigotes involves a single glycerol‐3‐phosphate Acyltransferase enzyme important for the synthesis of triacylglycerol but not essential for virulence
    Molecular microbiology, 2005
    Co-Authors: Rachel Zufferey, Choukri Ben Mamoun
    Abstract:

    The synthesis of the major phospholipids, including those that play an essential role in Leishmania virulence, initiates with the acylation of Glycerol-3-Phosphate and dihydroxyacetonephosphate at the sn-1 position by Glycerol-3-Phosphate and dihydroxyacetonephosphate Acyltransferases respectively. In this study, we show that Leishmania major promastigotes express a single Glycerol-3-Phosphate Acyltransferase activity important for triacylglycerol synthesis but not essential for virulence. The encoding gene, LmGAT, expressed in yeast results in full complementation of the lethality of a mutant, gat1Deltagat2Delta, lacking Glycerol-3-Phosphate activity. Biochemical analyses revealed that LmGAT is a low-affinity Glycerol-3-Phosphate Acyltransferase and exhibits higher specific activity with unsaturated long fatty acyl-CoA donors. A L. major null mutant, Deltalmgat/Deltalmgat, was created and a thorough analysis of its lipid composition was performed. Deletion of LmGAT resulted in a complete loss of Leishmania Glycerol-3-Phosphate Acyltransferase activity and a major reduction in triacylglycerol synthesis. Consistent with the specificity of LmGAT for Glycerol-3-Phosphate but not dihydroxyacetonephosphate, Deltalmgat/Deltalmgat mutant expressed normal levels of the ether-lipid derivatives and virulence factors, lipophosphoglycan and GPI-anchored proteins, gp63, and its virulence was not affected in mice.