Inhibition of glycerol-3-phosphate acyltransferase by analogs of glycerol-3-phosphate.

Analogs of glycerol-3-phosphate were tested as substrates or inhibitors of the glycerol-3-phosphate acyltransferases of mitochondria and microsomes. (rac)-3,4-Dihydroxybutyl-1-phosphonate, (rac)-glyceraldehyde 3-phosphate, (rac)-3-hydroxy-4-oxobutyl-1-phosphonate, (1S,3S)-1,3,4-trihydroxybutyl-1-phosphonate, and (1R,3S)-1,3,4 trihydroxybutyl-1-phosphonate were competitive inhibitors of both mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferase activity. An isosteric analog of dihydroxyacetone phosphate, 4-hydroxy-3-oxobutyl-1-phosphonate, was a much stronger competitive inhibitor of the microsomal than the mitochondrial enzyme. Phenethyl alcohol was a noncompetitive inhibitor of both the microsomal and the mitochondrial acyltransferases. The product of the mitochondrial acyltransferase reaction with (rac)-3,4-dihydroxybutyl-1- phosphonate was almost exclusively (rac)-4-palmitoyloxy-3-hydroxybutyl-1-phosphonate. The microsomal acylation reaction generated both the monoacyl product and (S)-3,4-dipalmitoyloxybutyl-1-phosphonate. The apparent Km for (S)-3,4-dihydroxybutyl-1-phosphonate was 2.50 and 1.38 mM for the mitochondrial and microsomal enzymes, respectively.     

The sequence of a human mitochondrial glycerol-3-phosphate dehydrogenase-encoding cDNA

A 2618-bp cDNA that encodes the human mitochondrial glycerol-3-phosphate dehydrogenase has been isolated from a HeLa cell cDNA library and the nucleotide sequence determined. An open reading frame encodes a protein of 727 amino acids that is 96% similar to the rat protein and, like the rat protein, contains sites homologous to the Ca²⁺ -binding sites of calmodulin, as well as FAD- and putative glycerol-phosphate-binding sites.     

Membrane topology of murine glycerol-3-phosphate acyltransferase 2

Glycerol-3-phosphate acyltransferase (GPAT) is a rate-limiting enzyme in mammalian triacylglycerol biosynthesis. GPAT is a target for the treatment of metabolic disorders associated with high lipid accumulation. Although the molecular basis for GPAT1 activation has been investigated extensively, the activation of other isoforms, such as GPAT2, is less well understood. Here the membrane topology of the GPAT2 protein was examined using an epitope-tag-based method. Exogenously expressed GPAT2 protein was present in the membrane fraction of transformed HEK293 cells even in the presence of Na(2)CO(3) (100 mM), indicating that GPAT2 is a membrane-bound protein. Trypsin treatment of the membrane fraction degraded the N-terminal (FLAG) and C-terminal (myc-epitope) protein tags of the GPAT2 protein. Bioinformatic analysis of the GPAT2 protein sequence indicated four hydrophobic sequences as potential membrane-spanning regions (TM1-TM4). Immunoblotting of the myc-epitope tag, which was inserted between each TM region of the GPAT2 protein, showed that the amino acid sequence between TM3 and TM4 was protected from trypsin digestion. These results suggest that the GPAT2 protein has two transmembrane segments and that the N-terminal and C-terminal regions of this protein face the cytoplasm. These results also suggest that the enzymatically active motifs I-III of the GPAT2 protein face the cytosol, while motif IV is within the membrane. It is expected that the use of this topological model of GPAT2 will be essential in efforts to elucidate the molecular mechanisms of GPAT2 activity in mammalian cells.     

Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity

Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28°C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.     

AGPAT6 is a novel microsomal glycerol-3-phosphate acyltransferase

AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [(13)C(7)]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wild-type mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT, and we propose renaming this enzyme GPAT4.     

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

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 88.8kDa 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 microM; 0.25 microCi) [1-(14)C]oleate for 6h increased incorporation of [(14)C]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 approximately 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.     

Aging and acyl-CoA binding protein alter mitochondrial glycerol-3-phosphate acyltransferase activity

It is well known that cellular function declines with age. Since phosphatidic acid (PtdOH) biosynthesis is central to the generation of membrane phospholipids, the hypothesis that aging decreases PtdOH biosynthesis was tested. Glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LAT) activities were examined in isolated mitochondria and microsomes from young and old rat liver. The results show that mitochondrial GPAT preference for palmitoyl-CoA over oleoyl-CoA was only observed if albumin or acyl-CoA binding protein (ACBP) were present in the assay in the young rats. Furthermore, mitochondrial GPAT activity was significantly reduced in the presence of albumin and ACBP in aged mitochondria using palmitoyl-CoA as the substrate. These data show, for the first time, that mitochondrial GPAT acyl-CoA preference is due to the presence of a protein that binds acyl-CoAs, not the enzyme itself, and that aging significantly reduces mitochondrial GPAT activity.     

Cloning and nucleotide sequence of placental hGH-V cDNA

We have previously demonstrated the presence in human placenta and maternal serum of a GH variant, called human placental growth hormone (hPGH). We have also shown that the hGH-V gene is expressed at the placental level thus possibly coding for hPGH. The hGH-V cDNA has now been isolated from a lambda gt 11 human placenta cDNA library. Its sequence has been determined which firmly establishes the GH-V gene mode of splicing as well as the GH-V protein structure. Our data give final evidence of placental hGH-V gene expression and reinforce the hypothesis of identity between the hGH-V protein and hPGH.     

Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato

A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.     

Cloning and nucleotide sequence of ovine prolactin cDNA

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.     

Identification and functional characterization of human glycerol-3-phosphate acyltransferase 1 gene promoters

Extracellular calcium is crucial for functioning of the epithelial barrier. Compounds that bind calcium, reducing its extracellular levels, have therefore been investigated as mucosal absorption enhancers. However, the conditions under which calcium reduction sufficiently modulates the epithelial barrier to result in meaningful improvements in mucosal drug absorption are unclear. Present work investigated the settings in which calcium depletion leads to optimal epithelial barrier-modulating effects. Using Calu-3 and Caco-2 cell layers and inducing calcium depletion site-specifically (apically, basolaterally or on both sides) we demonstrate that apical calcium removal produces a modest effect on the tight junctions (the extent of the effect being dependent on the duration of apical calcium unavailability), whilst basolateral calcium exhaustion leads to a prominent effect on the epithelial barrier. However, using polyacrylic acid as an example, we show that polymeric calcium-binding agents proposed as mucosal absorption-enhancing excipients alter calcium levels exclusively on the apical side of the epithelium, which explains their modest effect on epithelial barrier modulation (also demonstrated in our work). Therefore the use of calcium-depleting agents, especially those based on macromolecular polymers, is a relatively inefficacious strategy to promote mucosal absorption of macromolecules.     

Aging reduces glycerol-3-phosphate acyltransferase activity in activated rat splenic T-lymphocytes

T-lymphocyte proliferation declines with age. Phosphatidic acid (PA) is the precursor to all glycerophospholipids, which serve as important membrane structural components and signaling molecules. Therefore, we tested the hypothesis that aged T-lymphocyte proliferation may be reduced, in part, suppressing phosphatidic acid (PA) biosynthesis. We showed, for the first time, that anti-CD3 stimulation in rat splenic T-lymphocytes selectively increased mitochondrial glycerol-3-phosphate acyltransferase (GPAT) activity. GPAT activity could be further increased by the addition of recombinant acyl-CoA binding protein (rACBP), but the amplification of GPAT activity was blunted by aging. This is important because PA is the precursor lipid for phospholipid synthesis and GPAT is the rate-limiting enzyme in PA biosynthesis. The mechanism by which stimulation and rACBP increased GPAT activity may involve phosphorylation since incubating Jurkat T-lymphocyte mitochondria with casein kinase 2 in vitro significantly increased GPAT activity. The data presented here suggest a novel mechanism by which aging may reduce activation-dependent mitochondrial GPAT activity. This age-induced alteration would result in reduced PA biosynthesis and could explain, in part, the diminished phospholipid content of the membrane and subsequent loss of proliferative capacity in the aged T-lymphocyte.     

Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity

In an attempt to rationalize the relationship between structure and substrate selectivity of glycerol-3-phosphate acyltransferase (GPAT, 1AT, EC 2.3.1.15) we have cloned a number of cDNAs into the pET overexpression system using a PCR-based approach. Following assay of the recombinant enzyme we noted that the substrate selectivity of the squash (Cucurbita moschata) enzyme had altered dramatically. This form of GPAT has now been crystallized and its full three-dimensional structure elucidated. Since we now have two forms of the enzyme that display different substrate selectivities this should provide a powerful tool to determine the basis of the selectivity changes. Kinetic and structural analyses are currently being performed to rationalize the changes which have taken place.     

Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes

Arabidopsis thaliana was transformed with a plastidial safflower glycerol-3-phosphate acyltransferase (GPAT) and an Escherichia coli GPAT. The genes were used directly and in modified forms with, as applicable, the plastidial targeting sequence removed, and with an endoplasmic reticulum targeting sequence added. Seeds of plants transformed using only the vector were indistinguishable in oil content from wild-type control plants. All other gene constructs increased seed oil content. The unmodified safflower gene (spgpat) produced oil increases ranging from 10 to 21%. On average, the greatest increase (+22%) was observed in seeds of transformants carrying the spgpat with the targeting peptide removed. The E. coli plsB gene increased seed oil content by an average of 15%.