Publications

• Enzymatic activities of the human AGPAT isoform 3 and isoform 5: localization of AGPAT5 to mitochondria.

  Prasad S.S., Garg A., Agarwal A.K.

  The enzyme 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT) converts lysophosphatidic acid (LPA) to phosphatidic acid (PA). In this study, we show enzymatic properties, tissue distribution, and subcellular localization of human AGPAT3 and AGPAT5. In cells overexpressing these isoforms, the pro ... >> More

  The enzyme 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT) converts lysophosphatidic acid (LPA) to phosphatidic acid (PA). In this study, we show enzymatic properties, tissue distribution, and subcellular localization of human AGPAT3 and AGPAT5. In cells overexpressing these isoforms, the proteins were detected in the nuclear envelope and the endoplasmic reticulum. AGPAT5-GFP fusion protein was localized in the mitochondria of both Chinese hamster ovary and human epithelial cervical cancer cells. Using lysates of AD293 cells infected with AGPAT3 and AGPAT5 recombinant adenovirus, we show that AGPAT3 and AGPAT5 proteins have AGPAT activity. Both the isoforms have similar apparent V(max) of 6.35 and 2.42 nmol/min/mg protein, respectively, for similar LPA. The difference between the two isoforms is in their use of additional lysophospholipids. AGPAT3 shows significant esterification of lysophosphatidylinositol (LPI) in the presence of C20:4 fatty acid, whereas AGPAT5 demonstrates significant acyltransferase activity toward lysophosphatidylethanolamine (LPE) in the presence of C18:1 fatty acid. The AGPAT3 mRNA is ubiquitously expressed in human tissues with several-fold differences in the expression pattern compared with the closely related AGPAT4. In summary, we show that in the presence of different fatty acids, AGPAT3 and AGPAT5 prefer different lysophospholipids as acyl acceptors. More importantly, localization of overexpressed AGPAT5 (this study) as well as GPAT1 and 2 (previous studies) in mitochondria supports the idea that the mitochondria might be capable of synthesizing some of their own glycerophospholipids. << Less

  J. Lipid Res. 52:451-462(2011) [PubMed] [EuropePMC]

  This publication is cited by 21 other entries.

• Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils.

  Gijon M.A., Riekhof W.R., Zarini S., Murphy R.C., Voelker D.R.

  The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed ... >> More

  The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils. << Less

  J. Biol. Chem. 283:30235-30245(2008) [PubMed] [EuropePMC]

  This publication is cited by 26 other entries.

• Human 1-acylglycerol-3-phosphate O-acyltransferase isoforms 1 and 2: biochemical characterization and inability to rescue hepatic steatosis in Agpat2(-/-) gene lipodystrophic mice.

  Agarwal A.K., Sukumaran S., Cortes V.A., Tunison K., Mizrachi D., Sankella S., Gerard R.D., Horton J.D., Garg A.

  Loss-of-function mutations in 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 in humans and mice result in loss of both the white and brown adipose tissues from birth. AGPAT2 generates precursors for the synthesis of glycerophospholipids and triacylglycerols. Loss of adipose tissue, or lipo ... >> More

  Loss-of-function mutations in 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 in humans and mice result in loss of both the white and brown adipose tissues from birth. AGPAT2 generates precursors for the synthesis of glycerophospholipids and triacylglycerols. Loss of adipose tissue, or lipodystrophy, results in hyperinsulinemia, diabetes mellitus, and severe hepatic steatosis. Here, we analyzed biochemical properties of human AGPAT2 and its close homolog, AGPAT1, and we studied their role in liver by transducing their expression via recombinant adenoviruses in Agpat2(-/-) mice. The in vitro substrate specificities of AGPAT1 and AGPAT2 are quite similar for lysophosphatidic acid and acyl-CoA. Protein homology modeling of both the AGPATs with glycerol-3-phosphate acyltransferase 1 (GPAT1) revealed that they have similar tertiary protein structure, which is consistent with their similar substrate specificities. When co-expressed, both isoforms co-localize to the endoplasmic reticulum. Despite such similarities, restoring AGPAT activity in liver by overexpression of either AGPAT1 or AGPAT2 in Agpat2(-/-) mice failed to ameliorate the hepatic steatosis. From these studies, we suggest that the role of AGPAT1 or AGPAT2 in liver lipogenesis is minimal and that accumulation of liver fat is primarily a consequence of insulin resistance and loss of adipose tissue in Agpat2(-/-) mice. << Less

  J. Biol. Chem. 286:37676-37691(2011) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

• Functional characterization of human 1-acylglycerol-3-phosphate acyltransferase isoform 8: cloning, tissue distribution, gene structure, and enzymatic activity.

  Agarwal A.K., Barnes R.I., Garg A.

  Glycerophospholipids and triglycerides are synthesized de novo by cells through an evolutionary conserved process involving serial acylations of phosphorylated glycerol. Various isoforms of the enzyme, 1-acylglycerol-3-phosphate acyltransferase (AGPAT), acylate lysophosphatidic acid at the sn-2 po ... >> More

  Glycerophospholipids and triglycerides are synthesized de novo by cells through an evolutionary conserved process involving serial acylations of phosphorylated glycerol. Various isoforms of the enzyme, 1-acylglycerol-3-phosphate acyltransferase (AGPAT), acylate lysophosphatidic acid at the sn-2 position to produce phosphatidic acid. We cloned a cDNA predicted to be AGPAT isoform and designated it AGPAT8. Human and mouse AGPAT8 proteins are 89% homologous, and their gene structure is also highly conserved. AGPAT8 is most closely related to AGPAT5, and its cDNA is expressed most in the heart, while AGPAT5 is expressed more in the prostate and testis. In cell lysates, AGPAT8 shows moderate acyltransferase activity with [(3)H]oleoyl-CoA but lacks acyl-CoA:lysocardiolipin acyltransferase activity. In whole cells upon incubation with [(14)C]linoleic acid, most of the radioactivity was recovered in phosphatidyl ethanolamine, phosphatidyl choline and phosphatidic acid fraction. Of the two well conserved acyltransferase motifs, NHX(4)D is present in AGPAT8, whereas arginine in the EGTR motif is substituted by aspartate. However, mutation of EGTD to EGTR did not increase enzymatic activity significantly. Based on the X-ray crystallographic structure of a related acyltransferase, squash gpat, a model is proposed in which a hydrophobic pocket in AGPAT8 accommodates fatty acyl chains of both substrates in an orientation where the NHX(4)D motif participates in catalysis. << Less

  Arch. Biochem. Biophys. 449:64-76(2006) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Acylglycerophosphate acyltransferase 4 (AGPAT4) is a mitochondrial lysophosphatidic acid acyltransferase that regulates brain phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol levels.

  Bradley R.M., Marvyn P.M., Aristizabal Henao J.J., Mardian E.B., George S., Aucoin M.G., Stark K.D., Duncan R.E.

  The acylglycerophosphate acyltransferase/lysophosphatidic acid acyltransferase (AGPAT/LPAAT) family is a group of homologous acyl-CoA-dependent lysophospholipid acyltransferases. We performed studies to better understand the subcellular localization, activity, and in vivo function of AGPAT4/LPAATδ ... >> More

  The acylglycerophosphate acyltransferase/lysophosphatidic acid acyltransferase (AGPAT/LPAAT) family is a group of homologous acyl-CoA-dependent lysophospholipid acyltransferases. We performed studies to better understand the subcellular localization, activity, and in vivo function of AGPAT4/LPAATδ, which we found is expressed in multiple mouse brain regions. Endogenous brain AGPAT4 and AGPAT4 overexpressed in HEK293 or Sf9 insect cells localizes to mitochondria and is resident on the outer mitochondrial membrane. Further fractionation showed that AGPAT4 is present specifically in the mitochondria and not in the mitochondria-associated endoplasmic reticulum membrane (i.e. MAM). Lysates from Sf9 cells infected with baculoviral Agpat4 were tested with eight lysophospholipid species but showed an increased activity only with lysophosphatidic acid as an acyl acceptor. Analysis of Sf9 phospholipid species, however, indicated a significant 72% increase in phosphatidylinositol (PI) content. We examined the content of major phospholipid species in brains of Agpat4(-/-) mice and found also a >50% decrease in total levels of PI relative to wildtype mice, as well as significant decreases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), but no significant differences in phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid (PA). A compensatory upregulation of Agpats 1, 2, 3, 5, and 9 may help to explain the lack of difference in PA. Our findings indicate that AGPAT4 is a mitochondrial AGPAT/LPAAT that specifically supports synthesis of brain PI, PC, and PE. This understanding may help to explain apparent redundancies in the AGPAT/LPAAT family. << Less

  Biochim Biophys Acta 1851:1566-1576(2015) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Functional characterization of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 10/glycerol-3-phosphate acyltransferase isoform 3.

  Sukumaran S., Barnes R.I., Garg A., Agarwal A.K.

  Synthesis of phospholipids can occur de novo or via remodeling of the existing phospholipids. Synthesis of triglycerides, a form of energy storage in cells, is an end product of these pathways. Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) acylate lysophosphatidic acid (LPA) at th ... >> More

  Synthesis of phospholipids can occur de novo or via remodeling of the existing phospholipids. Synthesis of triglycerides, a form of energy storage in cells, is an end product of these pathways. Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) acylate lysophosphatidic acid (LPA) at the sn-2 (carbon 2) position to produce phosphatidic acid (PA). These enzymes are involved in phospholipids and triglyceride synthesis through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. We cloned a cDNA predicted to be an AGPAT isoform (AGPAT10). This cDNA has been recently identified as glycerol-3-phosphate-O-acyltransferase isoform 3 (GPAT3). When this AGPAT10/GPAT3 cDNA was expressed in Chinese Hamster ovary cells, the protein product localizes to the endoplasmic reticulum. In vitro enzymatic activity using lysates of human embryonic kidney-293 cells infected with recombinant AGPAT10/GPAT3 adenovirus show that the protein has a robust AGPAT activity with an apparent V(max) of 2 nmol/min per mg protein, but lacks GPAT enzymatic activity. This AGPAT has similar substrate specificities for LPA and acyl-CoA as shown for another known isoform, AGPAT2. We further show that when overexpressed in human Huh-7 cells depleted of endogenous AGPAT activity by sh-RNA-AGPAT2-lentivirus, the protein again demonstrates AGPAT activity. These observations strongly suggest that the cDNA previously identified as GPAT3 has AGPAT activity and thus we prefer to identify this clone as AGPAT10 as well. << Less

  J. Mol. Endocrinol. 42:469-478(2009) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers.

  Agarwal A.K., Garg A.

  The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they ... >> More

  The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well. << Less

  J. Lipid Res. 51:2143-2152(2010) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• YPR139c/LOA1 encodes a novel lysophosphatidic acid acyltransferase associated with lipid droplets and involved in TAG homeostasis.

  Ayciriex S., Le Guedard M., Camougrand N., Velours G., Schoene M., Leone S., Wattelet-Boyer V., Dupuy J.W., Shevchenko A., Schmitter J.M., Lessire R., Bessoule J.J., Testet E.

  For many years, lipid droplets (LDs) were considered to be an inert store of lipids. However, recent data showed that LDs are dynamic organelles playing an important role in storage and mobilization of neutral lipids. In this paper, we report the characterization of LOA1 (alias VPS66, alias YPR139 ... >> More

  For many years, lipid droplets (LDs) were considered to be an inert store of lipids. However, recent data showed that LDs are dynamic organelles playing an important role in storage and mobilization of neutral lipids. In this paper, we report the characterization of LOA1 (alias VPS66, alias YPR139c), a yeast member of the glycerolipid acyltransferase family. LOA1 mutants show abnormalities in LD morphology. As previously reported, cells lacking LOA1 contain more LDs. Conversely, we showed that overexpression results in fewer LDs. We then compared the lipidome of loa1Δ mutant and wild-type strains. Steady-state metabolic labeling of loa1Δ revealed a significant reduction in triacylglycerol content, while phospholipid (PL) composition remained unchanged. Interestingly, lipidomic analysis indicates that both PLs and glycerolipids are qualitatively affected by the mutation, suggesting that Loa1p is a lysophosphatidic acid acyltransferase (LPA AT) with a preference for oleoyl-CoA. This hypothesis was tested by in vitro assays using both membranes of Escherichia coli cells expressing LOA1 and purified proteins as enzyme sources. Our results from purification of subcellular compartments and proteomic studies show that Loa1p is associated with LD and active in this compartment. Loa1p is therefore a novel LPA AT and plays a role in LD formation. << Less

  Mol. Biol. Cell 23:233-246(2012) [PubMed] [EuropePMC]

• SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast.

  Benghezal M., Roubaty C., Veepuri V., Knudsen J., Conzelmann A.

  Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and ... >> More

  Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1Delta cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1Delta and slc4Delta cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1Delta and slc4Delta cells incubated with [14C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1Delta and slc4Delta cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg2+-dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids. << Less

  J. Biol. Chem. 282:30845-30855(2007) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase.

  Montero-Moran G., Caviglia J.M., McMahon D., Rothenberg A., Subramanian V., Xu Z., Lara-Gonzalez S., Storch J., Carman G.M., Brasaemle D.L.

  Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an alpha/beta-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acy ... >> More

  Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an alpha/beta-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids. << Less

  J. Lipid Res. 51:709-719(2010) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

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

  Yuki K., Shindou H., Hishikawa D., Shimizu T.

  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 a ... >> More

  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. << Less

  J. Lipid Res. 50:860-869(2009) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.