Publications

• Identification of a novel keratinocyte retinyl ester hydrolase as a transacylase and lipase.

  Gao J., Simon M.

  Retinoic acid influences epidermal morphology and function through its ability to control transcription. Because the circulation presents the epidermis with micromolar amounts of retinol that can be converted to retinoic acid, regulating retinol access is imperative. In keratinocytes the majority ... >> More

  Retinoic acid influences epidermal morphology and function through its ability to control transcription. Because the circulation presents the epidermis with micromolar amounts of retinol that can be converted to retinoic acid, regulating retinol access is imperative. In keratinocytes the majority of retinol is sequestered as long chain fatty acid esters. Although much has been learned about the major esterifying enzyme, little is known about the hydrolase that accesses retinol from its storage depot. Murine carboxylesterases and hormone sensitive lipase have been shown to have this activity. We found that their in vitro sensitivity to bis-p-nitrophenyl phosphate (BNPP), however, was not shared by the epidermal hydrolase activity. We therefore produced and screened two keratinocyte cDNA expression libraries and identified a previously sequenced gene (GS2) as a keratinocyte retinyl ester (RE) hydrolase insensitive to BNPP. The enzyme also catalyzes fattyacyl CoA-dependent and -independent retinol esterification. The hydrolysis reaction is greater at neutral pH, whereas the esterification reaction is greater at acidic pH. These activities are consistent with the increased RE content that accompanies epidermal maturation. In addition, this enzyme utilizes triolein as substrate and generates diacylglyceride and free fatty acid. << Less

  J. Invest. Dermatol. 124:1259-1266(2005) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Synthesis of neutral ether lipid monoalkyl-diacylglycerol by lipid acyltransferases.

  Ma Z., Onorato J.M., Chen L., Nelson D.W., Yen C.E., Cheng D.

  In mammals, ether lipids exert a wide spectrum of signaling and structural functions, such as stimulation of immune responses, anti-tumor activities, and enhancement of sperm functions. Abnormal accumulation of monoalkyl-diacylglycerol (MADAG) was found in Wolman's disease, a human genetic disorde ... >> More

  In mammals, ether lipids exert a wide spectrum of signaling and structural functions, such as stimulation of immune responses, anti-tumor activities, and enhancement of sperm functions. Abnormal accumulation of monoalkyl-diacylglycerol (MADAG) was found in Wolman's disease, a human genetic disorder defined by a deficiency in lysosomal acid lipase. In the current study, we found that among the nine recombinant human lipid acyltransferases examined, acyl-CoA:diacylglycerol acyltransferase (DGAT)1, DGAT2, acyl-CoA:monoacylglycerol acyltransferase (MGAT)2, MGAT3, acyl-CoA:wax-alcohol acyltransferase 2/MFAT, and DGAT candidate 3 were able to use 1-monoalkylglycerol (1-MAkG) as an acyl acceptor for the synthesis of monoalkyl-monoacylglycerol (MAMAG). These enzymes demonstrated different enzymatic turnover rates and relative efficiencies for the first and second acylation steps leading to the synthesis of MAMAG and MADAG, respectively. They also exhibited different degrees of substrate preference when presented with 1-monooleoylglycerol versus 1-MAkG. In CHO-K1 cells, treatment with DGAT1 selective inhibitor, XP-620, completely blocked the synthesis of MADAG, indicating that DGAT1 is the predominant enzyme responsible for the intracellular synthesis of MADAG in this model system. The levels of MADAG in the adrenal gland of DGAT1 KO mice were reduced as compared with those of the WT mice, suggesting that DGAT1 is a major enzyme for the synthesis of MADAG in this tissue. Our findings indicate that several of these lipid acyltransferases may be able to synthesize neutral ether lipids in mammals. << Less

  J. Lipid Res. 58:1091-1099(2017) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Characterization of the enzymatic activity and physiological function of the lipid droplet-associated triacylglycerol lipase AtOBL1.

  Mueller A.O., Ischebeck T.

  Similar to seeds, pollen tubes contain lipid droplets that store triacylglycerol (TAG), but the fate of this TAG as well as the enzymes involved in its breakdown are unknown. Therefore, two potential TAG lipases from tobacco and Arabidopsis, NtOBL1 (Oil body lipase 1) and AtOBL1, were investigated ... >> More

  Similar to seeds, pollen tubes contain lipid droplets that store triacylglycerol (TAG), but the fate of this TAG as well as the enzymes involved in its breakdown are unknown. Therefore, two potential TAG lipases from tobacco and Arabidopsis, NtOBL1 (Oil body lipase 1) and AtOBL1, were investigated, especially with respect to their importance for pollen tube growth. We expressed NtOBL1 and AtOBL1 as fluorescent fusion proteins to study their localization by confocal microscopy. Furthermore, we overexpressed AtOBL1 in Nicotiana benthamiana leaves to characterize it enzymatically. The obl1 mutant was studied in respect to its pollen tube growth in vivo and its seed germination. Both NtOBL1 and AtOBL1 localized to lipid droplets. AtOBL1 was abundant in pollen tubes and seedlings, and acted as a lipase on TAG, diacylglycerol and 1-monoacylglycerol at a pH optimum of 5.5. The obl1 mutant was hampered in pollen tube growth, whereas seedling establishment was not affected under optimal conditions, even though AtOBL1 accounted for a major lipase activity in seeds. TAG could be a direct precursor for the synthesis of membrane lipids in pollen tubes and proteins of the OBL family involved in the flux of acyl groups. << Less

  New Phytol. 217:1062-1076(2018) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Purification of the lysosomal acid lipase from human liver and its role in lysosomal lipid hydrolysis.

  Warner T.G., Dambach L.M., Shin J.H., O'Brien J.S.

  The lysosomal acid lipase has been purified 2,500-fold to near homogeneity from human liver. The enzyme was converted to a soluble form by extraction of frozen tissue with Triton X-100. The enzyme, which required Triton X-100 in buffers at all purification steps for optimal yields, was stabilized ... >> More

  The lysosomal acid lipase has been purified 2,500-fold to near homogeneity from human liver. The enzyme was converted to a soluble form by extraction of frozen tissue with Triton X-100. The enzyme, which required Triton X-100 in buffers at all purification steps for optimal yields, was stabilized by the inclusion of 33% ethylene glycol during purification. Lectin chromatography on concanavalin A-Sepharose followed by chromatography on carboxymethyl-cellulose and Sephadex G-150 provided the highly purified enzyme in 17% yield. Sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the minimum molecular weight was about 29,000 +/-1,000. Minor protein contaminants at Mr = 58,500, 14,700 and 13,900 were present in the final preparation. A single protein band, with enzyme activity, was observed in nondenaturing acrylamide gels containing Triton X-100. Gel filtration on Sephadex G-150 in the presence of Triton X-100 gave an apparent molecular weight of about 125,000 +/-13,000. Trioleoylglycerol, cholesterol oleate, and 1,2- and 1,3-dioleoylglycerols were substrates for the purified enzyme giving apparent Vmax values of 5,400, 1,400, 19,400, and 22,100 nmol min-1 mg of protein-1, respectively, and Km values of 0.8, 0.8, 0.9, and 1.2 mM, respectively. The recoveries of both trioleoylglycerol and cholesterol oleate hydrolytic activities were nearly identical at each purification step, suggesting that the acid lipase as single enzyme is responsible for lysosomal hydrolysis of the neutral lipids. Monooleoylglycerols were not substrates for the enzyme. << Less

  J. Biol. Chem. 256:2952-2957(1981) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Positional specificity of hormone-sensitive lipase from rat adipose tissue.

  Fredrikson G., Belfrage P.

  Hormone-sensitive lipase, purified from rat adipose tissue (Fredrikson, G., Strålfors, P., Nilsson, N. O., and Belfrage, P. (1981) J. Biol. Chem. 256, 6311-6320), has been incubated with tri-, di-, and monooleoyl[3H]glycerol, and the acylglycerol reaction products were isolated by thin layer chrom ... >> More

  Hormone-sensitive lipase, purified from rat adipose tissue (Fredrikson, G., Strålfors, P., Nilsson, N. O., and Belfrage, P. (1981) J. Biol. Chem. 256, 6311-6320), has been incubated with tri-, di-, and monooleoyl[3H]glycerol, and the acylglycerol reaction products were isolated by thin layer chromatography on silicic acid, impregnated with boric acid. Trioleoylglycerol was hydrolyzed with the intermediate accumulation of monooleoylglycerol, mainly the 2-isomer, and a small amount of 1,2(2,3)-, but no measurable, 1,3-dioleoylglycerol. 2-Monooleoylglycerol was also the major acylglycerol reaction product from 1,2(2,3)-dioleoylglycerol hydrolysis, which occurred at a Vmax of 60% of that with the 1,3-isomer. Part of the 1(3)-monooleoylglycerols found were formed by acyl migration, but 2-ester bond cleavage was directly demonstrated by the use of 1,3-dioleoyl-2-[14C]oleoyl[3H]glycerol as substrate, and by determination of the 14C/3H ratios of the acylglycerol reaction products. Based on the hydrolysis of specific monooleoylglycerol isomers, it was estimated that the 1(3)-ester bonds of the acylglycerols were hydrolyzed 3-to 4-fold faster than the 2-ester bonds. The main lipolytic reaction sequence catalyzed by hormone-sensitive lipase is thus triacylglycerol leads to 1,2(2,3)-diacylglycerol leads to 2-monoacylglycerol. However, the preference for the 1(3)-ester bonds is less marked than that of, e.g. pancreatic and lipoprotein lipase. << Less

  J. Biol. Chem. 258:14253-14256(1983) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase.

  Rodriguez J.A., Ben Ali Y., Abdelkafi S., Mendoza L.D., Leclaire J., Fotiadu F., Buono G., Carriere F., Abousalham A.

  Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, r ... >> More

  Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol. << Less

  Biochim. Biophys. Acta 1801:77-83(2010) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.

  Jenkins C.M., Mancuso D.J., Yan W., Sims H.F., Gibson B., Gross R.W.

  Genetic knockout of hormone-sensitive lipase in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Despite intense interest in these TAG lipases, their molecular identities thus far are largely unknown. Sequence data base s ... >> More

  Genetic knockout of hormone-sensitive lipase in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Despite intense interest in these TAG lipases, their molecular identities thus far are largely unknown. Sequence data base searches for proteins containing calcium-independent phospholipase A2 (iPLA2) dual signature nucleotide ((G/A)XGXXG) and lipase (GXSXG) consensus sequence motifs identified a novel subfamily of three putative iPLA2/lipase family members designated iPLA2epsilon, iPLA2zeta, and iPLA2eta (previously named adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA2 subfamily in Sf9 cells, and we demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA2epsilon, iPLA2zeta, and iPLA2eta also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(Bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, a mechanism-based suicide substrate inhibitor of all known iPLA2s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC50=0.1-0.5 microm). Quantitative PCR revealed dramatically increased expression of iPLA2epsilon and iPLA2zeta transcripts during the hormone-induced differentiation of 3T3-L1 cells into adipocytes and identified the presence of all three iPLA2 isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes. << Less

  J. Biol. Chem. 279:48968-48975(2004) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.