FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1982450156> ?p ?o ?g. }

• W1982450156 endingPage "1811" @default.
• W1982450156 startingPage "1803" @default.
• W1982450156 abstract "The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or “lid” covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL3 in the presence of the EL C-domain. The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or “lid” covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL3 in the presence of the EL C-domain. Endothelial lipase (EL) is a member of the triglyceride (TG) lipase gene family (1Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (425) Google Scholar) that is synthesized by endothelial cells and by other cell types such as macrophages and hepatocytes (1Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (425) Google Scholar, 2Hirata K. Dichek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T. Cloning of a unique lipase from endothelial cells extends the lipase gene family.J. Biol. Chem. 1999; 274: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 3Cohen J.C. Endothelial lipase: direct evidence for a role in HDL metabolism.J. Clin. Invest. 2003; 111: 318-321Crossref PubMed Scopus (28) Google Scholar). EL has a distinct substrate specificity profile and is relatively more active as a phospholipase than as a TG lipase (4McCoy M.G. Sun G-S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, the ratio of TG lipase to phospholipase activity of EL is markedly less than those of LPL and HL. Relative to phospholipase activity, LPL displays the highest TG lipase activity and EL the lowest TG lipase activity of the three enzymes. EL also has greater preference for HDL, and LPL has the greater preference for VLDL (4McCoy M.G. Sun G-S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar, 5Broedl U.C. Jin W. Fuki I.V. Glick J.M. Rader D.J. Structural basis of endothelial lipase tropism for HDL.FASEB J. 2004; 18: 1891-1893Crossref PubMed Scopus (13) Google Scholar). Thus, LPL, HL, and EL seem to have evolved with distinct substrate specificities to accommodate the full spectrum of circulating lipoproteins. The lid covering the active site has been implicated as a major source of substrate specificity for LPL and HL. Dugi et al. (6Dugi K.A. Dichek H.L. Talley G.D. Brewer H.B. Santamarina-Fojo S. Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates.J. Biol. Chem. 1992; 267: 25086-25091Abstract Full Text PDF PubMed Google Scholar) studied the hypothesis that the surface lid covering the catalytic pocket may modulate access of the substrate to the active site of LPL. Characterization of a number of mutants with altered amphipathic properties of the LPL lid showed that the disruption of the lid decreased its ability to hydrolyze an emulsified lipid substrate without affecting the ability to hydrolyze a water-soluble substrate. They proposed that the interaction between the lipoprotein substrates and the lid may in part determine substrate specificity. Chimeric lipases were also generated by exchanging the lid region between LPL and HL (7Dugi K.A. Dichek H.L. Santamarina-Fojo S. Human hepatic and lipoprotein lipase: the loop covering the catalytic site mediates lipase substrate specificity.J. Biol. Chem. 1995; 270: 25396-25401Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The lid of LPL conferred preferential TG hydrolysis, as opposed to augmenting phospholipase activity in the case of the lid of HL. Preferential in vivo hydrolysis of phospholipids (PLs) was demonstrated in HL-deficient mice injected with adenovirus-expressing lipases containing the HL lid (HL or LPL with the lid of HL) compared with lipases containing the LPL lid (LPL or HL with the lid of LPL) (8Kobayashi J. Applebraum-Bowden D. Dugi K.A. Brown D.R. Kashyap V.S. Parrott C. Duarte C. Maeda N. Santamarina-Fojo S. Analysis of protein structure-function in vivo. Adenovirus-mediated transfer of lipase lid mutants in hepatic lipase-deficient mice.J. Biol. Chem. 1996; 271: 26296-26301Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). These studies identified the lid as a major structural motif responsible for conferring different lipid substrate specificities of LPL and HL, a function that may modulate the distinct physiological roles of these two similar lipolytic enzymes in lipoprotein metabolism (8Kobayashi J. Applebraum-Bowden D. Dugi K.A. Brown D.R. Kashyap V.S. Parrott C. Duarte C. Maeda N. Santamarina-Fojo S. Analysis of protein structure-function in vivo. Adenovirus-mediated transfer of lipase lid mutants in hepatic lipase-deficient mice.J. Biol. Chem. 1996; 271: 26296-26301Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The LPL and HL lids are each 22 amino acids long, whereas the EL lid is only 19 amino acids long. Furthermore, the lid region of EL differs from the lids of LPL and HL not only in size but also in amino acid sequence and polarity. To determine the extent to which the lid plays a role in conferring substrate specificity for EL, we studied EL in comparison with LPL, its polar opposite with respect to the lipase activity spectrum. We created chimeric molecules of LPL and EL with exchanged lid regions as well as single mutants of the EL lid region. Based on the potential role of the C-terminal domain (C-domain) of lipases in binding substrate (5Broedl U.C. Jin W. Fuki I.V. Glick J.M. Rader D.J. Structural basis of endothelial lipase tropism for HDL.FASEB J. 2004; 18: 1891-1893Crossref PubMed Scopus (13) Google Scholar), we also addressed whether the C-domain of EL cooperates with the lid in determining substrate specificity by replacing both the C-domain and lid of LPL with the C-domain and lid of EL. Lipolytic activities of chimeras and mutants were determined in the presence of synthetic substrates and native lipoproteins. EL lid mutants include the glycine-241→arginine (EL-G241R) and glutamate-250→glutamine (EL-E250Q) substitutions as well as an inserted arginine at position 245 (EL-245R) to mimic the LPL lid sequence (Fig. 1). Sequences of primers used for these site-directed mutagenesis procedures were 5′-GT GGA CTC AAC GAT GTC TTG CGA TCA ATT GCA TAT GG-3′ for EL-G241R, 5′-GCA TAT GGA ACA ATC ACA CAG GTG GTA AAA TGT GAG C-3′ for EL-E250Q, and 5′-G GGA TCA ATT GCA TAT CGC GGA ACA ATC ACA GAG GTG G-3′ for EL-245R. All PCRs, including those for the single-amino acid changes for the lid mutants, were performed according to the Quikchange Site-Directed Mutagenesis Kit Protocol (Stratagene). All of the PCR products were sequenced to confirm accuracy. The EL/LPL-lid chimera consists of wild-type human EL backbone with the LPL lid, and the LPL/EL-lid chimera consists of wild-type human LPL backbone with the EL lid (Fig. 2). We engineered the exchange of the lid sequence between EL and LPL by overlap extension PCR (9Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.Gene. 1989; 77: 61-68Crossref PubMed Scopus (2638) Google Scholar). The primers were designed so that the original base pair sequence was conserved as much as possible. The primers included the sequences for the respective lid sequences flanked on each side by ∼20 bp. The sequences of these antisense primers were as follows: 5′-GCT CGC TCA TGC TCA CAT TTT ACC AGC TGG TCG ACG TCT CCA AGT CCT CTC TCT GCA ATC ACG CGG ATA GCT TCT CCA ATG TTA CAG CCT GGC TGG AAG TCA CC-3′ and 5′-GCG CTC GTG GGA GCA CTT CAC CAC CTC TGT GAT TGT TCC ATA TGC AAT TGA TCC CAA GAC ATC GTT GAG TCC ACA TCC TGG CTG AAA AGT ACC TCC-3′. They were used to introduce the LPL lid into the EL backbone and the EL lid into the LPL backbone, respectively. Briefly, the antisense lid primers were used with LPL or EL sense primers for the first PCR. The purified product from the first PCR was then used as a primer in the second PCR. To compare the level of expression of the chimeric and wild-type lipases, we constructed C-terminal myc-His-tagged proteins by inserting the full-length cDNA of each construct and the wild type into the pcDNA3.1/myc-His(−) plasmid expression vector (Invitrogen). Constructs were sequenced to confirm accuracy. The chimeric LPL/EL-Cdom lipase was synthesized by overlap extension polymerase chain reaction as described by Broedl et al. (5Broedl U.C. Jin W. Fuki I.V. Glick J.M. Rader D.J. Structural basis of endothelial lipase tropism for HDL.FASEB J. 2004; 18: 1891-1893Crossref PubMed Scopus (13) Google Scholar). The double chimera (LPL/EL-lid/Cdom) consists of wild-type human LPL backbone with the EL lid and C-domain (Fig. 2). This chimera was generated by double digestion of LPL/EL-Cdom and LPL/EL-lid chimeras with the restriction enzymes XbaI and Eco47III (all restriction enzymes were purchased from New England Biolabs, Inc.). After digestion of LPL/EL-Cdom, the DNA fragment containing the pcDNA3.1(−) plasmid and the 3′ end of LPL/EL-Cdom just downstream of the lid coding region was purified by gel extraction. Meanwhile, similar digestion was run on the LPL/EL-lid chimera. In this case, the 5′ DNA fragment including the chimeric lid region was purified by gel extraction. These two digested products were then ligated using T4 ligase and a Rapid Ligation Kit (Roche). HEK 293 cells were maintained in DMEM, 10% fetal bovine serum, and 1× antibiotic-antimycotic (Gibco). The cells were transfected in triplicate using Lipofectamine™ reagent (Invitrogen). Twenty-four hours after transfection, the medium was replaced with DMEM containing 10 U/ml heparin (Sigma) and incubated for another 24 h. Heparin was used to release the recombinant lipases that are bound to the cell surface via proteoglycans. Thirty minutes before harvesting this conditioned medium, more heparin was added to the medium, bringing the final concentration of heparin to 20 U/ml. Conditioned media were collected, clarified by low-speed centrifugation, and frozen in aliquots at −80°C. Ten microliters of conditioned media was resolved by 10% Bis-Tris SDS-PAGE (Invitrogen) and transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech). Proteins were detected using a monoclonal mouse anti-myc primary antibody (clone 9E10) and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). The mass of the chimeras (such as LPL/EL-Cdom and LPL/EL-lid/Cdom) could not be accurately measured with the available LPL and EL ELISAs. Therefore, the level of protein expression of these chimeras was estimated by Western blot. Emulsions of triolein or dipalmitoylphosphatidylcholine were used to measure TG lipase or phospholipase activity, respectively (4McCoy M.G. Sun G-S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar, 10Nilsson-Ehle P. Schotz M.C. A stable, radioactive substrate emulsion for assay of lipoprotein lipase.J. Lipid Res. 1976; 17: 536-541Abstract Full Text PDF PubMed Google Scholar, 11Belfrage P. Vaughan M. Simple liquid-liquid partition system for isolation of labeled oleic acid from mixtures with glycerides.J. Lipid Res. 1969; 10: 341-344Abstract Full Text PDF PubMed Google Scholar). For the TG lipase assay, the emulsion contained triolein and egg phosphatidylcholine containing glycerol-tri[9,10(n)-3H]oleate stabilized with glycerol. For the phospholipase assay, a similar glycerol-stabilized emulsion was used that contained radiolabeled PLs (dipalmitoylphosphatidylcholine) and cholesteryl oleate as the neutral lipid core. Conditioned medium containing the recombinant proteins was used as an enzyme source. Medium obtained from cells expressing green fluorescent protein was included in all experiments as a negative control, and the resulting background activity was subtracted. Two experimental conditions were necessary, one in the presence of serum (1.33%) as a source of apolipoprotein C-II for LPL activation, and one in the absence of serum as an inhibitory effect of serum on EL activity, which has already been reported (4McCoy M.G. Sun G-S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). Samples were incubated for 15 min at 37°C. All enzyme activities are reported as nanomoles of FFA liberated per hour per milliliter of conditioned medium as a source of enzyme. The ratio of triglyceride lipase activity to phospholipase activity (TG/PL ratio) reflects the substrate specificity of these lipases. To determine whether the mean of the TG/PL ratio of one mutant and the mean of the TG/PL ratio of the wild-type parental lipase (either EL or LPL, depending on the mutation) were statistically different, statistical analysis were performed using an unpaired t-test. The mean values were considered significantly different at P < 0.05. The values of the TG/PL ratio were measured across experiments run in triplicate on the conditioned media of independent transfections. The sample size (number of determinations) was at least six or greater. In this assay, two types of lipoprotein particles were used as lipase substrates, VLDL and HDL3. These human lipoproteins were isolated from pooled plasma samples (4McCoy M.G. Sun G-S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar) and incubated with medium containing lipases (4McCoy M.G. Sun G-S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar, 12Bamberger M. Lund-Katz S. Phillips M.C. Rothblat G.H. Mechanism of the hepatic lipase induced accumulation of high-density lipoprotein cholesterol by cells in culture.Biochemistry. 1985; 24: 3693-3701Crossref PubMed Scopus (100) Google Scholar). Each reaction tube contained 1.25 mM lipoprotein PLs, 40 or 65.3 μl of conditioned medium containing one of the various enzymes, 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 8 mM CaCl2, and 1% BSA in a final volume of 100 μl. Medium obtained from cells expressing green fluorescent protein was included in all experiments as a negative control, and the resulting background activity was subtracted. The reactions being linear with time for >4 h (data not shown), tubes were incubated for 4 h at 37°C. The released FFAs were measured enzymatically using a commercially available kit (NEFA C; Wako Pure Chemical Industries). Data are presented as nmol FFA produced/ml conditioned medium. The ratio of the enzymatic activity for VLDL to the enzymatic activity for HDL3 reflects the relative preference of each lipase for these two physiologic substrates. In the case of HDL3, we were also able to optimize the assay to run this experiment varying the concentration of PL present in the reaction. These data were then used to determine the apparent affinity of the enzyme for the PL present in HDL3 as substrate (appKm). These parameters were obtained after fitting the experimental data to the following equation: A=(Amax×[S])/(appKm+[S]) where Amax is the maximal activity of the enzyme (in nmol FFA/ml conditioned medium as a source of lipases), [S] is the concentration of PL present in HDL3 as substrate, and appKm is as defined above. The amphipathic properties of the two helices present in the lid have been reported to be important determinants of both LPL and HL substrate specificity (6Dugi K.A. Dichek H.L. Talley G.D. Brewer H.B. Santamarina-Fojo S. Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates.J. Biol. Chem. 1992; 267: 25086-25091Abstract Full Text PDF PubMed Google Scholar). Therefore, we analyzed the sequence of the EL lid compared with the lid of LPL (Fig. 1). Three amino acid residues within the lid of EL that differ substantially from LPL were mutated to the corresponding residue in LPL. EL-G241R and EL-E250Q were generated by substitution of a single residue, and EL-245R was generated by inserting an arginine at position 245. Each mutant (EL-G241R, EL-245R, and EL-E250Q) showed a small increase in the TG/PL ratio compared with wild-type EL (Table 1). Table 1 shows the results of one representative experiment. Pooling data collected at least in triplicate across four independent transfections, we showed the difference of the TG/PL ratio of each mutant to be statistically significant compared with wild-type EL using an unpaired t-test. The mean of the TG/PL ratio of EL-G241R, EL-245R, and EL-E250Q increased 1.52-, 1.55-, and 1.90-fold, respectively. This increase of the TG/PL ratio of these mutants compared with wild-type EL was statistically significant in each case (Table 1). Each mutation had the effect of slightly increasing the TG preference of EL, suggesting that the polarity of those residues plays a role in determining the difference in substrate specificity between LPL and EL.TABLE 1TG lipase and phospholipase activities of EL and three lid mutants of ELTG/PL RatioLipasesTG Lipase ActivityPhospholipase ActivityTG/PL RatioMean ± SDnPEL151 ± 21.9176 ± 39.10.861.05 ± 0.199EL-G241R84.3 ± 30.666.5 ± 11.41.271.52 ± 0.51aStatistically significant at P < 0.05.70.023EL-245R183 ± 16.5132 ± 23.61.391.55 ± 0.33aStatistically significant at P < 0.05.90.001EL-E250Q95.3 ± 11.455.0 ± 16.61.731.90 ± 0.43aStatistically significant at P < 0.05.60.0002EL, endothelial lipase; PL, phospholipid; TG, triglyceride; TG/PL, ratio of triglyceride lipase activity to phospholipase activity. The three lid mutants of EL studied are EL-G241R, EL-245R, and EL-E250Q. The TG lipase activity and phospholipase activity of each conditioned medium were measured in the absence of serum. All enzyme activities are expressed in nanomoles of product (FFA) formed per hour per milliliter of conditioned medium as a source of lipase and are reported are means ± SD. The TG/PL ratio data at left are results of one representative experiment. The TG/PL ratio data at right are means ± SD for EL and the three lid mutants of EL measured in a number (n) of different experiments. The significance of the difference of the mean value of the TG/PL ratio of wild-type EL versus each lid mutant of EL was determined using an unpaired t-test.a Statistically significant at P < 0.05. Open table in a new tab EL, endothelial lipase; PL, phospholipid; TG, triglyceride; TG/PL, ratio of triglyceride lipase activity to phospholipase activity. The three lid mutants of EL studied are EL-G241R, EL-245R, and EL-E250Q. The TG lipase activity and phospholipase activity of each conditioned medium were measured in the absence of serum. All enzyme activities are expressed in nanomoles of product (FFA) formed per hour per milliliter of conditioned medium as a source of lipase and are reported are means ± SD. The TG/PL ratio data at left are results of one representative experiment. The TG/PL ratio data at right are means ± SD for EL and the three lid mutants of EL measured in a number (n) of different experiments. The significance of the difference of the mean value of the TG/PL ratio of wild-type EL versus each lid mutant of EL was determined using an unpaired t-test. To determine the role of the lid covering the active site in conferring substrate specificity for EL, we studied EL in comparison with LPL, creating chimeric molecules of LPL and EL with exchanged lid regions (Fig. 2). Western blots of the conditioned media of transfected HEK 293 cells showed high-level expression of wild-type and chimeric lipases (Fig. 3). The TG/PL ratio for wild-type EL was 0.44 (Table 2). Placing the LPL lid in the EL backbone (EL/LPL-lid) resulted in an increase in the TG/PL ratio to 2.59, indicating a relative increase in substrate specificity for TG. In the presence of apolipoprotein C-II, the TG/PL ratio for wild-type LPL was 110 (Table 2). Placing the EL lid into the LPL backbone (LPL/EL-lid) resulted in a decrease of the TG/PL ratio to 32.TABLE 2TG lipase and phospholipase activities of LPL, EL, and their lid chimerasNo SerumSerumLipasesTG Lipase ActivityPhospholipase ActivityTG/PL RatioTG Lipase ActivityPhospholipase ActivityTG/PL RatioEL114 ± 7.3257 ± 14.20.4445.4 ± 8.24.14 ± 3.211.0EL/LPL-lid47.1 ± 5.518.2 ± 2.52.5914.1 ± 8.03.7 ± 6.83.8LPL1,030 ± 28.32.7 ± 0.73814,364 ± 26939.7 ± 3.9110LPL/EL-lid346 ± 13.52.2 ± 1.21571,940 ± 68.760.3 ± 3.832.2The TG lipase activity and phospholipase activity of each conditioned medium were measured both in the presence and absence of serum. All enzyme activities are expressed in nanomoles of product (FFA) formed per hour per milliliter of conditioned medium as a source of lipase and are reported as means ± SD. The values shown are results of 1 representative experiment out of 10 independent experiments. Open table in a new tab The TG lipase activity and phospholipase activity of each conditioned medium were measured both in the presence and absence of serum. All enzyme activities are expressed in nanomoles of product (FFA) formed per hour per milliliter of conditioned medium as a source of lipase and are reported as means ± SD. The values shown are results of 1 representative experiment out of 10 independent experiments. This trend was similar and statistically significant across experiments run on the conditioned media from 10 independent transfections (Table 3). The replacement of the EL lid by the LPL lid resulted in a substantial increase of 4.2-fold in the TG/PL ratio compared with wild-type EL but did not fully confer the high degree of preference of LPL for TG substrates (the mean of the TG/PL ratio of LPL across experiments being 83.8). The replacement of the LPL lid by the EL lid consistently generated a decrease in the TG/PL ratio to 32.7% of the ratio of wild-type LPL, indicating an increase of substrate specificity toward PL. However, the LPL/EL-lid chimera remained primarily a TG lipase like wild-type LPL, just as the EL/LPL-lid chimera did not become as active as a TG lipase as wild-type LPL.TABLE 3TG/PL ratios for LPL, EL, and their lid chimerasTG/PL RatioLipasesMean ± SDnPEL0.60 ± 0.3814EL/LPL-lid2.52 ± 1.52aStatistically significant at P < 0.05.140.0001LPL83.8 ± 40.422LPL/EL-lid27.4 ± 13.4aStatistically significant at P < 0.05.23<0.0001The values shown are means ± SD for LPL, EL, and their lid chimeras (EL/LPL-lid and LPL/EL-lid) measured across a number (n) of experiments. The significance of the difference of the mean value of the TG/PL ratio of each lid chimera compared with its parental lipase (EL or LPL) was determined using an unpaired t-test.a Statistically significant at P < 0.05. Open table in a new tab The values shown are means ± SD for LPL, EL, and their lid chimeras (EL/LPL-lid and LPL/EL-lid) measured across a number (n) of experiments. The significance of the difference of the mean value of the TG/PL ratio of each lid chimera compared with its parental lipase (EL or LPL) was determined using an unpaired t-test. We then asked whether the lid influenced the ability of EL to hydrolyze native lipoproteins (Table 4). Our results confirmed that wild-type LPL preferentially hydrolyzes the lipids of VLDL over HDL particles and that EL preferentially hydrolyzes the lipids of HDL particles over VLDL, as reported previously (4McCoy M.G. Sun G-S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). When the LPL lid was placed into the EL backbone, there was little effect on the relative ability to hydrolyze HDL3 versus VLDL, and this effect was determined to not be statistically significant (Table 4). Similarly, the ability to hydrolyze lipids of VLDL and HDL3 was not affected with the LPL/EL-lid chimera, indicating that the lid alone is not a determinant of native lipoprotein substrate preference.TABLE 4Lipolysis of lipoprotein particles by the lid chimerasVLDL/HDL3 RatioLipasesVLDL Lipolytic ActivityHDL3 Lipolytic ActivityVLDL/HDL3 RatioMean ± SDnPEL46.1 ± 7.755.4 ± 7.80.831.05 ± 1.368EL/LPL-lid41.0 ± 21.320.6 ± 4.41.992.08 ± 1.3550.209 (NS)LPL12,916 ± 1,86892.2 ± 3.7140104 ± 47.411LPL/EL-lid5,240 ± 44432.7 ± 7.4141129 ± 58.590.315 (NS)Lipase activities of conditioned media containing LPL, EL, or the lid chimeras (EL/LPL-lid and LPL/EL-lid) using isolated lipoprotein fractions (VLDL and HDL3) as substrates were measured as the amount of free fatty acid released after incubation at 37°C for 4 h. The lipolytic activities are expressed in nanomoles of product (FFA) formed per milliliter of conditioned medium as a source of lipase and are reported as means ± SD. The VLDL/HDL3 ratio data at left are results of one representative experiment. The VLDL/HDL3 ratio data at right are means ± SD for LPL, EL, and their lid chimeras (EL/LPL-lid and LPL/EL-lid) measured across a number (n) of experiments. The significance of the difference of the mean value of the VLDL/HDL3 ratio of each lid chimera compared with its parental lipase (EL, LPL, or LPL/EL-Cdom) was determined using an unpaired t-test. Two means were determined to be statistically not significant (NS) at P > 0.05. Open table in a new tab Lipase activities of conditioned media containing LPL, EL, or the lid chimeras (EL/LPL-lid and LPL/EL-lid) using isolated lipoprotein fractions (VLDL and HDL3) as substrates were measured as the amount of free fatty acid released after incubation at 37°C for 4 h. The lipolytic activities are expressed in nanomoles of product (FFA) formed per milliliter of conditioned medium as a source of lipase and are reported as means ± SD. The VLDL/HDL3 ratio data at left are results of one representative experiment. The VLDL/HDL3 ratio data at right are means ± SD for LPL, EL, and their lid chimeras (EL/LPL-lid and LPL/EL-lid) measured across a number (n) of experiments. The significance of the difference of the mean value of the VLDL/HDL3 ratio of each lid chimera compared with its parental lipase (EL, LPL, or LPL/EL-Cdom) was determined using an unpaired t-test. Two means were determined to be statistically not significant (NS) at P > 0.05. Using HDL3 as a substrate, we further studied the kinetics by measuring the amount of FFA generated after incubation of the lipase with different concentrations of HDL3 PLs. By fitting these experimental data, we estimated the apparent affinity of each of these enzymes (appKm) for HDL3 as substrate (Table 5). Wild-type EL showed an affinity for HDL3 of 0.18 mM. The apparent affinity for HDL3 of the EL/LPL-lid chimera remained similar to that of wild-type EL. Wild-type LPL showed an apparent affinity for HDL3 lipids of 0.81 mM. This result demonstrates that the affinity of LPL for HDL3 is substantially lower than the affinity of EL in vitro. In the case of the LPL/EL-lid chimera, the affinity for HDL3" @default.
• W1982450156 created "2016-06-24" @default.
• W1982450156 creator A5012223927 @default.
• W1982450156 creator A5014895058 @default.
• W1982450156 creator A5015029489 @default.
• W1982450156 creator A5026618894 @default.
• W1982450156 creator A5032720180 @default.
• W1982450156 creator A5067149045 @default.
• W1982450156 creator A5083757642 @default.
• W1982450156 date "2006-08-01" @default.
• W1982450156 modified "2023-09-23" @default.
• W1982450156 title "Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras" @default.
• W1982450156 cites W1490629978 @default.
• W1982450156 cites W1520339390 @default.
• W1982450156 cites W1520403560 @default.
• W1982450156 cites W1568019615 @default.
• W1982450156 cites W1587176444 @default.
• W1982450156 cites W1939640254 @default.
• W1982450156 cites W1967911168 @default.
• W1982450156 cites W1993613154 @default.
• W1982450156 cites W1997802367 @default.
• W1982450156 cites W1998591058 @default.
• W1982450156 cites W1999110412 @default.
• W1982450156 cites W2004724549 @default.
• W1982450156 cites W2007697108 @default.
• W1982450156 cites W2012176370 @default.
• W1982450156 cites W2013321895 @default.
• W1982450156 cites W2034308910 @default.
• W1982450156 cites W2039213421 @default.
• W1982450156 cites W2046346838 @default.
• W1982450156 cites W2049082732 @default.
• W1982450156 cites W2053081403 @default.
• W1982450156 cites W2057444257 @default.
• W1982450156 cites W2081817480 @default.
• W1982450156 cites W2093475422 @default.
• W1982450156 cites W2098469731 @default.
• W1982450156 cites W2099423532 @default.
• W1982450156 cites W2103933115 @default.
• W1982450156 cites W2105724958 @default.
• W1982450156 cites W2153637092 @default.
• W1982450156 cites W2154750450 @default.
• W1982450156 cites W2189314725 @default.
• W1982450156 cites W2220000844 @default.
• W1982450156 cites W2339303830 @default.
• W1982450156 doi "https://doi.org/10.1194/jlr.m500552-jlr200" @default.
• W1982450156 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16682746" @default.
• W1982450156 hasPublicationYear "2006" @default.
• W1982450156 type Work @default.
• W1982450156 sameAs 1982450156 @default.
• W1982450156 citedByCount "47" @default.
• W1982450156 countsByYear W19824501562012 @default.
• W1982450156 countsByYear W19824501562013 @default.
• W1982450156 countsByYear W19824501562014 @default.
• W1982450156 countsByYear W19824501562015 @default.
• W1982450156 countsByYear W19824501562016 @default.
• W1982450156 countsByYear W19824501562017 @default.
• W1982450156 countsByYear W19824501562018 @default.
• W1982450156 countsByYear W19824501562019 @default.
• W1982450156 countsByYear W19824501562020 @default.
• W1982450156 countsByYear W19824501562021 @default.
• W1982450156 countsByYear W19824501562022 @default.
• W1982450156 countsByYear W19824501562023 @default.
• W1982450156 crossrefType "journal-article" @default.
• W1982450156 hasAuthorship W1982450156A5012223927 @default.
• W1982450156 hasAuthorship W1982450156A5014895058 @default.
• W1982450156 hasAuthorship W1982450156A5015029489 @default.
• W1982450156 hasAuthorship W1982450156A5026618894 @default.
• W1982450156 hasAuthorship W1982450156A5032720180 @default.
• W1982450156 hasAuthorship W1982450156A5067149045 @default.
• W1982450156 hasAuthorship W1982450156A5083757642 @default.
• W1982450156 hasBestOaLocation W19824501561 @default.
• W1982450156 hasConcept C104317684 @default.
• W1982450156 hasConcept C181199279 @default.
• W1982450156 hasConcept C185592680 @default.
• W1982450156 hasConcept C18903297 @default.
• W1982450156 hasConcept C2777289219 @default.
• W1982450156 hasConcept C2779697368 @default.
• W1982450156 hasConcept C2994592520 @default.
• W1982450156 hasConcept C30278631 @default.
• W1982450156 hasConcept C55493867 @default.
• W1982450156 hasConcept C86803240 @default.
• W1982450156 hasConcept C94879076 @default.
• W1982450156 hasConceptScore W1982450156C104317684 @default.
• W1982450156 hasConceptScore W1982450156C181199279 @default.
• W1982450156 hasConceptScore W1982450156C185592680 @default.
• W1982450156 hasConceptScore W1982450156C18903297 @default.
• W1982450156 hasConceptScore W1982450156C2777289219 @default.
• W1982450156 hasConceptScore W1982450156C2779697368 @default.
• W1982450156 hasConceptScore W1982450156C2994592520 @default.
• W1982450156 hasConceptScore W1982450156C30278631 @default.
• W1982450156 hasConceptScore W1982450156C55493867 @default.
• W1982450156 hasConceptScore W1982450156C86803240 @default.
• W1982450156 hasConceptScore W1982450156C94879076 @default.
• W1982450156 hasIssue "8" @default.
• W1982450156 hasLocation W19824501561 @default.
• W1982450156 hasOpenAccess W1982450156 @default.
• W1982450156 hasPrimaryLocation W19824501561 @default.
• W1982450156 hasRelatedWork W2014956217 @default.

• next