BBA - Molecular and Cell Biology of Lipids (v.1484, #1)
Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source by A.F Ayyobi; A.G Lacko; K Murray; M Nair; M Li; H.O.F Molhuizen; P.H Pritchard (1-13).
Lecithin:cholesterol acyltransferase (LCAT) is an important plasma glycoprotein which plays a central role in lipid metabolism. This protein is responsible for generation of cholesteryl esters in plasma and it has been proposed to play a pivotal role in the reverse cholesterol transport pathway. Structural and functional studies of LCAT have employed various expression systems for production of recombinant LCAT (rLCAT). However, recent studies have shown some differences in the oligosaccharide structure and composition of rLCAT. In this study, we have generated a new hepatic based expression system using McArdle-RH7777 (Mc-7777) cells to produce a recombinant protein most similar to human plasma LCAT. The expressed glycoprotein was compared to the LCAT expressed in previously characterized baby hamster kidney (BHK) cells. Both proteins were compared on the basis of their carbohydrate structure and composition as well as their functional properties. Although the functional properties of both glycoproteins were similar, the carbohydrate structure was significantly different. While BHK-LCAT contained bi-, tri-, and tetraantennary structures, Mc-7777 LCAT presented only biantennary oligosaccharide structures. The difference in glycosylation pattern of rLCAT from Mc-7777 and BHK cells underlines the importance of appropriate expression system, both in vivo and in vitro.
Keywords: Lecithin:cholesterol acyltransferase; Reverse cholesterol transport; Carbohydrate structure; Enzyme kinetics; Nuclear magnetic resonance; Protein expression;
Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study by Alexander D. Dergunov; Elizaveta A. Smirnova; Aksam Merched; Sophie Visvikis; Gerard Siest; Vladimir V. Yakushkin; Vladimir Tsibulsky (14-28).
Slow refolding of human apolipoprotein E (apoE) in solution after guanidine- or cholate-induced denaturation followed by dialysis under controlled conditions was investigated using various spectroscopic properties of fluorescein- and dansyl-labeled apolipoprotein molecules. The results suggest that the last phase(s) of apoE refolding in solution include a slow (several hours at 24°C) interconversion of a self-associated ‘open’ conformer into a more dense ‘closed’ conformer. The hydrophobic interactions are primarily responsible for the formation of this more compact apoE structure. To visualize the contribution of apolipoprotein conformation and/or the number of ‘active’ lipid-bound apoE molecules in the reaction of binding to the low density lipoprotein receptor (LDLr) by solid-phase binding assay, the complexes of human plasma apolipoprotein or recombinant (rec) apoE3 with dipalmitoylphosphatidylcholine (DPPC) or palmitoyloleoylphosphatidylcholine (POPC) varying in size were used. For seven complexes with plasma protein (four DPPC and three POPC complexes), the final phosphatidylcholine (PC)/protein mole ratio ranged from 117 to 279; affinity constant K a averaged for both PCs and plotted against this ratio abruptly increased from 3.8×107 to 3.8×108 M−1 with a transition midpoint of 150–180 PC/apoE, mole ratio. Two DPPC complexes with rec protein bind much more efficiently. Complexes with both plasma and rec apoE were able to compete with very low density lipoproteins (VLDL) or low density lipoproteins (LDL) isolated from patients with E3/3 phenotype, for binding to the LDLr. Again, the competition efficiency abruptly increased at the increase in PC content with a transition midpoint of 130 PC/apoE, mole ratio. The transitions observed both in direct and competitive binding assay probably correspond to the abrupt increase in the number of ‘active’ apoE molecules on the complex surface accompanying the change in the size and/or in the shape of the complexes. The efficiency of apoE and apoB as the corresponding major ligands in the binding reaction of VLDL and LDL to the LDL receptor was compared. VLDL bind to LDLr following a simple encounter complex model, while LDL binding was characterized by a more complex two-step model with an additional isomerization step. The analysis of the binding data led us to suggest the existence of the continuum from several (2–3) apoE molecules on the surface of TG-rich particles that resulted in the increased binding affinity, on average 3.5-fold higher, compared to LDL. The existence of a complex equilibrium between aqueous and different lipid-bound forms of apoE is proposed, in particular, the formation of a transient disc-lipoprotein particle structure during the interaction with LDLr in vivo as well as in LPL-stimulated lipolysis of the lipid phase of the particle.
Keywords: Apolipoprotein B; Apolipoprotein E; Very low density lipoprotein; Low density lipoprotein; Low density lipoprotein receptor; Protein structure and refolding; Self-association; Hydrophobic interaction;
Structural peculiarities of the binding of very low density lipoproteins and low density lipoproteins to the LDL receptor in hypertriglyceridemia: role of apolipoprotein E by Alexander D. Dergunov; Elizaveta A. Smirnova; Aksam Merched; Sophie Visvikis; Gerard Siest; Vladimir V. Yakushkin; Vladimir Tsibulsky (29-40).
Very low (VLDL) and low density lipoproteins (LDL) were isolated from plasma of patients with the E3/3 phenotype which were divided into three groups based on their plasma triglyceride content: low (TG<200 mg/dl, TGl), intermediate (200<300 mg/dl, TGi) and high triglyceride content (TG>300 mg/dl, TGh). The protein density (PD) on the VLDL and LDL surface was calculated from lipoprotein composition and protein location was studied by tryptophan fluorescence quenching by I− anions at 25°C and 40°C. A comparison of the TGh with the TGl group revealed a significant (<0.05) increase of the PD parameter as much as 21% for VLDL, but not for LDL where this parameter did not change for any group; generally, PD(LDL) values were 3.2–3.8-fold lower than PD(VLDL). In accordance with this difference, the tryptophan accessibility f in VLDL vs. LDL was lower at both temperatures. There were temperature-induced changes of the f parameter in opposite directions for these lipoproteins. The difference in f value gradually decreased for VLDL in the direction TGl>TGi>TGh while for LDL there was a U-shaped dependence for these groups. The Stern-Volmer quenching constant K S-V which is sensitive to both temperature and viscosity, did not change for VLDL, but K S-V(LDL) was 2–3-fold higher for the TGi group compared to the other two. The efficiencies of VLDL and LDL binding to the LDL receptor (LDLr) in vitro were compared by solid-phase assay free of steric hindrance observed in cell binding. The maximal number of binding sites did not change for either type of particles and between groups. The association constant K a and apolipoprotein (apo) E/apoB mole ratio values all increased significantly for VLDL, but not for LDL, in comparison of the TGi+h with the TGl group. Based on VLDL and LDL concentrations in serum and on the affinity constant values obtained in an in vitro assay, VLDL concentrations corresponding to 50% inhibition of LDL binding (IC50) were calculated in an assumption of the competition of both ligands for LDLr in vivo; the mean values of IC50 decreased 2-fold when plasma TG exceeded 200 mg/dl. The functional dependences of K a(VLDL), IC50 and apoE content in VLDL (both fractional and absolute) and in serum on TG content in the whole concentration range studied were fitted to a saturation model. For all five parameters, the mean half-maximum values TG1/2 were in the range 52–103 mg/dl. The efficiency of protein-protein interactions is suggested to differ in normolipidemic vs. HTG-VLDL and apoE content and/or protein density on VLDL surface may be the primary determinant(s) of the increased binding of HTG-VLDL to the LDL receptor. ApoCs may compete with apoE for the binding to the VLDL lipid surface as plasma triglyceride content increases. The possible competition of VLDL with LDL for the catabolism site(s) in vivo, when plasma TG increases, could explain the atherogenic action of TG-rich lipoproteins. Moreover, the ‘dual action’ hypothesis on anti-atherogenic action of apoE-containing high density lipoproteins (HDL) in vivo is suggested: besides the well-known effect of HDL as cholesteryl ester catabolic outway, the formation of a transient complex of apoE-containing discs appearing at the site of VLDL TG hydrolysis by lipoprotein lipase with VLDL particles proposed in our preceding paper promotes the efficient uptake of TG-rich particles; in hypertriglyceridemia due to the diminished HDL content this uptake seems to be impaired which results in the increased accumulation of the remnants of TG-rich particles. This explains the observed increase in cholesterol and triglyceride content in VLDL and LDL, respectively, due to the CETP-mediated exchange of cholesteryl ester and triglyceride molecules between these particles.
Keywords: Apolipoprotein B; Apolipoprotein E; Hypertriglyceridemia; Very low density lipoprotein; Low density lipoprotein; Low density lipoprotein receptor;
Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin, hyperphagia, increased tissue lipid content, and TNF-α by Riaz A. Memon; Gökhan S. Hotamisligil; Sarah M. Wiesbrock; K.Teoman Uysal; Raffaella Faggioni; Arthur H. Moser; Kenneth R. Feingold; Carl Grunfeld (41-50).
Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-α in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-α has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-α.
Keywords: Uncoupling protein 2; Uncoupling protein 3; Energy expenditure; Leptin; Tumor necrosis factor α; Obesity;
LTA4-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB4 receptor of human polymorphonuclear leukocytes by Jean-Pierre Falgueyret; Denis Riendeau (51-58).
5-Oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A4 (LTA4) in addition to 5,12-dihydroxy-(6E,8E,10E,14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E,11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA4 was found to be pH-dependent. After incubation of LTA4 in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA4 in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC50 of 250 nM, as compared to values of 3.5 nM for leukotriene B4 (LTB4) and >500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB4 totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB4. The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB4 receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB4 receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB4.
Keywords: 5-Oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid; Leukotriene A4; Leukotriene B4; Leukotriene B4 receptor; Calcium mobilization;
Analysis of sulfatide from rat cerebellum and multiple sclerosis white matter by negative ion electrospray mass spectrometry by Beth Noelle Marbois; Kym F. Faull; Arvan L. Fluharty; Sujna Raval-Fernandes; Leonard H. Rome (59-70).
The accumulation of sulfatide (sulfatogalactosyl cerebroside) and changes in the sulfatide species present have been examined in the cerebellum of day 6–32 aged rats and in multiple sclerosis (MS) tissue samples. Negative ion electrospray mass spectrometry with daughter and parent ion analyses were used to distinguish the fatty acyl character in the amide linkage of sulfatide; measurement was done by selected ion and multiple reaction monitoring of individually identified sulfatide molecules. Sulfatide accumulation in rat cerebellum shows that 18:0- and hydroxylated 18:0-sulfatide are the first sulfatide molecules detectable. Very long fatty acyl chain sulfatide molecules (>20:0) are present at day 7 and the ratio of non-hydroxylated compared to hydroxylated sulfatide rises as the amount of non-hydroxylated sulfatide increases. 24:1-sulfatide accumulates at a ratio of about 3:1 over 24:0-sulfatide during active myelination. Analyses of the sulfatide in human tissue have shown differences between MS plaque tissues, normal appearing adjacent white matter and control tissues. The findings show that total sulfatide is reduced by 60% in the plaque matter and decreased 25% in adjacent normal appearing white matter. There are significant increases (P=0.05) in the amount of hydroxylation of sulfatide, demonstrated by an increase in the percentage of hydroxylated h24:0-sulfatide (hydroxy-lignoceroyl sulfatide).
Keywords: Sulfatide; Brain development; Myelination; Multiple sclerosis; Glycolipid; Electrospray mass spectrometry;
Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae 1 1 This work was supported in part by United States Public Health Service, National Institutes of Health Grant GM-28140. by Jamie M Furneisen; George M Carman (71-82).
The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-associated enzyme that catalyzes the Mg2+-independent dephosphorylation of phosphatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA). The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf-9 insect cells and used to examine the enzymological properties of the enzyme using PA, DGPP, and LPA as substrates. The optimum pH values for PA phosphatase, DGPP phosphatase, and LPA phosphatase activities were 7.5, 7.0, and 7.0, respectively. Divalent cations (Mn2+, Co2+, and Ca2+), NaF, heavy metals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA phosphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme. The inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-encoded enzyme were novel properties when compared with other Mg2+-independent lipid phosphate phosphatases from S. cerevisiae and mammalian cells. The LPP1-encoded enzyme exhibited saturation kinetics with respect to the surface concentrations of PA (K m=0.05 mol%), DGPP (K m=0.07 mol%), and LPA (K m=0.08 mol%). Based on specificity constants (V max/K m), the order of substrate preference was PA (4.2 units/mg/mol%)>DGPP (3.5 units/mg/mol%)>LPA (1.3 units/mg/mol%). DGPP (K i=0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K i=0.12 mol%) was a competitive inhibitor with respect to DGPP. This suggested that the binding sites for these substrates were the same. The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S. cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as substrates.
Keywords: LPP1-encoded lipid phosphatase; Phosphatidate; Diacylglycerol pyrophosphate; Lysophosphatidate; Yeast; Phospholipid;