Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

NMR observation of gramicidin A′ in phosphatidylcholine vesicles

Dimyristoyl phosphatidylcholine was prepared with perdeuterated hydrocarbon chains and sonicated into bilayer vesicles together with gramicidin A′. The ¹H NMR resonance from the tryptophan residues in the gramicidin has a linewidth of approximately 80 Hz, indicating significant local mobility for these residues. Paramagnetic lanthanides added to the aqueous medium cause a chemical shift of this signal indicating that some of the tryptophans may be located in the interfacial region of the bilayer.     

Deepening our understanding of HDL proteome

ABSTRACT

Introduction: High-density lipoprotein (HDL) particles are heterogeneous and their proteome is complex and distinct from HDL cholesterol. However, it is largely unknown whether HDL proteins are associated with cardiovascular protection.

Areas covered: HDL isolation techniques and proteomic analyses are reviewed. A list of HDL proteins reported in 37 different studies was compiled and the effects of different isolation techniques on proteins attributed to HDL are discussed. Mass spectrometric techniques used for HDL analysis and the need for precise and robust methods for quantification of HDL proteins are discussed.

Expert opinion: Proteins associated with HDL have the potential to be used as biomarkers and/or help to understand HDL functionality. To achieve this, large cohorts must be studied using precise quantification methods. Key factors in HDL proteome quantification are the isolation methodology and the mass spectrometry technique employed. Isolation methodology affects what proteins are identified in HDL and the specificity of association with HDL particles needs to be addressed. Shotgun proteomics yields imprecise quantification, but the majority of HDL studies relied on this approach. Few recent studies used targeted tandem mass spectrometry to quantify HDL proteins, and it is imperative that future studies focus on the application of these precise techniques.     

The effect of the bilayer phase transition on the carbonyl carbon-13 chemical shift anisotropy

Proton enhanced (PE), natural abundance carbon-13 magnetic resonance spectra have been obtained of the carbonyl groups in hydrated dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. A four-fold change in the overall linewidth results on passing from the fluid to crystalline phase. The carbonyl resonance provides a sensitive measure of the changes in mobility experienced by the lipid molecule above and below the phase transition temperature. The spectral shapes derived from both the fluid (T = 45°C) and crystalline (T = 15°C) phases indicate that even in the crystalline phase sufficient molecular motion is present to average the chemical shielding tensor. It is suggested that this motion in the L_β′ phase is a result of dislocations and packing faults diffusing in the plane of the bilayer. Because of the small size of the chemical shielding interaction (approx. 3 kHz for ω₀ = 22.63 MHz) lipid diffusion coefficients of order 10^?10 cm²/sec observed in the L_β′ phase [1] are effective in averaging the shielding tensor.A comparison is made with the perturbation suffered by the carbonyl groups in the L_α phase in the presence of substantial amounts of cholesterol or the polypeptide gramicidin A.     

Predictive value of different HDL particles for the protection against or risk of coronary heart disease

The inverse relationship between plasma HDL levels and the risk of developing coronary heart disease is well established. The underlying mechanisms of this relationship are poorly understood, largely because HDL consist of several functionally distinct subpopulations of particles that are continuously being interconverted from one to another. This review commences with an outline of what is known about the origins of individual HDL subpopulations, how their distribution is regulated, and describes strategies that are currently available for isolating them. We then summarise what is known about the functionality of specific HDL subpopulations, and how these findings might impact on cardiovascular risk. The final section highlights major gaps in existing knowledge of HDL functionality, and suggests how these deficiencies might be addressed. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion

Small-angle neutron scattering (SANS) with contrast variation was used to obtain the low-resolution structure of nascent HDL (nHDL) reconstituted with dimyristoyl phosphatidylcholine (DMPC) in the absence and presence of cholesterol, [apoA1:DMPC (1:80, mol:mol) and apoA1:DMPC:cholesterol (1:86:9, mol:mol:mol)]. The overall shape of both particles is discoidal with the low-resolution structure of apoA1 visualized as an open, contorted, and out of plane conformation with three arms in nascent HDL/dimyristoyl phosphatidylcholine without cholesterol (nHDL_DMPC) and two arms in nascent HDL/dimyristoyl phosphatidylcholine with cholesterol (nHDL_DMPC+Chol). The low-resolution shape of the lipid phase in both nHDL_DMPC and nHDL_DMPC+Chol were oblate ellipsoids, and fit well within their respective protein shapes. Modeling studies indicate that apoA1 is folded onto itself in nHDL_DMPC, making a large hairpin, which was also confirmed independently by both cross-linking mass spectrometry and hydrogen-deuterium exchange (HDX) mass spectrometry analyses. In nHDL_DMPC+Chol, the lipid was expanded and no hairpin was visible. Importantly, despite the overall discoidal shape of the whole particle in both nHDL_DMPC and nHDL_DMPC+Chol, an open conformation (i.e., not a closed belt) of apoA1 is observed. Collectively, these data show that full length apoA1 retains an open architecture that is dictated by its lipid cargo. The lipid is likely predominantly organized as a bilayer with a micelle domain between the open apoA1 arms. The apoA1 configuration observed suggests a mechanism for accommodating changing lipid cargo by quantized expansion of hairpin structures.     

Differential Stability of High-density Lipoprotein Subclasses: Effects of Particle Size and Protein Composition

High-density lipoproteins (HDLs) are complexes of proteins (mainly apoA-I and apoA-II) and lipids that remove cholesterol and prevent atherosclerosis. Understanding the distinct properties of the heterogeneous HDL population may aid the development of new diagnostic tools and therapies for atherosclerosis. Mature human HDLs form two major subclasses differing in particle diameter and metabolic properties, HDL₂ (large) and HDL₃ (small). These subclasses are comprised of HDL(A-I) containing only apoA-I, and HDL(A-I/A-II) containing apoA-I and apoA-II. ApoA-I is strongly cardioprotective, but the function of the smaller, more hydrophobic apoA-II is unclear. ApoA-II is thought to counteract the cardioprotective action of apoA-I by stabilizing HDL particles and inhibiting their remodeling. To test this notion, we performed the first kinetic stability study of human HDL subclasses. The results revealed that the stability of plasma spherical HDL decreases with increasing particle diameter; which may facilitate preferential cholesterol ester uptake from large lipid-loaded HDL₂. Surprisingly, size-matched plasma HDL(A-I/A-II) showed comparable or slightly lower stability than HDL(A-I); this is consistent with the destabilization of model discoidal HDL observed upon increasing the A-II to A-I ratio. These results clarify the roles of the particle size and protein composition in HDL remodeling, and help reconcile conflicting reports regarding the role of apoA-II in this remodeling.     

Functional group contributions to the partitioning of phenols between liposomes and water

The temperature dependency of the partitioning of p-alkylphenols and p-halophenols has been determined between dimyristoyl phosphatidylcholine liposomes and 0.15 M NaCl. Partition coefficients increased as a function of temperature below the endothermic phase transition temperature (T_c) of the phospholipid but decreased above this temperature. The transfer process was found to be entropy-dominated below and enthalpy-dominated above the T_c, although large negative entropy changes were observed. Regular changes in the thermodynamic functions, partition coefficients and functional group free energies occurred as a function of the alkyl chain length or size of the halogen substituent below but not above the T_c. This has tentatively been attributed to increased phenol-phospholipid interaction at the higher temperatures. The partitioning of p-fluorophenol behaved in a manner expected of fluorinated compounds, yielding relatively low partition coefficients, but it produced an additional effect of markedly lowering the T_c of dimyristoyl phosphatidylcholine. Good correlations of the partition coefficients in liposomes with those in bulk organic solvents and with molecular size of the solute have been obtained.     

卵磷脂胆固醇酰基转移酶缺乏综合征

卵磷脂胆固醇酰基转移酶（ｌｅｃｉｔｈｉｎｃｈｏｌｅｓｔｅｒｏｌａｃｙｌｔｒａｎｓｆｅｒａｓｅ,ＬＣＡＴ）缺乏综合征是一组独特的有关ＨＤＬ代谢的遗传性疾病,由ＬＣＡＴ基因的自发突变所致。近几年来,这方面的研究很多,特别是与动脉粥样硬化之间的关系引起人们的广泛关注。１．ＬＣＡＴ基因与蛋白质人类ＬＣＡＴ基因位于１６ｑ２１－２２区,含有４．２ｋｂ的碱基对,主要在肝脏表达。其成熟蛋白质分子量为４７．０９０ｋＤ,除含有一段极其疏水的多肽外,还含有４个Ｎ－连接的糖基化作用位点,这些位点的消除对酶的活性影响很大…     

Isoforms of apolipoprotein C-I associated with individuals with coronary artery disease

Apolipoprotein C-I (apoC-I) is a 6.6 kDa serum protein associated with high density lipoproteins (HDL) and triglyceride-rich lipoproteins. In this study, apoC-I was examined in high density lipoprotein subfractions from individuals with and without coronary artery disease (CAD). New isoforms of apoC-I, were detected in the cohort of individuals with CAD using mass spectrometry while the expected apoC-I isoforms were absent. In addition, the apoC-I mass spectra for the CAD cohort had satellite peaks indicative of the involvement of oxidative processes. Further analysis of the mass spectra of the CAD and non-CAD cohorts suggest that the origin of these new isoforms may be due to genetic mutations that could compromise the function of apoC-I.     

Rapid purification and activity of apolipoprotein CI on the proliferation of bovine vascular endothelial cells in vitro

The growth-promoting activity of human high-density lipoproteins (HDL) and of their apolipoprotein components on bovine vascular endothelial cells in vitro has been compared. When maintained on plastic culture dishes and exposed to medium containing lipoprotein-deficient serum and fibroblast growth factor, these cells do not proliferate. Addition of either HDL or the total HDL apolipoproteins induces significant cell proliferation. Apolipoprotein C_I, purified by chromatography on the ion-exchanger resin Polybuffer exchanger 94, has an effect on the cell growth similar to that of the total apolipoproteins of HDL.     

Volumetric determination of apolipoprotein stoichiometry of circulating HDL subspecies

Although HDL is inversely correlated with coronary heart disease, elevated HDL-cholesterol is not always protective. Additionally, HDL has biological functions that transcend any antiatherogenic role: shotgun proteomics show that HDL particles contain 84 proteins (latest count), many correlating with antioxidant and anti-inflammatory properties of HDL. ApoA-I has been suggested to serve as a platform for the assembly of these protein components on HDL with specific functions - the HDL proteome. However, the stoichiometry of apoA-I in HDL subspecies is poorly understood. Here we use a combination of immunoaffinity chromatography data and volumetric analysis to evaluate the size and stoichiometry of LpA-I and LpA-I,A-II particles. We conclude that there are three major LpA-I subspecies: two major particles, HDL[4] in the HDL₃ size range (d = 85.0 ± 1.2 Å) and HDL[7] in the HDL₂ size range (d = 108.5 ± 3.8 Å) with apoA-I stoichiometries of 3 and 4, respectively, and a small minor particle, HDL[1] (d = 73.8 ± 2.1Å) with an apoA-I stoichiometry of 2. Additionally, we conclude that the molar ratio of apolipoprotein to surface lipid is significantly higher in circulating HDL subspecies than in reconstituted spherical HDL particles, presumably reflecting a lack of phospholipid transfer protein in reconstitution protocols.     

Lipid-substituted cytochrome oxidase: no absolute requirement of cardiolipin for activity.

The endogenous lipid of yeast cytochrome oxidase has been replaced by dimyristoyl phosphatidylcholine. Thin layer chromatography of the total lipid extract from the substituted enzyme revealed phosphatidylcholine only and no cardiolipin. Gas-liquid chromatography showed that >99% of the lipid chains derived from the substituted lipid, and that cardiolipin must be <0.03 mole/mole enzyme. The activity of the lipid-substituted enzyme was 10% of the original activity and increased to 47% by addition of dimyristoyl phosphatidylcholine. Thus there is no absolute requirement of cardiolipin for oxidative activity.     

VLDL best predicts aortic root atherosclerosis in LDL receptor deficient mice

Hyperlipidemia is a major risk factor for developing atherosclerosis in humans, and epidemiological studies have correlated specific lipoprotein levels with cardiovascular disease risk. Murine models of atherosclerosis rely on the induction of hyperlipidemia for vascular lesions to form, but the pathogenic contributions attributed to different lipoprotein populations are not well defined. To address this issue, we analyzed over 300 LDL receptor (LDLR) deficient mice that have been fed a high-fat diet and for which a full lipoprotein profile and aortic root atherosclerosis values were assessed. Overall, aortic root atherosclerosis is best predicted by plasma VLDL cholesterol levels with less predictive value derived from either LDL or HDL cholesterol. Triglyceride levels are more atherogenic in female mice, especially immune competent females, and depletion of the adaptive immune system leads to a global reduction in plasma lipid levels and aortic root lesion size yet does not appear to alter the atherogenic potential of individual lipoprotein subspecies. In contrast, HDL-cholesterol is a better predictor of aortic root atherosclerosis in apoE-deficient mice. In summary, this large scale analysis of high-fat diet fed LDLR deficient mice highlight the relationship between different plasma lipid components, especially VLDL-cholesterol, and aortic root atherosclerosis.     

健脾方抗衰老作用的实验研究

中医学认为肾气和脾胃虚衰是人衰老的主要原因之一,而益肾健脾胃为培原固本养生的重要治则。我们应用健脾的中西药组方分组进行老龄Wistar大鼠的实验冶疗。实验结果表明:健脾方能升高SOD(P<0.001),降低血清总胆固醇(P<0.05)增加HDL(г<0.001),和升高HDL/TC比值的作用,并且提高大鼠的耐乏氧(P<0.05)和抗疲劳(P<(?).01)的功能。维生素E则有升高SOD(P<(?).001)和HDL(г<0.001)并提高耐乏氧的作用。腔睥胃有抗衰老的功效。     

一次密度梯度超速离心分离人血清四种主要脂蛋白的某些理化性质的研究

 本文对一次密度梯度超速离心获得的四种脂蛋白(VLDL、LDL、HDL_2、HDL_3)进行了理化性质的研究。超速离心分析LDL、HDL_2,HDL_3均呈现一个单一尖锐的上浮峰,上浮速率分别为S_1 6.9和F~0_(1.21) 5.7及2.6。等电点聚焦电泳显示,VLDL主要含载脂蛋白C族,E族和少量A-I,B。HDL_2、HDL_3二者载脂蛋白的种类很相似,但量上略有差异,均以载脂蛋白A-Ⅰ,A-Ⅱ为主,Apoc’s,E次之。VLDL、LDL、HDL_2和HDL_3的化学组分分析与文献报道相似。 作者用本法初步分析了不同性别的各脂蛋白分布图,获得有意义的结果。