Human Lipoprotein Research Articles

Apolipoprotein A-I Truncations in Chagas Disease Are Caused by Cruzipain, the Major Cysteine Protease of Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent of Chagas disease. Approximately 10 million people are infected worldwide. We have previously reported that in individuals infected with T. cruzi, apolipoprotein A-I (Apo A-I), the major structural component of host high-density lipoprotein, was truncated into fragments that are specific to Chagas disease and have the potential to be used as diagnostic biomarkers. We investigated the possibility that cruzipain, the major cysteine protease of T. cruzi, is responsible for truncating host Apo A-I. We found that due to Apo A-I truncation, the high-density lipoprotein subspecies profile is altered in individuals with Chagas disease compared with healthy controls. Western blot analysis revealed that both purified Apo A-I protein and Apo A-I in the high-density lipoprotein complex were susceptible to cruzipain cleavage, and the sizes of the truncation product in the latter matched the sizes of Apo A-I biomarkers. We also found that in vitro feeding T. cruzi-infected differentiated human adipocytes with purified human high-density lipoprotein led to the appearance of the biomarker fragments of Apo A-I. Cruzipain is found both on the cytoplasmic membrane and in the internal lysosomal structure of T. cruzi. We demonstrate that cruzipain from both sources contributes to the production of Apo A-I truncation in the biomarker set.

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.

Proteome of human plasma very low-density lipoprotein and low-density lipoprotein exhibits a link with coagulation and lipid metabolism

Apart from transporting lipids through the body, the human plasma lipoproteins very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) are also thought to serve as a modality for intra-organismal protein transfer, shipping proteins with important roles in inflammation and thrombosis from the site of synthesis to effector locations. To better understand the role of VLDL and LDL in the transport of proteins, we applied a combination of LTQ ORBITRAP-XL (nLC-MS/MS) with both in-SDS-PAGE gel and in-solution tryptic digestion of pure and defined VLDL and LDL fractions. We identified the presence of 95 VLDL- and 51 LDL-associated proteins including all known apolipoproteins and lipid transport proteins, and intriguingly a set of coagulation proteins, complement system and anti- microbial proteins. Prothrombin, protein S, fibrinogen γ, PLTP, CETP, CD14 and LBP were present on VLDL but not on LDL. Prenylcysteine oxidase 1, dermcidin, cathelicidin antimicrobial peptide, TFPI-1 and fibrinogen α chain were associated with both VLDL and LDL. Apo A-V is only present on VLDL and not on LDL. Collectively, this study provides a wealth of knowledge on the protein constituents of the human plasma lipoprotein system and strongly supports the notion that protein shuttling through this system is involved in the regulation of biological processes. Human diseases related to proteins carried by VLDL and LDL can be divided in three major categories: 1 - dyslipidaemia, 2 - atherosclerosis and vascular disease, and 3 - coagulation disorders.

Cognitive Deficits and Disruption of Neurogenesis in a Mouse Model of Apolipoprotein E4 Domain Interaction

Apolipoprotein E4 (apoE4) allele is the major genetic risk factor for sporadic Alzheimer disease (AD) due to the higher prevalence and earlier onset of AD in apoE4 carriers. Accumulating data suggest that the interaction between the N- and the C-terminal domains in the protein may be the main pathologic feature of apoE4. To test this hypothesis, we used Arg-61 mice, a model of apoE4 domain interaction, by introducing the domain interaction feature of human apoE4 into native mouse apoE. We carried out hippocampus-dependent learning and memory tests and related cellular and molecular assays on 12- and 3-month-old Arg-61 and age-matched background C57BL/6J mice. Learning and memory task performance were impaired in Arg-61 mice at both old and young ages compared with C57BL/6J mice. Surprisingly, young Arg-61 mice had more mitotic doublecortin-positive cells in the subgranular zone; mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB were also higher in 3-month-old Arg-61 hippocampus compared with C57BL/6J mice. These early-age neurotrophic and neurogenic (proliferative) effects in the Arg-61 mouse may be an inadequate compensatory but eventually detrimental attempt by the system to "repair" itself. This is supported by the higher cleaved caspase-3 levels in the young animals that not only persisted, but increased in old age, and the lower levels of doublecortin at old age in the hippocampus of Arg-61 mice. These results are consistent with human apoE4-dependent cognitive and neuro-pathologic changes, supporting the principal role of domain interaction in the pathologic effect of apoE4. Domain interaction is, therefore, a viable therapeutic/prophylactic target for cognitive impairment and AD in apoE4 subjects.

Human endothelial progenitor cells internalize high-density lipoprotein.

Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of “strings of pearl”- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568–treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular pathway and accumulated prominently in all parts of the Golgi apparatus and in lipid droplets. Subsequently, also the RER and mitochondria were involved. These studies demonstrated the different intracellular pathway of HDL-derived bodipy-cholesterol and HDL-derived bodipy-cholesteryl oleate by EPCs, with concomitant.

Separation of total lipids on human lipoproteins using surfactant‐coated multiwalled carbon nanotubes as pseudostationary phase in capillary electrophoresis

Surfactant-coated multiwalled carbon nanotubes (MWNTs) were used as pseudostationary phase (PSP) in CE to investigate the total lipids of high-density lipoproteins and low-density lipoproteins. To optimize the CE conditions, several experimental factors including carbon nanotube concentration, bile salt concentration, sodium phosphate (PB) concentration, organic modifier concentration and buffer pH value have been examined. In addition, the CE capillary temperature and applied voltage have also been examined. The optimal separation buffer selected was a mixture of 3.2 mg/L MWNT, 50 mM bile salt, 10 mM PB, 20% 1-propanol, pH 9.5. The optimal capillary temperature and applied voltage selected were 50°C and 20 kV, respectively. Phosphatidyl choline (PC) has been used as a model analyte and investigated by the optimal CE method. The linear range for PC was 0.1-3 mg/mL with a correlation coefficient of 0.9934, and the concentration LOD was 0.055 mg/mL. The optimal CE method has been used to characterize the total lipids of high-density lipoprotein and low-density lipoprotein. At absorbance 200 nm, one major peak and two or three minor peaks showed for the total lipids of lipoproteins within 13 minutes. Resolutions of the total lipids were enhanced using surfactant-coated MWNTs as PSPs in the CE separation buffer. However, resolutions of the total lipids were not enhanced using surfactant-coated single-walled carbon nanotubes as PSPs in the CE separation buffer.

Regulation of Plasma Lipoprotein Levels in Humans: New Insights Using Methods Pioneered by Bob Levy

In the early 1970s, Robert Levy and colleagues published a series of groundbreaking papers showing, in detail, the metabolic fates of the apolipoproteins on very low density lipoproteins after intravenous injection into normal and hyperlipidemic subjects. These studies were the foundation for the field of human lipoprotein kinetics that has provided very significant insights into the pathophysiology of lipid disorders as well as mechanisms of action of lipid altering diets and drugs. Early studies used mainly exogenously radiolabeled lipoproteins, but more recently, stable isotopes of amino acids have been used to endogenously label the apolipoproteins of interest. Our group has used both approaches for more than three decades and in this lecture, I will highlight the results of several of our studies including very recent unpublished work.

Mice with chimeric livers are an improved model for human lipoprotein metabolism.

ObjectiveRodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism.DesignFRG [ F ah(−/−) R ag2(−/−)Il2r g (−/−)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR.ResultsChimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal.ConclusionHumanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.

Glycation of human high density lipoprotein by methylglyoxal: Effect on HDL-paraoxonase activity

ObjectiveMethylglyoxal (MG), a reactive carbonyl compound formed primarily from triose phosphates, appears to be involved in the molecular mechanisms of diabetes, end-stage renal disease and neurodegenerative diseases. Methylglyoxal exerts several biological activities. Among these it promotes advanced glycation end products (AGEs), which are crucial in pathogenesis of human disease. Previous studies have demonstrated that MG reacts with proteins and compositional modifications reflect loss of biological activity. The aim of the study was to investigate the effect of in vitro MG-induced glycation on human high density lipoprotein (HDL) and on the activity of the enzyme paraoxonase-1 (PON1). MethodsHDL was incubated in the absence or in the presence of MG (0.2mmol/L and 1.0mmol/L) (MG-HDL) for different times (3, 6, 24h) at 37° C. We evaluated apoprotein compositional changes, in both control and MG treated HDL, using intrinsic fluorescence of tryptophan and monitoring the decrease of free amino groups. Furthermore we evaluated fluorescent advanced glycation end products (Ex=370nm, Em=440nm) and the activity of HDL-paraoxonase. ResultsWe demonstrated that human HDL is susceptible to glycation by MG (0.2mmol/L and 1mmol/L). The decrease of free amino groups and of intrinsic fluorescence of tryptophan demonstrates HDL apoprotein modifications in HDL incubated with MG. The compositional changes are associated with a significant increase in fluorescent advanced glycation end products and with a significant decrease of paraoxonase-1 enzyme activity associated with the HDL surface. ConclusionsHDL-associated paraoxonase is responsible for the anti-inflammatory and anti-oxidative properties of HDL and detoxification against homocysteine-thiolactone. Therefore, modifications of apoprotein composition and the decrease of paraoxonase-1 activity in MG-treated HDL could affect the protective effect exerted by HDL against oxidative damage and could contribute to complications in patients affected by diseases associated with aging and oxidative stress.

Stereoselective binding of chiral drugs to plasma proteins

Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed.

Effects of cholesteryl ester transfer protein inhibitors on human lipoprotein metabolism

To examine the recent advances in our knowledge of cholesteryl ester transfer protein (CETP) inhibitors, heart disease risk reduction, and human lipoprotein metabolism. CETP inhibitors block the transfer of cholesteryl ester from HDLs to triglyceride-rich lipoproteins (TRLs), thereby raising HDL cholesterol and lowering TRL cholesterol, and in some cases LDL cholesterol. Two CETP inhibitors, dalcetrapib and torcetrapib, have been tested in large clinical trials in statin-treated coronary heart disease patients and have shown no clinical benefit compared to placebo. Anacetrapib and evacetrapib, two potent CETP inhibitors, are now being tested in large clinical trials. Torcetrapib has been shown to decrease the fractional catabolic rate (FCR) of HDL apolipoproteins (apo) A-I and A-II, enhance the FCR of TRL apoB-100 and apoE, and decrease TRL apoB-48 production, but has no significant effects on fecal cholesterol excretion in humans. Anacetrapib also delays the FCR of HDL apoA-I. CETP inhibitors form a complex between themselves, CETP, and HDL particles, which may interfere with the many physiologic functions of HDL, including reverse cholesterol transport. Available data would suggest that CETP inhibitors will fail as lipid-altering medications to reduce coronary heart disease risk because of interference with normal human HDL metabolism.

Serum amyloid A is found on ApoB‐containing lipoproteins in obese humans with diabetes

In murine models of obesity/diabetes there is an increase in plasma SAA levels along with redistribution of SAA from high density lipoprotein (HDL) to apo-B containing lipoprotein particles, namely low density lipoprotein (LDL) and very low density lipoprotein (VLDL). The goal of this study was to determine if obesity is associated with similar SAA lipoprotein redistribution in humans. Three groups of obese individuals were recruited from a weight loss clinic: healthy obese (n=14), metabolic syndrome obese (n=8) and obese with type 2 diabetes (n=6). Plasma was separated into lipoprotein fractions by fast protein liquid chromatography (FPLC) and SAA was measured in lipid fractions using enzyme-linked immunosorbent assay (ELISA) and western blotting. Only the obese diabetic group had SAA detectable in apoB-containing lipoproteins, and SAA reverted back to HDL with active weight loss. In human subjects, SAA is found in apo-B containing lipoprotein particles only in obese subjects with type 2 diabetes, but not healthy obese, or obese subjects with metabolic syndrome.

Abstract 125: Weight Loss Selectively Increases High-density Lipoprotein without apoC-III, a Subfraction with Protective Association Against Coronary Heart Disease.

Human high-density lipoprotein (HDL) particles are highly heterogeneous in composition, and in relation to coronary heart disease (CHD). Specifically, HDL containing apoC-III predicts increased incidence of CHD, whereas HDL that does not contain apoC-III predicts decreased CHD incidence. Recently, we found that obese subjects had a higher concentration of HDL with apoC-III and a lower concentration of HDL without apoC-III than normal weight controls. However, the effects of weight loss on HDL with or without apoC-III are not known. We studied 39 obese participants (mean BMI of 34) in a weight-loss trial assigned to a high fat 40%/high protein 25%/low carbohydrate 35% diet for 2 years. We chose this diet group because HDL-cholesterol (-C) levels increased the most compared to the other diet groups with more carbohydrate and less fat. Participants with at least 8% loss of initial body weight at 2 years were assigned to the weight loss group (n=21), while those without a change in weight comprised the no-weight loss group (n=18). Subjects were matched by age (mean 51 years), gender, and race. HDL without apoC-III and HDL with apoC-III were separated from plasma by sequentially using anti-apoA-I and anti-apoC-III immunoaffinity chromatography. For both groups, the mean baseline HDL-C and total plasma apoA-I concentrations were 48 and 119mg/dL, respectively. Weight loss increased HDL-C concentration by an average of 25% (p<0.01 for changes between groups), and total apoA-I by 9% (p<0.02). In the weight-loss group, mean apoA-I concentrations of HDL without apoC-III increased by12% (from 99 to 111 mg/dL) compared to a decrease of 6% in the no-weight loss group (from 107 to 101mg/dL) (p<0.02). The apoA-I concentration of HDL with apoC-III, 15 mg/dL, remained unchanged. In conclusion , moderate weight loss selectively increased the concentration of HDL without apoC-III, the predominant HDL type that predicts reduced incidence of CHD. In contrast, weight loss did not affect HDL with apoC-III, an HDL type associated with increased CHD risk. This represents a partial normalization of HDL subtypes by moderate weight loss. This effect on HDL subpopulations defined by absence of apoC-III suggests a novel mechanism by which weight loss may reduce cardiovascular disease risk.

Osteoarthritis development is induced by increased dietary cholesterol and can be inhibited by atorvastatin in APOE*3Leiden.CETP mice—a translational model for atherosclerosis

ObjectiveHypercholesterolaemia, a risk factor for atherosclerosis (ATH), has been suggested to have a role in the development of osteoarthritis (OA). To test this hypothesis, the effect of cholesterol and different...

Interleukin-6 is a major regulator of lipoprotein(a) in humans in vivo

Interleukin-6 is a major regulator of lipoprotein(a) in humans in vivo

A Single Amino Acid Substitution in the Group 1 Trypanosoma brucei gambiense Haptoglobin-Hemoglobin Receptor Abolishes TLF-1 Binding

Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.

Functional genomics of the human high-density lipoprotein receptor scavenger receptor BI

The athero-protective role of scavenger receptor BI (SR-BI) is primarily attributed to its ability to selectively transfer cholesteryl esters from high-density lipoproteins (HDLs) to the liver during reverse cholesterol transport (RCT). In this review, we highlight recent findings that reveal the impact of SR-BI on lipid levels and cardiovascular disease in humans. Moreover, additional responsibilities of SR-BI in modulating adrenal and platelet function, as well as female fertility in humans, are discussed. Heterozygote carriers of P297S, S112F and T175A-mutant SR-BI receptors were identified in patients with high HDL-cholesterol levels. HDL from P297S-SR-BI carriers was unable to mediate macrophage cholesterol efflux, whereas hepatocytes expressing P297S-SR-BI were unable to mediate the selective uptake of HDL-cholesteryl esters. S112F and T175A-mutant receptors exhibited similar impaired cholesterol transport functions in vitro. Reduced SR-BI function in P297S carriers was also associated with decreased steroidogenesis and altered platelet function. Further, human population studies identified SCARB1 variants associated with female infertility. Identification of SR-BI variants confirms the key role of this receptor in influencing lipid levels and RCT in humans. A deeper understanding of the contributions of SR-BI to steroidogenesis, platelet function and fertility is required in light of exploration of HDL-raising therapies aimed at reducing cardiovascular risk.

Analysis of triacylglycerol hydroperoxides in human lipoproteins by Orbitrap mass spectrometer

Herein, we represent a simple method for the detection and characterization of molecular species of triacylglycerol monohydroperoxides (TGOOH) in biological samples by use of reversed-phase liquid chromatography with a LTQ Orbitrap XL mass spectrometer (LC/LTQ Orbitrap) via an electrospray ionization source. Data were acquired using high-resolution, high-mass accuracy in Fourier-transform mode. Platform performance, related to the identification of TGOOH in human lipoproteins and plasma, was estimated using extracted ion chromatograms with mass tolerance windows of 5 ppm. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO4 to generate oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No TGOOH molecular species were detected in the nLDL and nHDL, whereas 11 species of TGOOH molecules were detected in the oxLDL and oxHDL. In positive-ion mode, TGOOH was found as [M + NH4](+). In negative-ion mode, TGOOH was observed as [M + CH3COO](-). TGOOH was more easily ionized in positive-ion mode than in negative-ion mode. The LC/LTQ Orbitrap method was applied to human plasma and three molecular species of TGOOH were detected. The limit of detection is 0.1 pmol (S/N = 10:1) for each synthesized TGOOH.

Editorial introductions

Editorial introductions

Plasma Lipoproteins as a Transport Form of Extracellular DNA

We showed DNA-binding activity of different classes of plasma lipoproteins in rats and humans. Experiments with fluorescent dye Hoechst 33258 showed that about 12% extracellular plasma DNA is present in circulating lipoproteins complexes; 7-8% of them with HDL. Structural HDL protein apoA-I probably plays the major role in the interaction between extracellular DNA and lipoprotein particle. Participation of lipoproteins in the transport of extracellular DNA can be considered as an important mechanism for elimination of nucleic acids from blood plasma.