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Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

ABSTRACT: High-density lipoprotein (HDL) is regarded as atheroprotective because it provides antioxidant and anti-inflammatory benefits and plays an important role in reverse cholesterol transport. In this paper, we outline a novel methodology for studying the heterogeneity of HDL. Using anion-exchange chromatography, we separated HDL from 6 healthy individuals into five subfractions (H1 through H5) with increasing charge and evaluated the composition and biologic activities of each subfraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that apolipoprotein (apo) AI and apoAII were present in all 5 subfractions; apoCI was present only in H1, and apoCIII and apoE were most abundantly present in H4 and H5. HDL-associated antioxidant enzymes such as lecithin-cholesterol acyltransferase, lipoprotein-associated phospholipase A2, and paraoxonase 1 were most abundant in H4 and H5. Lipoprotein isoforms were analyzed in each subfraction by using matrix-assisted laser desorption-time-of-flight mass spectrometry. To quantify other proteins in the HDL subfractions, we used the isobaric tags for the relative and absolute quantitation approach followed by nanoflow liquid chromatography-tandem mass spectrometry analysis. Most antioxidant proteins detected were found in H4 and H5. The ability of each subfraction to induce cholesterol efflux from macrophages increased with increasing HDL electronegativity, with the exception of H5, which promoted the least efflux activity. In conclusion, anion-exchange chromatography is an attractive method for separating HDL into subfractions with distinct lipoprotein compositions and biologic activities. By comparing the properties of these subfractions, it may be possible to uncover HDL-specific proteins that play a role in disease.

SUBMITTER: Hsieh JY

PROVIDER: S-EPMC3919464 | biostudies-other | 2013 Dec

REPOSITORIES: biostudies-other

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Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

Hsieh Ju-Yi JY Chang Chiz-Tzung CT Huang Max T MT Chang Chia-Ming CM Chen Chia-Ying CY Shen Ming-Yi MY Liao Hsin-Yi HY Wang Guei-Jane GJ Chen Chu-Huang CH Chen Chao-Jung CJ Yang Chao-Yuh CY

Analytical chemistry 20131113 23

High-density lipoprotein (HDL) is regarded as atheroprotective because it provides antioxidant and anti-inflammatory benefits and plays an important role in reverse cholesterol transport. In this paper, we outline a novel methodology for studying the heterogeneity of HDL. Using anion-exchange chromatography, we separated HDL from 6 healthy individuals into five subfractions (H1 through H5) with increasing charge and evaluated the composition and biologic activities of each subfraction. Sodium do ...[more]

PMID: 24171625

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Project description:BackgroundSubclinical atherosclerosis is frequently observed in type 1 diabetes (T1D) although the mechanisms and markers involved in the evolution to established cardiovascular disease are not well known. High-density lipoprotein cholesterol in T1D is normal or even high, and changes in its functionality and proteomics are considered. Our aim was to evaluate the proteomics of HDL subfractions in T1D and control subjects and its association with clinical variables, subclinical atherosclerosis markers and HDL functionality.MethodsA total of 50 individuals with T1D and 30 matched controls were included. Carotid-femoral pulse wave velocity (PWV), flow-mediated vasodilation (FMD), cardiovascular autonomic neuropathy (CAN), and ten-year cardiovascular risk (ASCVDR) were determined. Proteomics (parallel reaction monitoring) was determined in isolated HDL2 and HDL3 that were also utilized to measure cholesterol efflux from macrophages.ResultsAmong 45 quantified proteins, 13 in HDL2 and 33 in HDL3 were differentially expressed in T1D and control subjects. Six proteins related to lipid metabolism, one to inflammatory acute phase, one to complement system and one to antioxidant response were more abundant in HDL2, while 14 lipid metabolism, three acute-phase, three antioxidants and one transport in HDL3 of T1D subjects. Three proteins (lipid metabolism, transport, and unknown function) were more abundant in HDL2; and ten (lipid metabolism, transport, protease inhibition), more abundant in HDL3 of controls. Individuals with T1D had higher PWV and ten-year ASCVDR, and lower FMD, Cholesterol efflux from macrophages was similar between T1D and controls. Proteins in HDL2 and HDL3, especially related to lipid metabolism, correlated with PWV, CAN, cholesterol efflux, HDLc, hypertension, glycemic control, ten-year ASCVDR, and statins use.ConclusionHDL proteomics can be predictive of subclinical atherosclerosis in type 1 diabetes. Proteins that are not involved in reverse cholesterol transport may be associated with the protective role of HDL.

Project description:High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the mo-lecular level. During HDL’s journey throughout the body, its functions are mediated through in-teractions with cell surface receptors on different cell types. To characterize and better under-stand the functional interplay between HDL particles and tissue, we analyzed the sur-faceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (au-to-CSC) and HATRIC-based ligand–receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors repre-senting potential HDL-receptor interaction candidates . Since vascular endotheli-al growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the au-to-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 addition-al receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated re-ceptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor ty-rosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.

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Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

Publications

Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

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