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Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/β hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli. Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity.

We recently demonstrated that daily whole egg consumption during moderate carbohydrate restriction leads to greater increases in plasma HDL-cholesterol (HDL-C) and improvements in HDL profiles in metabolic syndrome (MetS) when compared to intake of a yolk-free egg substitute. We further investigated the effects of this intervention on HDL composition and function, hypothesizing that the phospholipid species present in egg yolk modulate HDL lipid composition to increase the cholesterol-accepting capacity of subject serum. Men and women classified with MetS were randomly assigned to consume either three whole eggs (EGG, n = 20) per day or the equivalent amount of egg substitute (SUB, n = 17) throughout a 12-week moderate carbohydrate-restricted (25-30 % of energy) diet. Relative to other HDL lipids, HDL-cholesteryl ester content increased in all subjects, with greater increases in the SUB group. Further, HDL-triacylglycerol content was reduced in EGG group subjects with normal baseline plasma HDL-C, resulting in increases in HDL-CE/TAG ratios in both groups. Phospholipid analysis by mass spectrometry revealed that HDL became enriched in phosphatidylethanolamine in the EGG group, and that EGG group HDL better reflected sphingomyelin species present in the whole egg product at week 12 compared to baseline. Further, macrophage cholesterol efflux to EGG subject serum increased from baseline to week 12, whereas no changes were observed in the SUB group. Together, these findings suggest that daily egg consumption promotes favorable shifts in HDL lipid composition and function beyond increasing plasma HDL-C in MetS.

This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ∼ 12, 10, 7.5, and

The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr(-/-), apoA-I(-/-) (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 μg of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.

Comparative gene identification-58 (CGI-58) interacts with and co-activates adipose triglyceride lipase (ATGL). The H82R mutation in human CGI-58 causes a neutral lipid storage disorder (NLSD) characterized by ichthyosis and excessive triglyceride storage in many types of cells. We studied the comparable H84R mutation in mouse CGI-58, and H84A mutated CGI-58, to ask how CGI-58 function is impaired. The ectopic expression of wild-type (WT) CGI-58 in human NLSD fibroblasts reduced excessive triglyceride storage to normal levels, whereas H84R CGI-58 was significantly less effective. Additionally, neither H84R nor H84A CGI-58 co-activated ATGL in an in vitro triglyceride hydrolase assay. A co-immunoprecipitation assay demonstrated that H84R and H84A CGI-58 bound ATGL as well as WT CGI-58. Immunofluorescence microscopy revealed that H84R and H84A CGI-58 localized to ectopic perilipin 1A on lipid droplets of cultured NIH3T3CARΔ fibroblasts as well as WT CGI-58. Moreover, the addition of forskolin and isobutylme...

This report describes the 3-dimensional conformation of nascent HDL apoA-I particles generated via ATP binding cassette transporter A1 (ABCA1). Three main types of particles were consistently obtained from; overexpressing HEK293 cells, bone marrow derived mouse macrophages and HepG2 cells. Particles were separated by FPLC and sized using gel electrophoresis and electron microscopy. Total lipid compositions were obtained from HPLC and mass spectrometry and protein conformation by chemical cross-linking and mass spectrometry (CCLMS). The first particle type corresponded to a lipid-rich nascent HDL (nHDL) between 13-10 nm in diameter, containing 3 molecules of apoA-I, with 45% of their total lipid as cholesterol, 35% as phosphatidylcholine and 20% as sphingomyelin with a total of ∼250 molecules of lipid per particle. The second type corresponded to 7-8 nm diameter particle containing 2 molecules of apoA-I and 26 total lipid molecules, while the third particle type was a monomer of apoA-I with 3 lipid molecules. Three-dimensional information was derived from the 13-10 nm nHDL particles by first crosslinking these purified particles and separating the monomeric, dimeric and trimeric apoA-I bands by SDS-PAGE. These CCL products were subjected to in-gel trypsin digest and LC-MS/MS. Reconstructed MS total-ion chromatograms were evaluated for CCL peptides with identity verified by MS/MS sequencing. Using several lysine-specific crosslinkers with arm lengths from 12Å to 36Å we compiled the distribution of CCLs. The same analysis was performed for the 7-8 nm nHDL that only yielded dimmer and monomer. Several intramolecular, interpeptide and intermolecular crosslinks were identified for each type category of nHDL. CCLs from the 7-8 nm nHDL suggested that the 2 apoA-I molecules assumed antiparallel counter-rotating helices. Tighter bending of the double belt was required to cover the 26 lipid molecules. Crosslinking of the 13-10 nm nHDL suggested that all three molecules were antiparallel to one another. The conformation that fit the CCLs was a belt with the middle apoA-I molecule antiparallel to the two outer apoA-I molecules. Overall, these data suggest that apoA-I adapts its conformation to encompass available lipid, but with larger amounts of lipid a belt conformation is preferred.