Phosphatidylcholine-sterol Acyltransferase Research Articles

Proteome analysis develops novel plasma proteins classifier in predicting the mortality of COVID-19.

COVID-19 has been a global concern for 3 years, however, consecutive plasma protein changes in the disease course are currently unclear. Setting the mortality within 28 days of admission as the main clinical outcome, plasma samples were collected from patients in discovery and independent validation groups at different time points during the disease course. The whole patients were divided into death and survival groups according to their clinical outcomes. Proteomics and pathway/network analyses were used to find the differentially expressed proteins and pathways. Then, we used machine learning to develop a protein classifier which can predict the clinical outcomes of the patients with COVID-19 and help identify the high-risk patients. Finally, a classifier including C-reactive protein, extracellular matrix protein 1, insulin-like growth factor-binding protein complex acid labile subunit, E3 ubiquitin-protein ligase HECW1 and phosphatidylcholine-sterol acyltransferase was determined. The prediction value of the model was verified with an independent patient cohort. This novel model can realize early prediction of 28-day mortality of patients with COVID-19, with the area under curve 0.88 in discovery group and 0.80 in validation group, superior to 4C mortality and E-CURB65 scores. In total, this work revealed a potential protein classifier which can assist in predicting the outcomes of COVID-19 patients and providing new diagnostic directions.

Alteration in Lysophospholipids and Converting Enzymes in Glaucomatous Optic Nerves.

PurposeTo determine whether lysophospholipid (LPL) profiles and corresponding conversion enzymes in the LPL pathways are altered in the optic nerve (ON) between human control and glaucoma samples.MethodsLipids extracted from control (n = 11) and glaucomatous (n = 12) ON samples using the Bligh and Dyer method were subjected to high-resolution mass spectrometry on a Q-exactive mass spectrometer coupled with a high-performance liquid chromatography (Accela 600) system. Analysis was performed for LPLs (lysophosphatidylcholines, lysophosphatidylserines, lysophosphatidylethanolamines, lysophosphatidylinositols, and lysosphingomyelines) using LipidSearch v.4.1, MZmine v.2.0, and MetaboAnalyst v.4.0. LPL synthesis and degradation pathway maps, utilizing UniProt and BRENDA database entries as needed, were created using Kyoto Encyclopedia of Genes and Genomes (KEGG)–based tools. The mRNA expression level in normal and glaucomatous human ON were analyzed using Gene Expression Omnibus (GEO) entry GSE45570. Protein amounts were determined using PHAST gel and dot blot and were used for normalization of protein amounts across samples. Western blot, ELISA, and protein quantification were performed using established protocols.ResultsPrincipal component analysis of ON LPL profile placed control and glaucomatous ONs in two distinct separate groups. Mass spectrometric analysis of ON revealed decrease in lysophosphatidic acid, lysophosphatidylethanolamine, lysophosphatidylcholine, and significant increase in diacylglycerol in glaucomatous ON. Statistical analysis of LPL conversion enzymes revealed significant overexpression of phosphatidate phosphatase LPIN2, phospholipid phosphatase 3, phosphatidylcholine-sterol acyltransferase, and calcium-dependent phospholipase 2, and significant downregulation of glycerol-3-phosphate acyltransferase 4 at mRNA level in glaucomatous ON. Western blot and ELISA confirmed proteomic differences between normal and diseased ON.ConclusionsOur analysis revealed alterations in specific LPL levels and corresponding select enzyme-level changes in glaucomatous ON.

Diagnosis of epithelial ovarian cancer using a combined protein biomarker panel

BackgroundAn early detection tool for EOC was constructed from analysis of biomarker expression data from serum collected during the UKCTOCS.MethodsThis study included 49 EOC cases (19 Type I and 30 Type II) and 31 controls, representing 482 serial samples spanning seven years pre-diagnosis. A logit model was trained by analysis of dysregulation of expression data of four putative biomarkers, (CA125, phosphatidylcholine-sterol acyltransferase, vitamin K-dependent protein Z and C-reactive protein); by scoring the specificity associated with dysregulation from the baseline expression for each individual.ResultsThe model is discriminatory, passes k-fold and leave-one-out cross-validations and was further validated in a Type I EOC set. Samples were analysed as a simulated annual screening programme, the algorithm diagnosed cases with >30% PPV 1–2 years pre-diagnosis. For Type II cases (~80% were HGS) the algorithm classified 64% at 1 year and 28% at 2 years tDx as severe.ConclusionsThe panel has the potential to diagnose EOC one-two years earlier than current diagnosis. This analysis provides a tangible worked example demonstrating the potential for development as a screening tool and scrutiny of its properties. Limits on interpretation imposed by the number of samples available are discussed.

Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis

A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their isoforms or modifications.

Novel serum biomarkers for detection of excessive alcohol use.

Construct interview that correctly identifies those with alcohol use disorder have limitation, especially when the subjects are motivated to minimize the magnitude of drinking behavior. Current laboratory tests to detect excessive alcohol consumption are limited by marginal sensitivity/specificity. Excessive drinking has been shown to affect several organ systems, which may be reflected in changes in quantity of plasma proteins. Our aim was to employ novel proteomic analyses to identify potential markers for excessive alcohol use. A prospective case-control study included 49 controls and 54 excessive drinkers (discovery cohort). The serum proteomic analyses in these subjects were performed, and the results were tested in the verification cohort (40 controls and 40 excessive drinkers). Using the appropriate cutoff and confirmation with ELISA, we identified 4 proteins which were significantly elevated in the serum of excessive drinkers: AT-rich interactive domain-containing protein 4B (ARID4B), phosphatidylcholine-sterol acyltransferase (LCAT), hepatocyte growth factor-like protein (MST1), and ADP-ribosylation factor 6 (ARL6). The performance of the conventional markers (aspartate aminotransferase [AST], alanine aminotransferase [ALT], gamma-glutamyl transpeptidase [GGT], percentage of carbohydrate-deficient transferrin [%CDT], and mean corpuscular volume [MCV]) discriminating between excessive alcohol use and controls had an area under the curve (AUC) ranging from 0.21 (ALT) to 0.67 (MCV). The AUC of these novel proteins showed the improvement in the detection of excessive drinkers compared to conventional laboratory tests, ranging from 0.73 (for ARID4B) to 0.86 (for ARL6). We have identified 4 novel proteins that can discern subjects with excessive alcohol use. Further studies are needed to determine the clinical implications of these markers to detect excessive alcohol use and confirm abstinence.

Proteomic Signatures in Plasma during Early Acute Renal Allograft Rejection

Acute graft rejection is an important clinical problem in renal transplantation and an adverse predictor for long term graft survival. Plasma biomarkers may offer an important option for post-transplant monitoring and permit timely and effective therapeutic intervention to minimize graft damage. This case-control discovery study (n = 32) used isobaric tagging for relative and absolute protein quantification (iTRAQ) technology to quantitate plasma protein relative concentrations in precise cohorts of patients with and without biopsy-confirmed acute rejection (BCAR). Plasma samples were depleted of the 14 most abundant plasma proteins to enhance detection sensitivity. A total of 18 plasma proteins that encompassed processes related to inflammation, complement activation, blood coagulation, and wound repair exhibited significantly different relative concentrations between patient cohorts with and without BCAR (p value <0.05). Twelve proteins with a fold-change ≥1.15 were selected for diagnostic purposes: seven were increased (titin, lipopolysaccharide-binding protein, peptidase inhibitor 16, complement factor D, mannose-binding lectin, protein Z-dependent protease and β2-microglobulin) and five were decreased (kininogen-1, afamin, serine protease inhibitor, phosphatidylcholine-sterol acyltransferase, and sex hormone-binding globulin) in patients with BCAR. The first three principal components of these proteins showed clear separation of cohorts with and without BCAR. Performance improved with the inclusion of sequential proteins, reaching a primary asymptote after the first three (titin, kininogen-1, and lipopolysaccharide-binding protein). Longitudinal monitoring over the first 3 months post-transplant based on ratios of these three proteins showed clear discrimination between the two patient cohorts at time of rejection. The score then declined to baseline following treatment and resolution of the rejection episode and remained comparable between cases and controls throughout the period of quiescent follow-up. Results were validated using ELISA where possible, and initial cross-validation estimated a sensitivity of 80% and specificity of 90% for classification of BCAR based on a four-protein ELISA classifier. This study provides evidence that protein concentrations in plasma may provide a relevant measure for the occurrence of BCAR and offers a potential tool for immunologic monitoring.

Deep depletion of abundant serum proteins reveals low‐abundant proteins as potential biomarkers for human ovarian cancer

Epithelial ovarian cancer (EOC) ranks fifth as a cause of cancer deaths in women. Current diagnostic and monitoring markers have limited reliability for the detection of disease. We have tested the possibility of identifying candidate biomarkers present at low nanogram to picogram levels after removing both the 12 most abundant and 77 moderately abundant proteins from serum samples of EOC patients using antibody affinity columns. We showed that this approach allows the identification of proteins that are expressed at nanogram per liter levels in the serum. Using ICAT/MS/MS analysis, we identified 51 proteins that are differentially expressed by at least twofold. These proteins include leucine-rich alpha-2-glycoprotein, matrix metalloproteinase-9 (MMP-9), inter-alpha-trypsin inhibitor heavy chain H1, insulin-like growth factor-binding protein 6, insulin-like growth factor-binding protein 3, isoform 1 of epidermal growth factor receptor, angiopoietin-like protein 3 (ANGPTL3) and phosphatidylcholine-sterol acyltransferase. We confirmed the differential expression of MMP9 and ANGPTL3 in normal and ovarian cancer sera by ELISA assays. Further robust clinical evaluation of the candidate markers identified is necessary.

Lecithin:cholesterol acyltransferase: role of N-linked glycosylation in enzyme function.

Lecithin:cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) is a glycoprotein which is responsible for the formation of cholesteryl ester in plasma. The carbohydrate content has been estimated to be approx. 25% of the total LCAT mass, and four potential N-linked glycosylation sites have been predicted at residues 20, 84, 272 and 384 of the LCAT protein sequence. In the present study, we have examined which of these sites are utilized and how the N-glycosylation affects the secretion and function of the enzyme. Site-directed mutagenesis was performed to eliminate the glycosylation consensus sequence at each of the four potential sites, and the mutant proteins were expressed in COS cells. The amount of each mutant LCAT secreted was similar to that of the wild-type enzyme but the molecular mass was decreased by 3-4 kDa. The specific activity of each mutant LCAT was significantly different from the wild-type; however, the magnitude and direction of the change depended on the glycosylation site mutagenized. Loss of carbohydrate at position 20, 84 or 272 resulted in a decrease in the specific activity of the mutant enzymes by 18%, 82%, and 62% respectively. In contrast, the mutant protein without glycosylation at position 384 displayed a 2-fold increase in enzyme activity. In addition, a quadruple mutant was constructed such that all four potential glycosylation sites were eliminated. The amount of the unglycosylated LCAT secreted into the culture medium was less than 10% of the wild-type level and the specific activity of this enzyme was decreased to 5% of that of the wild type. The results demonstrate that all four potential N-glycosylation sites in LCAT are used and the presence of carbohydrate at each site has diverse effects on the enzyme activity.

Effects of site-directed mutagenesis at residues cysteine-31 and cysteine-184 on lecithin-cholesterol acyltransferase activity.

Native lecithin-cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase; phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) protein, and LCAT in which either or both of the enzyme free cysteines had been replaced with glycine residues by site-directed mutagenesis, has been expressed in cultured Chinese hamster ovary cells stably transfected with the human LCAT gene. The mass of LCAT secreted, determined by immunoassay, did not differ in the native and mutant species. LCAT specific activity was also unchanged in the mutant species. In particular, the cysteine-free double mutant, in which Cys-31 and Cys-184 had both been replaced, was fully active in the synthesis of cholesteryl esters. This result is not consistent with a catalytic role for LCAT free cysteine residues. The classical inhibitor of LCAT activity, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which strongly (89%) inhibited the native enzyme, had partial (45%) inhibitory activity with mutant enzyme species containing a single -SH residue, while the double mutant was not significantly inhibited by DTNB. These data are interpreted to suggest that Cys-31 and Cys-184 are vicinal both to each other and to the "interfacial binding site" at residues 177-182, and that DTNB exerts its effect by steric inhibition.

Polymorphisms in the Gene Coding for Cholesteryl Ester Transfer Protein are Related to Plasma High-Density Lipoprotein Cholesterol and Transfer Protein Activity

1. Cholesteryl ester transfer protein activity may have a physiological effect on high-density lipoprotein levels. 2. We examined restriction fragment length polymorphisms associated with the cholesteryl ester transfer protein gene and the apolipoprotein AI gene in a group of 60 unrelated subjects selected from an initial survey of 5000 subjects on the basis of their high-density lipoprotein levels being high or low at the extremes of the distribution. The activities of cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase (phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) were also determined. Analysis by selection of those with a low high-density lipoprotein cholesterol level (less than or equal to 1.1 for males, less than or equal to 1.2 for females) gave 32 individuals with 24% B2 alleles. Selection of subjects with a high-density lipoprotein cholesterol level (less than or equal to 2 mmol/l) gave 17 with 62% B2 alleles. 3. The group with low levels of high-density lipoprotein cholesterol had higher activity of cholesteryl ester transfer protein and significantly elevated triacylglycerol levels when compared with the group with high levels of high-density lipoprotein cholesterol. 4. A further significant finding was the correlation of the MspI restriction fragment length polymorphism detected by the apolipoprotein AI gene with lecithin:cholesterol acyltransferase activity.

Cloning and sequencing of human cholesteryl ester transfer protein cDNA.

The transfer of insoluble cholesteryl esters among lipoprotein particles is a vital step in normal cholesterol homeostasis and may be involved in the development of atherosclerosis. Extrahepatic tissues lack the enzymes required for the degradation of sterols to the excretable form of bile acids. Cholesterol synthesized in these tissues in excess of that needed for the synthesis of cell membranes or steroid hormones must accordingly be returned through the plasma to the liver for catabolism. The series of reactions involved has been termed reverse cholesterol transport. Catalysed steps of this pathway are believed to include an efflux from peripheral cells, which generates a diffusion gradient between these membranes and extracellular fluid; esterification of this cholesterol by lecithin-cholesterol acyltransferase (LCAT) (phosphatidylcholine-sterol acyltransferase) acting on species of high-density lipoproteins; transfer of the cholesteryl esters formed (largely to low- and very low-density lipoproteins) (LDL and VLDL) by a cholesteryl ester transfer protein (CETP); and removal of these lipoproteins, together with their cholesteryl ester content, by the liver through receptor-mediated and nonspecific endocytosis. Of these steps, the CETP reaction is the least characterized. Several laboratories have reported the purification from human plasma of proteins active on cholesteryl ester transfer between lipoprotein particles and possibly between cells and plasma. However, the reported relative molecular mass (Mr), abundance and specificity of the purified activities have differed considerably. We have recently described the preparation of a highly active CETP of Mr 74,000 purified about 100,000-fold from human plasma, which may represent the functional component of earlier preparations. Using a partial amino-acid sequence from this purified protein, CETP complementary DNA derived from human liver DNA has been cloned and sequenced and the cloned DNA used to detect CETP messenger RNA in a number of human tissues.

Activation of phosphatidylcholine-sterol acyltransferase by human apolipoprotein E isoforms.

The reaction catalysed by phosphatidylcholine-sterol acyltransferase (EC 2.3.1.43) is believed to be the major source of cholesteryl ester in human plasma; the enzyme requires a protein activator. Several human apolipoproteins were found to exhibit an activator function, the major one being apolipoprotein A-I. Human apolipoprotein E exists in the population mainly in three different genetic isoforms; apolipoprotein E-2, E-3 and E-4. These isopeptides were isolated from subjects homozygous for one of the isoforms, incorporated into phospholipid/cholesterol/[14C]cholesterol complexes by the cholate dialysis procedure and used to measure capacity to activate phosphatidylcholine-sterol acyltransferase in comparison to apolipoprotein A-I lipid substrate particles prepared by the same procedure. Acyltransferase activity was measured by the formation of [14C]cholesteryl ester from [14C]cholesterol using purified enzyme. With egg yolk phosphatidylcholine as acyl donor, apo E was 15-19% as efficient as apolipoprotein A-I for activation of the acyltransferase. Apo-E-stimulated cholesteryl ester formation by the enzyme was enhanced when 1-oleoyl-2-palmitoyl-glycerophosphocholine was used as a substrate phospholipid (45% of apo A-I/phosphatidylcholine control) and most pronounced with dimyristoylglycerophosphocholine (75% of apo A-I/phosphatidylcholine control). No significant difference in activation was found between apo E isoforms. It is concluded that apolipoprotein E activates phosphatidylcholine-sterol acyltransferase in vitro and that apolipoprotein E isoforms are similarly effective.