Enzyme Lecithin-cholesterol Acyltransferase Research Articles

Endocrinology for the hepatologist.

Endocrinology for the hepatologist.

Two novel variants in the lecithin:cholesterol acyltransferase gene resulted in classic LCAT deficiency

Background and aimsWe report the first two cases of familial lecithin:cholesterol acyltransferase (LCAT) deficiency in Croatia with classical clinical and biochemical features. Patients and methodsA 30-year-old man with nephrotic syndrome, corneal opacities, hepatosplenomegaly, anemia, low high-density lipoprotein (HDL)-cholesterol levels and arterial hypertension (blood pressure >200/100 mmHg) was admitted to our department. At admission, he had an elevated creatinine serum level (233 μmol/L), proteinuria of 12 g in 24-h urine (g/24 h), 3–7 erythrocytes in urine sediment and notable anemia (hemoglobin level 90 g/l). His HDL-cholesterol was significantly low (0.42 mmol/L). Besides chronic kidney disease (CKD), other secondary causes of hypertension were ruled out. The patient was previously diagnosed with membranous nephropathy and treated unsuccessfully with immunosuppressive agents (steroids, cyclosporine, cyclophosphamide). Re-evaluation of histopathological findings of kidney biopsy revealed massive deposition of lipid material in the glomerular basal membrane and in the mesangial region. His 4-year younger brother was also evaluated due to corneal opacities and new-onset arterial hypertension. Nephrotic range proteinuria with preserved global renal function was determined. He also had very low HDL-cholesterol levels. ResultsKidney biopsies from both patients were consistent with LCAT deficiency. The disease was confirmed by measurement of LCAT enzyme activity, plasma cholesterol esterification rate, and genetic testing. Two novel missense variants in the LCAT gene (c.496G > A and c.1138T > C) were found. ConclusionsTo our knowledge, the presented cases are the first reported cases of genetic LCAT deficiency in Croatia. Given the clinical presentation, the complete lack of LCAT activity and cholesterol esterification rate, diagnosis of familial LCAT deficiency was made.

Black Truffle Extract Exerts Antidiabetic Effects through Inhibition of Inflammation and Lipid Metabolism Regulation.

Black truffle, a culinary and medical fungus, is highly valued worldwide for its nutritional and therapeutic importance. To enhance the existing knowledge about the beneficial properties, this study investigates the antioxidant, antihyperlipidemic, and anti-inflammatory effects of black truffle extract in in vitro biochemical assays and animal study. Briefly, black truffle extract was administered orally to treat streptozotocin- (STZ-) induced diabetic Wistar rats for 45 days. At the end of the experimental duration, rats were sacrificed to perform biochemical and gene expression analyses related to lipid regulatory and inflammatory pathways. Our results indicated that total cholesterol, triglycerides, free fatty acids, phospholipids, and low-density lipoprotein in different tissues and circulation were significantly increased in diabetic rats. Furthermore, the β-hydroxy β-methylglutaryl-CoA enzyme was also significantly increased; lipoprotein lipase and lecithin–cholesterol acyltransferase enzymes were significantly decreased in diabetic rats. However, the above conditions were reversed upon black truffle extract feeding. Furthermore, black truffle extract was also found to downregulate the expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-6) and lipid regulatory genes (serum regulatory element-binding protein-1 and fatty acid synthase). The truffle extract-treated effects were comparable to glibenclamide and medication commonly used to treat diabetes mellitus. Overall, our results suggested that black truffle possesses strong antihyperlipidemic and anti-inflammatory effects on diabetic rats. These findings will enhance the current knowledge about the therapeutic importance of black truffles. They might be exploited as a possible food supplement or even as a natural source of pharmaceutical agents for diabetes prevention and treatment.

Current Status of Familial LCAT Deficiency in Japan

Lecithin cholesterol acyltransferase (LCAT) is a lipid-modification enzyme that catalyzes the transfer of the acyl chain from the second position of lecithin to the hydroxyl group of cholesterol (FC) on plasma lipoproteins to form cholesteryl acylester and lysolecithin. Familial LCAT deficiency is an intractable autosomal recessive disorder caused by inherited dysfunction of the LCAT enzyme. The disease appears in two different phenotypes depending on the position of the gene mutation: familial LCAT deficiency (FLD, OMIM 245900) that lacks esterification activity on both HDL and ApoB-containing lipoproteins, and fish-eye disease (FED, OMIM 136120) that lacks activity only on HDL. Impaired metabolism of cholesterol and phospholipids due to LCAT dysfunction results in abnormal concentrations, composition and morphology of plasma lipoproteins and further causes ectopic lipid accumulation and/or abnormal lipid composition in certain tissues/cells, and serious dysfunction and complications in certain organs. Marked reduction of plasma HDL-cholesterol (HDL-C) and corneal opacity are common clinical manifestations of FLD and FED. FLD is also accompanied by anemia, proteinuria and progressive renal failure that eventually requires hemodialysis. Replacement therapy with the LCAT enzyme should prevent progression of serious complications, particularly renal dysfunction and corneal opacity. A clinical research project aiming at gene/cell therapy is currently underway.

INDIVIDUAL CORRECTION OF THE TREATMENT OF PATIENTS WITH HEPATOSPLENOMEGALY SYNDROME, TAKING INTO ACCOUNT THE PARAMETERS OF LIPID AND PROTEIN METABOLISM

Summary. Purpose: to identify the features of lipid metabolism in patients with hepatosplenomegaly syndrome (HSS) of various origins to develop an individualized approach to the treatment of patients with this pathology.
 Materials and methods: blood serum of 73 patients with HSS on the background of liver cirrhosis complicated by portal hypertension, divided into 3 groups 1) with the etiological factor of hepatitis viruses HCV / HBV, 2) with autoimmune hepatitis with the etiological factor of the herpes group viruses CMV / VEB, 3) against the background of liver hepatosis and fermentopathies.
 Results and its discussion. In patients with HSS, multidirectional changes in the synthesis of lipid fractions were observed, also associated with impaired protein metabolism, namely:
 - with HSS against the background of viral hepatitis B and C, hypocholesterolemia and hypobetacholesterolemia were observed due to a decrease in cholesterol synthesis in hepatocytes; a decrease in the concentration of HDL is also associated with a violation of the process of cholesterol esterification due to a deficiency of the enzyme lecithin cholesterol acyltransferase, and an increase in the concentration of LDL is associated with a deficiency of the enzyme lipoprotein lipase in viral damage to hepatocytes.
 - with HSS against the background of autoimmune hepatitis, no significant changes were observed in the synthesis of lipid fractions;
 - in hepatosis (fermentopathies), type 5 hyperlipidemia with hypercholesterolemia and hypotriglyceridemia was found, which are a consequence of impaired formation and excretion of lipids from the liver, probably as a result of a decrease in the synthesis of apoproteins and indicate the accumulation of fat in hepatocytes and the formation of hepatosis.
 Conclusion:
 
 Surgical or endovascular treatment of HSS against the background of liver cirrhosis or hepatosis needs to be supplemented with individualized metabolic drug therapy, including lipid-lowering therapy.
 When detecting hypocholesterolemia and hypobetacholesterolemia in the first group, lipid-lowering therapy is not indicated, whereas if type 5 hyperlipidemia with hypercholesterolemia and hypotriglyceridemia is detected in the group of patients with fatty hepatosis, lipid-lowering therapy is necessary.

Novel LCAT (Lecithin:Cholesterol Acyltransferase) Activator DS-8190a Prevents the Progression of Plaque Accumulation in Atherosclerosis Models.

Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (2.0-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO×hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO×hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [3H]-cholesterol and confirmed significant increases of [3H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a-treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a-immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. In addition, this compound proves that a small-molecule direct LCAT activator can achieve HDL-C elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.

Lecithin- cholesterol acyltransferase and relationship with Platelet-activating factor in AB blood phenotype

Objectives: This study aimed to determine the association between Lecithin-cholesterol acyltransferase (LCAT) and Platelet-activating factor (PAF) with atherogenic potential in the ABO blood system.Background: The current study is the first study that focuses on healthy blood donors and investigating a relationship between ABO blood groups and LCAT enzyme and PAF, which plays a notable role in atherogenesis.Methods: ABO blood types characterize the role of the individualistic phenotype. Hence, it is generally assumed that ABO blood phenotypes are involved with many diseases. This cross-sectional study involved 176 healthy subjects with different ABO blood phenotypes. Measurements of routine lipids, LCAT, PAF and atherogenic plasma index (AIP) were performed in these subjects.Results: Our most important finding was that individuals with blood group AB have statistically significant lower LCAT levels in serum. Additionally, there was a moderately negative correlation between LCAT and triglycerides in the AB blood phenotype (r=-0.452, P=0.02). Nevertheless, there were statistically significant correlations between LCAT levels and PAF levels of subjects with only non –O blood phenotypes (A, B and AB). Serum AIP values were statistically significantly higher in the AB blood group.Conclusion: The present study suggests that blood group AB can be associated with decreased LCAT  levels when compared to the non- AB phenotypes. We suggest that LACT and AIP may be helpful biomarkers to illustrate atherogenic risk in AB blood phenotype, although this requires further investigation.

Recombinant LCAT (Lecithin:Cholesterol Acyltransferase) Rescues Defective HDL (High-Density Lipoprotein)-Mediated Endothelial Protection in Acute Coronary Syndrome.

Objective- Aim of this study was to evaluate changes in LCAT (lecithin:cholesterol acyltransferase) concentration and activity in patients with an acute coronary syndrome, to investigate if these changes are related to the compromised capacity of HDL (high-density lipoprotein) to promote endothelial nitric oxide (NO) production, and to assess if rhLCAT (recombinant human LCAT) can rescue the defective vasoprotective HDL function. Approach and Results- Thirty ST-segment-elevation myocardial infarction (STEMI) patients were enrolled, and plasma was collected at hospital admission, 48 and 72 hours thereafter, at hospital discharge, and at 30-day follow-up. Plasma LCAT concentration and activity were measured and related to the capacity of HDL to promote NO production in cultured endothelial cells. In vitro studies were performed in which STEMI patients' plasma was added with rhLCAT and HDL vasoprotective activity assessed by measuring NO production in endothelial cells. The plasma concentration of the LCAT enzyme significantly decreases during STEMI with a parallel significant reduction in LCAT activity. HDL isolated from STEMI patients progressively lose the capacity to promote NO production by endothelial cells, and the reduction is related to decreased LCAT concentration. In vitro incubation of STEMI patients' plasma with rhLCAT restores HDL ability to promote endothelial NO production, possibly related to significant modification in HDL phospholipid classes. Conclusions- Impairment of cholesterol esterification may be a major factor in the HDL dysfunction observed during acute coronary syndrome. rhLCAT is able to restore HDL-mediated NO production in vitro, suggesting LCAT as potential therapeutic target for restoring HDL functionality in acute coronary syndrome.

LCAT Enzyme Replacement Therapy Reduces LpX and Improves Kidney Function in a Mouse Model of Familial LCAT Deficiency.

Familial LCAT deficiency (FLD) is due to mutations in lecithin:cholesterol acyltransferase (LCAT), a plasma enzyme that esterifies cholesterol on lipoproteins. FLD is associated with markedly reduced levels of plasma high-density lipoprotein and cholesteryl ester and the formation of a nephrotoxic lipoprotein called LpX. We used a mouse model in which the LCAT gene is deleted and a truncated version of the SREBP1a gene is expressed in the liver under the control of a protein-rich/carbohydrate-low (PRCL) diet-regulated PEPCK promoter. This mouse was found to form abundant amounts of LpX in the plasma and was used to determine whether treatment with recombinant human LCAT (rhLCAT) could prevent LpX formation and renal injury. After 9 days on the PRCL diet, plasma total and free cholesterol, as well as phospholipids, increased 6.1 ± 0.6-, 9.6 ± 0.9-, and 6.7 ± 0.7-fold, respectively, and liver cholesterol and triglyceride concentrations increased 1.7 ± 0.4- and 2.8 ±0.9-fold, respectively, compared with chow-fed animals. Transmission electron microscopy revealed robust accumulation of lipid droplets in hepatocytes and the appearance of multilamellar LpX particles in liver sinusoids and bile canaliculi. In the kidney, LpX was found in glomerular endothelial cells, podocytes, the glomerular basement membrane, and the mesangium. The urine albumin/creatinine ratio increased 30-fold on the PRCL diet compared with chow-fed controls. Treatment of these mice with intravenous rhLCAT restored the normal lipoprotein profile, eliminated LpX in plasma and kidneys, and markedly decreased proteinuria. The combined results suggest that rhLCAT infusion could be an effective therapy for the prevention of renal disease in patients with FLD.

The level of 24-hydroxycholesteryl esters decreases in plasma of patients with Parkinson’s disease

24-hydroxycholesterol (24OH-C) is synthesized almost exclusively in neurons. This oxysterol is mostly present as ester form in both cerebrospinal fluid and plasma. The enzyme lecithin-cholesterol acyltransferase esterifies 24OH-C in the brain, and the level of 24OH-C esters in cerebrospinal fluid was found to be correlated with the level of 24OH-C esters in plasma. Decreased levels of 24OH-C esters levels were previously found in Alzheimer’s disease and Amyotrophic Lateral Sclerosis. This finding was attributed to the inhibitory effect of oxidative stress on lecithin-cholesterol acyltransferase activity in neurodegenerative conditions. Data reported here show that the plasma level of 24OH-C esters is decreased also in Parkinson’s disease. ROC analysis identified 69.0% of 24OH-C esterification as the threshold (AUC = 0.98) discriminating patients (N = 19) from healthy subjects (N = 19) with 100% specificity vs controls, 89.5% sensitivity, 94.7% accuracy, and 100% precision. The level of 24OH-C esters was not correlated with UPDRS I or UPDRS III when evaluated at the time of blood sampling. By contrast, it was negatively correlated with UPDRS I (r = −0.4984, p = 0.0299) after one year of follow up. Therefore, this level might represent a novel biomarker of neurodegeneration in Parkinson’s disease. The biomarker level is here proposed as a measure to evaluate the severity of disease, as well as to monitor the progression of this pathology.

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

The protozoan parasite Toxoplasma gondii develops within a parasitophorous vacuole (PV) in mammalian cells, where it scavenges cholesterol. When cholesterol is present in excess in its environment, the parasite expulses this lipid into the PV or esterifies it for storage in lipid bodies. Here, we characterized a unique T. gondii homologue of mammalian lecithin:cholesterol acyltransferase (LCAT), a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues. TgLCAT contains a motif characteristic of serine lipases "AHSLG" and the catalytic triad consisting of serine, aspartate, and histidine (SDH) from LCAT enzymes. TgLCAT is secreted by the parasite, but unlike other LCAT enzymes it is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity. TgLCAT uses phosphatidylcholine as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, but with a lower transesterification activity than mammalian LCAT. TgLCAT is stored in a subpopulation of dense granule secretory organelles, and following secretion, it localizes to the PV and parasite plasma membrane. LCAT-null parasites have impaired growth in vitro, reduced virulence in animals, and exhibit delays in egress from host cells. Parasites overexpressing LCAT show increased virulence and faster egress. These observations demonstrate that TgLCAT influences the outcome of an infection, presumably by facilitating replication and egress depending on the developmental stage of the parasite.

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

Lecithin-cholesterol acyltransferase in brain: Does oxidative stress influence the 24-hydroxycholesterol esterification?

24-Hydroxycholesterol (24OH-C) is esterified by the enzyme lecithin-cholesterol acyltransferase (LCAT) in the cerebrospinal fluid (CSF). We report here that the level of 24OH-C esters was lower in CSF of patients with amyotrophic lateral sclerosis than in healthy subjects (54% vs 68% of total 24OH-C, p=0.0005; n=8). Similarly, the level of 24OH-C esters in plasma was lower in patients than in controls (62% vs 77% of total 24OH-C; p=0.0076). The enzyme amount in CSF, as measured by densitometry of the protein band revealed by immunoblotting, was about 4-fold higher in patients than in controls (p=0.0085). As differences in the concentration of the LCAT stimulator Apolipoprotein E were not found, we hypothesized that the reduced 24OH-C esterification in CSF of patients might depend on oxidative stress. We actually found that oxidative stress reduced LCAT activity in vitro, and 24OH-C effectively stimulated the enzyme secretion from astrocytoma cells in culture. Enhanced LCAT secretion from astrocytes might represent an adaptive response to the increase of non-esterified 24OH-C percentage, aimed to avoid the accumulation of this neurotoxic compound. The low degree of 24OH-C esterification in CSF or plasma might reflect reduced activity of LCAT during neurodegeneration.

Efecto del ejercicio sobre las subpoblaciones HDL, la enzima lecitina-colesterol acil-transferasa y la proteína transportadora de ésteres de colesterol en estudiantes de Medicina

ObjectiveTo evaluate exercise effect on HDL subpopulations, lecithin-cholesterol acyltransferase enzyme and ester transfer protein cholesterol levels in medical students. MethodPopulation was divided voluntarily into 2 groups, exercise and no exercise. Waist circumference and body mass index were measured; subfractions HDL2 and HDL3 by ion precipitation and LCAT and CETP enzymes Enzyme-Linked Immuno-Sorbent Assay (ELISA). ResultsLipidrisk profile increased in both groups; HDL, HDL2 and HDL3 decreased in both groups, but only the HDL2 decreased significantly in students who exercised. LCAT and CETP remained without significant changes, however, in the exercise group, there was statistically significant decrease in HDL2 and LCAT in women. ConclusionsThis study shows that exercise does alter some variables such as waist circumference, body mass index and HDL3. These changes are dependent on gender, but despite the intervention of 3 months with an exercise program, it fails reducing lipid risk factors in this medical student populations, given their environment, which complicates their metabolic response to exercise.

Association of Lecithin Cholesterol Acyltransferase rs5923 Polymorphism in Iranian Individuals with Extremely Low High-Density Lipoprotein Cholesterol: Tehran Lipid and Glucose Study.

Background:The serum concentration of high-density lipoprotein cholesterol (HDL-C) is one of the important heritable risk factors for cardiovascular disease and is a target for therapeutic intervention. In this study, we aimed to evaluate the effects of lecithin cholesterol acyltransferase (LCAT) gene polymorphism rs5923 on LCAT enzyme activity and serum HDL-C concentration. Methods: The study population was selected from consecutive individuals with HDL-C ≤ 5th percentile (n = 73) and extremely high HDL-C ≥ 95th percentile (n = 57) who had participated in the Tehran Lipid and Glucose Study. The rs5923 polymorphism was genotyped using direct sequencing. LCAT activity was measured by fluorometric assay kit, and lipid concentrations were measured using the enzymatic colorimetric method. Results: The genotype frequencies were significantly different between the high HDL-C group (CC 94.7%, CT 5.3%) and the low HDL-C group (CC 83.6%, CT 16.4%) (P = 0.048). The T-allele frequencies in subjects with low and high HDL-C were 0.082 and 0.026, respectively (P = 0.16). The association of the single-nucleotide polymorphism rs5923 with low HDL-C was not statistically significant after adjustment for age, sex, and BMI (odd ratio = 2.65, 95% confidence interval = 0.32-21.5, P = 0.36, regression logistic analysis). Also, the effects of LCAT enzyme activity did not depend on the HDL-C level (P = 0.24). Conclusion:rs5923 polymorphism is not associated with low HDL-C levels in Iranian population.

9. AAV-Mediated Gene Transfer as a Therapeutic Approach for Familial LCAT Deficiency

Lecithin Cholesterol Acyltransferase (LCAT) is a plasma enzyme secreted by the liver and responsible for esterification of free cholesterol to cholesteryl ester in the blood compartment. Familial LCAT deficiency (FLD) is an autosomal recessive disorder caused by loss-of-function mutations of the LCAT gene and is characterized by extremely low levels of high density lipoprotein cholesterol (HDL-C), anemia, corneal opacities, and chronic progressive renal disease leading to end-stage kidney disease as early as the third decade of life. Currently, there is no specific treatment for FLD. The fact that patients with even very low levels of plasma LCAT activity do not develop kidney disease make FLD an attractive candidate for liver-directed gene therapy with adeno-associated viral (AAV) vectors. LCAT is activated by apolipoprotein A-I (apoA-I) and this interaction is highly species specific. Therefore, LCAT KO mice were crossed with human apoA-I transgenic (hApoAITg) mice in order to facilitate experiments involving expression of human LCAT. LCAT KO/hApoAI transgenic mice received a single IP injection of AAV8 encoding human LCAT in a dose range of 5e10 GC/kg to 5e11 GC/kg. HDL-C increased in a dose-dependent fashion, from 32 mg/dL in the control group to 103 mg/dL at the dose of 1.5e11 GC/kg and 473 mg/dL at the dose of 5e11 GC/kg. Plasma% cholesterol ester, a sensitive indicator of LCAT activity, was normalized at the low dose of 1.5e11 GC/kg. Thus, we demonstrate that low doses of AAV8-hLCAT provide stable expression of the LCAT enzyme and correct dyslipidemia in a ‘humanized’ mouse model of LCAT deficiency. Studies designed to demonstrate prevention of kidney disease in this mouse model are ongoing. These proof-of-concept studies provide a foundation for further development of AAV-based gene therapy for familial LCAT deficiency.

Corneal dystrophies related to cholesterol metabolism (fish eye and Schnyder dystrophy)

AbstractSchnyder corneal dystrophy (SCD) is a rare corneal dystrophy characterized by abnormally increased deposition of cholesterol and phospholipids in the cornea. SCD is inherited as an autosomal dominant trait with high penetrance and has been mapped to the UBIAD1 gene on chromosome 1p36.3. Diseases of systemic lipid metabolism that cause corneal opacification, such as lecithin‐ cholesterol acyltransferase (LCAT) deficiency and fish eye disease (FED), should be considered in the differential diagnosis. LCAT deficiency and FED are both entities that result from deficiency of the LCAT enzyme. LCAT deficiency is defined by deficient LCAT activity towards HDL and LDL whereas FED is defined by decreased LCAT activity against HDL only. When comparing LCAT deficiency and FED to SCD, there are numerous differences to help distinguish all 3 entities. Both LCAT deficiency and FED are inherited in an autosomal recessive manner while SCD has autosomal dominant inheritance. Systemically, SCD has a normal level of LCAT enzyme activity and normal HDL levels. Both LCAT deficiency and FED show abnormally low levels of HDL. SCD is often associated with a true arcus lipoides, unlike FED and LCAT deficiency.

The enzyme lecithin‐cholesterol acyltransferase esterifies cerebrosterol and limits the toxic effect of this oxysterol on SH‐SY5Y cells

Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)-hydroxycholesterol, 24(S)OH-C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin-cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH-C. Proteoliposomes containing apolipoprotein A-I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A-I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH-C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH-C esterification. When highly neurotoxic 24(S)OH-C was treated with enzyme and proteoliposomes before incubation with differentiated SH-SY5Y cells, the neuron survival improved. The esters of 24(S)OH-C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH-C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH-C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol-induced neurotoxic injuries to neurons in culture. 24-hydroxycholesterol (24(S)OH-C) is neurotoxic. The enzyme lecithin-cholesterol acyltransferase (LCAT) synthesizes monoesters of 24(S)OH-C in reaction mixtures with proteoliposomes containing phospholipids and apolipoprotein A-I or apolipoprotein E. The esters, also produced by incubation of cerebrospinal fluid only with tritiated 24(S)OH-C, are embedded into lipoproteins that do not enter neurons in culture. The enzyme activity limits the toxicity of 24-hydroxycholesterol in neuron culture.

Exploring Avenues for Raising HDL Cholesterol

Exploring Avenues for Raising HDL Cholesterol

Analysis of the haptoglobin binding region on the apolipoprotein A‐I‐derived P2a peptide

Apolipoprotein A-I (ApoA-I) is the main protein component of the high density lipoproteins and it plays an important role in the reverse cholesterol transport. In particular, it stimulates cholesterol efflux from peripheral cells toward liver and activates the enzyme lecithin-cholesterol acyltransferase (LCAT). Haptoglobin (Hpt), a plasma α2-glycoprotein belonging to the family of acute-phase proteins, binds to ApoA-I inhibiting the stimulation of the enzyme LCAT. Previously, we reported that a synthetic peptide, P2a, binds to and displaces Hpt from ApoA-I restoring the LCAT cholesterol esterification activity in the presence of Hpt. Here, we investigate the molecular determinants underlining the interaction between Hpt and P2a peptide. Analysis of truncated P2a analogs showed that P2a sequence can only be slight reduced in length at the N-terminal to preserve the ability of binding to Hpt. Binding assays showed that charged residues are not involved in Hpt recognition; actually, E146A and D157A substitutions increase the binding affinity to Hpt. Biological characterization of the corresponding P2a peptide analogs, Apo146 and Apo157, showed that the two peptides interfere with Hpt binding to HDL and are more effective than P2a peptide in rescue LCAT activity from Hpt inhibition. This result suggests novel hints to design peptides with anti-atherogenic activity.