LAL (Lysosomal Acid Lipase) Promotes Reverse Cholesterol Transport In Vitro and In Vivo.

OBJECTIVE: To explore the role of LAL (lysosomal acid lipase) in macrophage cholesterol efflux and whole-body reverse cholesterol transport. APPROACH AND RESULTS: Immortalized peritoneal macrophages from lal-/- mice showed reduced expression of ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1), reduced production of the regulatory oxysterol 27-hydroxycholesterol, and impaired suppression of cholesterol synthesis on exposure to acetylated low-density lipoprotein when compared with lal+/+ macrophages. LAL-deficient mice also showed reduced hepatic ABCG5 (ATP-binding cassette transporter G5) and ABCG8 (ATP-binding cassette transporter G8) expression compared with lal+/+ mice. LAL-deficient macrophages loaded with [3H]-cholesteryl oleate-labeled acetylated low-density lipoprotein showed impaired efflux of released [3H]-cholesterol to apoA-I (apolipoprotein A-I), with normalization of [3H]-cholesteryl ester levels and partial correction of ABCA1 expression and cholesterol efflux to apoA-I when treated with exogenous rhLAL (recombinant human LAL protein). LAL-deficient mice injected intraperitoneally with lal-/- macrophages cholesterol loaded and labeled in the same way exhibited only 1.55±0.35% total injected [3H]-cholesterol counts appearing in the feces for 48 h (n=30), compared with 5.38±0.92% in lal+/+ mice injected with labeled lal+/+ macrophages (n=27), P<0.001. To mimic the therapeutic condition of delivery of supplemental LAL in vivo, injection of labeled lal-/- macrophages into lal+/+ mice resulted in a significant increase in reverse cholesterol transport (2.60±0.46% of 3H-cholesterol counts in feces at 48 hours [n=19]; P<0.001 when compared with injection into lal-/- mice). CONCLUSIONS: These results indicate a critical role for LAL in promoting both macrophage and whole-body reverse cholesterol transport and the ability of supplemental LAL to be taken up and correct reverse cholesterol transport in vivo.

Properties, structure, and applications of microbial sterol esterases.

According to their substrate preferences, carboxylic ester hydrolases are organized in smaller clusters. Among them, sterol esterases (EC 3.1.1.13), also known as cholesterol esterases, act on fatty acid esters of cholesterol and other sterols in aqueous media, and are also able to catalyze synthesis by esterification or transesterification in the presence of organic solvents. Mammalian cholesterol esterases are intracellular enzymes that have been extensively studied since they are essential in lipid metabolism and cholesterol absorption, and the natural role of some microbial sterol esterases is supposed to be similar. However, besides these intracellular enzymes, a number of microbes produce extracellular sterol esterases, which show broad stability, selectivity, or wide substrate specificity, making them interesting for the industry. In spite of this, there is little information about microbial sterol esterases, and only a small amount of them have been characterized. Some of the most commercially exploited cholesterol esterases are produced by Pseudomonas species and by Candida rugosa, although in the last case they are usually described and named as "high substrate versatility lipases." From a structural point of view, most of them belong to the α/ß-hydrolase superfamily and have a conserved "catalytic triad" formed by His, an acidic amino acid and a Ser residue that is located in a highly conserved GXSXG sequence. In this review, the information available on microbial sterol esterases has been gathered, taking into account their origin, production and purification, heterologous expression, structure, stability, or substrate specificity, which are the main properties that make them attractive for different applications. Moreover, a comprehensive phylogenetic analysis on available sequences of cholesterol esterases has been done, including putative sequences deduced from public genomes.

Biochemical Characterization of a Lipolytic Enzyme From Aspergillus oryzae That Hydrolyzes Triacylglycerol and Sterol Esters.

A novel lipolytic enzyme-encoding gene, lipO745, from Aspergillus oryzae RIB40 was cloned and expressed in Pichia pastoris. Purified recombinant LipO745 (rLipO745) had a molecular mass of approximately 60 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. rLipO745 exhibited maximum activity at 40 °C and pH 7.0 and was stable at temperatures ≤ 40 °C. The substrate specificity of purified rLipO745 was analyzed using α-naphthyl esters as artificial substrates and various triacylglycerol and sterol esters as natural substrates. From among a panel of α-naphthyl esters (C2-C16), α-naphthyl butyrate (C4), with an activity of 269 ± 3.3 units/mg protein, was the optimal substrate for hydrolysis by the purified recombinant protein. The Km and kcat values of rLiO745 for the C4 substrate were 0.073 ± 0.0012 mM and 608 ± 108 s-1, respectively. The purified recombinant enzyme had considerable hydrolytic activity toward tributyrin, tripalmitin, and triolein, indicating lipase activity, and toward cholesteryl acetate, butyrate, palmitate, and oleate, indicating sterol esterase activity. Transesterification activities between tributyrin and cholesterol or between tributyrin and campesterol were also determined.

Optimization of lipase-catalyzed synthesis of ß-sitostanol esters by response surface methodology.

The esters of ß-sitostanol and fatty acids are known for their effect as cholesterol-lowering agents. In this work, the efficiency of three lipases as biocatalysts of the esterification of ß-sitostanol and C16 and C18 fatty acids was compared. The sterol esterase of Ophiostoma piceae (OPEr) yielded the highest esterification rates and was selected for further optimization of the reaction. The effects of four parameters (temperature, enzymatic dosage, acyl donor concentration, and reaction time) on ester synthesis were investigated and the process conditions were optimized using response surface methodology (RSM). The best conditions for esterification for each fatty acid were predicted using a second-order model, and experimentally validated. Very high esterification efficiencies (86-97%) were observed using the predicted values for the four variables. This approach was shown to be suitable for optimizing the enzymatic production of ß-sitostanol esters, which represents a green alternative to the chemical synthesis of these dietary complements.

Green synthesis of ß-sitostanol esters catalyzed by the versatile lipase/sterol esterase from Ophiostoma piceae.

ß-sitostanol esters, used as dietary complement for decreasing cholesterol absorption, have been synthesized at 28°C via direct esterification or transesterification catalyzed by the versatile lipase/sterol esterase from the ascomycete fungus O. piceae. Direct esterification was conducted in biphasic isooctane: water systems containing 10mM ß-sitostanol and lauric or oleic acid as acyl donors, reaching 90% esterification in 3h with the recombinant enzyme. The use of molar excesses of the free fatty acids did not improve direct esterification rate, and the enzyme did not convert one of the two fatty acids preferentially when both were simultaneously available. On the other hand, solvent-free transesterification was an extremely efficient mechanism to synthesize ß-sitostanyl oleate, yielding virtually full conversion of up to 80mM ß-sitostanol in 2h. This process may represent a promising green alternative to the current chemical synthesis of these esters of unquestionable nutraceutical value.

Structural traits and catalytic versatility of the lipases from the Candida rugosa-like family: A review.

Lipases and sterol esterases are enzymes with broad biotechnological applications, which catalyze the hydrolysis or synthesis of long-chain acylglycerols and sterol esters, respectively. In this paper, we review the current knowledge on the so-called Candida rugosa-like family of enzymes, whose members display in most cases affinity against the two substrates mentioned above. The family includes proteins with the α/ß-hydrolase folding, sharing conserved motifs in their sequences, and common structural features. We will go through their production and purification, relate their described structures and catalytic activity, and discuss the influence of the hydrophobic character of these lipases on their aggregation state and activity. On the basis of the few crystal structures available, the role of each of the functional areas in catalysis will be analyzed. Considering the particular characteristics of this group, we propose their classification as "Versatile Lipases" (EC 3.1.1.x).

Heterologous expression of a fungal sterol esterase/lipase in different hosts: Effect on solubility, glycosylation and production.

Ophiostoma piceae secretes a versatile sterol-esterase (OPE) that shows high efficiency in both hydrolysis and synthesis of triglycerides and sterol esters. This enzyme produces aggregates in aqueous solutions, but the recombinant protein, expressed in Komagataella (synonym Pichia) pastoris, showed higher catalytic efficiency because of its higher solubility. This fact owes to a modification in the N-terminal sequence of the protein expressed in Pichia pastoris, which incorporated 4-8 additional amino acids, affecting its aggregation behavior. In this study we present a newly engineered P. pastoris strain with improved protein production. We also produced the recombinant protein in the yeast Saccharomyces cerevisiae and in the prokaryotic host Escherichia coli, corroborating that the presence of these N-terminal extra amino acids affected the protein's solubility. The OPE produced in the new P. pastoris strain presented the same physicochemical properties than the old one. An inactive form of the enzyme was produced by the bacterium, but the recombinant esterase from both yeasts was active even after its enzymatic deglycosylation, suggesting that the presence of N-linked carbohydrates in the mature protein is not essential for enzyme activity. Although the yield in S. cerevisiae was lower than that obtained in P. pastoris, this work demonstrates the importance of the choice of the heterologous host for successful production of soluble and active recombinant protein. In addition, S. cerevisiae constitutes a good engineering platform for improving the properties of this biocatalyst.

Importância da biópsia hepática no diagnóstico da deficiência de lipase ácida lisossomal: relato de caso / Importance of liver biopsy in the diagnosis of lysosomal acid lipase deficiency: a case report

RESUMO Objetivo: Descrever a doença de depósito de ésteres de colesterol (DDEC) e a importância da biópsia hepática na realização do diagnóstico. Descrição do caso: Paciente feminina, dois anos e dez meses de idade, com queixa de aumento do volume abdominal secundário à hepatomegalia há quatro meses. Ultrassonografia abdominal demonstrou hepatomegalia e esteatose hepática. Exames laboratoriais mostraram aumento de enzimas hepáticas e dislipidemia. A biópsia hepática foi compatível com DDEC. Comentários: Embora a medida da atividade enzimática seja o padrão-ouro para o diagnóstico de DDEC, a biópsia hepática é muito útil na investigação de casos suspeitos, particularmente quando há outros diagnósticos diferenciais a serem considerados.

ABSTRACT Objective: To describe a case of cholesteryl ester storage disease (CESD) and discuss the importance of liver biopsy for diagnosis. Case description: A female patient, aged two years and ten months, presented with an increased abdominal volume following hepatomegaly for four months. Abdominal ultrasound demonstrated hepatomegaly and hepatic steatosis. Laboratory tests showed elevated liver serum enzymes and dyslipidemia. Liver biopsy was consistent with CESD. Comments: Although measuring enzyme activity is the gold standard for CESD diagnosis, liver biopsy is very helpful when investigating suspected cases of CESD, particularly upon other differential diagnoses to be considered.

Detección de la mutación E8SJM en el gen LIPA, por PCR en tiempo real, para la investigación de la enfermedad por almacenamiento de ésteres de colesterol / Detection of the E8SJM mutation in the LIPA gene, by real-time PCR, for the investigation of cholesteryl ester storage disease

Introducción: La enfermedad por Almacenamiento de Ésteres de Colesterol (CESD; Cholesteryl Ester Storage Disease) es una enfermedad de depósito lisosomal, su presentación es bastante variable y su diagnóstico constituye un desafío. Además, existe un número de anomalías observadas en los pacientes con CESD que se sobreponen a diagnósticos más comunes, siendo probable que sea subdiagnosticada. La mayoría de pacientes relatados hasta el momento son portadores de la mutación E8SJM en el gen LIPA. En este sentido, el auxilio en el diagnóstico es fundamental pues existen opciones de terapia en desarrollo. Diseño: Estudio observacional. Objetivo: Estandarizar la técnica de PCR en tiempo real para la detección de la mutación más frecuente, E8SJM, en muestras de sangre periférica y de biopsia hepática para el auxilio diagnóstico de CESD y futuros estudios de prevalencia de la mutación. Institución: Centro de Terapia Génica del Hospital de Clínicas de Porto Alegre (HCPA), Brasil. Material biológico: Muestras de ADN extraídas de sangre periférica y tejido hepático parafinado. Principales medidas de resultados: Presencia/Ausencia de la mutación E8SJM. Resultados: Se estandarizó la reacción de PCR en tiempo real, la mutación fue detectada correctamente y posteriormente validada por secuenciación de Sanger. La mutación fue analizada en 137 muestras y encontrada en apenas una paciente que ingresó al Servicio de Genética Médica del HCPA con diagnóstico clínico y bioquímico de CESD/Wolman. Conclusiones: La técnica de PCR en tiempo real es ideal para la detección rápida y en gran escala de la mutación frecuente asociada a CESD.

Introduction: Cholesteryl Ester Storage Disease (CESD) is a lysosomal storage disorder, its presentation is highly variable and its diagnosis challenging. In addition, there are several abnormalities observed in patients with CESD who overlap with more common diagnoses and are likely to be underdiagnosed. Most patients reported to date are carriers of the E8SJM mutation in the LIPA gene. In this sense, diagnostic assistance is essential because there are options for therapy in development, as well as mutation prevalence studies. Design: Observational research. Objective: To standardize the real-time PCR technique for the detection of the most frequent mutation, E8SJM, in peripheral blood and liver biopsy specimens for the diagnosis of CESD and future mutation prevalence studies. Institution: Center of Gene Therapy of the Hospital de Clinicas de Porto Alegre (HCPA), Brazil. Biological material: DNA samples extracted from peripheral blood and paraffinembedded liver tissue. Main outcome measures: Presence / Absence of E8SJM mutation. Results: The PCR reaction was standardized in real time; the mutation was correctly detected and validated by Sanger sequencing. The mutation was analyzed in 137 samples and found in only one patient who entered the Medical Genetics Service of the HCPA with clinical and biochemical diagnosis of CESD/Wolman. Conclusions: The real-time PCR technique is ideal for rapid and large-scale detection of the frequent CESD-associated mutation.