Enzyme studies on Epstein-Barr virus-transformed lymphoid cell lines from Wolman's disease. Lipases, cholesterol esterase and 4-methylumbelliferyl acyl ester hydrolases

(1) In lymphoid cell lines established by Epstein-Barr virus transformation of B-lymphocytes from normal subjects there exist two lipases hydrolysing triolein (the first one with acid optimum pH and the other one with alkaline optimum pH) and one cholesterol esterase (with acidic optimum pH). The acid triolein lipase (optimum pH 3.75-4.0) and the acid cholesterol esterase are activated by taurocholate (optimal concentration between 1 and 2.5 g/l) whereas alkaline triolein-lipase is inhibited by crude taurocholate. (2) Acid lipase deficiency is demonstrated in lymphoid cell lines from a Wolman's patient, using natural substrates, triolein and cholesteryl oleate (residual activity 5 and 8%, respectively). Thus, this similar deficiency demonstrates that, in lymphoid cell lines, triolein and cholesteryl esters are hydrolysed (under the conditions used here) by a single enzyme, i.e., lysosomal acid lipase muted in Wolman's disease. (3) pH profiles of synthetic substrate hydrolysis show marked differences between methylumbelliferyl oleate and methylumbelliferyl palmitate, and are greatly dependent on the assay conditions used. In the presence of optimal concentrations of taurocholate (1-2.5 g/l), nonspecific carboxylesterases are inhibited and acid lipase is activated: in this case, methylumbelliferyl oleate can be used to demonstrate the acid lipase deficiency in Wolman's lines (15-20% of residual activity). Methylumbelliferyl palmitate hydrolysis is less dependent on assay conditions and thus can be more accurately used for the diagnosis of Wolman's disease, with lower residual activity (10-15%) than using methylumbelliferyl oleate. Thus, Epstein-Barr virus-transformed lymphoid cell lines represent an accurate model system in culture for experimental studies of Wolman's disease.     

Acid lipase: a histochemical and biochemical study using triton X100-naphtyl palmitate micelles.

Hydrolsis of a-naphtyl palmitate dispersed with the detergent Triton X-100 at acid pH was studied by a histochemical diazocoupling technique in both fixed sections and cultures of primate tissues as well as by a biochemical assay employing the same chromogenic substrate. Evidence for the exclusive hydrolysis of this artificial fatty acid ester substrate by acid lipases was gathered from (1) comparison of isoelectric focusing zymograms developed with different substrates, (2) kinetic analysis of enzyme activity in the presence or absence of inhibitors, including a natural substrate of acid lipase, trioleylglycerol, (3) specific localization of marked enzyme activity in certain tissues, and (4) absence of detectable enzyme activity in a case of human acid lipase deficiency (Wolman's disease). Histochemically, acid lipase activity was most readily detected in cells active in the uptake and processing of neutral lipids, i.e., the phagocytes of the reticuloendothelial system, the adrenal cortex and the lipid-storing cells in the athero-sclerotic plaques of arteries.     

Prenatal monitoring for wolman's disease in a pregnancy at risk

Summary A pregnancy at risk for Wolman's disease was successfully monitored by assay of acid lipase activity in cultured amniotic fluid cells using the synthetic substrate 4-methylumbelliferyl oleate. A nonaffected fetus was detected showing heterozygosity for Wolman's disease. The healthy boy is now one year old.     

Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases

Fluorescent pyrene-methyl lauryl ester (PMLes) was synthesized and used for the determination of cellular lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or cholesteryl ester storage diseases (both exhibiting a deficiency of the lysosomal acid lipase). The hydrolysis of PMLes by acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use PMLes as a fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (i.e. pyrene-methanol) was read in the lower aqueous-ethanolic phase, at 378 nm. PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or cholesteryl ester storage diseases. This demonstrates that the fluorescent PMLes is hydrolyzed by the lysosomal acid lipase and can be used as a very sensitive fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.     

Characteristics of acid esterase in Wolman's disease

The characteristics of acid esterase from the patient with Wolman's disease, a rare familial lipidosis, were studied. Enzymatic analysis as well as mineral analysis were performed on the patient's liver, spleen, and adrenal glands. Acid esterase was low in the patient's leucocytes and other affected tissues. Further enzymatic study with subcellular fractions of the liver in both patient and control subject revealed that acid esterase was mostly localized in the membrane of lysosomes. The lysosomal esterase was unaffected by Ca2+, Mg2+, EDTA, E600 (microsomal esterase inhibitor), and it was less inhibited by NaCl than other fractions. Studies with those inhibitors showed that acid esterase has different properties compared to other lipases, such as lipoprotein lipase, adipose tissue lipase, and hepatic microsomal lipase. Studies with inhibitors also gave a negative view on a possible suppressive interaction of the high content of calcium in the target organs with acid esterase in Wolman's disease.     

Genetic lipid storage disease with lysosomal acid lipase deficiency in rats

We describe a new animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected Donryu rats, who inherited the disease in an autosomal recessive mode, manifested marked hepatosplenomegaly, lymph node enlargement, and thickened, dilated intestine. Morphologically, many characteristic foam cells were observed in livers and spleens. No adrenal calcification could be found in affected rats. Biochemical studies on spleen and liver tissues showed massive accumulation of esterified cholesterol and triglycerides, and deficiency of acid lipase for [14C]-cholesteryl oleate. This animal model could contribute greatly to the clarification of the physiological and pathological roles of lysosomal acid lipase in the metabolism of lipoproteins and cholesterol, and of the pathogenesis of atherosclerosis.     

Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity

The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.     

Multi-competitive enzymatic reactions in organic media: a simple test for the determination of lipase fatty acid specificity

Multiple substrate competition was kinetically analyzed to study lipase-catalyzed reactions in organic media. For each substrate, a competitive factor (the ratio of the specificity constants kcat/Km) was measured by reference to the best substrate using a mixture of fatty acid ethyl esters submitted to a solvolysis reaction by n-propanol. A scale of competitive factors was established which quantitatively described the lipase specificity. This principle was applied to the determination of the specificity of four commercial lipase preparations towards fatty acid chain length and degree of unsaturation. The results were not affected by changes in the physicochemical conditions of the reaction (water content, substrate concentration, nature of nucleophile, etc.). The simple test will be a useful tool to characterize lipase specificity.     

New spectrophotometric assays of acid lipase and their use in the diagnosis of Wolman and cholesteryl ester storage diseases

Trinitrophenylaminolauric acid (TNPAL) was linked to glycerol or cholesterol and the resulting yellow compounds were used as substrates for several lipases and cholesteryl esterase in cells from normal individuals and patients with Wolman's or cholesteryl ester storage diseases. Normal cells (lymphoid cell lines or skin fibroblasts) showed two peaks of lipase or cholesteryl esterase activity at about pH 4.0 and 6.0 each. The activity of the most acidic enzyme, which hydrolyzed natural or synthetic triacylglycerols as well as cholesteryl esters, was considerably reduced in cells derived from patients with Wolman's or cholesteryl ester storage diseases. Simple spectrophotometric procedures were developed for using tri-TNPAL glycerol or TNPAL cholesterol to identify homozygotes of these two respective diseases.     

Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade)

While invertebrates store neutral lipids as their major energy source, little is known about triacylglycerol (TAG) class composition and their differential catabolism in aquatic arthropods. This study focuses on the composition of the main energy source and its catabolism by lipase from the midgut gland (hepatopancreas) of the crustacean Macrobrachium borellii. Silver-ion thin-layer chromatography of prawn large TAG deposit (80% of total lipids) and its subsequent fatty acid analysis by gas chromatography allowed the identification of 4 major fractions. These are composed of fatty acids of decreasing unsaturation and carbon chain length, the predominant being 18:1n-9. Fraction I, the most unsaturated one, contained mainly 20:5n-3; fraction II 18:2n-6; fraction III 18:1n-9 while the most saturated fraction contained mostly 16:0. Hepatopancreas main lipase (Mr 72 kDa) cross-reacted with polyclonal antibodies against insect lipase, was not dependent on the presence of Ca²⁺ and had an optimum activity at 40 °C and pH 8.0. Kinetic analysis showed a Michaelis–Menten behavior. A substrate competition assay evidenced lipase specificity following the order: 18:1n-9-TAG > PUFA-enriched-TAG > 16:0-TAG different from that in vertebrates. These data indicate there is a reasonable correspondence between the fatty acid composition of TAG and the substrate specificity of lipase, which may be an important factor in determining which fatty acids are mobilized during lipolysis for oxidation in crustaceans.     

Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1.

The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus. The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.     

Alcohol inhibition and specificity studies of lipase B from candida antarctica in organic solvents

Alcohol inhibition of the lipase B from Candida antarctica has been studied through two different approaches: using the same inhibitor (1-butanol) in different organic solvents and using different inhibitors (differing in chain length) in the same solvent. The competitive inhibition constant values obtained in each case correlate with the calculated activity coefficients of the substrate, suggesting that desolvation of the alcohol is the major force changed. Data dispersion observed using the second approach has been interpreted to come from contributions of enzyme-inhibitor interactions to the binding energy. On the other hand, deacylation has been found to be much less influenced by the solvent variation than the acylation step, despite of the fact that solvation of the substrate involved in this step (the alcohol) is expected to change more than for the ester. Concerning the specificity behavior of the enzyme, a bimodal pattern was observed for the deacylation rate dependence on the alcohol chain length, with the highest values for hexanol (C6) and decanol (C10). With regard to the ester specificity, ethyl caproate (C6) is the preferred one. These results have been confronted with those reported for the lipase from Candida rugosa. Copyright 1998 John Wiley & Sons, Inc.     

Alteration of lipase chain length specificity in the hydrolysis of esters by random mutagenesis

The feasibility of altering the chain length specificity of industrially important Rhizomucor miehei lipase was investigated by randomly mutating Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate. The recombinant lipase was initially expressed in E. coli. Individual colonies were selected, grown, and the DNA sequence of the lipase gene determined. Fourteen of the 19 possible mutants were identified and each of these was transformed into Pichia pastoris which expresses the enzyme extracellularly. The yeast was grown and the supernatants assessed in several assays with long and short chain substrates. Based on this preliminary screen, one mutant, Phe94Gly, was selected and purified to homogeneity for further analysis. It was found that the substitution of phenylalanine 94 with glycine led to an enzyme which was about six times less active against resorufin ester but displayed 3-4 times higher activity with short chain substrates such as butyric acid esters. The observed alteration to the enzyme specificity was rationalised using the available 3D structure of the lipase.     

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">simulans</Emphasis> lipase expressed in <Emphasis Type="Italic">E. coli</Emphasis>

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.     

Lysosomal acid lipase deficiency in rats: lipid analyses and lipase activities in liver and spleen

We report the biological characterization of an animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected rats accumulated cholesteryl esters (13.3-fold), free cholesterol (2.8-fold), and triglycerides (5.4-fold) in the liver, as well as cholesteryl esters (2.5-fold) and free cholesterol (1.33-fold) in the spleen. Triglycerides did not accumulate, and the levels actually decreased in the spleen. Analysis of the fatty acid composition of the cholesteryl esters and triglycerides showed high percentages of linoleic acid (18:2) and arachidonic acid (20:4) in both organs, especially in the liver. No accumulation of phospholipids, neutral glycosphingolipids, or gangliosides was found in the affected rats. Acid lipase activity for [14C]triolein, [14C]cholesteryl oleate, and 4-methyl-umbelliferyl oleate was deficient in both the liver and spleen of affected rats. Lipase activity at neutral pH was normal in both liver and spleen. Heterozygous rats showed intermediate utilization of these substrates in both organs at levels between those for affected rats and those for normal controls, although they did not accumulate any lipids. These data suggest that these rats represent an animal counterpart of Wolman's disease in humans.     

Substrate specificity and positional preference of a lipoprotein lipase

The substrate specificity and positional preference of a membrane-bound lipoprotein lipase with and without reconstitution in liposomes were studied. The enzyme showed the same preference for the acyl groups in the 1- and 3-positions of triglycerides, while its activity toward the 2-position was one-third of that toward the 1- or 3-positions. The substrate specificity of the enzyme toward fatty acids of different chain length and degree of unsaturation was in the order of C:14>C:16>C:18:1≥C:18. The enzyme with and without reconstitution showed the same positional preference and substrate specificity.     

Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice

Fluorescent triacylglycerols containing pyrenedecanoic (P10) and pyrenebutanoic (P4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl-para-nitrophenyl phosphate (E600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate/detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.5 mmol/l), but was not significantly affected by serum albumin and E600 (10(-2) mmol/l). (2) The neutral pH7-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol. It is resistant to E600 and heat-stable, similarly to the acid pH4-lipase, but it is well discriminated from the acid enzyme by its substrate/detergent optimal molar ratios (1:2 and 1:3 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively), whereas higher detergent concentrations, optimal for the acid lipase, are strongly inhibitory for the neutral enzyme. (3) The pH5-lipase present in gastric juice as well as in stomach homogenate exhibited properties obviously discriminating it from the other lipolytic enzymes from stomach homogenate: broad substrate specificity for P10- as well as P4-triacylglycerols, activation by low concentrations of amphiphiles (with optimal ratios triacylglycerols/taurocholate, triacylglycerols/taurocholate and triacylglycerols/phosphatidylcholine around 1:1, 1:3 and 1:0.1, respectively), heat-lability, strong activation by serum albumin and inhibition by E600 (10(-2) mmol/l). This pH5-lipase is the sole lipolytic enzyme present in gastric juice and is probably identical with the well-known 'gastric' lipase. (4) A pH7.5-enzyme is characterized by its specificity for P4-triacylglycerols, its heat-lability at 50 degrees C and its strong inhibition by E600 (10(-2) mmol/l).     

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or "lid" covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL(3) in the presence of the EL C-domain.     

Glycerol Ester Hydrolase Activity of Microbacterium thermosphactum

Microbacterium thermosphactum possesses a significant glycerol ester hydrolase (lipase, EC 3.1.1.3) activity and a weak but definite carboxylic ester hydrolase (esterase, EC 3.1.1.1) activity. Harvested whole cell preparations contained 53 units of lipase activity with tripropionin as the substrate. This activity decreased with an increasing chain length of fatty acid in the triglyceride to 13 units with trilaurin as the substrate and no activity with tripalmitin. Maximum lipase activity was found at a temperature of 35 to 37 C and at a pH of 7.1 to 7.3. Lipase activity was associated with three different protein peaks when the protein of cell-free extract was fractionated by polyacrylamide gel electrophoresis.     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.