Molecular cloning and expression of cDNA for rat pancreatic cholesterol esterase

A full-length cDNA complementary to the rat pancreatic cholesterol esterase mRNA was isolated by screening a rat pancreatic cDNA expression library in lambda gt11 vector with antibodies against the porcine pancreatic cholesterol esterase. The isolated cholesterol esterase cDNA is 2050 bp in length and contains an open reading frame coding for a protein of 612 amino acids. A 20-amino acid hydrophobic leader sequence is predicted, based on the position of the first ATG initiation codon upstream from the sequenced amino terminus of the isolated cholesterol esterase. The cholesterol esterase cDNA was subcloned into a mammalian expression vector, pSVL, for transfection studies. Expression of the cDNA in COS cells resulted in the production of bile salt-stimulated cholesterol esterase. Comparison of the cholesterol esterase cDNA sequence with other proteins revealed that the pancreatic cholesterol esterase is identical to rat pancreatic lysophospholipase. The primary structure of cholesterol esterase displayed no significant homology with other lipases, although the putative lipid interfacial recognition site of G-X-S-X-G is present in the cholesterol esterase sequence. However, the cholesterol esterase sequence revealed a 63-amino-acid domain which is highly homologous to the active site domain of other serine esterases. These data suggest that cholesterol esterase may be a member of the serine esterase supergene family. Analysis of the cholesterol esterase structure also revealed a repetitive sequence enriched with Pro, Asp, Glu, Ser, and Thr residues at the C-terminal end of the protein. This sequence is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that cholesterol esterase may have a short half-life in vivo. Northern blot hybridization showed that the bile salt-stimulated cholesterol esterase mRNA is present in liver suggesting that this protein may also be synthesized by liver cells.     

Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

The activities of pancreatic cholesterol esterase from calf and cow pancreas were examined in detail. A 1300-fold enhancement of enzymatic activity was found after maturation, even though cholesterol esterase activity levels in other organs did not change from the juvenile to the adult species. Radioimmunoassays also showed that the calf pancreas contained at least 100-fold less cholesterol esterase protein. Decreased amounts of protein were not due to enhanced proteolysis, since cytosol from cow pancreas degrades exogenously added cholesterol esterase faster than that from calf pancreas. Rather, enhancement of pancreatic cholesterol esterase activity associated with bovine maturation was the result of specific, increased synthesis of a 72-kDa enzyme. This labile 72-kDa cholesterol esterase species was purified to homogeneity by a two-step process in 75% yield and is the major form of bovine pancreatic cholesterol esterase (99%). A much less abundant 67-kDa species, accounting for less than 1% of total pancreatic cholesterol esterase activity, was also purified to homogeneity in a similar two-step process. These results demonstrate that a specific form of pancreatic cholesterol esterase is induced during maturation, and they bear importantly on understanding juvenile cholesterol metabolism as related to dietary absorption of this sterol.     

Purification and characterization of rat pancreatic fatty acid ethyl ester synthase and its structural and functional relationship to pancreatic cholesterol esterase

Formation of fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) has been implicated in the pathogenesis of chronic pancreatitis. In previous studies, we demonstrated that FAEE synthase, purified from rat liver microsomes, is identical to rat liver carboxylesterase (pI 6.1), and structurally and functionally different than that from pancreas. In this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with rat pancreatic cholesterol esterase (ChE). Since most of the serine esterases express p-nitrophenyl acetate (PNPA)-hydrolyzing activity as well as synthetic activity to form fatty acid esters or amides with a wide spectrum of alcohols and amines, respectively, we used PNPA-hydrolyzing activity to monitor the purification of FAEE synthase during various chromatographic purification steps. Synthesizing activity towards FAEEs, fatty acid methyl esters, and fatty acid anilides was measured only in the pooled fractions. At each step of purification (ammonium sulfate saturation, Q Sepharose XL, and heparin-agarose column chromatographies, and high performance liquid chromatography (anion exchange and gel filtration)) synthetic as well as hydrolytic activities copurified. Using ethanol, methanol, or aniline as substrates, the ester or anilide synthesizing activity of the purified protein was found to be 8709, 13000, and 2201 nmol/h/mg protein, respectively. The purified protein displayed a single band with an estimated molecular mass of approximately 68 kD upon SDS-PAGE under reduced denaturing conditions, cross-reacted with antisera against rat pancreatic ChE and showed 100% N-terminal sequence homology of the first 15 amino acids to that of rat pancreatic ChE. These results suggest that the purified protein has broad substrate specificity towards the conjugation of endogenous long chain fatty acids with substrates having hydroxyl and amino groups and is identical to ChE.     

Identification of the active site serine in pancreatic cholesterol esterase by chemical modification and site-specific mutagenesis

Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic cholesterol esterase. In the first approach, purified porcine pancreatic cholesterol esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled cholesterol esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat cholesterol esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat cholesterol esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized cholesterol esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic cholesterol esterase.     

Immunological comparison of cholesterol esterases.

Rat pancreas cholesterol esterase has been immunologically compared with rat intestinal cholesterol esterase. Monospecific precipitating antisera against purified rat pancreas cholesterol esterase were produced in rabbits. Immune IgG, isolated from the antisera, crossreacted with the cholesterol esterase of intestine in the immunodiffusion assay with a pattern of complete identity. Titration of the pancreatic and intestinal enzyme with immune IgG revealed a maximum precipitation (99 and 98%) and maximum inhibition of enzyme activity (66 and 65%) when the ratio of enzyme activity (units) to immune IgG (mg) was 4.1 and 4.0, respectively. The immunological identity demonstrated in these studies lend support to the concept that intestinal cholesterol esterase is derived from the pancreatic enzyme. In additional studies, the immune IgG was employed in the immunodiffusion assay to test for cross-reaction with cholesterol esterases prepared from rat aorta, adrenal, and liver and with cholesterol esterases prepared from the pancreas of rabbit, dog, cow, and guinea pig. There was no evidence of cross-reaction in any case. Further, cholesterol esterase prepared from the pancreas of rabbit, dog, and cow retained full enzymatic activity when titrated with immune IgG.     

Site-specific mutagenesis of an essential histidine residue in pancreatic cholesterol esterase

The histidine residue essential for the catalytic activity of pancreatic cholesterol esterase (carboxylester lipase) has been identified in this study using sequence comparison and site-specific mutagenesis techniques. In the first approach, comparison of the primary structure of rat pancreatic cholesterol esterase with that of acetylcholinesterase and cholinesterase revealed two conserved histidine residues located at positions 420 and 435. The sequence in the region around histidine 420 is quite different between the three enzymes. However, histidine 435 is located in a 22-amino acid domain that is 47% homologous with other serine esterases. Based on this sequence homology, it was hypothesized that histidine 435 is the histidine residue essential for catalytic activity of cholesterol esterase. The role of His435 in the catalytic activity of pancreatic cholesterol esterase was then studied by the site-specific mutagenesis technique. Substitution of the histidine in position 435 with glutamine, arginine, alanine, serine, or aspartic acid abolished the ability of cholesterol esterase to hydrolyze p-nitrophenyl butyrate and cholesterol [14C]oleate. In contrast, mutagenesis of the histidine residue at position 420 to glutamine had no effect on cholesterol esterase enzyme activity. The results of this study strongly suggested that histidine 435 may be a component of the catalytic triad of pancreatic cholesterol esterase.     

Inhibitors of sterol synthesis. Oleate ester of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one as a substrate for pancreatic cholesterol esterase

5 alpha-Cholest-8(14)-en-3 beta-yl-15-one oleate (15-ketosteryl oleate), the oleate ester of a compound with the capacity to lower serum cholesterol, was effectively hydrolyzed by partially purified porcine pancreatic cholesterol esterase with an apparent Km of 0.28 +/- 0.01 mM and a Vmax of 0.62 +/- 0.01 mumol/min per mg protein compared to an apparent Km of 0.19 +/- 0.02 mM and a Vmax of 0.37 +/- 0.02 mumol/min per mg protein for cholesteryl oleate. The 15-ketosteryl oleate was also hydrolyzed by highly purified rat pancreatic cholesterol esterase with an apparent Km of 0.20 +/- 0.01 mM and a Vmax of 86.7 +/- 3.0 mumol/min per mg protein compared to an apparent Km of 0.43 +/- 0.01 mM and a Vmax of 119.8 +/- 2.6 mumol/min per mg protein for cholesteryl oleate. 15-Ketosteryl oleate is, therefore, a good substrate for pancreatic cholesterol esterase from either source. The 15-ketosterol is a weak competitive inhibitor of partially purified porcine pancreatic cholesterol esterase when cholesteryl oleate is the substrate.     

Inhibition of rat liver cholesterol esterase by local anaesthetics.

1. A number of local anaesthetics was shown to inhibit rat liver cholesterol esterase activity towards radioactively labelled cholesterol oleate. The anaesthetics inhibited in the order dibucaine greater than chlorpromazine greater than tetracaine greater than benzocaine greater than procaine greater than lidocaine greater than cocaine. 2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca2+. 3. The inhibition by tetracaine is partially reversed by sodium deoxycholate. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration dependent manner. Only sodium taurocholate stimulates enzymic activity.     

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.     

Burkholderia cepacia胆固醇酯酶及其分子伴侣基因的串联表达及酶学性质的研究

为实现胆固醇酯酶的可溶性表达,以Burkholderia cepacia ST-200胆固醇酯酶为研究对象,将酶及其分子伴侣通过人工添加的SD序列在大肠杆菌中进行串联表达。通过SDS-PAGE电泳分析,胆固醇酯酶成功获得可溶性表达,摇瓶水平的总酶量为1 077 U/L。经过镍柱纯化之后可以获得一条相对分子量约为37 kDa的单一条带。利用圆二色谱解析胆固醇酯酶的二级结构,并测定Tm值,从最适温度、最适pH、热稳定性、pH稳定性及有机溶剂耐受性等方面对胆固醇酯酶进行研究,结果表明该酶在pH为7.0,45℃条件下表现出最大活力。     

Purification of pancreatic cholesterol esterase expressed in recombinant baculovirus-infected Sf9 cells.

A cDNA clone encoding the entire coding sequence of rat pancreatic cholesterol esterase (bile salt-stimulated lipase) was subcloned into the Baculovirus transfer vector pVL1392 and used to co-transfect Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. Two recombinant proteins (M(r) 74 kDa and 64 kDa) reactive with anti-cholesterol esterase IgG were produced and secreted by the infected Sf9 cells in large quantities in a time-dependent manner. The 74-kDa protein was detectable in the cultured medium at the second day post-infection and increased progressively, reaching a level of 50 micrograms/ml of culture medium after 8 days. Amino-terminal sequencing of this recombinant protein showed that the signal peptide of cholesterol esterase was correctly cleaved, resulting in the production of mature protein. The 64-kDa recombinant protein was not detected in the medium until Day 5 post-infection and accumulated to a level of 25 micrograms/ml at Day 8. Both the 74- and the 64-kDa cholesterol esterases were biologically active and hydrolyzed the artificial substrate p-nitrophenyl butyrate. Results of this study demonstrated that Baculovirus-infected Sf9 cells can be used for high-level expression of pancreatic cholesterol esterase. The recombinant enzyme will be useful for further characterization of this protein.     

Cholesterol esterase bound to intestinal brush border membranes does not accelerate incorporation of micellar cholesterol into absorptive cells

We confirmed that cholesterol esterase accelerated the incorporation of unesterified cholesterol solubilized in bile salt micelles into differentiated Caco-2 cells under various experimental conditions. Rat pancreatic juice and bovine cholesterol esterase increased the incorporation of micellar cholesterol into rat intestinal brush border membranes. The incorporation of micellar cholesterol was not changed in the brush border membranes enriched in and depleted of cholesterol esterase. The results suggest that the accelerated incorporation of micellar cholesterol by cholesterol esterase into absorptive cells is not mediated by the enzyme bound to the brush border membranes.     

Inhibition of liver lysosomal cholesterol esterase activity in rats by dextran blue in vivo and in vitro

Dextran blue decreases the activity of lysosomal acid cholesteryl esterase of rat liver at a concentration from 0.25 M to 10 M without altering acid phosphatase, acid beta-galactosidase and beta-glucosidase activities. The dextran blue filled lysosomes with a high degree of purity prepared by centrifugation over the linear sucrose density gradient contained insignificant impurities (up to 19%) of protein from other organelles. The specific activity of acid phosphatase, beta-galactosidase and beta-glucosidase was increased 35-40-fold in this fraction, whereas the activity of acid cholesteryl esterase rose but 14.7-fold. Chromatography on a Sepharose 2B column of the digitonin-digested native and dextran-containing lysosomes attests to the formation of large dextran aggregates with lysosomal matrix proteins. Since aggregation of dextran blue with acid phosphatase, beta-galactosidase and beta-glucosidase does not affect their activities, it is concluded that to bring about hydrolysis of lipoprotein cholesterol esters, it is necessary that cholesteryl esterase be associated with hydrophobic macromolecules. Moreover, dextran blue can be used for simulation cholesterol esters deposition in lysosomes.     

The hydrolysis of cholesterol esters in plasma lipoproteins by hormone-sensitive cholesterol esterase from adipose tissue.

Adipose tissue contains a high level of neutral esterase active against emulsions of cholesteryl oleate. The present studies show that this enzyme can also effectively hydrolyze the cholesterol esters in native rat plasma high density lipoproteins (HDL) and low density lipoproteins (LDL). The hydrolysis of lipoprotein cholesterol esters by a pH 5.2 isoelectric precipitate fraction from the freshly prepared 100,000 X g supernatant of chicken adipose tissue was low, but increased more than 50-fold on activation with cyclic AMP-dependent protein kinase. Rat adipose tissue homogenates were also very active against lipoprotein cholesterol esters, hydrolyzing as much as 60% of the total labeled cholesterol ester in HDL or LDL in 1 h. Activity was optimal at pH 7 and very low at pH 4. No protease activity was detected at pH 7 and, since assays were done in 2 mM EDTA, phospholipase A activity was presumably negligible. The results show that hormone-sensitive cholesterol esterase of adipose tissue has ready access to the neutral lipid core of plasma lipoproteins, either because the enzyme penetrates the polar shell or because the cholesterol ester in the core is exposed, at least intermittently, to allow enzyme-substrate complex formation. Whether or not this enzyme activity plays a role in lipoprotein degradation by adipose tissue remains to be determined.     

The nature of the inhibition of cholesterol esterase by delta 1-tetrahydrocannabinol

An improved assay for cholesterol esterase based on the use of fatty acid radiolabelled cholesterol esters has been developed. The method was used to demonstrate the effects of delta 1-tetrahydrocannabinol on a crude Leydig cell esterase preparation and on crystalline pancreatic esterase. Both enzymes were inhibited and the Km values determined (6.6 mumol/1 for the Leydig cell esterase and 6.25 mumol/1 for the pancreatic enzyme). While the former exhibited a mixed type of inhibition, the latter clearly was competitive.     

p-Nitrophenyl and cholesteryl-N-alkyl carbamates as inhibitors of cholesterol esterase

p-Nitrophenyl and cholesteryl-N-alkyl carbamates are good inhibitors of porcine pancreatic cholesterol esterase-catalyzed hydrolysis of p-nitrophenyl butyrate. p-Nitrophenyl-N-butyl and N-octyl carbamates (compounds 1 and 2, respectively) are potent active site-directed irreversible inhibitors of this enzyme. The inhibition of cholesterol esterase by compound 1 or 2 shows saturation kinetics with increasing inhibitor concentration. The activity of cholesterol esterase in the presence of compound 1 or 2 can be protected by the competitive inhibitor, phenylboronic acid. First-order decreases in cholesterol esterase activity effected by compound 1 or 2 are also observed in the presence of taurocholate/phosphatidylcholine micelles. Dilution of the inhibited enzyme results in a gradual return of activity, the rate of which is increased in the presence of the nucleophile hydroxylamine. Hence, inhibition of cholesterol esterase-catalyzed hydrolysis of p-nitrophenyl butyrate by compound 1 or 2 in the aqueous or micellar phase occurs via a carbamyl-cholesterol esterase mechanism. The turnover of the butyl carbamylenzyme is increased in the presence of micelles, which indicates that the micelles have a direct effect on the catalytic activity of the enzyme. However, this effect is dependent on the structure of the substrate as the turnover of the octyl carbamylenzyme is unaffected in the presence of micelles. A comparison of the second-order rate constants for the inhibition of cholesterol esterase by compound 1 or 2 indicates that the octyl derivative is the more potent inhibitor. Cholesteryl-N-alkyl carbamates do not carbamylate cholesterol esterase but instead act as reversible inhibitors. This is due to the stability of cholesteryl carbamates relative to p-nitrophenyl carbamates.     

Metabolic fate of pancreas-derived cholesterol esterase in intestine: an in vitro study using Caco-2 cells

Bile salt-stimulated cholesterol esterase is synthesized in the pancreatic acinar cells and is released into the intestinal lumen where it catalyzes cholesterol absorption. In the current study, Caco-2 cells were used as an in vitro model to study the interaction between the pancreatic cholesterol esterase with intestinal cells. Results showed that addition of increasing concentrations of cholesterol esterase in the incubation medium increased the uptake of micellar cholesteryl oleate by Caco-2 cells. The cholesterol esterase also increased the cellular uptake of the nonhydrolyzable cholesteryl linoleoyl ether. However, maximum uptake of the cholesteryl ether analog was 50% of that for cholesteryl oleate. The initial interaction of cholesterol esterase with Caco-2 cells was mediated by binding of the protein to a low affinity and high capacity binding site on the cell surface. Cholesterol esterase bound to the cell surface could be internalized via a monensin-sensitive mechanism. The cholesterol esterase taken up by the cells had a short residence time and was either degraded or was rapidly re-secreted from the cells. Chloroquine had no effect on the degradation or re-secretion of cholesterol esterase by Caco-2 cells, indicating that lysosomes were not involved with these processes. The cholesterol esterase taken up by the cells was not available to mediate further cholesterol uptake. These results indicated that the bile salt-stimulated cholesterol esterase secreted from pancreas could facilitate intestinal lipid absorption only transiently. The data suggest that the regulation of cholesterol esterase synthesis and secretion by the pancreas may be important for regulation of cholesterol absorption.     

Activation of myocardial neutral triglyceride lipase and neutral cholesterol esterase by cAMP-dependent protein kinase

Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.     

Decreased activity of peptide-elongation factors after treatment with cholesterol esterase.

1. Peptide-elongation factors were purified from rat liver and treated with cholesterol esterase and phospholipase A2 immobilized on Sepharose 4B. 2. Binding of L-[3H]-phenylalanyl-tRNA to 40S ribosomal subunits was decreased by approx. 70% and to polyribosomes by 30% in the presence of the binding factor incubated with cholesterol esterase. Treatment of this factor with immobilized phospholipase A2 decreased the binding to smaller ribosomal subunits by only about 15%. 3. Poly(U)-dependent phenylalanine polymerization by ribosomal subunits was decreased to approx. 30% of its original value by treatment of both elongation factors with cholesterol esterase. 4. The normal activity of esterase-treated elongation factor in both the binding reaction and peptide-elongation assay was fully recovered by the addition of cholesteryl 14-methyl-hexadecanoate. 5. Different classes of lipids present in peptide-elongation factor 1 have apparently different functions. Whereas phospholipids are required to maintain the strcture of heavy aggregates of this factor, the presence of cholesteryl 14-methylhexadecanoate is obviously necessary for the normal function of peptide-elongation factors.