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The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.
The purpose of this study was to examine whether an absence of triglyceride-rich lipoproteins (chylomicrons and very-low-density lipoproteins) in plasma is associated with any changes in the enzyme activity of lipoprotein lipase or hepatic lipase after heparin administration. To study this, the activities of hepatic lipase and lipoprotein lipase were determined in control subjects, in two patients with heterozygous hypobetalipoproteinemia, and in three patients with phenotypic abetalipoproteinemia after administration of heparin. Both enzymes showed normal activity in the patients with hypobetalipoproteinemia, but showed consistently reduced activity in the patients with abetalipoproteinemia. Hepatic lipase activity in plasma samples from these three patients obtained 15 minutes after intravenous injection of heparin was 55%, 87%, and 46% of that of the controls, whereas corresponding values in plasma samples obtained 30 minutes after heparin were 47%, 70%, and 57%, respectively. Lipoprotein lipase activity in the three patients with abetalipoproteinemia was 46%, 29%, and 34% of that of the controls in the samples obtained 15 minutes after heparin injection, whereas the values obtained after 30 minutes were 53%, 64%, and 47% of that of the controls. We conclude that an inherent absence of triglyceride-rich lipoproteins, as occurs in abetalipoproteinemia, is associated with reduced enzyme activity of both hepatic lipase and lipoprotein lipase in plasma after heparin administration.
We have determined the genotypes of two common polymorphisms in the lipoprotein lipase (S447X) and hepatic lipase (-480C/T) genes in a cohort of 285 representative selected Czech probands (131 male and 154 female), examined in 1988 and reinvestigated in 1996. The genotype distributions of both polymorphisms were in Hardy-Weinberg equilibrium and did not differ between male and female subjects. The rare allele frequency of the lipoprotein lipase polymorphism did not differ significantly from the other European populations. Compared to the German populations, the frequency of the hepatic lipase -480T allele was significantly higher in the Czech group (20% vs. 36%, p<0.0001). There were no significant associations between the lipoprotein lipase gene variants and lipid parameters measured either in 1988, or in 1996 or with changes of lipid parameters over the 8-year period. The carriers of the T-480 allele of the hepatic lipase polymorphism were found to have higher HDL cholesterol levels (p=0.02). However, this difference was confined to female subjects only. The male carriers of the -480T allele had higher concentrations of total cholesterol (p=0.03) as compared to CC-480 subjects. Both associations were observed in 1996 only. In the Slavic Czech population, a common polymorphism in the hepatic lipase gene (-480C/T), but not in the lipoprotein lipase gene (S447X), is a significant determinant of plasma HDL cholesterol in females and plasma total cholesterol in males and indicates the importance of gender-associated effects in the genetic determinations of plasma lipids.
Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.
Lipoprotein lipase and pancreatic lipase have about 30% sequence identity, suggesting a similar tertiary fold. Three-dimensional models of lipoprotein lipase were constructed, based upon two recently determined x-ray crystal structures of pancreatic lipase, in which the active site was in an open and closed conformation, respectively. These models allow us to propose a few hypotheses on the structural determinants of lipoprotein lipase which are responsible for heparin binding, dimer formation, and phospholipase activity. The folding of the protein assembles a number of positive charge clusters at the back of the molecule, opposite the active site. These clusters probably form the heparin binding site, as confirmed by recent site-directed mutagenesis experiments. The active sites of lipoprotein lipase and pancreatic lipase look very similar, except for the lid (a surface loop covering the catalytic serine in the inactive state). A different open (active) conformation of the lid in both enzymes may be responsible for their differing substrate specificities. Predictions of the nature of the lipoprotein lipase dimer remain elusive, although our model enabled us to propose a few possibilities.
An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.
Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B. Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides. This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent. Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1. SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K. Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein. Furthermore, the N-terminal amino acid sequence of the C. viscosum lipase was identical to the one of Pseudomonas glumae lipase.
The catalytic mechanism for members of the lipase gene family incorporates a serine-histidine-acidic group triad. In general, the acidic group is an aspartate, Asp177 in human pancreatic lipase, but glutamate is found in some lipases. Previously, we demonstrated that site-specific mutagenesis of Asp177 to Glu177 produced a mutant human pancreatic lipase with near normal activity against triolein, thereby, raising questions about the role of Asp177 in the catalytic triad and about the evolutionary pressure which selected Asp over Glu in the catalytic mechanism. To address these questions, we constructed and expressed mutants of Asp177 and Asp206, another acidic residue that could participate in the catalytic triad. The Glu177 mutant had a substrate specificity, specific activity, pH profile, colipase dependance, and interfacial activation comparable to the native lipase, Asp177. Several mutants of Asp206 were normally active, thus, confirming the important role of Asp177 in pancreatic lipase function. Additionally, we found that the Glu177 mutant had increased susceptibility to proteases and to urea denaturation. These findings demonstrated decreased conformational stability of the mutant lipase and provided an explanation for the preference of aspartate in the catalytic triad of human pancreatic lipase.
UNLABELLED: An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity. KEYWORDS: Bacter
BACKGROUND: The guinea pig pancreatic lipase-related protein 2 (GPLRP2) differs from classical pancreatic lipases in that it displays both lipase and phospholipase A1 activities; classical pancreatic lipases have no phospholipase activity. The sequence of GPLRP2 is 63 % identical to that of human pancreatic lipase (HPL), but the so-called lid domain, is much reduced in GPLRP2. A phospholipase A1 from hornet venom (Dolml PLA1) is very similar to HPL and GPLRP2 but is devoid of lipase activity; Dolml PLA1 also contains a reduced lid domain and lacks a region termed the beta9 loop, which is located in the vicinity of the HPL and GPLRP2 active sites. The structure determination of a chimera of GPLRP2 and HPL and domain building of Dolml PLA1 were undertaken to gain a better understanding of the structural parameters responsible for the differences in lipase versus phospholipase activity among these structurally related enzymes. RESULTS: The crystal structure of a chimeric mutant of GPLRP2, consisting of the catalytic domain of GPLRP2 and the C-terminal domain of HPL, has been solved and refined to 2.1 A resolution. This enzyme belongs to the alpha/beta hydrolase fold family and shows high structural homology with classical pancreatic lipases. The active site is closely related to those of serine esterases, except for an unusual geometry of the catalytic triad. Due to the reduced size of the lid domain, the catalytic serine is fully accessible to solvent. Part of the beta9 loop, which stabilizes the lid domain in the closed conformation of the classical HPL, is totally exposed to the solvent and is not visible in the electron-density map. CONCLUSIONS: The structures of the related enzymes, GPLRP2 and HPL and the model of Dolml PLA1, provide insights into the role played by the loops located above the active site in controlling substrate selectivity towards triglycerides or phospholipids. In GPLRP2, the lid domain is reduced in size compared to HPL, and hydrophilic residues are exposed to solvent. GPLRP2 is thus able to accommodate the polar head of phospholipids. The beta9 loop is still present in GPLRP2, making it possible for this enzyme to still accommodate triglycerides. In Dolml PLA1, the beta9 loop is absent, and this enzyme is unable to process triglycerides retaining only the phospholipase A1 activity.
Reaction of lipase with diethyl pyrocarbonate results in the modification of three histidine residues. One is highly reactive, although without affecting the activity, while the two others react more slowly with a concomitant loss of activity on both dissolved and emulsified substrates. As previously shown, lipase can also be modified either by reaction of five carboxyl groups with carbodiimide (5N-lipase) or by esterification of one serine residue with diethyl p-nitrophenyl phosphate (DP-lipase). In the three cases, the activity on emulsified substrates is abolished. The modification of histidine residues results also in a loss of activity on dissolved substrates, suggesting that the essential histidine is at (or close to) the active site. The ability of lipase to be adsorbed on siliconized glass beads is not impaired in this reaction. By contrast, 5N-lipase is still able to hydrolyze dissolved monomeric substrates and to adsorb on siliconized glass beands. Therefore, the essential carboxyl group is assumed to play an important role in the interfacial activation. Finally, since DP-lipase is still fully active on dissolved p-nitrophenyl acetate, the serine residue, which has been previously suggested to be the acylable one, is more likely implicated in the recognition and the binding to interfaces, as confirmed by the inability of DP-lipase to be adsorbed on siliconized glass beads.
The human hormone-sensitive lipase (HSL) gene encodes a 786-aa polypeptide (85.5 kDa). It is composed of nine exons spanning approximately 11 kb, with exons 2-5 clustered in a 1.1-kb region. The putative catalytic site (Ser423) and a possible lipid-binding region in the C-terminal part are encoded by exons 6 and 9, respectively. Exon 8 encodes the phosphorylation site (Ser551) that controls cAMP-mediated activity and a second site (Ser553) that is phosphorylated by 5'-AMP-activated protein kinase. Human HSL showed 83% identity with the rat enzyme and contained a 12-aa deletion immediately upstream of the phosphorylation sites with an unknown effect on the activity control. Besides the catalytic site motif (Gly-Xaa-Ser-Xaa-Gly) found in most lipases, HSL shows no homology with other known lipases or proteins, except for a recently reported unexpected homology between the region surrounding its catalytic site and that of the lipase 2 of Moraxella TA144, an antarctic psychrotrophic bacterium. The gene of lipase 2, which catalyses lipolysis below 4 degrees C, was absent in the genomic DNA of five other Moraxella strains living at 37 degrees C. The lipase 2-like sequence in HSL may reflect an evolutionarily conserved cold adaptability that might be of critical survival value when low-temperature-mobilized endogenous lipids are the primary energy source (e.g., in poikilotherms or hibernators). The finding that HSL at 10 degrees C retained 3- to 5-fold more of its 37 degrees C catalytic activity than lipoprotein lipase or carboxyl ester lipase is consistent with this hypothesis.
A lipase from the latex of Euphorbia characias was purified using a method involving extraction with apolar solvent and adsorption chromatography on silica gel. The lipase (specific activity, 1500 international units/mg of protein) was eluted from silica gel complexes with a lipid. The main protein fraction, which had a molecular mass of 38 kDa, was inactive when dissociated from the lipid fraction. When the lipid and protein fractions were reassociated, 72% of the lipolytic activity was recovered. This lipolytic activity was inhibited by diethyl p-nitrophenyl phosphate, which was shown to bind the lipase with a molar ratio of 0.75. High specific activities (1000 international units/mg) were measured for the lipase of E. characias on lipid extracts rich in galactosyl diacylglycerols. The apolipase was sequenced up to residue 23. The B chain of ricin has a strong homology (43.5%) with that sequence and cross-reacted with antibodies raised against the purified lipase from E. characias. The activity of the B chain of ricin was comparable (54 international units/mg) to that of the apolipase of E. characias (100 international units/mg) mixed with the same lipid cofactor complex. The primary structure (residues 68-72) of the B chain of ricin contains the lipase consensus sequence Gly-Xaa-Ser-Xaa-Gly. Its reactivity with diethyl p-nitrophenyl phosphate indicates the presence of an activated serine that, in addition to its well-documented lectin activity for galactosides, suggests that the B chain of ricin may be a galactosyl diacylglycerol lipase, closely analogous to the lipase from E. characias.
A new lipoprotein lipase-like gene has been cloned from endothelial cells through a subtraction methodology aimed at characterizing genes that are expressed with in vitro differentiation of this cell type. The conceptual endothelial cell-derived lipase protein contains 500 amino acids, including an 18-amino acid hydrophobic signal sequence, and is 44% identical to lipoprotein lipase and 41% identical to hepatic lipase. Comparison of primary sequence to that of lipoprotein and hepatic lipase reveals conservation of the serine, aspartic acid, and histidine catalytic residues as well as the 10 cysteine residues involved in disulfide bond formation. Expression was identified in cultured human umbilical vein endothelial cells, human coronary artery endothelial cells, and murine endothelial-like yolk sac cells by Northern blot. In addition, Northern blot and in situ hybridization analysis revealed expression of the endothelial-derived lipase in placenta, liver, lung, ovary, thyroid gland, and testis. A c-Myc-tagged protein secreted from transfected COS7 cells had phospholipase A1 activity but no triglyceride lipase activity. Its tissue-restricted pattern of expression and its ability to be expressed by endothelial cells, suggests that endothelial cell-derived lipase may have unique functions in lipoprotein metabolism and in vascular disease.
Very low levels of lipase can easily be measured by a new serum lipase assay method (the BALB-DTNB method), using BAL-tributyrate (BALB) as a substrate, 5,5'-dithiobis(2-nitrobenzoic acid) as a chromogenic SH reagent, phenylmethylsulfonylfluoride as an inhibitor of esterases and sodium dodecyl sulfate as a surfactant. The BALB-DTNB method has a higher sensitivity than the conventional serum lipase assay methods, and proved useful for analyzing the properties of serum lipases in combination with gel-filtration on a Sephacryl S 200 column and isoelectrofocusing in an Ampholine column. Serum samples containing high levels of lipases from patients with pancreatic diseases or patients in whom the pancreatic exocrine gland had been stimulated by injecting caerulein and secretin were analyzed by these methods. The lipolytic profiles obtained indicated the presence of a lipase with an estimated molecular weight of 46,000 and isoelectric points of 7.4, 6.8, or/and 6.4. A lipase with properties similar to those of the serum lipase was found to be present in human pancreatic juice.
The function of the gene, act, which encodes lipase activator and that is downstream from the gene that encodes lipase, lip, in Pseudomonas sp. strain KWI-56, was studied with Escherichia coli as the host organism. E. coli carrying both the lip and act genes in either cis or trans produced an active lipase, but E. coli carrying only the lip gene produced an equal amount of inactive lipase protein. The active and inactive lipases had the same molecular weight and the same N-terminal amino acid sequence, so their primary structures were the same. The inactive lipase was not activated spontaneously, but it was activated in vitro by the addition of a crude cell extract of E. coli carrying an expression plasmid of the act gene. Lipase from Pseudomonas sp. strain KWI-56 was completely without enzymatic activity when denatured, but regained activity in vitro upon addition of the crude cell extract. These results indicate that the act gene affects the activation involved in the conformational change of the lipase protein to its active form.