Gene_locus Report for: human-LIPF

The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.

We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.

The cDNA encoding human gastric lipase (HGL) was integrated into the genome of Pichia pastoris using the pGAPZalpha A transfer vector. The HGL signal peptide was replaced by the yeast alpha-factor to achieve an efficient secretion. Active rHGL was produced by the transformed yeast but its levels and stability were dependent on the pH. The highest activity was obtained upon buffering the culture medium at pH5, a condition that allowed preserving enzyme activity over time. A large fraction (72+/-2%) of secreted rHGL remained however bound to the yeast cells, and was released by washing the cell pellet with an acid glycine-HCl buffer (pH2.2). This procedure allowed establishing a first step of purification that was completed by size exclusion chromatography. N-terminal sequencing and MALDI-ToF mass spectrometry revealed that rHGL was produced in its mature form, with a global mass of 50,837+/-32Da corresponding to a N-glycosylated form of HGL polypeptide (43,193Da). rHGL activity was characterized as a function of pH, various substrates and in the presence of bile salts and pepsin, and was found similar to native HGL, except for slight changes in pH optima. We then studied by site-directed mutagenesis the role of three key residues (K4, E225, R229) involved in salt bridges stabilizing the lid domain that controls the access to the active site and is part of the interfacial recognition site. Their substitution has an impact on the pH-dependent activity of rHGL and its relative activities on medium and long chain triglycerides.

Gastric cancer (GC) is a common and deadly digestive tract tumor worldwide. Unfortunately, diagnosis of GC is usually confused and misleading because of atypical symptoms or incomplete complaints. Accordingly, exploring gene expression profile and identifying genes with analogical variance trend will bring new perspective into the diagnosis and treatment of GC. Herein, a RNASeq dataset from Caucasian GC and their matched noncancerous samples [Gene Expression Omnibus (GEO): SRP049809] and datasets from four microarrays constituted with tumor and nontumor tissues (GEO: GSE13911, GSE19826, GSE29272, GSE33335) were analyzed to explore the differentially expressed genes (DGEs). As a result, we identified a core set of 373 DGEs. Among these genes, we found that most downregulated genes were related to lipidmetabolic functions. Especially, the gastric lipase (LIPF) gene, which was connected with various lipid metabolism processes, was significantly decreased among all datasets. We then performed immunohistochemistry experiments using gastric tissue arrays to investigate the clinical effects, and the expression of a LIPF target gene, diacylglycerol kinase alpha (DGKA). Among the 90 samples of gastric adenocarcinoma, the LIPF and DGKA levels were both decreased in cancer tissues [LIPF, 59.1% (53/90); DGKA, 77.8% (70/90)] compared to normal tissues [LIPF, 94.4% (85/90); DGKA, 90% (81/90)]. The expression level of these two proteins in GC was associated with local invasion and disease stage. Cox regression identified high DGKA expression (HR, 0.49; 95% CI, 0.260.94; P=0.03) as a predictor of good prognosis and LNM status (HR, 4.63; 95% CI, 1.3915.51; P=0.01) as a predictor of poor prognosis. Thus we speculated that LIPFDGKA might serve as a potential possible biomarkers for diagnosis of GC, and their downregulation may bring new perspective into the investigation of GC prognosis.

Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5mg recombinant hGL per gram of infiltrated leaf tissue were obtained. Proteins expressed from two hGL constructs, wild type (wt-hGL) and with a Histidine tag at its C terminal (hGL-His), were purified from leaf tissue using Immobilized Lectin Affinity chromatography and Immobilized Metal Affinity chromatography. Both variants were glycosylated, enzymatically active, and had an apparent molecular weight similar to native hGL (approx. 50kDa). The recombinant hGLs were stable under acidic conditions and in the presence of gastric pepsin. Moreover, as found with the naturally occurring enzyme, the activity of recombinant hGL on the short chain lipid, tributyrin, was higher than on long chain Intralipid 30% emulsion. The maximum specific activity measured on tributyrin was 310 U/mg of protein and the maximum yield was 193 U/g of infiltrated leaf tissue. These results show that transient expression in plants can be used to produce active hGL that could be efficiently purified using established techniques. The approach provides a means of generating large quantities of hGL that could be of use for a number of applications both in vitro and in vivo.

At least six families of mammalian acid lipases (E.C. 3.1.1.-) catalyse the hydrolysis of triglycerides in the body, designated as LIPA (lysosomal), LIPF (gastric), LIPJ (testis) and LIPK, LIPM and LIPN (epidermal), which belong to the AB hydrolase superfamily. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for acid lipase genes and encoded proteins using data from several mammalian genome projects. Mammalian acid lipase genes were located within a gene cluster for each of the 8 mammalian genomes examined, including human (Homo sapiens), chimpanzee (Pons troglodytes), rhesus monkey (Macacca mulatta), mouse (Mus musculus), rat (Rattus norvegicus), cow (Bos taurus), horse (Equus caballus) and dog (Canis familaris), with each containing 9 coding exons. Human and mouse acid lipases shared 44-87% sequence identity and exhibited sequence alignments and identities for key amino acid residues and conservation of predicted secondary and tertiary structures with those previously reported for human gastric lipase (LIPF) (Roussel et al., 1999). Evidence for a new family of acid lipase genes is reported for mouse and rat genomes, designated as Lipo. Mouse acid lipase genes are subject to differential mRNA tissue expression, with Lipa showing wide tissue expression, while others have a more restricted tissue expression in the digestive tract (Lipf), salivary gland (Lipo) and epidermal tissues (Lipk, Lipm and Lipn). Phylogenetic analyses of the mammalian acid lipase gene families suggested that these genes are products of gene duplication events prior to eutherian mammalian evolution and derived from an ancestral vertebrate LIPA gene, which is present in the frog, Xenopus tropicalis.

Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

In the human stomach, gastric lipase hydrolyzes only 10 to 30% of ingested triacylglycerols because of an inhibition process induced by the long chain free fatty acids generated, which are mostly protonated at gastric pH. The aim of this work was to elucidate the mechanisms by which free fatty acids inhibit further hydrolysis. In vitro experiments examined gastric lipolysis of differently sized phospholipid-triolein emulsions by human gastric juice or purified human gastric lipase, under close to physiological conditions. The lipolysis process was further investigated by scanning electron microscopy, and gastric lipase and free fatty acid movement during lipolysis were followed by fluorescence microscopy. The results demonstrate that: 1) free fatty acids generated during lipolysis partition between the surface and core of lipid droplets with a molar phase distribution coefficient of 7.4 at pH 5.40; 2) the long chain free fatty acids have an inhibitory effect only when generated during lipolysis; 3) inhibition of gastric lipolysis can be delayed by the use of lipid emulsions composed of small-size lipid droplets; 4) the release of free fatty acids during lipolysis induces a marked increase in droplet surface area, leading to the formation of novel particles at the lipid droplet surface; and 5) the gastric lipase is trapped in these free fatty acid-rich particles during their formation. In conclusion, we propose a model in which the sequential physicochemical events occurring during gastric lipolysis lead to the inhibition of further triacylglycerol lipolysis.

Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We have solved the structure of recombinant human gastric lipase at 3.0 A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain, belonging to the alpha/beta hydrolase fold family, and an extrusion domain. It possesses a classical catalytic triad (Ser 153, His 353, Asp 324) and an oxyanion hole (NH groups of Gln 154 and Leu 67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 30 residues, which can be defined as a lid. Its displacement is necessary for the substrates to access the active site. A phosphonate inhibitor was positioned in the active site which clearly suggests the location of the hydrophobic substrate binding site.

Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We report here the structure of recombinant human gastric lipase at 3.0-A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain belonging to the alpha/beta hydrolase-fold family and a "cap" domain, which is analogous to that present in serine carboxypeptidases. It possesses a classical catalytic triad (Ser-153, His-353, Asp-324) and an oxyanion hole (NH groups of Gln-154 and Leu-67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under a segment consisting of 30 residues, which can be defined as a lid and belonging to the cap domain. The displacement of the lid is necessary for the substrates to have access to Ser-153. A phosphonate inhibitor was positioned in the active site that clearly suggests the location of the hydrophobic substrate binding site. The lysosomal acid lipase was modeled by homology, and possible explanations for some previously reported mutations leading to the cholesterol ester storage disease are given based on the present model.

Human gastric lipase (HGL; triacylglycerol lipase; EC 3.1.1.3) plays an important role in the digestion of dietary triglycerides in the gastrointestinal tract, especially in patients suffering from pancreatic lipase deficiencies. The enzyme is secreted by the fundic mucosa of the stomach and hydrolyzes the ester bonds of triglycerides under acidic pH conditions, while cholesteryl esters are not attacked. The 379-amino acid protein is highly homologous to two other acidic lipases, rat lingual lipase (RLL; triacylglycerol lipase; EC 3.1.1.3) and human lysosomal acid lipase (HLAL; cholesteryl esterase; EC 3.1.1.13). To determine whether this remarkable similarity is also present at the genomic level, we have elucidated the respective gene structures by screening three bacteriophage lambda libraries and by polymerase chain reaction-based intron amplification. The genes encoding HGL, RLL, and HLAL are composed of 10 exons interrupted by nine introns and span about 14 kb, 18.7 kb, and 38.8 kb of genomic DNA, respectively. The HGL and RLL gene organizations are identical, suggesting that RLL is the rat gastric lipase expressed in the serous von Ebner glands of the tongue. The positions of the HLAL intervening sequences are also absolutely conserved, except for the location of intron 1. Our results support the concept that HLAL and HGL/RLL are members of a gene family of lipases that most likely have evolved by duplication of an ancestral gene and subsequently assumed distinct roles in neutral lipid metabolism due to sequence divergence and different expression patterns.

Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.

Human gastric lipase subjected to limited tryptic proteolysis lost its ability to hydrolyze emulsified long-chain triacylglycerol. Activity against a water-soluble substrate was however retained, indicating that proteolysis did not affect the active site. Sequence analysis revealed that trypsin specifically cleaved the linkage between lysine-4 and leucine-5. This cleavage rendered the enzyme unable to bind to emulsified triacylglycerol particles, e.g. human milk fat globules. We suggest that the N-terminal tetrapeptide, in particular lysine-4, is essential for the binding of human gastric lipase to lipid/water interfaces, and hence, for its physiological function.

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.