Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions

Gargouri, Youssef; Piéroni, G; Rivière, C.; Sauniere, Jean-Frédéric; Lowe, Peter A.; Sarda, Louis; Verger, Robert

doi:10.1016/0016-5085(86)90695-5

Cited by 218 publications

(80 citation statements)

References 30 publications

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“…HGL is a glycoprotein with a molecular mass of around 50 kDa, which is located in the chief cells of fundic mucosa [4]. In-vitro studies have shown that HGL hydrolyses both short-chain and long-chain triacylglycerols at comparable rates [5]. The enzyme is highly stable and active under acidic pH conditions.…”

Section: Epitope Mapping and Immunoinactivation Of Human Gastric Lipamentioning

confidence: 99%

“…Assays on tributyrin (Fluka) and soybean oil (commercial grade) emulsified in gum arabic were carried out with the bulk pH-stat method under standard conditions as described by Gargouri et al [5]. The kinetics of the hydrolysis of 1,2-didecanoyl-sn-glycerol films by HGL [21] were recorded, with or without incubation of the enzyme with each mAb, using the barostat technique previously described by Verger and de Haas [22].…”

Section: Enzymic Activity Assaysmentioning

confidence: 99%

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Epitope mapping and immunoinactivation of human gastric lipase using five monoclonal antibodies

Aoubala

Daniel²,

et al. 1993

European Journal of Biochemistry

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Five monoclonal antibodies (mAb) directed against human gastric lipase (HGL) have been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. All these mAb belong to the IgGl class with a K light chain. The effects of these mAb on the enzymic activity of HGL were studied and used to define three classes of antibodies, depending upon their immunoinactivation properties. As determined by ELISA and immunoinactivation studies, four overlapping epitopes were found to be part of the functional sites of the enzyme. The mAb appear to be suitable probes for studying the lipid binding and catalytic domains of HGL. The results of the ELISA additivity test were used to describe tentatively the epitopes of HGL in terms of a schematic spatial map.In humans, the hydrolysis of dietary triacylglycerols begins in the stomach and is catalyzed by one major enzyme; human gastric lipase (HGL) [l-31. HGL is a glycoprotein with a molecular mass of around 50 kDa, which is located in the chief cells of fundic mucosa [4]. In-vitro studies have shown that HGL hydrolyses both short-chain and long-chain triacylglycerols at comparable rates [5]. The enzyme is highly stable and active under acidic pH conditions. The optimal pH for HGL activity is 5.4 in the case of long-chain and 6 in the case of short-chain triacylglycerols.The amino acid sequence of HGL, deduced from the cDNA, consists of 379 residues [6] and shows a conservative pentapeptide (Gly-Xaa-Ser-Xaa-Gly) common to all the known lipases. . In order to further characterize these functional domains of HGL, an immunological approach was adopted here using mAb against HGL as probes for the spatial identification of the domains.In the present paper, we describe the production and functional characterization of five mAb against HGL. MATERIALS AND METHODS ProteinsHGL was purified in the laboratory from human gastric juice, using procedures described previously [15]. The protein had a specific activity of 1000 U . mg-' with tributyrin as substrate and showed a single band upon SDS/gel electrophoresis [16]. The bovine serum albumin and anti-mouseIgG -peroxidase conjugate used were from Sigma. Preparation of mAbTwo young female BALB/c mice were immunized with HGL as follows. The first and the second immunizations were carried out with 100 pg HGL emulsified in complete Freund's adjuvant, injected subcutaneously with a 10-day interval. Two weeks after the second injection, the mice were bled and the serum tested in a direct binding ELISA test. The mouse with the highest titre was selected and 100 pg pure antigen were again injected. 10 days later, three fusion-priming intraperitoneal injections of 50 pg HGL, each in 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 (buffer A) were given on three subsequent days. Spleen cells were obtained 1 day after the last injection and fused with non-secreting mouse myeloma

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Section: Epitope Mapping and Immunoinactivation Of Human Gastric Lipamentioning

confidence: 99%

Section: Enzymic Activity Assaysmentioning

confidence: 99%

Epitope mapping and immunoinactivation of human gastric lipase using five monoclonal antibodies

Aoubala

Daniel²,

et al. 1993

European Journal of Biochemistry

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“…The substrates used were either a defined mixture of longchain triacylglycerols (Intralipid 30%; Amersham Biosciences, Inc.) or a short-chain triacylglycerol (tributyrin). With both substrates, the lipolytic activity was measured at 37°C under the conditions described by Gargouri et al (5). One unit of r-DGL activity corresponds to the release of 1 mol of fatty acid/min.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of the Open Form of Dog Gastric Lipase in Complex with a Phosphonate Inhibitor

Roussel

Miled

Berti-Dupuis

et al. 2002

Journal of Biological Chemistry

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114

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Fat digestion in humans and some mammals such as dogs requires the successive intervention of two lipases: gastric lipase, which is stable and active despite the highly acidic stomach environment, followed by the classical pancreatic lipase secreted into the duodenum. We previously solved the structure of recombinant human gastric lipase (HGL) at 3.0-Å resolution in its closed form; this was the first structure to be described within the mammalian acid lipase family. Among the mammalian lipases, the preduodenal and lysosomal lipases belong to a family of enzymes that are able to withstand acidic conditions, under which they continue to be active. This family shows no sequence homology with any other known lipase families. The preduodenal lipases form a group of closely related enzymes originating from the stomach, the tongue, or the pharynx, and none of them requires any specific protein cofactor for its activity (1-3).The state of malnutrition of patients suffering from exocrine pancreatic insufficiency, which is often associated with cystic fibrosis, can be improved by prescribing dietary supplements prepared from pancreatic extracts of animal origin. The use of gastric lipases may lead to considerable progress in the treatment of exocrine pancreatic insufficiency (4) because of their resistance to the gastric medium and their high lipolytic activity at the low pH values observed in the duodenum A screening program (1) and biochemical studies (5-7) led to the conclusion that dog gastric lipase (DGL), 1 a glycoprotein with a molecular mass of 49 kDa, was the most active enzyme when tested on long-chain triacylglycerols. These lipids are the main components of human dietary fats. The first attempts to produce an active recombinant DGL (r-DGL) in procaryotes (4) were unsuccessful. Joliff et al. (8) described the expression and secretion of an active r-DGL in the baculovirus insect cell system. However, with a view to therapeutic applications, r-DGL production by a safe eukaryotic system of expression such as transgenic plants provides an attractive alternative. Transgenic plants offer considerable advantages, such as large production yields and the fact that they entail no risk of contamination by pathogens infectious to humans, whereas they contain the cell machinery mediating eukaryotic protein modifications. The first transgenic plants producing r-DGL were constructed in tobacco (9) and then in maize by Meristem therapeutics in collaboration with the Institut de Recherche Jouveinal/Parke-Davis during the last decade.All the triglyceride lipases for which the three-dimensional structures have been determined so far belong to the serine esterase class (10 -14). The active serine is part of a catalytic triad, which is similar to that observed in the case of serine proteases. The three-dimensional structure of the Candida rugosa lipase, which is inhibited by two enantiomers of a phosphonate inhibitor, provided the structural basis for establishing the chiral preferences of lipases (15). Covalent adducts of Rhizomu...

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“…Short-chain triacylglycerol (tributyrin, Fluka, St. Louis, MO, USA) was used as substrate. Lipolytic activity was measured at 37 ‡C in the conditions described by Gargouri et al [19]. One lipase unit is the amount of enzyme which catalyzes the release of 1 Wmol of fatty acid per min.…”

Section: Determination Of Speci¢c Lipase Activitymentioning

confidence: 99%

“…The catalytic activities of recombinant rGLs after transient expression were measured on short-chain triacylglycerols [19]. s-rGL is the most active recombinant protein with a speci¢c activity of 360 U/mg.…”

Section: Comparison Of Catalytic Activities Of S-rgl R-rgl and V-rglmentioning

confidence: 99%

A transient tobacco expression system coupled to MALDI‐TOF‐MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants

Mokrzycki-Issartel¹,

Bouchon

Farrer³

et al. 2003

FEBS Letters

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Tobacco-based transient expression was employed to elucidate the impact of di¡erential targeting to subcellular compartments on activity and quality of gastric lipase as a model for the production of recombinant glycoproteins in plants. Overall N-linked glycan structures of recombinant lipase were analyzed and for the ¢rst time sugar structures of its four individual N-glycosylation sites were determined in situ by matrix-assisted laser desorption/ionization time-of-£ight mass spectrometry (MALDI-TOF-MS) on a trypsin digest without isolation or deglycosylation of the peptides. Three glycosylation sites contain both complex-type N-glycans and high-mannose-type structures, the fourth is exclusively linked to high-mannose glycans. Although the overall pattern of glycan structures is in£uenced by the targeting, our results show that the type of glycans found linked to a given Asn residue is largely in£uenced by the physico-chemical environment of the site. The transient tobacco system combined with MALDI-TOF-MS appears to be a useful tool for the evaluation of glycoprotein production in plants. ß

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Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions

Cited by 218 publications

References 30 publications

Epitope mapping and immunoinactivation of human gastric lipase using five monoclonal antibodies

Epitope mapping and immunoinactivation of human gastric lipase using five monoclonal antibodies

Crystal Structure of the Open Form of Dog Gastric Lipase in Complex with a Phosphonate Inhibitor

A transient tobacco expression system coupled to MALDI‐TOF‐MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants

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