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

Abstract

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...

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

“…A hallmark feature of many phospholipases is to be minimally active on monomeric lipid substrates but undergo a substantial activation on binding to the surface of phospholipid membranes or micelles, a phenomenon known as interfacial activation (28)(29)(30)(31). This behavior has been attributed to a flexible lid that at the lipid-water interface facilitates substrate diffusion to the catalytic site rather than being allosterically modulated through distant ligand binding (25).…”

Adsorption of gastric lipase onto multicomponent model lipid monolayers with phase separation

“…Under the open conformation of the lid, the 3D structure of rDGL shows a large hydrophobic surface surrounding the entrance of the active site [6]. It was checked by in silico titration that the large apolar ring surrounding the active-site cavity is preserved whatever the pH and that the protein was positively charged below pH 5 [10].…”

“…Like other lipases, HGL belongs to the α/β hydrolase family and possesses a Ser-His-Asp catalytic triad [3]. To catalyze such interfacial reaction, lipases undergoe a conformational change consisting in the opening of an amphiphilic lid that gives access to the active site while it generates a large hydrophobic surface surrounding the catalytic cleft and part of the interfacial recognition site (IRS) [3][4][5][6]. HGL has been called "extremophilic" as it is stable and active in the acid environment of the stomach, it is resistant to pepsin hydrolysis and it is not inhibited by the bile salts present in the gastro-intestinal tract [7].…”

Enzymatic Assay Method for Evaluating the Lipase Activity in Complex Extracts from Transgenic Corn Seed

“…Since butyric acid is a weak acid with a pK a of 4.83 at 25°C (21), butyric acid produced during the hydrolysis of nitrophenyl butyrate could potentially change the solution pH. The need to ensure a stable pH during measurements also came from the fact that lipase activity was a strong function of pH (14). Additionally, samples to be tested may have a pH other than 5.5, and their addition may affect the pH of the emulsion.…”

“…As a therapeutic protein, recombinant human gastric lipase has been expressed and produced in insects (10,11), yeast (12), and recombinant dog gastric lipase in transgenic tobacco (13). Transgenic corn has been used as a host to express and produce recombinant dog gastric lipase in seeds (14).…”