Francine Ferrato

A critical reevaluation of the phenomenon of interfacial activation.

Publisher Summary Lipases are carboxylic ester hydrolases and have been termed glycerolester-hydrolase in the international system of classification. They greatly differ with respect to both of their origins and their kinetic properties. They can catalyze in vitro the hydrolysis, or synthesis, of a wide range of different carboxylic esters; however, they all show a higher specific activity toward glyceridic substrates. Under physiological conditions, because natural triacylglycerols are water insoluble, lipases that are generally soluble in water, catalyze the hydrolysis of carboxylic ester bonds at lipid/water interfaces. Some lipases, such as gastric lipases, rapidly become denatured at an interface with a pure tributyrin or tributanoylglycerol emulsion. Consequently, it is impossible to assess experimentally what interfacial activation may have occurred. The three-dimensional structures of lipases suggested that the interfacial activation phenomenon might be due to the presence of an amphiphilic surface loop covering the active site of the enzyme in solution, just like a lid. When contact occurs with a lipid/water interface, this lid might undergo a conformational rearrangement as the result of which the active site becomes accessible.

Human preduodenal lipase is entirely of gastric fundic origin

Lipase activity was measured in supernatant homogenates from various anatomic regions in the upper part of the human digestive tract of two organ donors. It is shown unambiguously that lipase activity occurs only in the fundic mucosa of the stomach, whereas no significant activity takes place in the antral, pharyngeal, or lingual areas, including the circumvallate papillae. In adults, the potential activity of human gastric lipase, as measured using tributyrin as substrate, amounts to 20% of its pancreatic counterpart. Lipase activity was also determined on human gastric biopsy samples taken during gastrofibroscopy tests on healthy adults. These results confirmed the finding that a lipolytic activity of gastric origin occurs uniformly and only in the fundic mucosa. Triacylglycerol hydrolysis is associated with a genuine gastric lipase activity that is clearly distinct from the classical esterase observed using p-nitrophenyl acetate as substrate. Lipase activity decreases significantly with age: it ranges on average from 4700 U/g of fresh mucosa in subjects aged up to 50 yr to 700 U/g of fresh mucosa in persons over 60 yr of age.

Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa

Lipase from Pseudomonas aeruginosa is a M r, 29 kDa protein with a single functional disulfide bond as shown by a shift in electrophoretic mobility after treatment with dithiothreitol and iodoacetamide. Limited proteolysis of lipase with Staphylococcus aureus protease V8 resulted in cleavage after amino acid residues Asp38 and Glu46. Comparison of the lipase amino acid sequence with those of other hydrolases with known 3D structures indicated that the folding pattern might be compatible with the α/β hydrolase fold, thereby allowing us to construct a 3D model which fitted the biochemical properties. The model predicts a catalytic triad consisting of Ser82, Asp229 and His251, and contains a disulfide bond connecting residues Cys183 and Cys235. Residues Asp38 and Glu46 are located at the surface of the enzyme, whereas the disulfide bond is rather inaccessible, which is in agreement with the finding that the protein needed to be partly unfolded before a reduction of the disulfide bond could take place. A striking prediction from the model was the lack of a lid‐like α‐helical loop structure covering the active site which confers to other well‐characterized lipases a unique property known as interfacial activation. Experimental determination of lipase activity under conditions where the substrate existed either as monomeric solutions or aggregates confirmed the absence of interfacial activation.

Structure and activity of rat pancreatic lipase-related protein 2.

The pancreas expresses several members of the lipase gene family including pancreatic triglyceride lipase (PTL) and two homologous proteins, pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Despite their similar amino acid sequences, PTL, PLRP1, and PLRP2 differ in important kinetic properties. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, bile acid inhibition, colipase requirement, and interfacial activation. To begin understanding the structural explanations for these functional differences, we solved the crystal structure of rat (r)PLRP2 and further characterized its kinetic properties. The 1.8 Å structure of rPLRP2, like the tertiary structure of human PTL, has a globular N-terminal domain and a β-sandwich C-terminal domain. The lid domain occupied the closed position, suggesting that rPLRP2 should show interfacial activation. When we reexamined this issue with tripropionin as substrate, rPLRP2 exhibited interfacial activation. Because the active site topology of rPLRP2 resembled that of human PTL, we predicted and demonstrated that the lipase inhibitors E600 and tetrahydrolipstatin inhibit rPLRP2. Although PTL and rPLRP2 have similar active sites, rPLRP2 has a broader substrate specificity that we confirmed using a monolayer technique. With this assay, we showed for the first time that rPLRP2 prefers phosphatidylglycerol and ethanolamine over phosphatidylcholine. In summary, we confirmed and extended the observation that PLRP2 lipases have a broader substrate specificity than PTL, we demonstrated that PLRP2 lipases show interfacial activation, and we solved the first crystal structure of a PLRP2 lipase that contains a lid domain.

In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases.

The stereoselectivity of dog gastric and dog pancreatic lipases was investigated both in vitro, under simulated physiological conditions, and in vivo, during the digestion of a liquid test meal. In vitro it was observed that although both lipases had a stereopreference for the sn-3 position in triglycerides, it was about three times higher in the case of the gastric lipase. On the other hand, both lipases clearly showed a comparable enantioselectivity for the sn-1 position when a racemic diolein was used as the substrate. In the case of pancreatic lipase, the enantiomeric excess of 1,2-sn-diolein generated in vitro by the hydrolysis of triolein was found to decrease significantly, and even to be slightly reversed, at high rates of hydrolysis (above 50%) due to the further stereoselective hydrolysis of diglycerides into monoglycerides. This finding may explain the low enantiomeric excess of the diglycerides observed in vivo during the early phase of intraduodenal digestion when pancreatic lipase plays a predominant role and the rate of triolein hydrolysis is already high. On the other hand, a large enantiomeric excess of 1,2-sn-diolein generated from triolein was always the fingerprint of the gastric lipase in vitro even at high hydrolysis rates. This fingerprinting of gastric lipase was observed during both the intragastric phase and the late intestinal phase of lipolysis. This feature was therefore taken as an index to determine the respective roles of gastric and pancreatic lipases during in vivo lipolysis. To the best of our knowledge, this is the first time that stereoselectivity has been used as a tool to discriminate between the activities of two enzymes hydrolyzing the same substrate in vivo.

Further biochemical characterization of human pancreatic lipase-related protein 2 expressed in yeast cells

Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major protein with a molecular mass of 50 kDa was purified from the culture medium using SP-Sepharose and Mono Q chromatography. The protein was found to be highly sensitive to the proteolytic cleavage of a peptide bond in the lid domain. The proteolytic cleavage process occurring in the lid affected both the lipase and phospholipase activities of rHPLRP2. The substrate specificity of the nonproteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All of the enzyme activities were maximum at alkaline pH values and decreased in the pH 5–7 range corresponding to the physiological conditions occurring in the duodenum. rHPLRP2 was found to act preferentially on substrates forming small aggregates in solution (monoglycerides, egg phosphatidylcholine, and galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on monogalactosyldiglyceride and digalactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2, which shows a large deletion in the lid domain. The presence of a full-length lid domain in rHPLRP2 makes it possible for enzyme activity to occur at higher surface pressures. The finding that the inhibition of nonproteolyzed rHPLRP2 by tetrahydrolipstatin and diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media.

Isoform purification of gastric lipases : towards crystallization

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.

Purification and molecular characterization of lamb pregastric lipase

Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. The purification procedure was based on an aqueous extract containing 0.7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchanger and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel AcA-54. The final enzymatic preparation, where the overall activity recovery was 3%, showed a single protein band on SDS-PAGE with a molecular mass of 50 kDa. LPGL is a glycoprotein containing approx. 14% (w/w) of carbohydrate. Extensive deglycosylation using peptide N-glycosidase F yielded a protein with an apparent molecular mass of 43 kDa. An uncontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HLAL) and rabbit gastric lipase (RGL). The labile bond X54-Leu55 was identified. Isoelectric focusing of LPGL reveals a major band corresponding to an isoelectric point of 4.8. The pure enzyme displayed specific activities of 950 U mg-1, 300 U mg-1 and 30 U mg-1 at pH 6.0, using tributyroylglycerol, trioctanoylglycerol and trioleoylglycerol as substrates, respectively. Using Western blot analysis, a cross-immunoreactivity of LPGL was observed with purified anti-human gastric lipase polyclonal antibodies. Determination of the amino-acid sequence of 62 residues revealed a high degree of homology with other known preduodenal lipases.

The influence of bile salts and bile lipoprotein complex on pancreatic lipase hydrolysis of monomolecular films

We report a new technique which allows us to follow the lipolysis of monomolecular films in the presence of bile salts by using a zero-order trough (Verger, R. and de Haas, G.H. (1973) Chem. Phys. Lipids 10, 127). The effects of bile salts, the bile lipoprotein complex and colipase on pancreatic lipase hydrolysis of rac-1,2-didodecanoylglycerol films were studied at different surface pressures. Taking into account previous studies, lipase activity was interpreted as a function of its degree of binding to the bile lipoprotein complex.

Regulation by the “interfacial quality” of some biological activities

Abstract A few examples are given of regulation by the “interfacial quality” of some biological activities. Experiments with monolayers have the unique advantage that the arrangement and packing of the molecules can be easily measured and controlled. The first part is devoted to proteins which do not degrade lipids. Soluble cardiotoxins are generally injected into the subphase below a preformed lipid monolayer and measurements are taken either at constant surface area or at constant surface pressure. These experiments can give information on the penetration capacity of the protein into the interface and its lipid specificity, with direct access to the area of the protein segment interacting with lipids. Most intrinsic membrane proteins are insoluble in water. In the absence of detergent they aggregate and display no affinity for lipid interfaces. These proteins can be spread from an organic solvent solution but with the risk of being denatured. In order to circumvent this difficulty a method for spreading an aqueous suspension of lipoproteins or natural membrane vesicles was developed. This spreading method allows the formation of lipoprotein films retaining biological activities and native membrane constituents. In the second part, the use of lipid monolayers as substrates for lipolytic enzymes is reviewed. The monolayer technique permits an accurate study to be made of the influence of surface pressure and protein cofactors on the hydrolysis velocity and lag time in lipolysis. Two examples are developed: first, the assistance provided by colipase during the penetration of phospholipid films by pancreatic lipase; second the activation by apolipoprotein CII of phospholipid-monolayer hydrolysis by lipoprotein lipase. The monolayer technique is ideally suited to the study of the mode of action of lipolytic enzymes on monolayers of controlled “interfacial quality”.