Domain Of Lipase Research Articles

Characterization and evaluation of MFG-E8 protein-derived peptides: From isolation to functional assessment in skeletal muscle lipid metabolism

This research investigated preparation, identification and relative properties of milk fat globule epidermal growth factor Ⅷ (MFG-E8) protein-derived peptides. MFG-E8 protein was extracted from bovine milk and peptides were isolated and obtained using an in vitro simulating gastrointestinal digestion model, the peptide sequence was then identified. High-abundance MFG-E8 peptides bind well to the protein domain of pancreatic lipase was selected. Enzyme kinetic studies exhibited that the inhibition effect of KDPG and YAR on pancreatic lipase activity was reversible and non-competitive inhibition. Fluorescence spectroscopic analysis showed that KDPG and YAR acted as fluorescence quenchers of pancreatic lipase and exhibited certain dose-dependent effect. In vivo animal experiments further verified the regulatory effects of MFG-E8 peptides on lipid metabolism and skeletal muscle profiles, which provided the potential role of KDPG and YAR in regulating lipid metabolism and alleviating skeletal muscle dysfunction via AMPK/SREBP-1/SCD-1 signaling pathway.

Molecular cages encapsulating lipase and the effect of cage hydrophobicity and cage size

To firmly immobilize enzyme on carrier surface without covalent attachment as well as improve enzyme stability, an effective method of enzyme immobilization was developed by encapsulating enzyme into nano-molecular cages on the surface of magnetic microspheres. Compared with physical adsorption, molecular cage encapsulation could fix lipase much more firmly and make lipase more active. Then, this study focused on investigating the effects of cage hydrophobicity and cage size on lipase encapsulation respectively by adjusting the log P value (from −0.85 to 1.28) and chain length (from 2.93 to 5.41 nm) of crosslinkers. It showed that lipase activity was dependent on the hydrophobicity of cage. Moreover, the size of cage had important effect on lipase loading amount, and a big cage was conducive to house lipase molecule. The structural analysis indicated that molecular cages could retain the active conformation of lipase and increase its structural rigidity. Molecular dynamics simulation revealed the interaction between the lid domain of lipase and molecular cage, leading to the higher activity of encapsulated lipase (63.6 U/mg) than that of free lipase (50.6 U/mg). Besides, the encapsulated lipase showed more than twice higher thermal stability and almost 8 times higher tolerance to denaturant than the adsorbed lipase.

Substitution and mutation of the C-terminal loop domain of Penicillium expansum lipase significantly changed its regioselectivity and activity

In order to explore the effect of the C-terminal loop domain of Penicillium expansum lipase (PEL) on its regioselectivity and activity, homologous substitution and site-directed saturation mutagenesis were used. Two mutants PEL-loopAO and M234F were selected to catalyze the hydrolysis of tricaprylin and the molar ratios of 1,2-dicaprylin to 1,3-dicaprylin in the products were 25.57 and 8.57 respectively, showing obvious sn-1,3 regioselectivity of the mutants. Bioinformatics analysis showed that the structures of the substrate binding pocket of the mutants changed significantly. Moreover, due to the different positions of sn-1(3) and sn-2 ester bonds in the substrate molecule, it is easier for the substrate molecule to adapt to the significant changes in the substrate binding pocket and form the correct sn-1(3) catalytic conformation instead of sn-2 catalytic conformation, which makes the mutants exhibit apparent sn-1,3 position selectivity. In addition, the decrease of hydrophobicity of C-terminal loop domain will cause the decrease of lipase activity, whereas maintaining or improving the hydrophobicity can maintain or improve the activity. These findings are of great value in elucidating the relationship between the C-terminal loop domain of the lipase and its regioselectivity and activity, and also provide some reliable clues for obtaining lipases with better regioselectivity.

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including β2, β3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3-α3 and progress to β5 and β4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O-glycans that can be modified by ABO blood group determinants

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.

Characterization of a peptide containing the major heparin binding domain of human hepatic lipase.

Human hepatic lipase (hHL) is a cell surface associated enzyme that hydrolyzes triacylglycerols and phospholipids within circulating lipoproteins. We hypothesized that an amino acid sequence mimicking the major heparin binding domain (HBD) of hHL will displace hHL from cell surfaces. To test this hypothesis, we generated a recombinant protein of thioredoxin linked with a cleavable, tagged sequence containing amino acids 442 to 476 of the mature hHL sequence, which contains the major HBD of hHL. The recombinant protein associated with heparin-sepharose, and its peak elution from heparin-sepharose occurred in the presence of 0.5M NaCl. We cleaved and purified the tagged sequence containing the HBD from the recombinant protein and tested the ability of the peptide to displace full-length hHL from HEK-293 cells. The peptide indeed displaced hHL from cell surfaces, while no significant displacement was observed in the presence of a peptide with a scrambled sequence. Finally, we obtained structural information for the peptide containing the HBD. 1 H- and 15 N-NMR spectra of the peptide indicate the peptide is largely unstructured, although not completely random coil. The addition of heparin to the peptide induced some changes in chemical shift, suggesting changes in peptide structure and/or specific interactions with heparin. Molecular simulations confirm the largely unstructured nature of the isolated peptide, but they also indicate weak tendencies for both α- and β-structure formation in different parts of the chain. Overall, these data provide a proof-of-principle for the use of mimetic peptides for the displacement of cell surface associated lipases.

Binding orientation and interaction of bile salt in its ternary complex with pancreatic lipase-colipase system

The interfacial activity of pancreatic lipases (PL) depends on the presence of colipase and bile salt. The activity of PL is inhibited by micellar concentrations of bile salt which can be restored by the addition of colipase. Though the formation of 1:1:1 tertiary complex by lipase-colipase-bile salt micelle is well accepted, the residue-level interactions between lipase-colipase and bile salt are yet to be clearly understood. Molecular dynamic simulations of lipase-colipase complex, lipase and colipase were performed in the presence of a model bile salt, sodium taurocholate (NaTC), at its near-CMC and supra-micellar concentrations. From the interactions obtained from the molecular dynamic simulations, the ternary complex was modelled and compared with earlier reports. The analysis suggested that a micelle of NaTC consisting of nine monomers was formed at the concave groove between lipase and colipase chain and it mainly interacted with the fourth finger of colipase. This complex was mainly stabilized by van der Waals interactions. Interestingly, the C-terminal domain of lipase which holds the colipase did not show any significant role in formation or stabilization of NaTC micelle.

Bis-indole alkaloids from Tabernaemontana divaricata as potent pancreatic lipase inhibitors: molecular modelling studies and experimental validation

Obesity is the major leading cause of global mortality among metabolic disorders. Orlistat, a pancreatic lipase inhibitor, is the only approved drug of choice for the long term treatment of obesity. However, recent findings reported severe adverse effects with long term administration of orlistat. Plant-based natural products represent a vast reservoir of chemical entities that have the potential to treat various metabolic disorders. In the present study, we have performed a preliminary screening of local flora for pancreatic lipase inhibition assay, which highlighted the methanol extract of Tabernaemontana divaricata leaves (IC50 of 12.73 µg/mL). Molecular docking of the 38 alkaloids, reported from the leaves of T. divaricata, into the active site of pancreatic lipase led to the identification of furan bridged bis-indole alkaloids viz., Conophylline (1), Conophyllinine (2) and Conophyllidine (3) as potential leads, while Taberhanine (4), a monomeric indole alkaloid was found to exhibit comparatively poor docking score. Further, molecular docking analysis of the top three molecules (1–3) highlighted the importance of hydrophobic interactions of the dimeric extension with the lid domain of pancreatic lipase, which was not found with 4. Molecular dynamics simulations of 1–4 in complex with pancreatic lipase, further confirmed the docking results, wherein compound 4 was comparatively less stable (RMSD ≈ 1.5 A). Liquid chromatography–mass spectrometry analysis of the alkaloid-rich fraction of T. divaricata leaves indicated the presence of 1, while 2–4 were found to be absent. Molecule 1 was tested for pancreatic lipase inhibition potential, where it exhibited potent IC50 of 3.31 µM, comparable to that of orlistat (IC50 of 0.99 µM). Enzyme kinetic studies validated the in silico analyses, wherein compound 1 exhibited a competitive reversible inhibition of pancreatic lipase. The present study identified the bis-indole alkaloids of T. divaricata leaves as a new class of potent pancreatic lipase inhibitors.

A Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming Intracellular Aggregates That Activate Apoptosis

Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.

Isolation and characterization of a thermostable lipase from Bacillus thermoamylovorans NB501.

Two thermophilic bacterial strains, Bacillus thermoamylovorans NB501 and NB502, were isolated from a high-temperature aerobic fermentation reactor system that processes tofu refuse (okara) in the presence of used soybean oil. We cloned a lipase gene from strain NB501, which secretes a thermophilic lipase. The biochemical characteristics of the recombinant enzyme (Lip501r) were elucidated. Lip501r is monomeric in solution with an apparent molecular mass of 38 kDa on SDS-PAGE. The optimal pH and apparent optimal temperature of Lip501r were 8 and 60°C, respectively. Supplementation of 5 mM Ca2+ enhanced the thermostability, and the enzyme retained 56% of its activity for 30 min at 50°C. Lip501r was active on a wide range of substrates with different lengths of p-nitrophenyl (pNP) esters, and showed a remarkably higher activity with pNP-myristate. The Km and Vmax values for pNP-butyrate in the presence of 5 mM CaCl2 were 1.8 mM and 220 units/mg, respectively. The possible industrial use of the thermophilic lipase in modifying edible oil was explored by examining the degradation of soybean oil. A TLC analysis of the degraded products indicated that Lip501r is an 1,3-position specific lipase. A homology modeling study revealed that helix α6 in the lid domain of NB501 lipase was shorter than that of lipases from the Geobacillus group.

Crystal structure of vespid phospholipase A1 reveals insights into the mechanism for cause of membrane dysfunction

Vespid phospholipase A1 (vPLA1) from the black-bellied hornet (Vespa basalis) catalyzes the hydrolysis of emulsified phospholipids and shows potent hemolytic activity that is responsible for its lethal effect. To investigate the mechanism of vPLA1 towards its function such as hemolysis and emulsification, we isolated vPLA1 from V. basalis venom and determined its crystal structure at 2.5 Å resolution. vPLA1 belongs to the α/β hydrolase fold family. It contains a tightly packed β-sheet surrounded by ten α-helices and a Gly-X-Ser-X-Gly motif, characteristic of a serine hydrolyase active site. A bound phospholipid was modeled into the active site adjacent to the catalytic Ser-His-Asp triad indicating that Gln95 is located at hydrogen-bonding distance from the substrate's phosphate group. Moreover, a hydrophobic surface comprised by the side chains of Phe53, Phe62, Met91, Tyr99, Leu197, Ala167 and Pro169 may serve as the acyl chain-binding site. vPLA1 shows global similarity to the N-terminal domain of human pancreatic lipase (HPL), but with some local differences. The lid domain and the β9 loop responsible for substrate selectivity in vPLA1 are shorter than in HPL. Thus, solvent-exposed hydrophilic residues can easily accommodate the polar head groups of phospholipids, thereby accounting for the high activity level of vPLA1. Our result provides a potential explanation for the ability of vPLA1 to hydrolyze phospholipids of cell membrane.

N-Terminal Domain of Turkey Pancreatic Lipase is Active on Long Chain Triacylglycerols and Stabilized by Colipase

The gene encoding the TPL N-terminal domain (N-TPL), fused with a His6-tag, was cloned and expressed in Pichia pastoris, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The recombinant protein was successfully expressed and secreted with an expression level of 5 mg/l of culture medium after 2 days of culture. The N-TPL was purified through a one-step Ni-NTA affinity column with a purification factor of approximately 23-fold. The purified N-TPL, with a molecular mass of 35 kDa, had a specific activity of 70 U/mg on tributyrin. Surprisingly, this domain was able to hydrolyse long chain TG with a specific activity of 11 U/mg using olive oil as substrate. This result was confirmed by TLC analysis showing that the N-TPL was able to hydrolyse insoluble substrates as olive oil. N-TPL was unstable at temperatures over 37°C and lost 70% of its activity at acid pH, after 5 min of incubation. The N-TPL exhibited non linear kinetics, indicating its rapid denaturation at the tributyrin–water interface. Colipase increased the N-TPL stability at the lipid-water interface, so the TPL N-terminal domain probably formed functional interactions with colipase despite the absence of the C-terminal domain.

The minimal domain of adipose triglyceride lipase (ATGL) ranges until leucine 254 and can be activated and inhibited by CGI-58 and G0S2, respectively.

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis. ATGL specifically hydrolyzes triacylglycerols (TGs), thereby generating diacylglycerols and free fatty acids. ATGL's enzymatic activity is co-activated by the protein comparative gene identification-58 (CGI-58) and inhibited by the protein G0/G1 switch gene 2 (G0S2). The enzyme is predicted to act through a catalytic dyad (Ser47, Asp166) located within the conserved patatin domain (Ile10-Leu178). Yet, neither an experimentally determined 3D structure nor a model of ATGL is currently available, which would help to understand how CGI-58 and G0S2 modulate ATGL's activity. In this study we determined the minimal active domain of ATGL. This minimal fragment of ATGL could still be activated and inhibited by CGI-58 and G0S2, respectively. Furthermore, we show that this minimal domain is sufficient for protein-protein interaction of ATGL with its regulatory proteins. Based on these data, we generated a 3D homology model for the minimal domain. It strengthens our experimental finding that amino acids between Leu178 and Leu254 are essential for the formation of a stable protein domain related to the patatin fold. Our data provide insights into the structure-function relationship of ATGL and indicate higher structural similarities in the N-terminal halves of mammalian patatin-like phospholipase domain containing proteins, (PNPLA1, -2,- 3 and -5) than originally anticipated.

Structural Homologies, Importance for Catalysis and Lipid Binding of the N-Terminal Peptide of a Fungal and a Pancreatic Lipase

Lipases share an overall alpha/beta hydrolase fold structure characteristic of serine hydrolases. Nevertheless, each lipases group possesses its characteristic 3-D structure and catalytic properties. The purified N-terminal truncated forms of a pancreatic (from ostrich) and a fungal (from Rhizopus oryzae, ROL32) (sayari et al., 2005) lipases displayed much lower activities as compared to the native proteins. The aim of this study is to explain this common functional feature on a structural basis. The molecular modelling showed that the N-terminal peptide of the fungal lipase displays an extended "V" shaped structure motif (sayari et al., 2005). We observed that the N-terminal peptide of a pancreatic lipase shares the same extended structure with that of the ROL32, despite the low sequence homology between the two peptides. Upon superimposition of the 3-D structure of the N-terminal catalytic domain of the pancreatic lipase with the model of the ROL32, we have shown that the N-terminal peptide and the open lid domain, of each lipase, are located distally within the putative interfacial binding surface. In particular, two hydrophobic residues, Leu and Ile belonging to the N-terminal peptide of each lipase are well placed to interact with the lipidic substrate. Furthermore, the N-terminal peptide of each lipase seems to be well placed to interact with the loop bearing the catalytic aspartic acid. All these observations might explain the fact that the loss of the N-terminal peptide affects the lipase activity. This work shows that the two lipases share striking structural and functional features with respect to their N-terminal peptide despite the fact that they belong to very distant kingdoms such as fungal and higher animals' ones.

The Lack of the C-Terminal Domain of Adipose Triglyceride Lipase Causes Neutral Lipid Storage Disease through Impaired Interactions with Lipid Droplets

The molecular mechanisms by which triglycerides in lipid droplets (LDs) are synthesized, stored, and degraded need to be elucidated. The objectives were to report siblings with neutral lipid storage disease with myopathy (NLSDM) with a novel mutation of adipose triglyceride lipase (ATGL) and determine whether the C-terminal part of ATGL containing the hydrophobic region plays a role in the interaction with LDs. Skin fibroblasts and peripheral blood leukocytes were obtained from NLSDM patients. In vitro experiments were performed with fibroblasts and COS7 cells. Transfection studies were used to assess the effects of various recombinant ATGL proteins on lipase activities and lipid contents. Fluorescence microscopy were used for determination of intracellular distribution of ATGL proteins. The direct sequence of ATGL cDNA reveals that a patient is a homozygote for the 4-bp deletion, leading to a premature stop codon and causes the lack of the C terminus of the protein including the hydrophobic domain. Overexpressed control ATGL in NLSDM fibroblasts was found around the rims of LDs and caused significantly reduced cellular lipid accumulation. In contrast, NLSDM ATGL was homogeneously located in the cytoplasm despite the presence of LDs and had almost no effect on LD degradation despite its similar lipase activity. A series of C-terminal truncated ATGLs without the intact hydrophobic domain failed to localize around and degrade LDs. These findings indicate that the domain including the hydrophobic region of ATGL was essential for association with LDs.

Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy

Recently, mutations in PNPLA2 encoding adipose triglyceride lipase (ATGL) were reported to underlie a neutral lipid storage disease (NLSD) subgroup characterized by mild myopathy and the absence of ichthyosis. In the present study a novel homozygous PNPLA2 mutation c.475_478dupCTCC (p.Gln160ProfsX19) in the patatin domain, the ATGL active site, was detected in a woman with NLSD and severe myopathy. The present results suggest that a premature truncation mutation in the patatin domain causes NLSD with severe myopathy.

Biochemical and structural comparative study between bird and mammal pancreatic colipases

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of approximately 10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact.

Mapping the heparin-binding domain of human hepatic lipase

Human hepatic lipase (HL) is known to bind to the cell surface of hepatocytes and the sinusoidal endothelium of the liver. In each case, it appears that the enzyme remains associated with the cell surface through an ionic interaction with heparan sulfate proteoglycans. However, it remains unclear as to which residues are responsible for this critical function of the enzyme. In the present study, we have used a systematic approach to map the heparin-binding regions of human HL by utilizing peptide arrays spanning the complete sequence of the mature protein. Following probing with biotin–heparin, six peptides spanning residues 301–320 and 465–476 were identified as regions binding to heparin. Probing of an additional array containing these six parent peptides and a comprehensive series of mutant peptides identified two putative HL heparin-binding domains. The first was composed of residues R310, K312, K314, and R315 at the distal N-terminal domain and the second was composed of residues R473, K474, and R476 at the C-terminal end of the protein.

Val-407 and Ile-408 in the β5′-Loop of Pancreatic Lipase Mediate Lipase-Colipase Interactions in the Presence of Bile Salt Micelles

In a previous study, we demonstrated that the beta5'-loop in the C-terminal domain of human pancreatic triglyceride lipase (hPTL) makes a major contribution in the function of hPTL (Chahinian et al. (2002) Biochemistry 41, 13725-13735). In the present study, we characterized the contribution of three residues in the beta5'-loop, Val-407, Ile-408, and Leu-412, to the function of hPTL. By substituting charged residues, aspartate or lysine, in these positions, we altered the hydrophilic to lipophilic ratio of the beta5'-loop. Each of the mutants was expressed, purified, and characterized for activity and binding with both monolayers and emulsions and for binding to colipase. Experiments with monolayers and with emulsions suggested that the interaction of hPTL with a phospholipid monolayer differs from the interaction of the hPTL-colipase complex with a dicaprin monolayer or a triglyceride emulsion (i.e. neutral lipids). Val-407, Ile-408, and Leu-412 make major contributions to interactions with monolayers, whereas only Val-407 and Ile-408 appear essential for activity on triglyceride emulsions in the presence of bile salt micelles. In solutions of taurodeoxycholate at micellar concentrations, a major effect of the beta5'-loop mutations is to change the interaction between hPTL and colipase. These observations support a major contribution of residues in the beta5'-loop in the function of hPTL and suggest that a third partner, bile salt micelles or the lipid interface or both, influence the binding of colipase and hPTL through interactions with the beta5'-loop.

La digestion des graisses : des aspects moléculaires à la pathologie

Obesity is a severe public health problem in industrialised as well in emergent countries. Considering that dietary fat, because of its high calorific value, plays an important role in the development of obesity, reduction of fat digestion through pancreatic lipase inhibition is now considered as a novel approach in obesity treatment. The isolated C-terminal domain of pancreatic lipase acting as a “protein lure” toward colipase offers a new way for inhibiting intestinal lipolysis by competing with lipase for colipase. In this respect, the C-terminal domain is a very specific inhibitor of pancreatic lipase, compared to the leading obesity drug, orlistat which inhibits the various lipases of the digestive tract. Administration of C-terminal domain to rats fed a high fat diet reduces diet-induced body weight gain and induces an amelioration of fatty liver. Therefore, the C-terminal domain is a strong candidate for an agent that impedes intestinal absorption of dietary fat by inhibiting specifically pancreatic lipase activity.