Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

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.

Lid dynamics of porcine pancreatic lipase in non-aqueous solvents.

BACKGROUND: Understanding the dynamics of enzymes in organic solvents has wider implications on their industrial applications. Pancreatic lipases, which show activity in their lid open-state, demonstrate enhanced activity in organic solvents at higher temperatures. However, the lid dynamics of pancreatic lipases in non-aqueous environment is yet to be clearly understood. METHODS: Dynamics of porcine pancreatic lipase (PPL) in open and closed conformations was followed in ethanol, toluene, and octanol using molecular simulation methods. In silico double mutant D250V and E254L of PPL (PPLmut-Cl) was created and its lid opening dynamics in water and in octanol was analyzed. RESULTS: PPL showed increase in solvent accessible surface area and decrease in packing density as the polarity of the surrounded solvent decreased. Breaking the interactions between D250-Y115, and D250-E254 in PPLmut-Cl directed the lid to attain open-state conformation. Major energy barriers during the lid movement in water and in octanol were identified. Also, the trajectories of lid movement were found to be different in these solvents. CONCLUSIONS: Only the double mutant at higher temperature showed lid opening movement suggesting the essential role of the three residues in holding the lid in closed conformation. The lid opening dynamics was faster in octanol than water suggesting that non-polar solvents favor open conformation of the lid. GENERAL SIGNIFICANCE: This study identifies important interactions between the lid and the residues in domain 1 which possibly keeps the lid in closed conformation. Also, it explains the rearrangements of residue-residue interactions during lid opening movement in water and in octanol.

Lid closure dynamics of porcine pancreatic lipase in aqueous solution.

BACKGROUND: Pancreatic lipases hydrolyze fatty acids in dietary pathway. The activity of porcine pancreatic lipase (PPL) is controlled by lid domain along with a coenzyme, colipase. The active open-state conformation of the protein could be induced by detergents or bile salts which would be further stabilized by binding of colipase. In the absence of these interactions, the lid preferably attains a closed conformation in water. METHODS: Molecular dynamic simulation was used to monitor the lid movement of PPL in open and closed conformations in water. Free energy surface was constructed from the simulation. Energy barriers and major structural changes during lid opening were evaluated. RESULTS: The lid closure of PPL in water from its open conformation might be initiated by columbic interactions which initially move the lid away from domain 1. This is followed by major dihedral changes on the lid residues which alter the trajectory of motion. The lid then swirls back towards domain 1 to attain closed conformation. This is accompanied with conformational changes around ß5- and ß9-loops as well. However, PPL in closed conformation shows only the domain movements and the lid remains in its closed conformation. CONCLUSIONS: PPL in closed conformation is stable in water and the open conformation is driven towards closed state. The lid follows a swirling trajectory during the closure. GENERAL SIGNIFICANCE: Conformational state of the lid regulates the activity and substrate specificity of PPL. Hence, it is essential to understand the lid dynamics and the role of specific amino acid residues involved.

The adsorption-desorption behaviour and structure function relationships of bile salts.

The digestion of dietary components in the human gastrointestinal (GI) tract is a complex, dynamic, inherently heterogeneous process. A key aspect of the digestion of lipid in the GI tract is the combined action of bile salts, lipase and colipase in hydrolysing and solubilising dispersed lipid. The bile salts are a mixture of steroid acid conjugates with surfactant properties. In order to examine whether the different bile salts have different interfacial properties their dynamic interfacial behaviour was characterised. Differences in the adsorption behaviour to solid hydrophobic surfaces of bile salt species were studied using dual polarisation interferometry and atomic force microscopy (AFM) under physiological conditions. Specifically, the cholates adsorbed more slowly and a significant proportion were irreversibly adsorbed following buffer rinsing; whereas the deoxycholates and chenodeoxycholates adsorbed more rapidly and desorbed to a greater extent following buffer rinsing. The conjugating groups (taurine, glycine) did not influence the behaviour. AFM showed that the interfacial structures that remained following buffer rinsing were also different between these two groups. In addition, the adsorption-desorption behaviour affected the adsorption of colipase to a solid surface. This supports the idea that cooperative adsorption occurs between certain bile salts and colipase to facilitate the adsorption and activity of pancreatic lipase in order to restore lipolytic activity in the presence of bile salts. This study provides insights into how differences in bile salt structure could affect lipase activity and solubilisation of lipolysis products and other lipid-soluble bioactive molecules.

Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles.

The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile-salt-oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I330/I350 ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.

The Arg92Cys colipase polymorphism impairs function and secretion by increasing protein misfolding.

Colipase is essential for efficient fat digestion. An arginine-to-cysteine polymorphism at position 92 of colipase (Arg92Cys) associates with an increased risk for developing type-2 diabetes through an undefined mechanism. To test our hypothesis that the extra cysteine increases colipase misfolding, thereby altering its intracellular trafficking and function, we expressed Cys92 colipase in HEK293T cells. Less Cys92 colipase is secreted and more is retained intracellularly in an insoluble form compared with Arg92 colipase. Nonreducing gel electrophoresis suggests the folding of secreted Cys92 colipase differs from Arg92 colipase. Cys92 colipase misfolding does not trigger the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress. The ability of secreted Cys92 colipase to stimulate pancreatic triglyceride lipase (PTL) is reduced with all substrates tested, particularly long-chain triglycerides. The reaction of Cys92 colipase with triolein and Intralipid has a much longer lag time, reflecting decreased ability to anchor PTL on those substrates. Our data predicts that humans with the Arg92Cys substitution will secrete less functional colipase into the duodenum and have less efficient fat digestion. Whether inefficient fat digestion or another property of colipase contributes to the risk for developing diabetes remains to be clarified.

Biochemical properties of pancreatic colipase from the common stingray Dasyatis pastinaca.

BACKGROUND: Pancreatic colipase is a required co-factor for pancreatic lipase, being necessary for its activity during hydrolysis of dietary triglycerides in the presence of bile salts. In the intestine, colipase is cleaved from a precursor molecule, procolipase, through the action of trypsin. This cleavage yields a peptide called enterostatin knoswn, being produced in equimolar proportions to colipase. RESULTS: In this study, colipase from the common stingray Dasyatis pastinaca (CoSPL) was purified to homogeneity. The purified colipase is not glycosylated and has an apparent molecular mass of around 10 kDa. The NH2-terminal sequencing of purified CoSPL exhibits more than 55% identity with those of mammalian, bird or marine colipases. CoSPL was found to be less effective activator of bird and mammal pancreatic lipases than for the lipase from the same specie. The apparent dissociation constant (Kd) of the colipase/lipase complex and the apparent Vmax of the colipase-activated lipase values were deduced from the linear curves of the Scatchard plots. We concluded that Stingray Pancreatic Lipase (SPL) has higher ability to interact with colipase from the same species than with the mammal or bird ones. CONCLUSION: The fact that colipase is a universal lipase cofactor might thus be explained by a conservation of the colipase-lipase interaction site. The results obtained in the study may improve our knowledge of marine lipase/colipase.

Adsorption of bile salts and pancreatic colipase and lipase onto digalactosyldiacylglycerol and dipalmitoylphosphatidylcholine monolayers.

It is increasingly recognized that changes in the composition of the oil-water interface can markedly affect pancreatic lipase adsorption and function. To understand interfacial mechanisms determining lipase activity, we investigated the adsorption behavior of bile salts and pancreatic colipase and lipase onto digalactosyldiacylglycerol (DGDG) and dipalmitoylphosphatidylcholine (DPPC) monolayers at the air-water interface. The results from Langmuir trough and pendant drop experiments showed that a DGDG interface was more resistant to the adsorption of bile salts, colipase, and lipase compared to that of DPPC. Atomic force microscopy (AFM) images showed that the adsorption of bile salts into a DPPC monolayer decreased the size of the liquid condensed (LC) domains while there was no visible topographical change for DGDG systems. The results also showed that colipase and lipase adsorbed exclusively onto the mixed DPPC-bile salt regions and not the DPPC condensed phase. When the colipase and lipase were in excess, they fully covered the mixed DPPC-bile salt regions. However, the colipase and lipase coverage on the mixed DGDG-bile salt monolayer was incomplete and discontinuous. It was postulated that bile salts adsorbed into the DPPC monolayers filling the gaps between the lipid headgroups and spacing out the lipid molecules, making the lipid hydrocarbon tails more exposed to the surface. This created hydrophobic patches suitable for the binding of colipase and lipase. In contrast, bile salts adsorbed less easily into the DGDG monolayer because DGDG has a larger headgroup, which has strong intermolecular interactions and the ability to adopt different orientations at the interface. Thus, there are fewer hydrophobic patches that are of sufficient size to accommodate the colipase on the mixed DGDG-bile salt monolayer compared to the mixed DPPC-bile salt regions. The results from this work have reinforced the hypothesis that the interfacial molecular packing of lipids at the oil-water interface influences the adsorption of bile salts, colipase, and lipase, which in turn impacts the rate of lipolysis.

Computational study of colipase interaction with lipid droplets and bile salt micelles.

Colipase is a key element in the lipase-catalyzed hydrolysis of dietary lipids. Although devoid of enzymatic activity, colipase promotes the pancreatic lipase activity in physiological intestinal conditions by anchoring the enzyme at the surface of lipid droplets. Analysis of structures of NMR colipase models and simulations of their interactions with various lipid aggregates, lipid droplet, and bile salt micelle, were carried out to determine and to map the lipid binding sites on colipase. We show that the micelle and the oil droplet bind to the same side of colipase 3D structure, mainly the hydrophobic fingers. Moreover, it appears that, although colipase has a single direction of interaction with a lipid interface, it does not bind in a specific way but rather oscillates between different positions. Indeed, different NMR models of colipase insert different fragments of sequence in the interface, either simultaneously or independently. This supports the idea that colipase finger plasticity may be crucial to adapt the lipase activity to different lipid aggregates.

Structure and orientation of pancreatic colipase in a lipid environment: PM-IRRAS and Brewster angle microscopy studies.

Colipase is a key element in lipase-catalyzed dietary lipids hydrolysis. Although devoid of enzymatic activity, colipase promotes pancreatic lipase activity in the physiological intestinal conditions by anchoring the enzyme on the surface of lipid droplets. Polarization modulation infrared reflection absorption spectroscopy combined with Brewster angle microscopy studies was performed on colipase alone and in various lipid environments to obtain a global view of both conformation and orientation and to assess lipid perturbations. We clearly show that colipase fully inserts into a dilaurin monolayer and promotes the formation of lipid/protein domains, whereas in a phospholipid environment its insertion is only partial, limited to the polar head group. In a mixed 70% phosphatidylcholine/30% dilaurin environment, colipase adsorbs to but does not penetrate deeply into the film. It triggers the formation of diglyceride domains under which it would form a rather uniform layer. We also clearly demonstrate that colipase adopts a preferred orientation when dilaurin is present at the interface. In contrast, at a neutral phospholipid interface, the infrared spectra suggest an isotropic orientation of colipase which could explain its incapacity to reverse the inhibitory effects of these lipids on the lipase activity.

Probing the opening of the pancreatic lipase lid using site-directed spin labeling and EPR spectroscopy.

Access to the active site of human pancreatic lipase (HPL) is controlled by a surface loop (the lid) that undergoes a conformational change in the presence of amphiphiles and lipid substrate. The question of how and when the lid opens still remains to be elucidated, however. A paramagnetic probe was covalently bound to the lid via the D249C mutation, and electron paramagnetic resonance (EPR) spectroscopy was used to monitor the conformational change in solution. Two EPR spectral components, corresponding to distinct mobilities of the probe, were attributed to the closed and open conformations of the HPL lid, based on experiments performed with the E600 inhibitor. The open conformation of the lid was observed in solution at supramicellar bile salt concentrations. Colipase alone did not induce lid opening but increased the relative proportions of the open conformation in the presence of bile salts. The opening of the lid was found to be a reversible process. Using various colipase to lipase molar ratios, a correlation between the proportion of the open conformation and the catalytic activity of HPL was observed.

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.

Val-407 and Ile-408 in the beta5'-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.

Real-time measurement of solute partitioning to lipid monolayers.

The interaction of a peripheral protein with a lipid-water interface can show a pronounced dependence on the composition and two-dimensional packing density of the lipids that comprise the interface. We report a novel optical method for measuring the adsorption of macromolecules, such as proteins and nucleic acids, and smaller solutes, such as drugs, to lipid monolayers at the gas-liquid interface. Using fluorescence emission from proteins and a small molecule, we demonstrate that the emissions from these solutes when in the aqueous phase and when associated with the monolayer can be temporally separated. Such separation allows measurement of the extent of solute adsorption, spectral characterization of the adsorbed solute, and characterization of lipid organization using adsorption kinetics. The method does not require, but is compatible with, the solute having different spectral properties in the bulk and surface phases. Indeed, if optical signals from adsorbed and soluble solute are the same or their relationship is known, absolute surface excess of adsorbed solute can be calculated without independent calibration. With appropriate instrumental configuration, the method should be adaptable for screening solutes for interaction with planar monolayers having both well-defined composition and adjustable lipid packing density.

Human pancreatic lipase-related protein 2 is a galactolipase.

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.

Transfer of orlistat through oil-water interfaces.

The transfer of radiolabelled orlistat ([14C]orlistat), a potent gastrointestinal lipase inhibitor, through an oil-water interface from a single oil droplet to an aqueous phase was investigated, using an oil drop tensiometer. The absolute transfer fluxes were found to be very low, even in the presence of micellar concentrations of bile salts, which increased their values from 0.2 to 2.5 and 6.5 pmol cm(-2) min(-1) in the presence of 0, 4 and 15 mM NaTDC, respectively. Adding either a lipid emulsion or pure human pancreatic lipase (HPL) or human serum albumin or beta-lactoglobulin had no effect on the flux of transfer of orlistat. The presence of colipase or a mixture of colipase and HPL was found, however, to reduce the flux of orlistat transfer, probably because it partly covered the single oil drop surface, even in the presence of bile salts. Using a finely emulsified system, we investigated the partitioning of orlistat between the aqueous and oil phases, in the absence or presence of bile salts above their CMC (4 mM NaTDC, final concentration). Under these emulsified conditions, orlistat was found to be mostly associated with the oil phase, since more than 98.8% of the total radioactivity was recovered after decantation with the oil phase. The low transfer rates of orlistat, as well as its partitioning coefficient between the oil and the aqueous phases, should help us to better understand the inhibitory effects of orlistat on lipid digestion in humans.

Purification of classical pancreatic lipase from dog pancreas.

The purification of canine classical pancreatic lipase from canine pancreatic juice, but not from pancreatic tissue, has been reported previously. Given the logistic difficulties associated with collection of pancreatic juice in dogs and efforts to minimize experiments in live animals the objective of this project was to purify canine classical pancreatic lipase from dog pancreas. Dog pancreata were collected from research dogs that had been sacrificed for unrelated research projects. Pancreatic tissue was delipidated using organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamindine and phenylmethylsulfonyl fluoride (PMSF), followed by anion exchange chromatography, gel-filtration, and cation exchange chromatography. The purified protein showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of approximately 50.7. Isoelectric focusing showed isoelectric points ranging from 6.0 to 6.2. N-terminal amino acid sequencing of the first 25 amino acid residues showed the sequence Lys-Glu-Val-X-Phe-Pro-Arg-Leu-Gly-X-Phe-Ser-Asp-Asp-Ser-Pro-Trp-Ala-Gly-Ile-Val-Glu-Arg-Pro-Leu. This sequence showed close homology with classical pancreatic lipase in pigs, horses, and human beings. We conclude that canine classical pancreatic lipase can be successfully purified from canine pancreatic tissue.

Lipid lateral organization in fluid interfaces controls the rate of colipase association.

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.