Minireview on pancreatic lipase and colipase

By hydrolyzing the dietary triacylglycerols, pancreatic lipase causes catalysis in heterogeneous medium. In vivo, lipase action cannot take place without colipase due to the presence of bile salts. The cofactor enables lipase anchoring to the water-lipid interface. The lipase-colipase system furnishes an excellent example of specific interactions (protein-protein and protein-lipid). The studies of lipase catalytic properties brought to light the importance of certain parameters related to the 'quality of the interface'. The structure-function relationship analyses revealed a certain number of functional amino acid residues in lipase and colipase involved either in the catalytic site of the enzyme or in the recognition sites (lipase-colipase and protein-interface). Comparisons of the sequences of lipases derived from different sources display interesting similarities in certain cases.     

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.     

Characterization of turkey pancreatic lipase

Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 degrees C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 degrees C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 degrees C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (beta-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals.     

Comparative studies of canine colipase and lipases from bovine, porcine, canine, human and rat pancreases.

1. Colipase was purified from canine pancreatic juice and found to have certain specificity in its reaction with various pancreatic lipases. 2. This colipase will stimulate the lipolytic activities of lipases isolated from canine, bovine and porcine pancreas but not lipases from a fungus, or from human and rat pancreases. 3. Characterization of these lipases showed (a) the molecular dimension of rat lipase is very different from the other lipases; (b) the pIs of canine, porcine and bovine lipases are almost identical but different from the pIs of rat, human and Candida (a fungus) lipases; and (c) the antiserum prepared against canine lipase will also react with lipases from human, hog and cow pancreases but not with rat and Candida lipases. 4. These physical differences can explain partly the difference in reaction between the various lipases and the canine colipase.     

Sequence of horse pancreatic lipase as determined by protein and cDNA sequencing. Implications for p-nitrophenyl acetate hydrolysis by pancreatic lipases.

The complete sequence of the horse pancreatic lipase was elucidated by combining polypeptide chain and cDNA sequencing. Among the structural features of horse lipase, it is worth mentioning that Lys373 is not conserved. This residue, which is present in human, porcine and canine lipases, has been assumed to be involved in p-nitrophenyl acetate hydrolysis by pancreatic lipases. Kinetic investigation of the p-nitrophenyl acetate hydrolysis by the various pancreatic lipases and by the C-terminal domain (336-449) of human lipase reveals that this hydrolysis is the result of the superimposition of independent events; a specific linear hydrolysis occurring at the active site of lipase, a fast acylation depending on the presence of Lys373 and a non-specific hydrolysis most likely occurring in the C-terminal domain of the enzyme. This finding definitely proves that pancreatic lipase bears only one active site and raises the question of a covalent catalysis by pancreatic lipases. Moreover, based on sequence comparison with the above-mentioned pancreatic lipases, three residues located in the C-terminal domain, Lys349, Lys398 and Lys419, are proposed as possible candidates for lipase/colipase binding.     

Egg yolk lipoproteins as substrates for lipases

Egg yolk emulsions containing phospholipids (about 31%, w/w) are classically used as substrates for measuring phospholipase A2 activity using the pH-stat method. Here we investigated the susceptibility of egg yolk lipoproteins to lipolysis by various highly purified lipases of animal or microbial origin. Egg yolk lipoproteins, which contain up to 65% triacylglycerols, were found to be effective substrates for all the lipases tested. The specific activities measured on egg yolk lipoproteins using the pH-stat technique were found to be 8000, 1000, 1250 and 1700 U/mg in the case of human pancreatic lipase, horse pancreatic lipase, porcine pancreatic lipase and Humicola lanuginosa lipase, respectively. No activity was detected in the absence of colipase with any of the pancreatic lipases tested. Consequently, the classical egg yolk assay cannot be considered as a specific phospholipase A2 assay.     

Various lipases for producing products enriched with polyenic acids in fish fat hydrolysis]

The enzymic hydrolysis of fish with lipases from various sources was studied. The lipase from the fungus Rhizopus microsporus preferentially removes saturated fatty acids, while lipase from the pyloric caeca of salmon unsaturated fatty acids upon hydrolysis of fish fats. The enzymes can be used to obtain fatty products enriched with eicosanopentaenoic acid, mono- and diacylglycerols by enzymic hydrolysis of the ivasi fat.     

Crab digestive lipase acting at high temperature: purification and biochemical characterization

In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates have taken place and this had led to the emergence of some interesting new applications of these enzymes. However, much less is known about lipases from crustaceans. A lipolytic activity was located in the crab digestive glands (hepatopancreas), from which a crab digestive lipase (CDL) was purified. Pure CDL has a molecular mass of 65kDa as determined by SDS/PAGE analysis. Unlike known digestive lipases, CDL displayed its maximal activity on long and short-chain triacylglycerols at a temperature of 60 degrees C. A specific activity of 500U/mg or 130U/mg was obtained with TC(4) or olive oil as substrate, respectively. Only 10% of the maximal activity was detected at 37 degrees C. The enzyme retained 80% of its maximal activity when incubated during 10 min at 60 degrees C, and was completely inactivated at a temperature higher than 65 degrees C. Interestingly, neither colipase, nor bile salts were detected in the crab hepatopancreas. Which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produce bile salts. No similarity between the 13 N-terminal amino acid residues of CDL was found with those of known other digestive lipases.     

Purification and characterization of a novel lipase from the digestive glands of a primitive animal: the scorpion

Higher animal's lipases are well characterized, however, much less is known about lipases from primitive ones. We choose the scorpion, one of the most ancient invertebrates, as a model of a primitive animal. A lipolytic activity was located in the scorpion digestive glands, from which a scorpion digestive lipase (SDL) was purified. Pure SDL, a glycosylated protein, has a molecular mass of 50 kDa, it presents the interfacial activation phenomenon. It was found to be more active on short-chain triacylglycerols than on long-chain triacylglycerols. SDL is a serine enzyme and possesses one accessible sulfhydryl group which is not essential for the catalysis. Among the NH2-terminal 33 residues, a 17 amino acids sequence shows similarities with sequence of Drosophila melanogaster putative lipase. Interestingly, neither colipase, nor bile salts were detected in the scorpion hepatopancreas. This indicates that colipase evolved in vertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produces bile salts. Furthermore, polyclonal antibodies directed against SDL failed to recognise the classical digestive lipases. Altogether, these results suggest that SDL is a member of a new group of digestive lipases belonging to invertebrates.     

Pancreatic bile salt dependent lipase from cod (Gadus morhua): purification and properties.

The enzymatic basis for cod digestive lipolysis has been investigated. Lipase activity was found in aqueous extracts from pyloric caeca as well as in pancreatic tissue surrounding the caeca and the bile duct. A bile salt-dependent lipase (BSDL) was purified from either defatted powder of cod pyloric caeca or aqueous pancreatic extracts by combined affinity chromatography on cholate-Sepharose and gel filtration on Sephacryl S-200 HR. By SDS-PAGE analysis the molecular weight of purified cod BSDL was estimated to 60 kDa. The enzyme was totally dependent on bile salts for hydrolysis of insoluble fatty acid esters. Antiserum raised against purified cod BSDL reacted specifically with selected mammalian pancreatic BSDLs by Western blot analysis. Results presented in this paper strongly suggest that the bile salt-dependent lipase is the only pancreatic enzyme involved in lipid digestion in cod. The enzyme has been characterized and compared to human pancreatic BSDL with respect to substrate specificity, temperature- and pH-dependence and inhibitors. Both soluble and insoluble fatty acid esters were hydrolysed and the enzyme was 1,3-specific in hydrolysis of triolein. The enzyme was inhibited by di-isopropyl fluorophosphate and phenyl boronic acid, but not significantly by phenyl methyl sulfonyl fluoride. The cod BSDL is probably homologous to mammalian pancreatic BSDLs.     

Cloning and characterization of human pancreatic lipase cDNA

Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen. In the presence of bile acids, the activity of lipase is stimulated by colipase. As a prelude to studying the relationship of the protein structures to the functional properties of lipase and colipase, a cDNA encoding human pancreatic lipase was isolated from a lambda gt11 cDNA library screened with a rabbit polyclonal anti-human pancreatic lipase antibody. The full length cDNA clone of 1477 base pairs contained an open reading frame encoding a 465-amino acid protein, including a 16-amino acid signal peptide. The nucleotide sequence was 69% identical to the dog pancreatic lipase cDNA. The predicted NH2-terminal protein sequence agreed with the published NH2-terminal sequence of human pancreatic lipase and the predicted protein sequence was 85 and 70% identical to the protein sequences of pig and dog pancreatic lipase, respectively. A region of homology around Ser-153 is conserved in a number of lipid-binding proteins. Human hepatic lipase and lipoprotein lipase share extensive homology with pancreatic lipase, suggesting that the three proteins are members of a small gene family. In vitro translation of mRNA transcribed from the cDNA resulted in a protein of the expected molecular size that could be processed by microsomal membranes to yield a glycolated protein with proper signal peptide cleavage. RNA blot analysis demonstrated tissue specificity for pancreatic lipase. Thus, for the first time, a full length human pancreatic lipase cDNA has been isolated and characterized. The demonstrated regions of homology with other lipases will aid definition of interactions with substrate and colipase through site-specific mutagenesis.     

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis.

In the intestine, the hydrolysis of triglycerides by pancreatic lipase is performed only in the presence of colipase, whose function is to anchor lipase to the bile-salt-coated lipid interface. Biochemical and crystallographic data on porcine and human lipases have shown that the molecule is made of two well-delimited domains. In order to get more information on the role of the domains in catalysis and colipase binding, we performed limited proteolysis on lipase from various species and obtained different patterns of cleavage. In the case of porcine and human lipases, only the C-terminal domain (12 kDa) could be obtained after chymotryptic attack, whereas in the horse enzyme the cleavage of the Leu410-Thr411 bond gave rise to a large N-terminal (45 kDa) and a small C-terminal (4 kDa) fragment. The isolated porcine and human C-terminal domains were completely inactive towards emulsified tributyrin, though were able to bind colipase. Conversely, the horse 45 kDa fragment retained the lipase activity but failed to correctly bind colipase. This work definitely proves that catalysis and colipase binding are separate events involving topographically distinct regions of the molecule and focuses attention on the role of the C-terminal domain in colipase binding.     

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.     

Fatty acids generated by gastric lipase promote human milk triacylglycerol digestion by pancreatic colipase-dependent lipase

The concerted action of purified bovine gastric lipase and human pancreatic colipase-dependent lipase and colipase, or crude human pancreatic juice, in the digestion of human milk triacylglycerols was explored in vitro. Gastric lipase hydrolyzed milk triacylglycerol with an initially high rate but became severely inhibited already at low concentration of released fatty acid. In contrast, colipase-dependent lipase could not, by itself, hydrolyze milk triacylglycerol. However, a short preincubation of milk with gastric lipase, resulting in a limited lipolysis, made the milk fat triacylglycerol available for an immediate and rapid hydrolysis by pancreatic juice, and also for purified colipase-dependent lipase, provided colipase and bile salts were present. The same effect was obtained when incubation with gastric lipase was replaced by addition of long-chain fatty acid. Long-chain fatty acid increased the binding of colipase-dependent lipase to the milk fat globule. Binding was efficient only in the presence of both fatty acid and colipase. We conclude that a limited gastric lipolysis of human milk triacylglycerol, resulting in a release of a low concentration of long-chain fatty acids, is of major importance for the subsequent hydrolysis by colipase-dependent lipase in the duodenum.     

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.     

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.     

The open lid mediates pancreatic lipase function

Pancreatic triglyceride lipase (PTL) and the homologous pancreatic lipase related protein 2 (PLRP2) provide a unique opportunity to understand the molecular mechanism of lipolysis. They differ in substrate specificity, sensitivity to bile salts, and colipase dependence despite their close amino acid and tertiary structure identity. One important structure, present in both lipases, is the lid which occupies different positions in the inactive and active forms of PTL. We investigated the role of the lid in lipase function by site-specific mutagenesis. By exchanging the lids between PTL and PLRP2, we created two chimeric lipases. Additionally, we made multiple substitution mutations in the PTL lid. PLRP2 with the PTL lid had kinetic properties similar to PLRP2. PTL with the PLRP2 lid was greatly impaired and had no activity at micellar bile salt concentrations even in the presence of colipase. Both chimeras showed interfacial activation suggesting that the closed lid position was maintained. A series of substitution mutations were made in positions Arg257 and Asp258. These mutations demonstrated the importance of these two residues to maintaining the normal activity, triglyceride acyl chain specificity, and colipase interaction of PTL. The preserved interfacial activation in the chimeras, the similar crystal structure of the two lids in the closed position, and the importance of Arg257 and Asp258 in mediating the open conformation of the lid argue that the position of the open lid influences the differences in activity against triglycerides, in sensitivity to bile salts, and in colipase dependence between PTL and PLRP2.     

Inhibition of pancreatic and microbial lipases by proteins

We have compared the effect of several proteins, including melittin, beta-lactoglobulin A, serum albumin, ovalbumin and myoglobin, on the hydrolysis of tributyrin and triolein by lipases from various origins. All proteins tested inactivate pancreatic lipase in absence of colipase and bile salt. Inhibition is not significantly reversed by colipase in absence of bile salt except in systems containing tributyrin and melittin or triolein and beta-lactoglobulin A. In all other cases, activation of pancreatic lipase by colipase in presence of inhibitory protein requires the presence of bile salt. Lipase from Rhizopus delemar is also inhibited by the proteins that inactivate pancreatic lipase. In contrast, the activity of lipase from Rhizopus arrhizus is not affected by the proteins in the same concentration range. Inhibition of lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon not directly related to a decrease in tension at the triacylglycerol-water interface. Inhibition could be the result of desorption of lipase from its substrate due to a change in interfacial quality.     

Purification and biochemical properties of Hexaplex trunculus digestive lipase

Enzymes from fish and aquatic invertebrates have recently been characterized and their study has led to the emergence of some new applications of these classes of enzymes. However, very little is known about lipases from mollusks. A lipolytic activity was located in the marine snail digestive glands (hepatopancreas), from which a marine snail digestive lipase named mSDL was purified. Pure mSDL has a molecular mass of about 70 kDa as determined by SDS/PAGE analysis. Unlike the known digestive lipases while acting at 37 °C, the mSDL displayed its maximal activity on long and short-chain triacylglycerols at 50 °C. A specific activity of 400 U/mg and 100 U/mg was obtained with TC4 or olive oil as substrate respectively. Only 25% of the maximal activity was measured at 37 °C. Interestingly, neither colipase, nor bile salts were detected in the marine snail hepatopancreas, which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produces bile salts. No similarity was found between the N-terminal amino acids sequence of the mSDL and those of the known digestive lipases. Altogether, these results suggest that the mSDL is a member of a new group of digestive lipases belonging to invertebrates.     

Insectivore to frugivore: ontogenetic changes in gut morphology and digestive enzyme activity in the characid fish Brycon guatemalensis from Costa Rican rain forest streams

Brycon guatemalensis , a Neotropical characid fish, consumes an entirely terrestrial diet, shifting from eating insects as juveniles to fruits and leaves as adults. Juvenile and larger‐sized fish collected in the Rio Puerto Viejo at the La Selva Biological Station in Costa Rica were studied to test the hypotheses that, with ontogeny, (1) relative gut length increases, (2) pyloric caeca arrangement and number remain unchanged and (3) pepsin, trypsin and lipase activities decrease, while α‐amylase activity increases. These hypotheses were mainly supported in that larger fish had longer guts, unchanged pyloric caeca arrangement but fewer caeca, and, at both environmental and standard temperatures for the enzyme assays, lower pepsin and trypsin activities but higher α‐amylase activities than the juveniles. Only lipase, among the digestive enzymes, exhibited the unexpected outcome of either not differing significantly in activity (per g of tissue) between juveniles and larger fish or being significantly higher (per mg of protein) in larger fish. The overall results support the view that B. guatemalensis is specialized morphologically and biochemically to function first as a carnivore and then as a herbivore during its life history.