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Mark E. Lowe - One of the best experts on this subject based on the ideXlab platform.

  • identification of amino acids in human Colipase that mediate adsorption to lipid emulsions and mixed micelles
    Biochimica et Biophysica Acta, 2013
    Co-Authors: Leah E Ross, Xunjun Xiao, Mark E. Lowe
    Abstract:

    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
    Journal of Lipid Research, 2013
    Co-Authors: Xunjun Xiao, Michael R Ferguson, Kelsey Magee, Pamela Hale, Yan Wang, Mark E. Lowe
    Abstract:

    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.

  • val 407 and ile 408 in the β5 loop of pancreatic lipase mediate lipase Colipase interactions in the presence of bile salt micelles
    Journal of Biological Chemistry, 2006
    Co-Authors: Angela B Freie, Frédéric Carrière, Franciine Ferrato, Mark E. Lowe
    Abstract:

    In a previous study, we demonstrated that the β5′-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 β5′-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 β5′-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 β5′-loop mutations is to change the interaction between hPTL and Colipase. These observations support a major contribution of residues in the β5′-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 β5′-loop.

  • Pancreatic Lipase-related Protein 2 Is the Major Colipase-Dependent Pancreatic Lipase in Suckling Mice
    The Journal of nutrition, 2004
    Co-Authors: Dymphna D'agostino, Mark E. Lowe
    Abstract:

    Suckling mice express Colipase before the expression of pancreatic triglyceride lipase. Yet, efficient fat digestion in newborns requires Colipase, suggesting that Colipase may act as a cofactor for another lipase such as pancreatic lipase-related protein 2 (PLRP2). We determined whether PLRP2 or another lipase depends on Colipase for maximal activity in newborn mice by analyzing extracts from the pancreas of 4-d-old Colipase-deficient and PLRP2-deficient mice. Pancreatic extracts from Colipase-deficient pups had lipase activity that was stimulated onefold by the addition of exogenous Colipase (P < 0.001). The activity was completely inhibited by an antibody against pancreatic triglyceride lipase that also recognizes PLRP2. In contrast, pancreatic extracts from PLRP2-deficient pups had significantly lower baseline activity and no Colipase-dependent activity. The baseline activity was not inhibited by the anti-pancreatic triglyceride lipase antibody or an antibody against carboxyl ester lipase. We next separated the extracts into two fractions, one containing PLRP2 and the other devoid of PLRP2. All of the Colipase-dependent activity segregated with the PLRP2-containing fraction, consistent with the conclusion that PLRP2 is the major Colipase-dependent lipase in the pancreas of newborns.

  • The triglyceride lipases of the pancreas
    Journal of Lipid Research, 2002
    Co-Authors: Mark E. Lowe
    Abstract:

    : Pancreatic triglyceride lipase (PTL) and its protein cofactor, Colipase, are required for efficient dietary triglyceride digestion. In addition to PTL, pancreatic acinar cells synthesize two pancreatic lipase related proteins (PLRP1 and PLRP2), which have a high degree of sequence and structural homology with PTL. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, behavior in bile salts and dependence on Colipase. Each protein has a globular amino-terminal (N-terminal) domain, which contains the catalytic site for PTL and PLRP2, and a beta-sandwich carboxyl-terminal (C-terminal) domain, which includes the predominant Colipase-binding site for PTL. Inactive and active conformations of PTL have been described. They differ in the position of a surface loop, the lid domain, and of the beta5-loop. In the inactive conformation, the lid covers the active site and, upon activation by bile salt micelles and Colipase or by lipid-water interfaces, the lid moves dramatically to open and configure the active site. After the lid movement, PTL and Colipase create a large hydrophobic plateau that can interact with the lipid-water interface. A hydrophobic surface loop in the C-terminal domain, the beta5' loop, may also contribute to the interfacial-binding domain of the PTL-Colipase complex.

Charlotte Erlansonalbertsson - One of the best experts on this subject based on the ideXlab platform.

  • pancreatic lipase Colipase binds strongly to the thylakoid membrane surface
    Journal of the Science of Food and Agriculture, 2013
    Co-Authors: Sinan Cem Emek, Charlotte Erlansonalbertsson, Hanserik Akerlund, Perake Albertsson
    Abstract:

    BACKGROUND: Isolated thylakoid membranes, i.e. the photosynthetic membranes of green leaves, inhibit the activity of pancreatic lipase and Colipase during hydrolysis of fat in vitro. This inhibition has been demonstrated to cause reduced food intake and improved hormonal and lipid profile in vivo. One of the reasons suggested for the inhibiting effect is binding of lipase-Colipase to the thylakoid membrane surface. This prompted a study of the binding of lipase and Colipase to thylakoids. RESULTS: The results showed that lipase and Colipase strongly bind to the thylakoid membrane surface. The dissociation constant was determined at 1.2 × 10(-8) mol L(-1) ; binding decreased after treatment of thylakoids with pepsin/trypsin to 1.0 × 10(-7) and to 0.6 × 10(-7) mol L(-1) after treatment with pancreatic juice. Similarly, delipidation of thylakoids caused a decrease in binding, the dissociation constant being 2.0 × 10(-7) mol L(-1) . CONCLUSION: The binding of pancreatic lipase-Colipase to the thylakoid membrane is strong and may explain the inhibition of lipase-Colipase activity by thylakoids. After treatment with proteases to mimic intestinal digestion binding is decreased, but is still high enough to explain the observed metabolic effects of thylakoids in vivo. © 2013 Society of Chemical Industry.

  • feeding appetite suppressing thylakoids to pigs alters pancreatic lipase Colipase secretion
    Livestock Science, 2010
    Co-Authors: Rickard Kohnke, Linnea Svensson, Jose Luis Valverde Piedra, Stefan Pierzynowski, Bjorn Westrom, Charlotte Erlansonalbertsson
    Abstract:

    The mechanism for a new appetite suppressor named thylakoids (membrane proteins derived from spinach leaves) was examined in vivo in pigs. Thylakoids inhibit the lipase/Colipase hydrolysis of triacylglycerols (TG) in vitro and suppress food intake, decrease body weight gain and raise the circulating satiety hormone cholecystokinin (CCK) in rats but its mechanism in vivo remains unclear. We hypothesized that a thylakoid-enriched diet prolongs intestinal digestion of food and therefore promote satiety signaling. Five pigs were surgically prepared with a fistula in the duodenum for collection of digesta and with two catheters, one in v. jugularis and one in v. porta, for blood collection. After 1 week of recovery and an overnight fast the pigs were fed a high-fat diet with and without supplementation with thylakoids. Duodenal content and blood samples were taken before and 15, 30, 60, 120, 240 and 360 min after feeding. Pancreatic lipase and Colipase enzymes were measured in duodenal digesta. Blood samples were analyzed for the satiety hormone CCK as well as insulin and glucose. We found that pancreatic lipase/Colipase level increased and stayed elevated for a longer time in the duodenum in the pigs receiving thylakoids compared to the control. CCK levels were unchanged. Insulin levels were significantly reduced by the thylakoid treatment without any change in blood glucose. In conclusion, thylakoids increased lipase/Colipase secretion. The mechanism for this secretion appears not to be related to CCK and may be an effect of vagal activation. Thylakoids gave reduced insulin levels without any change in glucose levels. (C) 2010 Published by Elsevier B.V. (Less)

  • identification of enterostatin and the relation between lipase and Colipase in various species
    Nutritional Neuroscience, 1998
    Co-Authors: Catarina Rippe, Charlotte Erlansonalbertsson
    Abstract:

    Enterostatin, the N-terminal activation peptide of pancreatic proColipase, has been identified in three different forms in rat: VPDPR (Val-Pro-Asp-Pro-Arg), APGPR (Ala-Pro-Gly-Pro-Arg) and VPGPR (Val-Pro-Gly-Pro-Arg). We investigated the possibility for a species to have several isoforms of enterostatin. Pancreas was purified from four different species (rat, mouse, cat and pig) and the enterostatin sequences were identified. At the same time, the activities of pancreatic lipase and Colipase were measured. In rat and mouse pancreas APGPR was the only form of enterostatin identified. The Colipase activity was 188 ± 25 U/mg protein in rat and 189 ± 16 U/mg in mouse and the lipase activity 354 ± 33 U/mg and 292 ± 19 U/mg respectively. Rat and mouse had a Colipase/lipase ratio close to 0.5. In pancreas from cat and pig we only detected the form VPDPR (Val-Pro-Asp-Pro-Arg). We found the Colipase activity in cat to be 493 ± 92 U/mg, while the lipase activity was three times lower, 167 ± 18 U/mg. Pig pancreas concentrations of Colipase was 110 ± 8 U/mg and of lipase 38 ± 5 U/mg. In both cat and pig the Colipase/lipase ratio was close to 3. This suggests that Colipase might have an additional role than to restore the activity of lipase. Our hypothesis is that an overproduction of Colipase and hence also enterostatin is involved in the regulation of fat metabolism.

  • evidence of a stimulatory effect of cyclic amp on pancreatic lipase and Colipase synthesis in rats
    Scandinavian Journal of Gastroenterology, 1992
    Co-Authors: Ruidong Duan, Charlotte Erlansonalbertsson
    Abstract:

    The effects of endogenous and exogenous cyclic AMP on the synthesis of pancreatic lipase, Colipase, and amylase were studied. Pancreatic lobules were prepared and incubated with forskolin, dibutyryl cyclic AMP (dbcAMP), and dibutyryl cyclic GMP (dbcGMP), respectively, in the presence of “S-cysteine”. The individual pancreatic enzymes were isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactive cysteine into lipase, Colipase, and amylase was determined. Incubation with forskolin (25 uM) rapidly increased lipase synthesis rate within 30 min, followed by an increase in Colipase synthesis rate after 60 min of incubation. Amylase synthesis rate did not change during the 1st h of incubation but decreased slightly when incubated for 2h. Incubation of pancreatic lobules with dbcAMP (1 mM) for 1 h also stimulated the incorporation of cysteine into lipase and Colipase by 21% and 25%, respectively, whereas incubation with dbcGMP had no effect on the synthesis rates of lipase and Colipase...

  • gastric inhibitory polypeptide stimulates pancreatic lipase and Colipase synthesis in rats
    American Journal of Physiology-gastrointestinal and Liver Physiology, 1992
    Co-Authors: Ruidong Duan, Charlotte Erlansonalbertsson
    Abstract:

    Effects of short-term infusion and long-term injection of gastric inhibitory polypeptide (GIP) on changes in pancreatic lipase and Colipase contents in rats were studied, and mRNAs encoding for lipase and Colipase were determined by Northern blot hybridization with specific cDNA probes. GIP infused at a dose of 3 micrograms/h for 24 h significantly increased the pancreatic lipase content by 34% (P less than 0.05) but had no significant effect on Colipase and amylase contents. No change in mRNAs encoding for these proteins was found after infusion of GIP for 24 h. Injection of GIP (5-60 micrograms/kg) three times a day for 5 days dose dependently increased the contents of lipase and Colipase, with the increase in Colipase being more prominent. Injection of GIP for 5 days at a dose of 30 micrograms.kg-1.day-1 increased Colipase and lipase contents by 52 and 25%, and their corresponding mRNAs by 60 and 160%, respectively. The amylase mRNA was not changed by injection of GIP. It is concluded that GIP has a specific stimulatory effect on the synthesis of pancreatic lipase and Colipase at both pretranslational and translational levels.

Youssef Gargouri - One of the best experts on this subject based on the ideXlab platform.

  • biochemical characterization cloning and molecular modeling of a digestive lipase from red seabream pagrus major structural explanation of the interaction deficiency with Colipase and lipidic interface
    Engineering in Life Sciences, 2017
    Co-Authors: Nabil Smichi, Youssef Gargouri, Ahmed Fendri, Soumaya Triki, Vincent Arondel, Ahmed Rebai, Nabil Miled
    Abstract:

    Red seabream digestive lipase (RsDL) was purified from fresh pyloric caeca. Pure RsDL has an apparent molecular mass of 50 kDa. The RsDL is more active on short-chain triacylglycerols (TC4), and enzymatic activity decreases when medium (TC8) or long-chain (olive oil) triacylglycerols were used as substrates. The specific activities of RsDL are very weak as compared to those obtained with classical pancreatic lipases. No Colipase was detected in the red seabream pyloric caeca. Furthermore, the RsDL was not activated by a mammal Colipase. Similar results were reported for annular seabream lipase. In order to explain structurally the discrepancies between sparidae and mammal lipases, genes encoding mature RsDL and five other lipases from sparidae fish species were cloned and sequenced. Phylogenetic studies indicated the closest homology of sparidae lipases to bird pancreatic ones. Structural models were built for annular seabream and RsDL under their closed and open forms using mammal pancreatic lipases as templates. Several differences were noticed when analyzing the amino acids corresponding to those involved in HPL binding to Colipase. This is likely to prevent interaction between the fish lipase and the mammalian Colipase and may explain the fact that mammalian Colipase is not effective in activating sparidae lipases. In addition, several hydrophobic residues, playing a key role in anchoring pancreatic lipase onto the lipid interface, are replaced by polar residues in fish lipases. This might explain the reason why the latter enzymes display weak activity levels when compared to mammalian pancreatic lipases.

  • from the common stingray Dasyatis pastinaca
    2013
    Co-Authors: Abir Ben Bacha, Youssef Gargouri, Aida Karray, Lobna Daoud, Emna Bouchaala, Madiha Bou Ali, Yassine Ben Ali
    Abstract:

    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

  • Biochemical properties of pancreatic Colipase from the common stingray Dasyatis pastinaca
    Lipids in Health and Disease, 2011
    Co-Authors: Abir Ben Bacha, Lobna Daoud, Aida Karray, Emna Bouchaala, Youssef Gargouri
    Abstract:

    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 NH_2-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.

Frédéric Carrière - One of the best experts on this subject based on the ideXlab platform.

  • val 407 and ile 408 in the β5 loop of pancreatic lipase mediate lipase Colipase interactions in the presence of bile salt micelles
    Journal of Biological Chemistry, 2006
    Co-Authors: Angela B Freie, Frédéric Carrière, Franciine Ferrato, Mark E. Lowe
    Abstract:

    In a previous study, we demonstrated that the β5′-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 β5′-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 β5′-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 β5′-loop mutations is to change the interaction between hPTL and Colipase. These observations support a major contribution of residues in the β5′-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 β5′-loop.

  • human pancreatic lipase related protein 2 is a galactolipase
    Biochemistry, 2004
    Co-Authors: Barbara Sias, Robert Verger, Francine Ferrato, Philippe Grandval, Dominique Lafont, Paul Boullanger, Alain De Caro, B Leboeuf, Frédéric Carrière
    Abstract:

    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.

  • human pancreatic lipase Colipase dependence and interfacial binding of lid domain mutants
    Biochemistry, 1999
    Co-Authors: Sofiane Bezzine, Robert Verger, Francine Ferrato, Margarita Ivanova, Veronique Lopez, Frédéric Carrière
    Abstract:

    Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by Colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and Colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with Colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-Colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by Colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of Colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-Colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-Colipase complex. The effects of Colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one Colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of Colipase. Reactivation of HPL(-lid) by Colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-Colipase interactions.

  • lipid binding and activating properties of porcine pancreatic Colipase split at the ile79 thr80 bond
    Biochimica et Biophysica Acta, 1995
    Co-Authors: Nathalie Rugani, Frédéric Carrière, Bengt Borgström, Lars Thim, Louis Sarda
    Abstract:

    Abstract Porcine Colipase, the protein cofactor of pancreatic lipase, was isolated from pancreas freshly collected on animals and from a side fraction from the production of insulin (Novo Nordisk A/S). Samples of purified Colipase were analyzed for homogeneity by polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography (RPLC), quantitative N-terminal sequence determination and mass spectrometry. The activating properties of Colipase preparations were assayed against tributyrin, triolein or the commercial Intralipid emulsion, in presence of bile salt. Two fractions of Colipase with the same specific activity were purified from fresh pancreas. The major fraction (85%) contained one single protein corresponding to fragment 1–93 of the 95-residue form of Colipase (proColipase) previously characterized in porcine pancreatic juice. The other fraction (15%) corresponded to fragment 1–91 of proColipase. Also, two fractions of Colipase were purified from the side fraction supplied by Novo. These fractions consisted of the 95-residue proform of Colipase and of fragment 1–93, respectively, both specifically cleaved at the Ile 79 -Thr 80 peptide bond with partial removal of isoleucine at position 79 and serine at position 78. ProColipase split at the 79–80 bond retained full activity on tributyrin and triolein and on the Intralipid emulsion but the kinetics of hydrolysis of triacylglycerol substrates showed much longer lag periods than those observed with native proColipase. Also, all forms of proColipase split at the 79–80 bond showed one peak in RPLC but their retention time was markedly decreased as compared to that of native proColipase which indicated a weaker hydrophobic binding capacity. The value of the retention time was of the same order of magnitude as that of inactive reduced proColipase. Treatment of native proColipase by pancreatic endopeptidases showed that elastase is likely responsible for specific cleavage at the 79–80 bond of proColipase purified from the Novo extract. Limited proteolysis by trypsin of the proforms of Colipase split at the 79–80 bond reduced the lag period. Results presented in this communication provide the first direct evidence showing that the finger-shaped peptide segment between half-cystine residues at positions 69 and 87 is involved in Colipase-lipid interaction as previously hypothesized from the three-dimensional structure of the protein.

Catherine Chapus - One of the best experts on this subject based on the ideXlab platform.

  • stabilization of the c terminal part of pig and horse Colipase by carboxypeptidase and trypsin inhibitors
    FEBS Journal, 2005
    Co-Authors: Catherine Chapus, P Desnuelle, Edith Foglizzo
    Abstract:

    Pig and horse Colipases have been purified by a common procedure using trypsin and carboxypeptidase inhibitors as stabilizers. Two forms of pig Colipase were identified: a predominant A1 form with about 103–105 residues, and a minor slightly degraded A2 form in which the last two C-terminal residues, Asp and Ser, were lacking. This type of degradation is considerably slowed down by carboxypeptidase inhibitors. A total of four forms of the horse cofactor were characterized: two (A1 and B1) were probably isoColipases which differed by only a few substitutions. Both contained the same number of residues (about 96), an N-terminal valine and an Arg-Ser-Glu-(Glx)1,2-Arg C-terminal sequence. A2 and B2 were slightly degraded forms probably resulting from tryptic cleavage of the Arg-Ser bond in the above sequence. The presence of methioriine in the horse cofactor allowed fragmentation by cyanogen bromide. The C-terminal fragment was composed of 16 or 17 residues and contained no histidine. The single histidine of horse B1 was found in the intermediary fragment between Met-18 and Met-(n-16) or Met-(n-I7). These data show that the C-terminal parts of both pig and horse Colipases are still more exposed to proteolytic degradations than the N-terminal parts. Preliminary attempts to crystallize B1 were carried out.

  • interactions of bile salt micelles and Colipase studied through intermolecular noes
    FEBS Letters, 2000
    Co-Authors: Cyril Dominguez, Corinne Sebbankreuzer, Olivier Bornet, Brigitte Kerfelec, Catherine Chapus, Francoise Guerlesquin
    Abstract:

    Colipase is a small protein (10 kDa), which acts as a protein cofactor for the pancreatic lipase. Various models of the activated ternary complex (lipase-Colipase-bile salt micelles) have been proposed using detergent micelles, but no structural information has been established with bile salt micelles. We have investigated the organization of sodium taurodeoxycholate (NaTDC) micelles and their interactions with pig and horse Colipases by homonuclear nuclear magnetic resonance (NMR) spectroscopy. The NMR data supply evidence that the folding of horse Colipase is similar to that already described for pig Colipase. Intermolecular nuclear Overhauser effects have shown that two conserved aromatic residues interact with NaTDC micelles.

  • ion pairing between lipase and Colipase plays a critical role in catalysis
    Journal of Biological Chemistry, 1998
    Co-Authors: Laurence Ayvazian, Catherine Chapus, Isabelle Crenon, Juan A Hermoso, David Pignol, Brigitte Kerfelec
    Abstract:

    Among the polar interactions occurring in pancreatic lipase/Colipase binding, only one ion pair involving lysine 400 on lipase and glutamic acid 45 on Colipase has been described. These residues are strictly conserved among species, suggesting that the ion pair is likely to play an important role. Therefore, in order to prevent this interaction, mutations intended to neutralize or inverse the charge of these residues have been introduced in the cDNAs encoding horse lipase and Colipase. The recombinant proteins have been expressed in insect cells, and their catalytic properties have been investigated. In all cases, preventing the formation of the correct ion pair Lys400/Glu45 leads to lipase-Colipase complexes of reduced affinity unable to perform an efficient catalysis, notably in the presence of bile salt micelles. Diethyl p-nitrophenyl phosphate inhibition experiments with either mutant lipase or mutant Colipase indicate a poor stabilization of the lipase flap. These results suggest that the ion pair plays a critical role in the active conformation of the lipase-Colipase-micelle ternary complex by contributing to a correct orientation of Colipase relative to lipase resulting in a proper opening of the flap.

  • neutron crystallographic evidence of lipase Colipase complex activation by a micelle
    The EMBO Journal, 1997
    Co-Authors: Juan A Hermoso, Catherine Chapus, David Pignol, Simon Penel, Michel Roth, Juan C Fontecillacamps
    Abstract:

    The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. Here, we present the neutron structure of the activated lipase-Colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of Colipase and the distal tip of the C-terminal domain of lipase. Since the micelle- and substrate-binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by Colipase and a micelle.

  • lipase activation by nonionic detergents the crystal structure of the porcine lipase Colipase tetraethylene glycol monooctyl ether complex
    Journal of Biological Chemistry, 1996
    Co-Authors: Juan A Hermoso, Brigitte Kerfelec, Catherine Chapus, Isabelle Crenon, David Pignol, Juan C Fontecillacamps
    Abstract:

    The crystal structure of the ternary porcine lipase-Colipase-tetra ethylene glycol monooctyl ether (TGME) complex has been determined at 2.8 A resolution. The crystals belong to the cubic space group F23 with a = 289.1 A and display a strong pseudo-symmetry corresponding to a P23 lattice. Unexpectedly, the crystalline two-domain lipase is found in its open configuration. This indicates that in the presence of Colipase, pure micelles of the nonionic detergent TGME are able to activate the enzyme; a process that includes the movement of an N-terminal domain loop (the flap). The effects of TGME and Colipase have been confirmed by chemical modification of the active site serine residue using diisopropyl p-nitrophenylphosphate (E600). In addition, the presence of a TGME molecule tightly bound to the active site pocket shows that TGME acts as a substrate analog, thus possibly explaining the inhibitory effect of this nonionic detergent on emulsified substrate hydrolysis at submicellar concentrations. A comparison of the lipase-Colipase interactions between our porcine complex and the human-porcine complex (van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R., and Cambillau, C. (1993) Nature 362, 814-820) indicates that except for one salt bridge interaction, they are conserved. Analysis of the superimposed complexes shows a 5.4° rotation on the relative position of the N-terminal domains excepting the flap that moves in a concerted fashion with the C-terminal domain. This flexibility may be important for the binding of the complex to the water-lipid interface.