The lipase C-terminal domain. A novel unusual inhibitor of pancreatic lipase activity.

In vertebrates, dietary fat digestion mainly results from the combined effect of pancreatic lipase, colipase, and bile. It has been proposed that in vivo lipase adsorption on oil-water emulsion is mediated by a preformed lipase-colipase-mixed micelle complex. The main lipase-colipase binding site is located on the C-terminal domain of the enzyme. We report here that in vitro the isolated C-terminal domain behaves as a potent noncovalent inhibitor of lipase and that the inhibitory effect is triggered by the presence of micelles. Lipase inhibition results from the formation of a nonproductive C-terminal domain-colipase-micelle ternary complex, which competes for colipase with the active lipase-colipase-micelle ternary complex, thus diverting colipase from its lipase-anchoring function. The formation of such a complex has been evidenced by molecular sieving experiments. This nonproductive complex lowers the amount of active lipase thus reducing lipolysis. Preliminary experiments performed in rats show that the C-terminal domain also behaves as an inhibitor in vivo and thus could be considered a potential new tool for specifically reducing intestinal lipolysis.

Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering.

In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.

Ion pairing between lipase and colipase plays a critical role in catalysis.

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.

The lipase/colipase complex is activated by a micelle: neutron crystallographic evidence.

The catalytic activity of most lipases depends on the aggregation state of their substrates. It is supposed that lipase activation requires the unmasking and structuring of the enzyme's active site through conformational changes involving the presence of oil-in-water droplets. This phenomenon has been called interfacial activation. Here, we report the crystal structure of the pancreatic activated lipase/colipase/micelle complex as determined using the D2O/H2O contrast variation low resolution neutron diffraction method. We find that a disk-shaped micelle interacts extensively with the concave face of colipase (CL) and the distal tip of the C-terminal domain of lipase away from the active site of the enzyme. Such interaction appears to help stabilizing the lipase-CL interaction. Consequently, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and it is mediated by CL and a micelle.

Pancreatic lipase-related protein type 1: a double mutation restores a significant lipase activity.

Besides the active pancreatic lipase (PL) which plays a major role in dietary fat digestion, the presence of a pancreatic lipase related protein 1 (PLRP1) displaying a very low lipolytic activity has been reported in vertebrates. It has been suggested that the reduced lipolytic activity of PLRP1 results from specific features of the N-terminal domain of the protein. Therefore, based on sequence comparison between PL and PLRP1 and modelling experiments, several residues located in the vicinity of the active site pocket of both enzymes have been mutated. In this paper, we report that, as regards to PL, two substitutions in positions 179 and 181 in PLRP1 account for the very low lipolytic activity of the protein. Indeed, substituting these residues (V179 and A181) in PLRP1 for those found in PL (A179 and P181), restores a significant lipolytic activity for PLRP1.

Pancreatic lipase-related protein type I: a specialized lipase or an inactive enzyme.

The existence of pancreatic lipase-related protein 1 (PLRP1) in vertebrates has been postulated based on the screening of pancreatic cDNA libraries from different species. In this paper, we report the presence of variable amounts of PLRP1 relative to colipase-dependent lipase (PL) in adults from several species. Only a very low lipase activity could be detected for native or recombinant PLRP1 using a large variety of substrates and conditions. Interestingly, this activity is dependent on the presence of bile salts and colipase and PLRP1 is shown to possess the same affinity as PL for colipase. Modelling investigations revealed some interesting differences between PLRP1 and PL, notably concerning substitutions in the C-terminal domain which might affect the bending motion of this domain relative to the N-terminal domain in PLRP1. The potential impact of these differences on the lack of lipase activity of PLRP1 was investigated using chimeric proteins designed by C-terminal domain exchange between dog PLRP1 and horse PL. Analysis of the catalytic properties of the chimera clearly indicated that the C-terminal domain exchange neither inactivates the horse enzyme nor results in an active dog PLRP1. From these findings, it can be concluded that the PLRP1 C-terminal domain is fully functional with respect to colipase binding. The lack of lipase activity or the still undetermined function of PLRP1 is likely to result mainly from particular features of the N-terminal domain.

Recombinant C-terminal domain of pancreatic lipase retains full ability to bind colipase.

The organization of the pancreatic lipase in two well defined domains has been correlated to a specific function for each domain, catalytic activity for the N-terminal domain and colipase binding for the C-terminal domain. In order to see if such an organization implies that the two domains can behave as separate entities, we expressed the N- and C-terminal domains in insect cells. The recombinant proteins secreted in the cell supernatants present the expected molecular properties. However, whereas the C-terminal domain retains its function of colipase binding, the N-terminal domain appears to be unable to ensure catalysis. The lack of activity of the recombinant N-terminal domain could result either from a (partially) incorrect folding or from an incapacity to function by itself. These results suggest that, although both are structurally well defined, the two domains of the pancreatic lipase behave differently when they are expressed as separate entities.

Lipase activation by nonionic detergents. The crystal structure of the porcine lipase-colipase-tetraethylene glycol monooctyl ether complex.

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 degrees 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.

Molecular cloning and expression of two horse pancreatic cDNA encoding colipase A and B.

Pancreatic colipase plays an essential role in the intestinal fat digestion by anchoring lipase on lipid/water interfaces in the presence of bile salts. In contrast to other species, two molecular forms of colipase, A and B, have been found in horse. The two corresponding cDNAs were isolated from a horse pancreatic library and their nucleotide sequences were determined. Moreover, for the first time, active colipase has been obtained after transfection of COS cells by either colipase A or B cDNA.

The transcriptionally active factors mediating the effect of the HTLV-I Tax transactivator on the IL-2R alpha kappa B enhancer include the product of the c-rel proto-oncogene.

The transactivator HTLV-I Tax activates the promoter of the gene coding for the interleukin 2 alpha-chain receptor (IL-2R alpha) via a kappa B site that can bind several protein species of the rel family. Tax1 strongly activates the enhancer activity of this motif, in both epithelial HeLa and lymphoid Jurkat cells. This activation was not observed in undifferentiated embryocarcinoma F9 cells. Overexpression of the p50, p65 and Rel proteins in these cells showed that significant activation of the IL-2R alpha kappa B site was observed only with Rel and Rel plus p65. Moreover, whereas both Tax and phorbol 12-myristate 13-acetate (PMA) are able to efficiently induce the binding of NF-kappa B to the IL-2R alpha kappa B site, PMA is functionally inactive. Using the DNA affinity precipitation assay, we observed that Tax1 is able to efficiently induce the binding of Rel, whereas PMA is not. This established a clear difference between both stimuli, indicating that Rel is the functionally active factor. We conclude from these results that the functional activity of members of the rel family is regulated by their interaction with DNA and that Rel can be a potent transcriptional activator on specific kappa B sites.

Tax1 induction of the HTLV-I 21 bp enhancer requires cooperation between two cellular DNA-binding proteins.

Activation of the HTLV-I promoter by the viral Tax1 transactivator is mediated by a 21 bp sequence motif imperfectly repeated three times and composed of three exactly conserved domains (A, B and C from 5' to 3'). We show here that the Tax1 response requires the integrity of the B domain and of at least one of the flanking A or C domains. We have identified three cellular proteins which bind specifically to the 21 bp motif. One of these is the already well-characterized transcription factor ATF. The other two, namely HEB1 and HEB2, are specific for the 21 bp motif. HEB1 can bind to either domain A or C, but binding of ATF and HEB2 is determined by domain B. However, neither domain B alone, nor ATF/CREB binding sites respond significantly to Tax1. We therefore propose that Tax1 induction of the 21 bp enhancer element requires interaction with the two different cellular proteins identified in this study: HEB1 and HEB2, rather than binding of the ATF factor.

Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay.

The extracellular adenylate cyclase of Bordetella pertussis was purified either as a free enzyme or as a complex with calmodulin. The purified enzyme has a specific activity of 1600 mumol of cAMP min-1 X mg-1 and exists under two molecular forms of 45 and 43 kDa which are apparently structurally related. Calmodulin increased considerably the resistance of adenylate cyclase to inactivation by trypsin. Although trypsin cleaved the adenylate cyclase-calmodulin complex, the digested fragments remained associated by noncovalent interactions in an active conformation. Specific mouse anti-adenylate cyclase antibodies inhibit adenylate cyclase activity and were used to develop a specific radioimmunoassay that allows detection of as little as 5 ng of adenylate cyclase in culture supernatants.

Characterization of a beta-galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase.

A hybrid protein of Escherichia coli, exhibiting both adenylate cyclase and beta-galactosidase activities, was purified and characterized. This protein, obtained by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth-residue of beta-galactosidase through a pentapeptide Val-Gly-Asp-Pro-Val. The fusion protein was less stable than the native beta-galatosidase. Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w). The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and beta-galactosidase. 'Truncated' adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild-type adenylate cyclase. Photoirradiation of the hybrid protein with 8-azidoadenosine 5'-triphosphate inactivated the adenylate cyclase activity, leaving intact the beta-galactosidase activity. A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.