Báo cáo khoa học: Role of the structural domains in the functional properties of pancreatic lipase-related protein 2 pot

Role of the structural domains in the functional properties of pancreatic lipase-related protein ´ Amelie Berton, Corinne Sebban-Kreuzer and Isabelle Crenon ´ ´ ´ ´ ´ UMR, INSERM 476, INRA 1260, Universite de la Mediterranee, Nutrition Humaine et Lipides, Faculte de Medecine de la Timone, Marseille, France Keywords chimera; colipase; domain; pancreatic lipase; PLRP2 Correspondence I Crenon, UMR, 476 INSERM ⁄ 1260 INRA, ´ ´ Faculte de Medecine, 27 Boulevard Jean-Moulin, 13385 Marseille Cedex 5, France Fax: +33 91 78 21 01 Tel: +33 91 29 41 10 E-mail: Isabelle.Crenon@medecine univ-mrs.fr (Received August 2007, revised 10 September 2007, accepted October 2007) doi:10.1111/j.1742-4658.2007.06123.x Although structurally similar, classic pancreatic lipase (PL) and pancreatic lipase-related protein (PLRP)2, expressed in the pancreas of several species, differ in substrate specificity, sensitivity to bile salts and colipase dependence In order to investigate the role of the two domains of PLRP2 in the function of the protein, two chimeric proteins were designed by swapping the N and C structural domains between the horse PL (Nc and Cc domains) and the horse PLRP2 (N2 and C2 domains) NcC2 and N2Cc proteins were expressed in insect cells, purified by one-step chromatography, and characterized NcC2 displays the same specific activity as PL, whereas N2Cc has the same as that PLRP2 In contrast to N2Cc, NcC2 is highly sensitive to interfacial denaturation The lipolytic activity of both chimeric proteins is inhibited by bile salts and is not restored by colipase Only N2Cc is found to be a strong inhibitor of PL activity, due to competition for colipase binding Active site-directed inhibition experiments demonstrate that activation of N2Cc occurs in the presence of bile salt and does not require colipase, as does PLRP2 The inability of PLRP2 to form a high-affinity complex with colipase is only due to the C-terminal domain Indeed, the N-terminal domain can interact with the colipase PLRP2 properties such as substrate selectivity, specific activity, bile salt-dependent activation and interfacial stability depend on the nature of the N-terminal domain In 1992, Giller et al isolated mRNA coding for two novel human pancreatic lipase-related proteins (PLRPs) showing a high level of identity with the human classic pancreatic lipase [1] On the basis of amino acid sequence comparisons, Giller et al proposed the classification of pancreatic lipases in three subgroups: classic pancreatic lipase (PL), PLRP1 and PLRP2 Numerous PLRP sequences have been identified in several species by isolating mRNA [2–11] Furthermore, by using classic protein purification procedures, the presence of PLRP1 and ⁄ or PLRP2 has been demonstrated in the pancreas or in the pancreatic juice from different species and also in other secretions [8,11–15] PLRP and PL differ in enzymatic properties such as substrate specificity, sensitivity to inhibition by bile salts and colipase dependence [16] Pancreatic lipases are highly active and selective for triglyceride substrates Under physiological conditions, the PL activity is dependent on the presence of colipase, which able to overcome the inhibitory effect of bile salts [17,18] Despite extensive studies on a large variety of substrates, only very low lipolytic activity against triglycerides has been reported with PLRP1 [1,4,14,15] The Abbreviations E600, diethyl p-nitrophenyl phosphate; ho, horse; NaTDC, sodium taurodeoxycholate; PL, classic pancreatic lipase; PLRP, pancreatic lipase-related protein FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works 6011 PLRP2 and colipase ) no interaction PLRP2s are distinguishable from classical lipases by their substrate specificity, because, besides triglycerides, they are able to hydrolyze phospholipids, galactolipids and esters of vitamins [3,8,9,19,20] Moreover, the activities of PL and PLRP2 seem to be different according to the vehicles in which the substrate is solubilized [21] Concerning the effect of bile salts and colipase on the activity of PLRP2, there is no clear conclusion, because the results appear to change according to the different species and seem to depend on the triglyceride substrate and the bile salt [9,16] Indeed, some of them, such as horse PLRP2 and human PLRP2, are inhibited on tributyrin substrate by the presence of bile salts, and this inhibition is only slightly overcome or not overcome by the presence of an excess of colipase [8,13,22] Another PLRP2 group including guinea pig and coypu PLRP2s is affected neither by the bile salt concentration nor by the addition of colipase on tributyrin substrate, but is strongly inhibited on trioctanoin substrate [3] Concerning the rat PLRP2, because of contradictory results, no conclusions can be drawn [4,23] The three-dimensional structure resolution of pancreatic lipases provides important information concerning the structure–function relationship of the PL [24–28] In agreement with biochemical data, these structures demonstrate the functional organization of the PL into two structural domains: a large N-terminal domain, which contains the active site with the catalytic triad formed by Ser152, Asp176 and His263, and a smaller C-terminal domain, which is important for colipase binding In the inactivated state, the PL catalytic site is inaccessible to substrate, being covered by a surface loop called the lid domain (residues 237–261) In particular conditions, the lid must move to accommodate a lipid substrate The closed PL conformation converts into the open form upon interaction with lipid [26] The principal elements that undergo space reorganization during the activation of the enzyme are the lid (residues 238–262) and loop b5 of the N-terminal domain (residues 77–86) The functional consequences of the structural reorganization are as follows: (a) the active site is accessible to the substrate; (b) the oxyanion hole is created; (c) an important hydrophobic surface is formed; and (d) a new binding site is generated between the colipase and the open lid Some studies have questioned whether lipase activation is even interfacial in the presence of bile salt and colipase, on the basis of attaining an activated ternary complex of PL, colipase and a small micelle in the absence of any interface [29] Three-dimensional structures of canine PLRP1 and rat PLRP2 have also been reported [30,31] These data 6012 A Berton et al indicate that, as predicted by high primary structure homology, the three-dimensional structure of the PLRP members can be superimposed on that of PL, which cannot explain the particular features of the PLRPs Indeed, they possess an N-terminal domain with the same catalytic triad, a C-terminal domain in which the residues implicated in colipase binding are conserved, and a lid domain (except for guinea pig PLRP2, which has a naturally truncated lid [3]), which must move during the activation process Previous data indicate that the motion of the PLRP2 lid is dependent on the presence of bile salts and does not require the presence of colipase [32] The two-domain structural organization of the pancreatic lipases allowed the development of the domainexchange strategy to provide further insights into the structure–function relationships of pancreatic lipases [14,33–36] These studies show that the lid domain alone is responsible neither for the substrate selectivity nor for the activation process They did not show whether the PLRP2 C-terminal domain could or could not bind colipase The differences in kinetic properties of the various PLRP2s imply that these proteins should not be grouped together and that it is important to obtain new information about the properties of PLRP2 family members In the present study, we produced, purified and characterized chimeric proteins designed by N-terminal and C-terminal domain exchange between horse PLRP2 (hoPLRP2) and horse PL (hoPL) in order to investigate the role of the two domains in the function of hoPLRP2 The influence of bile salt and colipase on the lipase activity of the different chimeras was investigated using tributyrin as substrate Experiments were performed to investigate active site-directed inhibition and competition for colipase binding The properties of the chimeras were compared to those of the original proteins bearing the modifications induced by the constructions in the chimeric proteins and compared with the properties of the native hoPL and hoPLRP2 proteins This work provided new information about the ability of the hoPLRP2 C-terminal domain to bind colipase and the respective contribution of each PLRP2 domain to the activation process, the substrate specificity and the interfacial stability of this protein Results Expression and purification of chimeric proteins Chimeric proteins designed by domain exchange between hoPL and hoPLRP2 were constructed and expressed in insect cells The strategy that we followed FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works A Berton et al to construct plasmids expressing the two chimeric cDNAs was to exchange the cDNA fragment encoding the C-terminal domain of the two lipases between plasmids pVLhoPL and pAcGP67hoPLRP2, which carry the cDNA of hoPL and hoPLRP2, respectively This exchange could be done because an Eag1 site was engineered in each plasmid at the junction between the N-terminal and C-terminal domain sequences of each protein The chimeric protein composed of the N-terminal domain of hoPL and of the C-terminal domain of hoPLRP2 was named NcC2 Conversely, the other chimeric protein, bearing the N-terminal domain of hoPLRP2 and the C-terminal domain of hoPL, was named N2Cc This construction procedure induced substitutions in the amino acid sequence of each C-terminal domain, as shown in Fig To ensure that these substitutions did not influence the behavior of the C-terminal domain as compared to the wild-type proteins, we expressed NcCc and N2C2 as controls The chimeric proteins were expressed in insect cells using the Baculovirus Expression System The four proteins were secreted into the culture medium with yields reaching 10–40 mg of recombinant proteinỈL)1 After days of culture, the secreted proteins were purified from the dialyzed supernatant by a one-step anionic exchange chromatography procedure, with a recovery yield of 50% The four purified recombinant proteins were analyzed and compared to native hoPL and hoPLRP2 by SDS ⁄ PAGE followed by Coomassie blue staining (Fig 2A) or western blot (Fig 2B,C) PLRP2 and colipase ) no interaction In the absence of dithiothreitol in the sample buffer, native hoPL and hoPLRP2 ran as a single band of about 50 kDa (Fig 2A, lanes and 2) In the presence of dithiothreitol in the sample buffer, in contrast to hoPL (Fig 2A, lane 3), hoPLRP2 ran as two fragments of 27.5 and 22.5 kDa (Fig 2A, lane 4), in agreement with previous results demonstrating the high sensitivity of the hoPLRP2 Ser244–Thr245 bond to proteolytic cleavage [32] The four chimeric proteins ran as a major single band with a molecular mass of about 50 kDa (Fig 2A, lanes 5–8) Nevertheless, the two chimeras bearing the N2 domain (N2C2, lane 6, and N2Cc, lane 8) had a slightly higher molecular mass than the chimera bearing the Nc domain (NcCc, lane 5, and NcC2, lane 7) according to the theoretical value, as indicated in Table Microsequencing of purified NcCc and NcC2 yielded the N-terminal sequence NEVCY, corresponding to the N-terminal sequence of the mature hoPL The N-terminal sequence of N2C2 and N2Cc was ADLKE, corresponding to the three terminal amino acid extension resulting from the construction strategy, followed by the N-terminal sequence of the mature hoPLRP2 These results indicated that the cleavage of the signal sequence by the insect signal peptidase was correct Despite crossreactions due to the strong homology between the two proteins, hoPL antibodies recognized hoPL and NcCc better than hoPLRP2 and N2C2, and conversely, hoPLRP2 antibodies reacted better with hoPLRP2 and N2C2 than they did with hoPL and Fig Functional maps of the plasmids expressing natural and chimeric isoforms of hoPLRP2 and hoPL A novel EagI site was engineered (see Experimental procedures) Above each plasmid map, the nucleotide and amino acid sequences of the region at the junction between the two protein domains are reported The EagI site is underlined FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works 6013 PLRP2 and colipase ) no interaction A Berton et al of N2C2 and N2Cc to proteolytic cleavage generating one fragment detected by hoPLRP2 antibodies and corresponding to the larger proteolytic fragment of native hoPLRP2 (Fig 2C, lanes and 8) Using different preparations of N2C2 and N2Cc, we checked that this proteolysis did not have an effect on either the activity or the behavior of the proteins A Kinetic properties of chimeric proteins ) effects of bile salts and colipase B C Fig Analysis of purified protein by SDS ⁄ PAGE 12% Coomassie blue staining (A) and western blots using hoPL antibodies (B) and hoPLRP2 antibodies (C) Lanes and 2: protein migration without dithiothreitol Lanes 3–8: protein migration with dithiothreitol Lanes and 3: hoPL Lanes and 4: hoPLRP2 Lane 5: NcCc Lane 6: N2C2 Lane 7: NcC2 Lane 8: N2Cc Table Theorical biochemical properties of the chimeric proteins Proteins N-terminal sequence Molecular mass (Da) Amino acids Isoelectric point hoPL hoPLRP2 NcCc N2C2 NcC2 N2Cc NEVCY ADLKE NEVCY ADLKE NEVCY ADLKE 49 50 49 50 49 50 449 455 449 455 451 453 5.19 5.50 5.19 5.62 5.80 5.10 710.47 362.16 696.45 387.23 559.32 524.36 NcCc (Fig 2B,C) Concerning the chimera, NcC2 was recognized better by hoPL antibodies than by hoPLRP2 antibodies, and conversely, N2Cc was recognized better by hoPLRP2 antibodies than by hoPL antibodies This suggests that both antibodies were preferentially raised against the N-terminal domain of the respective proteins We observed slight sensitivity 6014 The lipolytic activity of the different chimeric proteins was investigated by titrimetry using emulsified tributyrin as substrate In a first experiment, the assays were performed in the absence of bile salts [sodium taurodeoxycholate (NaTDC)], in the absence or in the presence of colipase As shown in Fig 3, the kinetic rate for NcCc (3000 mg)1) rapidly decreased in the absence of colipase and bile salts NcCc was probably irreversibly inactivated at the surface of tributyrin droplets Prior addition of colipase enhanced the kinetic rate (7200 mg)1) and prevented this inactivation This well-known phenomenon, named interfacial inactivation, has been extensively described with several pancreatic classic lipases, and in particular with hoPL [37] The kinetic rate for N2C2 in the absence of bile salt was constant (560 mg)1), as it was in the absence and in the presence of colipase (Fig 3) Similar data were observed with hoPLRP2 [8] These results indicated that NcCc and N2C2 behaved like native hoPL and hoPLRP2, respectively [8,35,37] Thus, the modifications introduced at the junction between the N-terminal and C-terminal domains as compared to the native proteins influence neither their stability nor their activity In the absence of bile salts and colipase, the kinetic rates of both NcC2 (6000 mg)1) and N2Cc (650 mg)1) decreased, and this decrease was even more rapid for NcC2 (Fig 3) These results indicated that both the N-terminal and C-terminal domains of hoPLRP2 contributed to the stability of the protein in the presence of the water–lipid interface Also, both the N-terminal and C-terminal domains of hoPL were involved in the inactivation of the protein at the water–triglyceride interface The inactivation of N2Cc in the absence of bile salts was prevented by prior addition of colipase, suggesting that N2Cc was able to bind the colipase In contrast, the inactivation of NcC2 was not prevented by prior addition of colipase, suggesting that NcC2 was not able to bind colipase These results indicated that only the proteins possessing the PL C-terminal domain are able to bind colipase FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works A Berton et al PLRP2 and colipase ) no interaction Fig Kinetics of hydrolysis of tributyrin by chimeric proteins without bile salt and in presence or absence of colipase Lipolytic activity was measured titrimetrically at pH 7.5 with NcCc (0.99 · 10)9 M), NcC2 (0.89 · 10)9 M), N2C2 (1.75 · 10)9 M) or N2Cc (0.5 · 10)9 M) without (in black) and with (in gray) colipase (5 · 10)9 M) In a second experiment, the activities of the chimeras were tested on emulsified tributyrin in the presence of increasing concentrations of bile salts (0–6 mm), in the absence or in the presence of colipase As seen in Fig 4, increasing the concentration of NaTDC inhibited the activity of NcCc (1500 mg)1 at mm NaTDC versus 3200 mg)1 at mm NaTDC) and N2C2 (300 mg)1 at mm NaTDC versus 560 mg)1 at mm NaTDC) In the presence of colipase, only NcCc activity was increased, even in the presence of bile salt (8000 mg)1 at 0.1 mm NaTDC and 5600 mg)1 at mm NaTDC) These results were similar to those obtained for the native proteins [8,35,37] The activity of NcC2 was slightly increased at a very low NaTDC concentration (8000 mg)1 at 0.1 mm NaTDC) and inhibited when the NaTDC concentration increased The inhibitory effect was complete for NaTDC concentrations above mm The colipase was not able to restore the NcC2 activity For N2Cc, a slight activator effect was observed at a very Fig Bile salt and colipase dependence of the chimeric protein activity The assays were done using 10)9 M each lipases in the pH-stat at various concentrations of NaTDC and in the absence (in black) or presence (in gray) of a M excess of colipase FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works 6015 PLRP2 and colipase ) no interaction A Berton et al low NaTDC concentration (700 mg)1 at 0.1 mm NaTDC) An inhibitory effect then appeared, increased up to the NaTDC critical micellar concentration, and stabilized at a plateau value corresponding to 400 mg)1 Interestingly, the colipase failed to restore the maximal activity for N2Cc The colipase effect on the lipase activity in the presence of bile salt depended not only on the presence of the classic C-terminal domain, but also on the nature of the N-terminal domain Inhibition of the PL by the chimeras In the presence of a supramicellar concentration of NaTDC (4 mm), PL needs the colipase to develop its full activity In the same conditions, the N2Cc and NcC2 activities were inhibited and not restored in the presence of colipase (see above) The influence of increasing concentrations of NcC2, N2Cc, hoPL inactive forms [diethyl p-nitrophenyl phosphate (E600)hoPL] and hoPLRP2 inactive forms (E600-hoPLRP2) on the native PL activity was investigated In these experiments, the concentrations of lipase (10)9 m) and colipase (10)9 m) were constant Inactive forms of hoPL and hoPLRP2 were prepared as previously described [32] using high concentrations of E600, which covalently binds to the active site serine As shown in Fig 5A, E600-hoPL was found to be an excellent inhibitor of the lipase activity, as 50% inhibition was obtained with an [E600-hoPL] ⁄ [PL] molar ratio of 0.5 Only 18% of residual activity remained when E600-hoPL was used at a molar excess of Interestingly, no inhibition of the lipase test activity was observed when E600-hoPLRP2 was added, even at a molar excess of 1800 As shown in Fig 5B, the inhibitory effect with N2Cc was similar to that of E600-hoPL Fifty per cent inhibition was obtained with an [N2Cc] ⁄ [PL] molar ratio of about 0.5, and complete inhibition was observed when N2Cc was used at a molar excess of 10 The inhibitory effect of E600hoPL and N2Cc was abolished when an excess of colipase was added during the assay, and was observed only in the presence of NaTDC (data not shown) In the case of NcC2, no inhibition of the lipase activity was observed, as 100% of the lipase activity still remained even at a molar excess of 45 The effect of NcC2 was similar to that of E600-hoPLRP2 The same results were obtained using human or porcine lipase and colipase The proteins bearing the C-terminal domain of PL were efficient inhibitors of the lipase activity, whereas the proteins bearing the C-terminal domain of PLRP2 had no effect on the lipase activity The inhibitory 6016 Fig Competition for colipase between PL and inactive or chimeric proteins Colipase (10)9 M) was incubated with increasing concentrations of inhibitor protein in the presence of a tributyrin emulsion and bile salts at a final concentration of mM After min, PL (10)9 M) was added The activity was determined and expressed as a percentage compared to the lipase activity measured in the absence of inhibitor protein (A) E600-hoPL (d) and E600-hoPLRP2 (.) (B) N2Cc (d) and NcC2 (.) effect of E600-hoPL and N2Cc is probably due to competition for colipase binding These data suggested that the C-terminal domain of PL is able to bind colipase whatever the nature of the N-terminal domain Moreover, the C-terminal domain of hoPLRP2 was not able to bind colipase even in the presence of the PL N-terminal domain Influence of NaTDC and colipase on chimera inhibition by E600 The activation of the pancreatic lipase is a mechanism allowing accessibility of the active site to the substrate FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works PLRP2 and colipase ) no interaction A Berton et al Table Influence of bile salt and colipase on the chimeric protein inhibition by E600 Chimeric proteins, at · 10)6 M, were incubated in the presence of E600 in the absence or in the presence of bile salt (NaTDC 0.5 mM or mM) or colipase (10)5 M) T50% is the time needed to reach 50% inhibition ND, not determined T50% (min) Without colipase With colipase Proteins E600 (mM) – NaTDC 0.5 mM NaTDC mM NaTDC – NaTDC mM NaTDC NcCc N2C2 NcC2 N2Cc NcCc NcC2 0.05 0.05 0.05 0.05 2.5 2.5 > 1440 75 > 1440 90 > 1440 > 1440 > 1440 16 > 1440 30 ND ND > 1440 > 1440 > 1440 75 > 1440 70 > 1440 64 > 1440 > 1440 > 1440 > 1440 70 79 and resulting in the unmasking of the catalytic triad of the enzyme induced by the motion of the flap The accessibility of the active site can be tested using the ability of an organophosphate, E600, to react with the active site serine only when the enzyme adopts an opened flap conformation E600 inhibition experiments were carried out to investigate the influence of NaTDC and colipase on the activation of the chimeric proteins Table shows T50%, corresponding to the time needed to reach 50% inhibition With 0.05 mm E600, no inhibition was observed for NcCc and NcC2 At 2.5 mm E600, inhibition of NcCc activity was observed after incubation in the presence of bile salt and colipase (T50% ¼ 70 min) In contrast, inhibition of NcC2 activity was observed in the presence of bile salt alone (T50% ¼ 75 min), and the colipase addition had no effect on the rate of inhibition In the absence of bile salt, noticeable inhibition of N2C2 by 0.05 mm E600 was observed (T50% ¼ 75 min), and the addition of colipase had no significant effect (T50% ¼ 70 min) In contrast, the rate of inhibition was significantly increased in the presence of NaTDC monomers (T50% ¼ 16 min) At NaTDC concentrations beyond the critical micellar concentration, the rate of inhibition of N2C2 increased (T50% ¼ min) The addition of colipase still had no significant influence (T50% ¼ min) These results were in agreement with previous experiments on inhibition by E600 performed on native hoPLRP2 [32] Significant inhibition by E600 was observed for N2Cc in the absence of colipase and bile salt (T50% ¼ 90 min) The rate of inhibition was increased in the presence of NaTDC, concentrations beyond the critical micellar concentration having a higher efficiency than the monomer concentration (T50% ¼ versus T50% ¼ 30 min) The addition of colipase alone had a slight influence on N2Cc inhibition, as the rate of inhibition was increased (T50% ¼ 64 versus T50% ¼ 90 min), in contrast to N2C2 Blank experiments performed in the absence of E600 showed that, in any case, proteins retained at least 85% of activity after 24 h, indicating that the enzymes were stable under the conditions used for the study These results indicated that the NcCc active site was accessible to high E600 concentrations only in the presence of colipase and bile salt, whereas the accessibility of the NcC2 active site depended only on the presence of bile salt The accessibility of the N2C2 and N2Cc active sites to E600 was possible even in the absence of colipase and bile salt, and was considerably increased by the presence of bile salt Therefore, the concentration of E600 needed to obtain clear inhibition of NcC2 and NcCc was 50 times higher than that used for N2C2 and N2Cc In conclusion, the accessibility of the active site was better in the protein bearing the N2 domain than in the protein bearing the Nc domain Thus, the accessibility of the active site in the N2 proteins was independent of the nature of the C-terminal domain, in contrast to the situation with Nc proteins Indeed, the C2 domain induced sensitivity of the Nc active site to E600 inhibition in the presence of bile salt Discussion Despite their structural similarities, the PLRP2s form a subfamily that is clearly distinct from the classic lipase subfamily, notably concerning their functional properties Moreover, considerable variability is observed among the members of the PLRP2 subfamily The aim of our study was to investigate the contribution of the N-terminal and C-terminal domains to the particular behavior of hoPLRP2 The structural organization of the pancreatic lipases is completely suitable FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works 6017 PLRP2 and colipase ) no interaction for the domain-exchange strategy, which has previously been used successfully in the study of the structure–function relationships of different lipases [35,38] Chimeric proteins, named NcC2 and N2Cc, were designed by N and C structural domain exchange between hoPL (Nc and Cc domains) and hoPLRP2 (N2 and C2 domains) NcC2 and N2Cc were produced as secreted proteins and purified Their properties were compared to those of NcCc and N2C2, corresponding to the original proteins PL and PLRP2, respectively, bearing the modifications induced by the construction in the chimeric proteins These modifications have no effects on the behavior of the proteins [8,35,37] The kinetic characterizations of proteins in the absence of bile salts and colipase show that, in contrast to N2C2, the NcCc, NcC2 and N2Cc proteins undergo irreversible inactivation, which is thought to result from denaturation of these enzymes in the lipid– water interface [39] These observations underline the fact that the proteins possessing at least one of the two domains of hoPL are more sensitive to interfacial denaturation The involvement of the C-terminal domain in the interfacial denaturation of the PL was ` already proposed by Carriere et al [35] Indeed, these authors showed that, in the absence of bile salts, a chimeric protein composed of the N-terminal domain of guinea pig PLRP2 and of the C-terminal domain of human PL (gpN2 ⁄ huCc) was inactivated at the interface Moreover, it was reported that the C-terminal domain of PL bound efficiently to a triglyceride–water interface and was an absolute requirement for possible interfacial binding of PL [40] In the case of the gpN2 ⁄ huCc chimera, the rate of denaturation was higher, indicating that the C-terminal domain of hoPL is less sensitive to the interface than that of huPL In the present work, the similarities between NcCc and NcC2 with regard to the rate of inactivation show for the first time the dominant role of the N-terminal domain of PL in the phenomenon of interfacial denaturation The N-terminal domain of PLRP2 does not possess this feature PLRP2 is not sensitive to interfacial denaturation; either the two domains confer high stability on the lipid–water interface, or PLRP2 has no affinity for the lipid–water interface We recently showed that PL and PLRP2 hydrolyzed retinyl esters Moreover, PL preferentially hydrolyzed the substrate when it was included in droplets, and PLRP2 was more efficient when it was included in micelles of smaller size [21] Even if PL and PLRP2 hydrolyze triglycerides, it is probable that the physical property of the substrate is specific for each enzyme: droplet for PL, and a water-soluble structure for PLRP2 It has also been previously reported that PLRP2 does not display 6018 A Berton et al interfacial activation [3,9], and preferentially hydrolyzes triglycerides with short chains hoPLRP2 is characterized by a specific activity on TC4 of about 600–700 mg)1 in the absence of bile salts The specific activity of hoPL is 8000 mg)1 (in the presence of colipase) In the absence of bile salts, the proteins containing the same N-terminal domain show a similar specific activity on tributyrin (in the presence of colipase) (500–700 mg)1 for N2 proteins and 6000–8000 mg)1 for Nc proteins) In the presence of bile salts, we observed that the behavior of chimeric proteins is also related to the nature of the N-terminal domain Indeed, the specific activities of NcCc and NcC2 are very strongly decreased, whereas those of N2C2 and N2Cc are much less sensitive to the inhibitory effect of bile salts This indicates that in the absence or in the presence of bile salts, the specific activity of hoPL and hoPLRP2 depends on the nature of their N-terminal domain In contrast to NcCc, NcC2 was not protected from the interfacial denaturation by colipase and not reactivated by colipase in the presence of bile salt, suggesting that NcC2 is not able to form a stable complex with colipase Competition experiments on colipase binding reveal that NcC2, like hoPLRP2, is a very bad competitor This observation indicates that NcC2 and N2C2 not bind well to colipase, probably due to the C2 domain However, as shown in Fig 6, the residues of the C-terminal domain involved in the primary interaction of PL with colipase are preserved in the C-terminal domain of hoPLRP2 (Asn366, Gln369, Lys400) It is possible that these residues are not in an ideal conformation to allow either binding to colipase or correct orientation of colipase, in particular for stabilizing the lid Although N2Cc activity was not restored by colipase in the presence of bile salt, there are substantial arguments in favor of N2Cc–colipase complex formation N2Cc is protected from interfacial denaturation by colipase and behaves as an excellent inhibitor of colipase binding Indeed, the [N2Cc] ⁄ [lipase] molar ratio needed to obtain 50% inhibition is the same as that found with E600-hoPL competitor or with other inactive forms of PL used as competitors by Miled et al [41] This result indicates that N2Cc binds to colipase as well as hoPL Experiments previously carried out with the C-terminal domain of PL as inhibitor showed that a [C-terminal domain] ⁄ [lipase] molar ratio of 1000 was needed to give 50% inhibition [42] It was assumed that the whole lipase is a better inhibitor than the C-terminal domain alone, because new interactions, which stabilize the lipase–colipase complex, were created between colipase and the lid of lipase in the opened conformation The results FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works PLRP2 and colipase ) no interaction A Berton et al A B Fig (A) Amino acid sequence comparison between hoPLRP2 and hoPL The residues of the catalytic triad are in red, the lid sequence is in blue, and the amino acids of the C-terminal domain involved in colipase binding are in green (B) Superimposition of hoPLRP2 (1W52) and hoPL (1HPL) Ca traces are displayed in red and blue, respectively obtained with N2Cc as a competitor mean that the C-terminal domain in this context is able to bind colipase, but especially that the complex formed would probably be stabilized by the open lid of the N2 domain The movement of the lid making it possible to adopt an open conformation is a crucial stage in the mechanism of action of lipase The motion of the flap makes the active site accessible to the substrate, simultaneously forming a functional oxyanion hole and generating lipase interfacial binding It has been previously proposed from active site-directed inhibition experiments with an organophosphate that the accessibility of the active site of pancreatic lipase, in the absence of interface, could be obtained in the presence of colipase and bile salts, probably through the formation of a ternary lipase–colipase–micelle complex of biliary compounds [29] The same type of experiment indicates that the lid of PLRP2 is already more mobile than that of PL, and especially that it moves and uncovers the active site in the presence only of the bile compounds [32] The accessibility of E600 to the N2 active site is considerably increased in the presence of bile salts, which masks the slight influence of colipase observed with N2Cc in the absence of bile salt The unmasking of the active site of the Nc domain absolutely requires colipase and bile salts in micellar concentrations in the presence of the Cc domain and bile salt only in the presence of the C2 domain The inhibition of the active site serine by E600 needs both the motion of the flap and the recognition of the vehicle in which E600 was solubilized It was clearly established that soluble E600 can be included in bile salt micelles, and that this inclusion is a prerequisite for inhibition of PL [43] Our work indicates that E600 included in bile salt micelles is a better inhibitor of the N2 active site than of the Nc active site This observation supports the idea that PLRP2 preferentially hydrolyzes substrates that are soluble or included in micelles, as was proposed by Reboul et al [21] Nevertheless, the mechanism of activation of PLRP2, which involves the C2 domain, is different from that of PL, which involves the Cc domain and colipase The C-terminal domain alone is involved in the affinity of the PLRP2 for micellar substrates, and probably allows the interaction of the enzyme with the substrate structure Whether the motion of the lid promotes the recognition of the substrate structure, or the recognition of this structure promotes the displacement of the lid, is still questionable Three structures of PLRP2 are now available in the Protein Data Bank: rat PLRP2 (Protein Data Bank code 1BU8 [31], human PLRP2 (Protein Data Bank code 2OXE, to be published), and hoPLRP2 (Protein Data Bank code 1W52 [44]) These three PLRP2 structures are comparable to the hoPL structure in the closed conformation, or to the porcine PL structure in the opened conformation [25,26] With respect to the conformation of loop b5 of the N-terminal domain, hoPLRP2 and human PLRP2 are probably in the opened conformation, in contrast to rat PLRP2, which is in the closed conformation For the human and rat proteins, it is not possible to draw conclusions about the exact position of the lid, because a sequence of approximately 20 amino acids is missing In the case of hoPLRP2, the lid is partially opened (Fig 6) A comparison of the exposed surface between PLRP2 and PL in the opened conformation would explain the difference in behavior between PL and PLRP2 with respect to the interface The resolution of the structure of N2Cc would be very useful to determine the position of the opened lid, whether it can be stabilized by FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works 6019 PLRP2 and colipase ) no interaction colipase, and what the nature is of the amino acids that correspond to the exposed surface The superposition of hoPL and hoPLRP2 (Fig 6) shows that loop b5¢ of the C-terminal domain (residues 405–414) is oriented differently This observation is very interesting, because this loop was shown to play an important role in lipase function and could influence the binding of colipase [45] No conclusion is possible about the orientation of this b5¢ loop in the other PLRP2s, as this fragment was found to have no interpretable electron density In conclusion, the studies on the functional properties of the two structural N-terminal and C-terminal domains of hoPLRP2 show that the enzyme stability in the presence of the lipid–water interface, the motion of the lid and the substrate specificity are properties that are mainly related to the nature of the N-terminal domain On the other hand, PLRP2 is not able to form a stable complex with colipase, and its C-terminal domain is responsible for this feature Structural analysis of this domain will provide new information to enable a better understanding of the role of the C-terminal domain in the function of PLRP2, mainly with regard to the orientation of the residues essential for colipase binding and the behavior of PLRP2 towards the lipid–water interface or water-soluble micelles These structural data will be very important to determine the real contribution of PLRP2 to intestinal lipolysis Experimental procedures Reagents The BaculoGold Starter Package pVL1393 and pAcGP67 transfer vectors were purchased from Pharmingen (San Diego, CA) X-Press medium and fetal bovine serum were supplied by BioWhittaker (Walkersville, MD, USA) Antibiotics were obtained from Invitrogen (Carlsbad, CA, USA) Alkaline phosphatase-labeled goat anti-(rabbit IgG), E600, tributyrin and NaTDC were purchased from Sigma-Aldrich (St Louis, MO, USA) Taq polymerase, restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA, USA) or Eurogentec (Seraing, Belgium) Construction of chimeric proteins The constructions encoding the chimeric proteins composed of a PL domain and a PLRP2 domain are described in Fig First, pVLhoPL, previously described, resulted in the integration of the nucleotide sequence encoding hoPL, including the peptide signal, at the EcoR1 restriction site of pVL1393 transfer vector Thereafter, an Eag1 restriction site was introduced by site-directed mutagenesis at the 6020 A Berton et al junction between the N-terminal and C-terminal domain sequences (named Nc and Cc, respectively) that induced the substitution A337G [14] The resulting vector pVLNcCc encoded the protein named NcCc The N-terminal and C-terminal domain sequences of hoPLRP2 (named N2 and C2, respectively) were amplified by PCR using pAcGP67hoPLRP2, previously described [8], as template This plasmid resulted in the insertion of the mature hoPLRP2 sequence into the BamH1 ⁄ EcoR1 restriction site of the pAcGP67 transfer vector, downstream of the signal sequence of the baculovirus glycoprotein GP67 The two oligonucleotides 5¢-N2 (5¢-GGAATTCAGATCTCAAAGA GGTTTGCTATACCCC-3¢) and 3¢-N2 (5¢-CCCGGCCG TAGTCACCACTTTCTCC-3¢) were used as 5¢ and 3¢ primer, respectively, to amplify the N2 sequence The sequences in bold correspond to the Bgl2 restriction site for primer 5¢-N2 and Eag1 restriction site for primer 3¢-N2 The underlined sequences in the primers correspond to the sequences encoding the first and the last residues of N2, respectively To amplify the C2 sequence, the two oligonucleotides (5¢-C2) 5¢-CCCGGCCGTTGGAGATATAGAGTATC-3¢ and (3¢-C2) 5¢-GGTTCTTGCCGGGTCCCCAGG-3¢ were used The sequence in bold corresponds to the Eag1 restriction site The sequence in italic corresponds to the sequence encoding the first residue of C2 The 3¢-C2 primer corresponds to the end of the multiple cloning site of the pAcGP67 vector The underlined sequence corresponds to the substitutions introduced in the C2 domain as compared to the wild-type PLRP2 The PCR reactions were carried out under standard conditions, with 0.5 at 95 °C, at 50 °C and at 72 °C for 25 cycles After the PCR reaction, the N2 and C2 PCR fragments were purified and digested by Bgl2–Eag1 and by Eag1, respectively, and introduced into the BamH1 ⁄ Eag1 restriction site of the pAcGP67 transfer vector The resulting construction pAcN2C2 encoded the protein named N2C2 NcCc and N2C2 were used as controls The pVLNcCc and pAcN2C2 vectors were digested by Eag1 and subjected to electrophoresis on polyacrylamide gel, and the Eag1 fragments were electroeluted The small Eag1 fragments corresponding to the Cc and C2 domains were interchanged and cloned in the Eag1 pAcN2 and pVLNc fragments, respectively The resulting constructions pVLNcC2 and pAcN2Cc encoded the chimeric proteins NcC2 and N2Cc All the constructions were propagated in the JM101 Escherichia coli strain and checked by DNA sequencing carried out by Genome Express (Grenoble, France) Expression of chimeric proteins using the Baculovirus Expression System After purification using the Qiagen (Hilden, Germany) plasmid purification protocol, the different constructions were used with linearized genomic DNA from Autographa FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works A Berton et al californica virus (BaculoGold DNA from the BaculoGold transfection kit) for cotransfection into Sf21 insect cells as described in the Baculovirus Expression Vector System Manual (Pharmingen) The SF21 cells were grown in a monolayer at 27 °C in tissue culture flasks using X-press medium containing 5% fetal bovine serum, 50 UIỈmL)1 penicillin and 50 mgỈmL)1 streptomycin Recombinant viruses were purified by plaque assay and amplified by an additional SF21 cell infection cycle For the production of the chimera, six 162 cm2 tissue culture flasks were seeded with · 107 cells per flask in complete X-press medium When the cells were attached, the complete medium was removed and replaced with 20 mL of serum-free X-press medium The high-titer stock solutions of recombinant baculoviruses were added to the cells at a multiplicity of infection close to In all cases, the chimeric proteins were secreted into the culture media, as observed by electrophoresis on SDS ⁄ PAGE After days of culture at 27 °C, the cells were pelleted by centrifugation at 900 g for °C, and the supernatants were kept at °C for further purification Purification of chimeric protein All the chimeric proteins were purified following the onestep procedure reported previously for the purification of horse recombinant PLRP2 expressed in insect cells [8] The culture supernatants were dialyzed overnight at °C against 20 mm Tris ⁄ HCl buffer (pH 8) containing mm benzamidine and loaded onto a Q-sepharose Fast Flow column equilibrated in the same buffer Elution was performed using a linear NaCl concentration gradient (from to 200 mm NaCl) The fractions were analyzed by measuring the lipase activity and by SDS ⁄ PAGE The fractions containing the protein of interest were pooled, dialyzed overnight at °C against distilled water, lyophilized or not, and kept at ) 20 °C Native hoPL and native hoPLRP2 were purified as previously described [8] N-terminal sequence analysis The purified chimeric proteins were submitted to N-terminal microsequencing Stepwise Edman degradation was performed using an automatic sequencer, model Procise 494, from Applied Biosystems (Foster City, CA, USA) Gel electrophoresis and western blotting Electrophoresis on 12% polyacrylamide gels was carried out in the presence of SDS as described by Laemmli [46] Western blots were performed according to Burnette [47] After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane Membranes were incubated with rabbit polyclonal antibodies raised against either hoPL or hoPLRP2 The rabbit sera were used at a PLRP2 and colipase ) no interaction : 5000 dilution, and the reacting antibodies were detected with goat anti-rabbit IgG conjugated with alkaline phosphatase at a : 5000 dilution Activity measurements and protein assays The lipase activity was measured titrimetrically at 25 °C using emulsified 0.11 m tributyrin in mm Tris ⁄ HCl buffer (pH 7.5) containing 0.1 m NaCl and mm CaCl2 The assays were performed either in the absence or in the presence of bile salt (NaTDC) 0.1–4 mm and ⁄ or a five-fold molar excess of colipase One unit corresponds to the release of lmol fatty acidỈmin)1 Protein concentrations were determined with the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL, USA) Chimeric protein inhibition by E600 The inhibition experiments were performed in 50 mm sodium acetate buffer (pH 6.0) containing 0.1 m NaCl Proteins (2 · 10)6 m) were treated with E600 (0.05 or 2.5 mm as indicated), either in the absence or in the presence of bile salt and ⁄ or colipase (five-fold molar excess) The mixture was incubated at 25 °C, and aliquots were withdrawn at various time intervals and used to determine the remaining lipase activity as described above Control experiments were also performed without E600 to check protein stability Competition experiments The lipase activity was measured as described above in the presence of mm NaTDC, colipase and increasing concentrations of chimeric proteins The lipase and colipase concentrations were 10)9 m The colipase and the chimeric proteins were added at the beginning of the test, and the lipase was added during the test Under these conditions, the lipase activity was determined and expressed as a percentage of the lipase activity measured in the absence of chimeric protein Native hoPLRP2 and hoPL previously inactivated by E600 as described in [32] were used as controls Acknowledgements This research was supported by grants from the Insti´ ´ tut National de la Sante et de la Recherche Medicale and from the Institut National de la Recherche Agron´ omique The PhD work of Miss Amelie Berton was supported by a grant from Institut National de la Recherche Agronomique and ARILAIT RECHER´ CHE Industry We thank Regine Lebrun and Danielle Moinier for performing the sequence analyses, and Mouhcine Louaste for helpful technical assistance We FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works 6021 PLRP2 and colipase ) no interaction thank Dr Catherine Chapus for helpful advice and fruitful discussions We are very grateful to Dr Franc¸ oise Guerlesquin for critical reading of the manuscript References Giller T, Buchwald P, Blum-Kaelin D & Hunziker W (1992) Two novel human pancreatic lipase related proteins, hPLRP1 and hPLRP2 Differences in colipase dependence and in lipase activity J Biol Chem 267, 16509–16516 Kerfelec B, LaForge KS, Puigserver A & Scheele G (1986) Primary structures of canine pancreatic lipase and phospholipase A2 messenger RNAs Pancreas 1, 430–437 Hjorth A, Carriere F, Cudrey C, Woldike H, Boel E, Lawson DM, Ferrato F, Cambillau C, Dodson GG, Thim L et al (1993) A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho) lipase Biochemistry 32, 4702–4707 Payne RM, Sims HF, Jennens ML & Lowe ME (1994) Rat pancreatic lipase and two related proteins: enzymatic properties and mRNA expression during development Am J Physiol 266, G914–G921 Wicker-Planquart C & Puigserver A (1992) Primary structure of rat pancreatic lipase mRNA FEBS Lett 296, 61–66 Remington SG, Lima PH & Nelson JD (1999) Pancreatic lipase-related protein mRNA in female mouse lacrimal gland Invest Ophthalmol Vis Sci 40, 1081–1090 Wishart MJ, Andrews PC, Nichols R, Blevins GT Jr, Logsdon CD & Williams JA (1993) Identification and cloning of GP-3 from rat pancreatic acinar zymogen granules as a glycosylated membrane-associated lipase J Biol Chem 268, 10303–10311 Jayne S, Kerfelec B, Foglizzo E, Chapus C & Crenon I (2002) High expression in adult horse of PLRP2 displaying a low phospholipase activity Biochim Biophys Acta 1594, 255–265 Thirstrup K, Verger R & Carriere F (1994) Evidence for a pancreatic lipase subfamily with new kinetic properties Biochemistry 33, 2748–2756 10 Grusby MJ, Nabavi N, Wong H, Dick RF, Bluestone JA, Schotz MC & Glimcher LH (1990) Cloning of an interleukin-4 inducible gene from cytotoxic T lymphocytes and its identification as a lipase Cell 60, 451–459 11 Sias B, Ferrato F, Pellicer-Rubio MT, Forgerit Y, Guillouet P, Leboeuf B & Carriere F (2005) Cloning and seasonal secretion of the pancreatic lipase-related protein present in goat seminal plasma Biochim Biophys Acta 1686, 169–180 12 Fauvel J, Bonnefis MJ, Sarda L, Chap H, Thouvenot JP & Douste-Blazy L (1981) Purification of two lipases with high phospholipase A1 activity from guinea-pig pancreas Biochim Biophys Acta 663, 446–456 6022 A Berton et al 13 De Caro J, Sias B, Grandval P, Ferrato F, Halimi H, Carriere F & De Caro A (2004) Characterization of pancreatic lipase-related protein isolated from human pancreatic juice Biochim Biophys Acta 1701, 89–99 14 Crenon I, Foglizzo E, Kerfelec B, Verine A, Pignol D, Hermoso J, Bonicel J & Chapus C (1998) Pancreatic lipase-related protein type I: a specialized lipase or an inactive enzyme Protein Eng 11, 135–142 15 De Caro J, Carriere F, Barboni P, Giller T, Verger R & De Caro A (1998) Pancreatic lipase-related protein (PLRP1) is present in the pancreatic juice of several species Biochim Biophys Acta 1387, 331–341 16 Lowe ME (2002) The triglyceride lipases of the pancreas J Lipid Res 43, 2007–2016 17 Borgstrom B (1975) On the interactions between pancreatic lipase and colipase and the substrate, and the importance of bile salts J Lipid Res 16, 411–417 18 Brockman H (2000) Kinetic behavior of the pancreatic lipase–colipase–lipid system Biochimie 82, 987–995 19 Andersson L, Carriere F, Lowe ME, Nilsson A & Verger R (1996) Pancreatic lipase-related protein but not classical pancreatic lipase hydrolyzes galactolipids Biochim Biophys Acta 1302, 236–240 20 Sias B, Ferrato F, Grandval P, Lafont D, Boullanger P, De Caro A, Leboeuf B, Verger R & Carriere F (2004) Human pancreatic lipase-related protein is a galactolipase Biochemistry 43, 10138–10148 21 Reboul E, Berton A, Moussa M, Kreuzer C, Crenon I & Borel P (2006) Pancreatic lipase and pancreatic lipase-related protein 2, but not pancreatic lipase-related protein 1, hydrolyze retinyl palmitate in physiological conditions Biochim Biophys Acta 1761, 4–10 22 Sebban-Kreuzer C, Deprez-Beauclair P, Berton A & Crenon I (2006) High-level expression of nonglycosylated human pancreatic lipase-related protein in Pichia pastoris Protein Expr Purif 49, 284–291 23 Jennens ML & Lowe ME (1995) Rat GP-3 is a pancreatic lipase with kinetic properties that differ from colipase-dependent pancreatic lipase J Lipid Res 36, 2374– 2382 24 Winkler FK, D’Arcy A & Hunziker W (1990) Structure of human pancreatic lipase Nature 343, 771–774 25 Bourne Y, Martinez C, Kerfelec B, Lombardo D, Chapus C & Cambillau C (1994) Horse pancreatic lipase The crystal structure refined at 2.3 A resolution J Mol Biol 238, 709–732 26 van Tilbeurgh H, Egloff MP, Martinez C, Rugani N, Verger R & Cambillau C (1993) Interfacial activation of the lipase–procolipase complex by mixed micelles revealed by X-ray crystallography Nature 362, 814–820 27 van Tilbeurgh H, Sarda L, Verger R & Cambillau C (1992) Structure of the pancreatic lipase–procolipase complex Nature 359, 159–162 28 Hermoso J, Pignol D, Kerfelec B, Crenon I, Chapus C & Fontecilla-Camps JC (1996) Lipase activation by FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works A Berton et al 29 30 31 32 33 34 35 36 37 nonionic detergents The crystal structure of the porcine lipase–colipase–tetraethylene glycol monooctyl ether complex J Biol Chem 271, 18007–18016 Pignol D, Hermoso J, Kerfelec B, Crenon I, Chapus C & Fontecilla-Camps JC (1998) The lipase ⁄ colipase complex is activated by a micelle: neutron crystallographic evidence Chem Phys Lipids 93, 123–129 Roussel A, de Caro J, Bezzine S, Gastinel L, de Caro A, Carriere F, Leydier S, Verger R & Cambillau C (1998) Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations Proteins 32, 523–531 Roussel A, Yang Y, Ferrato F, Verger R, Cambillau C & Lowe M (1998) Structure and activity of rat pancreatic lipase-related protein J Biol Chem 273, 32121– 32128 Jayne S, Kerfelec B, Foglizzo E, Granon S, Hermoso J, Chapus C & Crenon I (2002) Activation of horse PLRP2 by bile salts does not require colipase Biochemistry 41, 8422–8428 Crenon I, Jayne S, Kerfelec B, Hermoso J, Pignol D & Chapus C (1998) Pancreatic lipase-related protein type 1: a double mutation restores a significant lipase activity Biochem Biophys Res Commun 246, 513–517 Bezzine S, Roussel A, de Caro J, Gastinel L, de Caro A, Carriere F, Leydier S, Verger R & Cambillau C (1998) An inactive pancreatic lipase-related protein is activated into a triglyceride-lipase by mutagenesis based on the 3-D structure Chem Phys Lipids 93, 103–114 Carriere F, Thirstrup K, Hjorth S, Ferrato F, Nielsen PF, Withers-Martinez C, Cambillau C, Boel E, Thim L & Verger R (1997) Pancreatic lipase structure–function relationships by domain exchange Biochemistry 36, 239–248 Yang Y & Lowe ME (2000) The open lid mediates pancreatic lipase function J Lipid Res 41, 48–57 Gargouri Y, Bensalah A, Douchet I & Verger R (1995) Kinetic behaviour of pancreatic lipase in five species using emulsions and monomolecular films of synthetic glycerides Biochim Biophys Acta 1257, 223–229 PLRP2 and colipase ) no interaction 38 Wong H, Davis RC, Nikazy J, Seebart KE & Schotz MC (1991) Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase Proc Natl Acad Sci USA 88, 11290–11294 39 Rietsch J, Pattus F, Desnuelle P & Verger R (1977) Further studies of mode of action of lipolytic enzymes J Biol Chem 252, 4313–4318 40 Chahinian H, Bezzine S, Ferrato F, Ivanova MG, Perez B, Lowe ME & Carriere F (2002) The beta 5¢ loop of the pancreatic lipase C2-like domain plays a critical role in the lipase–lipid interactions Biochemistry 41, 13725– 13735 41 Miled N, Berti-Dupuis L, Riviere M, Carriere F & Verger R (2003) In vitro lipolysis by human pancreatic lipase is specifically abolished by its inactive forms Biochim Biophys Acta 1645, 241–246 42 Ayvazian L, Kerfelec B, Granon S, Foglizzo E, Crenon I, Dubois C & Chapus C (2001) The lipase C-terminal domain A novel unusual inhibitor of pancreatic lipase activity J Biol Chem 276, 14014–14018 43 Rouard M, Sari H, Nurit S, Entressangles B & Desnuelle P (1978) Inhibition of pancreatic lipase by mixed micelles of diethyl p-nitrophenyl phosphate and bile salts Biochim Biophys Acta 530, 227–235 44 Mancheno JM, Jayne S, Kerfelec B, Chapus C, Crenon I & Hermoso JA (2004) Crystallization of a proteolyzed form of the horse pancreatic lipase-related protein 2: structural basis for the specific detergent requirement Acta Crystallogr D Biol Crystallogr 60, 2107–2109 45 Freie AB, Ferrato F, Carriere F & Lowe ME (2006) Val-407 and Ile-408 in the beta5¢-loop of pancreatic lipase mediate lipase–colipase interactions in the presence of bile salt micelles J Biol Chem 281, 7793–7800 46 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 47 Burnette WN (1981) ‘Western blotting’: electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A Anal Biochem 112, 195–203 FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS No claim to original French government works 6023 ... for N2C2 and N2Cc In conclusion, the accessibility of the active site was better in the protein bearing the N2 domain than in the protein bearing the Nc domain Thus, the accessibility of the active... both the N-terminal and C-terminal domains of hoPLRP2 contributed to the stability of the protein in the presence of the water–lipid interface Also, both the N-terminal and C-terminal domains of. .. active site in the N2 proteins was independent of the nature of the C-terminal domain, in contrast to the situation with Nc proteins Indeed, the C2 domain induced sensitivity of the Nc active