Hindawi BioMed Research International Volume 2020, Article ID 3064290, 10 pages https://doi.org/10.1155/2020/3064290 Research Article Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights Neila Achouri,1 Màrius Tomàs-Gamisans,2 Soumaya Triki,3 Francisco Valero,2 Nabil Miled,4,5 Ahmed Fendri,1 and Nabil Smichi 1,6 1 University of Sfax, ENIS, Laboratory of Biochemistry and Enzymatic Engineering of Lipases, Road of Soukra, BPW 1173-3038 Sfax, Tunisia 2 UAB, Universitat Autònoma de Barcelona, Departament d’Enginyeria Química, Biològica i Ambiental, Bellaterra Barcelona, Spain 3 University of Sfax, Center of Biotechnology of Sfax, Laboratory of Molecular and Cellular Screening Processes, BP 111 7 Road Sidi Mansour km 6, Sfax, Tunisia 4 University of Jeddah, College of Science, Department of Biological Sciences, Jeddah, Saudi Arabia 5 Functional Genomics and Plant Physiology Unit, Higher Institute of Biotechnology of Sfax, 3038 Sfax, Tunisia 6 Mayo Clinic Arizona, 13400 Shea Boulevard, Scottsdale, AZ 85259, USA Correspondence should be addressed to Nabil Smichi; smichi.nabil@mayo.edu Received 7 September 2019; Revised 23 January 2020; Accepted 29 January 2020; Published 13 March 2020 Academic Editor: Marcelo A. Soares Copyright © 2020 Neila Achouri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL. 1. Introduction sequence of the porcine pancreatic lipase was first published in 1981 [6], and since then, several mammalian pancreatic Pancreatic lipases (triacylglycerol hydrolase, EC 3.1.1.3) are lipases have been isolated and characterized [7–10]. Thanks carboxylester hydrolases that catalyse the hydrolysis of fats to the resolution of numerous three-dimensional structures, and oils into glycerol and free fatty acids at the oil/water a better comprehension has been reached for the pancre- interfaces [1–4]. They are much more active on insoluble atic lipase structure-function relationships [10–12]. Van substrates than on soluble esters; this property has been des- Tilbeurgh et al.were the first to determine the 3-D structure ignated as “interfacial activation” [5]. In the presence of bile of HPL, followed directly by the 3-D structure of a HPL- salts, mammalian pancreatic lipase activity requires a small procolipase complex in the presence or in the absence of protein coenzyme called colipase that allows the enzyme to mixed phospholipid/bile salt micelles, as well as in complex bind to the triacylglycerol/water interface. The amino acid with a C11 alkyl phosphonate inhibitor [12]. The lipase is a 2 BioMed Research International single polypeptide chain of 449 amino acids and is folded into Table 1: Primers used to amplify genes encoding for SmDL and two structural domains [10]: a large N-terminal domain (res- EeDL. idues 1-336) and a smaller C-terminal domain (residues 337- 449). The N-terminal domain belongs to the α/β-hydrolase Primers Sequences 5 ′ -3 ′ fold [13] and contains a catalytic triad formed by serine, pSmDL-F GCC GAA ATC TGC TAT AGC AG aspartic acid, and histidine that is analogous to that of serine pSmDL-R TTA GCA AGG CAA AAC AGT TTG proteases [14, 15]. While the N-terminal domain carries the pEeDL-F GCC GAG GTG TGC TAT GAA AAC catalytic activity, the β-sandwich C-terminal domain plays pEeDL-R TCA GCA CGG AGG AAC AGT CTG an important structural role as it binds specifically to the coli- pase. This cofactor exposes the hydrophobic tips of its fingers at the opposite side of its lipase-binding site through nonco- 2. Materials and Methods valent interactions [10, 13]. In recent years, researchers have been seeking to widen the study of pancreatic lipases from 2.1. Strains, Plasmids, and Chemicals. E. coli strain XL1 blue birds [8, 16] and invertebrates [14, 17, 18]. The turkey pan- was used as the cloning host and was grown in Luria-Bertani creatic lipase (TPL) shows the same features of HPL, and (LB) medium [28], supplemented with 10 μg/mL tetracycline its three-dimensional structure consists of two domains sta- and 100 μg/mL ampicillin. A pGEM-T Easy Cloning Vector bilized by six disulphide bridges [8]. Moreover, other studies was from Promega (France). The PCR products were purified have reported new enzymes isolated from aquatic species using a QIAquick PCR Purification Kit (Qiagen). IPTG and with interesting potential in food processing [3, 19–21]. To X-Gal (isopropyl-thio-β-d-galactopyranoside and 5-bromo- provide an overview on pancreatic lipases, we have tried to 4-chloro-3-indolyl-beta-D-galactopyranoside) were bought extend our research on the fish lipolytic system in order to from Global Biotechnology. All oligonucleotides and enzymes understand the evolutionary aspects of fish lipases as com- used in DNA manipulations were from Invitrogen. THL pared to those of mammals. Previously, many studies have (tetrahydrolipstatin) was a generous gift from Pr. A.F. FIS- been carried out on digestive lipases of bony fish from the CHLI (Buchs, Switzerland). NaDC (sodium deoxycholate) grass carp [22] and Pengze crucian carp [23]. The Sparidae was from Sigma Chemical (St. Louis, USA). family has been considered with regard to lipolytic enzymes involved in lipid digestion; Smichi et al. [24] have purified a 2.2. Biological Materials. Fresh common smooth-hound (M. lipase from the annular seabream (AsDL). The character- mustelus) and European eel (A. anguilla) were caught and ized enzyme shows distinct properties to TPL [8] and transported forthright in cold ice (4°C) to the laboratory. lipases from other marine species [19, 21]. From the same Fresh pancreases from each species were dissected and family, a lipase from the red seabream has been isolated immediately used for total RNA extractions. and purified and its functional properties have been per- 2.3. Pancreatic Lipases. The pancreatic lipases SmDL and formed to explain the differences with pancreatic lipases TPL were purified as previously described [16, 27]. [25]. Yet, little information is available regarding structure- function relationships of cartilaginous fish (chondrichthyans) 2.4. Pancreatic Colipase Preparation. In order to check the lipases. presence of colipase in the common smooth-hound’s pan- The common smooth-hound (Mustelus mustelus) as one creas, the delipidated powder homogenate was treated during of the chondrichthyans and the most used for human con- 15 min at pH 2 and at 60°C to inactivate lipase and maintain sumption in Tunisia [26] was used as a model in this study. only colipase activity. To study the lipase interaction with This species possesses a complex digestive system with a pan- colipase, SmDL and TPL activities were measured using olive creas secreting digestive enzymes. Recently, we have purified oil emulsion as a substrate in the presence and absence of and characterized the SmDL from the delipidated pancreatic colipase, using the pH stat method under optimal conditions powder of smooth-hound [27]. The SmDL molecular weight of pH and temperature as described previously [16, 27]. was around 50 kDa. The purified enzyme hydrolyses effi- ciently triacylglycerols with 2200 U/mg on tributyrin as a 2.5. RNA Extraction and Cloning of SmDL and EeDL. Total substrate and was found to be stable at 50°C. Unlike known RNA was isolated from 0.1 g of both fish pancreases using a mammal pancreatic lipases, this enzyme was not activated TRIzol reagent as described by the manufacturer’s protocol by the pancreatic colipase. There is a little information about (Invitrogen). First-strand cDNAs were synthesized in 20 μL pancreatic lipases from chondrichthyans. Here, we report for by reverse transcribing 2 μg of total RNA, using 1 μL of M- the first time the isolation and the cloning of the gene coding MLV as the reverse transcriptase (200 unites) and 10 μM for the SmDL. For the sake of comparison, the gene coding oligo (dT)18 as the primer according to the manufacturer’s the mature lipase from the European eel A. anguilla (EeDL) instructions. First-strand cDNAs were treated with 1 μL was cloned and sequenced. A phylogenetic tree has been gen- RNase inhibitor (40 U/μL), and the part of the cDNAs encod- erated in order to determine the evolutionary line of chon- ing for mature lipases was amplified using the oligonucleo- drichthyan lipases with other digestive lipases. Using the tides listed in Table 1. Primers were predicted from putative three-dimensional structure of the HPL as a template, struc- pancreatic lipases of elephant shark (Callorhinchus milii, tural models of the SmDL and EeDL were built and used to GenBank accession number PRJNA18361) and Japanese eel provide explanation on the poor interaction of the SmDL (Anguilla japonica, GenBank accession number AB070722.1) with the classical colipase. for amplification of smooth-hound (SmDL) and European BioMed Research International 3 eel (EeDL) genes coding for digestive lipases, respectively. Lid domain PCR amplifications were realized by Pfu DNA Polymerase (Promega) according to the enzyme instructions. The PCR mixture (20 μL) contained 1 μg of 1st-strand cDNA, 1.25 μL of each specific oligonucleotide primer (25 μM) (Table 1), 0.25 μL of Pfu DNA Polymerase (2-3 U/μL), and 0.2 mM of deoxynucleoside triphosphate. Amplification was realized in a Bio-Rad thermal cycler, utilizing the reaction settings as follows: initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturing at 95°C for 1 min, annealing tem- perature for 30 s, extension at 72°C for 3 min, and a final extension at 72°C for 5 min. PCR fragments (approximately 1350 bp) were purified and ligated into the dephosphorylated pGEM-T Easy Vector according to the manufacturer’s proto- col. After transformation into the competent XL1 blue cells, Figure 1: Cartoon presentation of structure models of SmDL in red the recombinant colonies were identified through blue-white superimposed with the human pancreatic lipase (in cyan). The color selection in IPTG, X-Gal, and ampicillin-containing LB catalytic triad residues are shown as green sticks, and the lid plates and confirmed by PCR colony followed by sequencing domain under its open form is indicated. on both strands using an ABI PRISM 3100 sequencer. The nucleotide sequences of the two mature lipases (SmDL and 3. Results and Discussion EeDL) determined in this study were deposited in the Gen- Bank database under accession numbers KY548033 and 3.1. Identification of SmDL and EeDL Lipase Coding Genes. KY548034, respectively. Total RNAs were obtained from M. mustelus (Figure S1A, lane 1) and A. anguilla (Figure S1A, lane 2) pancreases. The 2.6. Database Research and Phylogenetic Studies. The reading analyzed products showed RNA high qualities that will be frame of the two sequenced cDNAs was translated using the used as templates for gene amplification. Full-length coding ExPASy translate tool program, and the deduced fish diges- sequences for the SmDL and EeDL were obtained from M. tive lipase amino acid sequences were compared using the mustelus and A. anguilla products, respectively, by the reverse BLASTP algorithm with sequences already existing in the transcriptase-PCR technique and using the appropriate NCBI Protein Database. Sixteen homologous digestive lipase primers as described in Material and Methods. Amplified sequences from different mammals, fish, and birds were used cDNA fragments (about 1350 bp) were electrophoretically for multiple sequence alignment using BioEdit v.7.2.5 and separated (Figure S1B). These cDNA products were ligated the default settings [29]. The output of the BioEdit multiple in a pGEM-T Easy Vector. The recombinant vector was sequence alignment was color coded according to their iden- transformed into E. coli cells. The white colonies (positives tity. The amino acid sequences of lipases from different spe- clones) were randomly selected, and the presence of SmDL cies were aligned. Using the maximum parsimony method, and EeDL inserts in the plasmid was checked by colony the phylogenetic trees were carried out in Molecular Evolu- PCR. Vectors having each insert were sequenced using tionary Genetics Analysis (MEGA6) [30, 31]. The branch T7 and SP6 primers. The sequences of all fragments were robustness was assessed by bootstrap analysis of 100 repli- aligned by BioEdit v.7.2.5 [29]. The complete sequences of cates of resampling, and only values that were highly signif- SmDL and EeDL (Figure S2) consisting of 1.35 kb were icant (≥70) are shown [32]. All positions containing gaps submitted in the GenBank and were assigned the accession and missing data were eliminated. There were a total of numbers KY548033 and KY548034, respectively. The 442 positions in the final dataset. Evolutionary analyses were nucleotide BLAST analysis for SmDL showed that this carried out in MEGA6 [31]. enzyme shared high identity (77%–87%) with pancreatic lipase genes from other cartilaginous fish: 87% with the 2.7. 3-D Structure Modeling. The 3-D structure models of whale shark (Rhincodon typus), 82% with the common SmDL and EeDL were built under an active (open) form stingray (Dasyatis pastinaca) [36], and 77% with the using the 3-D structure of the active form of human pancre- elephant shark (Callorhinchus milii). The EeDL (Figure S2B) atic lipase (1LPB) as a template. The models were built by shared high identities of 96% and 75% with the Japanese eel homology by submitting the alignment generated by BioEdit (Anguilla japonica) and hareng (Clupea harengus) lipase v.7.2.5 [29] to the automated structure-modeling SWISS- genes, respectively. The deduced polypeptide sequences MODEL workspace [33]. In order to optimize the structures, of SmDL and European eel digestive lipases comprise the model was subjected to three cycles of minimization, each 451 and 452 amino acids, respectively. High amino acid containing 50 steps of conjugate gradient, using the GRO- sequence identities were found between SmDL and other MOS96 software implemented to Swiss-PdbViewer [34]. cartilaginous fish lipases like Callorhinchus milii (68%) and The cutoff was set to 10 Ǻ and a harmonic constraint was Dasyatis pastinaca (79%) [36]. The EeDL amino acid used. The geometry quality of the final model was checked sequence displayed prominent similarity with those of other using the PROCHECK program [35]. The figure was gener- fish species like Anguilla japonica (97%) and red seabream ated using PyMOL (http://www.pymol.org). (63%). SmDL and EeDL structures superposition with the 4 BioMed Research International Figure 2: Alignment of the amino acid sequences of the mature forms of SmDL and EeDL with HPL. Boxes in light grey indicate positions at which the amino acids are identical in the three lipases. The closed arrows indicate the Ser, Asp, and His which form the catalytic triad. The dashes represent gaps introduced during the alignment process. The secondary structures of Sparidae lipases are indicated. Homology alignment was performed using the software BioEdit v.7.2.5. human pancreatic lipase (HPL) is shown in Figure 1. Both Sequences corresponding to the HPL lid domain and β5 enzymes possess two and three supplementary amino acids, loop [13] are conserved for the two fish lipases. The twelve respectively, as compared to the HPL (449 amino acids). cysteine residues involved in disulphide bridges in the case Residues of the HPL catalytic triad (Ser153, Asp177, and of HPL are also conserved for SmDL and EeDL, suggesting His 264) are conserved in both fish lipases. The highest also the presence of 6 disulphide bridges in the two fish homology was observed around the active-site serine for digestive lipase structures (Figure 2). the three lipases (Figure 2). A comparison of the N- and C- terminal domains of SmDL and EeDL with that of HPL 3.2. Phylogenetic Analysis of Fish Digestive Lipases. We shows that the N-terminal domain is more conserved performed the phylogenetic analysis using 16 sequences: (about 63%) than the C-terminal domain (about 47%). 11 classical pancreatic lipases (PL) and 5 fish digestive BioMed Research International 5 47 Human/Homo sapiens 39 Pig/Sus scrofa Cow/Bos taurus 50 100 Sheep/Ovis aries musimon 86 Dromedary/Camelus ferus 100 Horse/Equus caballus 65 Mouse/Mus musculus 90 Dog/Canis lupus familiaris 99 Rat/Rattus norvegicus Turkey/Meleagris gallopavo 100 Chicken/Gallus gallus Smooth-hound/Mustelus mustelus Eel/Anguilla anguilla Tuna/Thunnus orientalis 81 100 Red seabream/Pagrus major 100 Annular seabream/Diplodus annularis 50 Figure 3: Maximum parsimony analysis of taxa. The evolutionary history was inferred using the maximum parsimony method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches [32]. The tree is drawn to scale with branch lengths calculated using the average pathway method [30] and being in the units of the number of changes over the whole sequence. The analysis involved 16 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 442 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [29]. lipases (DL). A phylogenetic tree was constructed based on the HPL structure (Figure 1). The root-mean-squared the alignment of the whole sequences (Figure 3). Based on deviations (RMSD) using alpha carbons were only 1.2 Å the bootstrap values, all the obtained phylograms showed (419 atoms implicated) between the open forms of HPL the same clustering of the sequences into two major clusters. and the fish digestive lipase. Like the HPL, the model of According to the established phylogeny, the polypeptide SmDL (open form) showed the presence of two domains: sequences of smooth-hound and European eel digestive the catalytic N-terminal domain (residues 1 to 332) which lipases were gathered together with those of tuna (Thunnus is folded into a central parallel β-sheet consisting of 11 orientalis) and Sparidae lipases in one fish digestive lipase strands bounded by alpha helices on both sides and the gene family (Figure 3). The phylogenetic tree also showed that β-sandwich C-terminal domain characterized by two anti- lipases from annular and red seabreams and tuna took a sim- parallel β-sheets (Figure 1). The 3-D models of SmDL and ilar evolutionary line separated in the early evolution from EeDL show that Ser153, Asp176, and His263 residues are those of European Eel and smooth-hound which belong, located in a catalytic triad-like configuration (Figure 1). Like respectively, to teleosts and the cartilaginous fish lipase sub- the HPL structure [10], the model of both enzymes contains group. In 1999, Rasmussen and Amason have described the six disulphide bridges. All structural features required for teleosts and the chondrichthyans as sister groups [37]. The HPL activity and substrate recognition such as lid domain phylogenetic tree showed that birds (chicken and turkey) and β5 and β9 regions are also conserved in SmDL and EeDL are the closest to the cartilaginous fish; this behavior seems structures (Figure 1). The fact that all the classical structural to be similar to those reported by Smichi et al. [24]. features are conserved in the 3-D models of SmDL and EeDL altogether suggest that these enzymes may share the same 3.3. Modeling of SmDL and EeDL. The 3-D structure of the docket and the catalytic mechanism toward lipids. Thus, we active human pancreatic lipase form (PDB code 1LPA) was have tried to bring more information on the digestive process utilized as a template to the build model for the open form of the SmDL. of SmDL using the automated structure-modeling SWISS- MODEL workspace [34]. The generated 3-D models were 3.4. SmDL Is a Serine Enzyme. The 3-D model of SmDL then optimized by energy minimization. The Ramachandran shows also that the amino acid key which is involved in the plot statistics of the SmDL was carried out by the PRO- nucleophilic mechanism is Ser153 (Figure 4) which suggests CHECK program and revealed that 97% of the residues were that SmDL may be a serine enzyme. In order to verify if either in the most-favoured or in the additional authorized SmDL is a serine enzyme, we have used a serine inhibitor, regions. A similar result (97.1%) was shown for the HPL THL, which acts as a potent inhibitor of gastric and pancre- structure. The fish structural model is well superposed to atic lipases, reacting with these enzymes’ catalytic serine 6 BioMed Research International 2500 120 TPL+Col SmDL±Col 2000 100 Specific activity (U/mg) Residual activity (%) 80 1500 60 1000 TPL 40 500 20 0 0 20 40 60 80 100 0 Time (mn) 0 2 4 6 8 10 NaDC (mM) THL THL+NaDC Figure 5: Effect of increasing the concentration of bile salts: NaDC. Figure 4: Inhibition of SmDL by THL in the absence and the Lipolytic activity of SmDL and TPL was measured at pH 9 and 40°C presence of 4 mM NaDC. SmDL was incubated at 25°C with THL using olive oil emulsion as a substrate in the presence and in the (molar ratio THL/SmDL = 100). SmDL activity was measured at absence of a molar excess of colipase. pH 9 and 40°C using TC4 as a substrate. Table 2: Main residues situated in the flap and the C-terminal domain involved in the binding of HPL to the porcine colipase. [38]. We studied the effect of THL on the SmDL activity [4]. Corresponding residues in SmDL, PPLRP2, and HPLRP2, in The lipase lost 63% of its initial activity when incubated dur- porcine and cartilaginous fish colipase, are reported. ing 60 min with THL at a molar excess of 100. As described previously [39], NaDC addition (final concentration 4 mM) Porcine Fish HPL SmDL AsDL HPLRP2 in the incubation medium accelerates significantly the inhib- colipase colipase itory effect of THL. The mixed NaDC-THL micelles seem to Asn241 Asn241 Asn238 Asn242 Glu15 Glu15 be more appropriate than the THL alone for SmDL inhibi- Ser244 Ser244 Gly240 Ser245 Leu16 Leu16 tion (Figure 4). We can conclude that SmDL is a serine Val247 Ile247 Ser243 Thr248 Arg38 Arg38 enzyme, as all known lipases characterized from several Asp248 Asp248 Asp244 Asp248 Arg38 Arg38 sources [40, 41]. Gln369 Gln369 Glu367 Gln371 Glu64 Glu64 Lys400 Lys400 Lys398 Lys403 Glu45 Glu45 3.5. SmDL-Colipase Complex Ile402 Leu402 Arg400 Leu404 Arg65 Arg65 3.5.1. Biochemical Approach. It is well established that all Tyr404 Asn404 Asn402 Asn406 Arg65 Arg65 amphiphiles are strong inhibitors of pancreatic lipases [42]. Unlike classical lipases (HPL and TPL), the SmDL retained its maximal activity even at high bile salt concentrations that the SmDL does not require interactions with colipase reaching 8 mM [27]. Under these conditions, NaDC acts as to be fully active. Similar results were reported for some bony an inhibitor of TPL activity when TC4 or olive oil emulsion fish digestive systems like grey mullet and Sparidae [43]. is used as the substrate, but this inhibition is reversed by However, this behavior differs from that of mammal [16], the addition of colipase. Thus, to check the presence of the avian [8], and stingray pancreatic [36] lipases, which are classical colipase in the common smooth-hound pancreas, strongly inhibited by bile salts and reactivated by pancreatic the homogenate was treated at pH 2 during 30 min, then at colipase. As far as colipase activity was detected in the com- 60°C to retain only colipase activity. This colipase prepara- mon smooth-hound pancreas, this may suggest that it pro- tion succeeded to reactivate the classical TPL inhibited by bile duces another classical lipase, apart from SmDL, which is salts (Figure 5). These results suggest that the common inactivated by bile salts and reactivated by colipase. smooth-hound pancreas contains the classically known coli- pase. Considered as the most primitive living jawed verte- 3.5.2. Structural Approach: Colipase Potential Binding Sites. brates, cartilaginous fish, represented by sharks, might be Bile salts (NaDC) are physiological inhibitors of pancreatic considered as the oldest vertebrates having a complex diges- lipases due to enzyme desorption. In the classical system, tive system the same as that of mammals. Surprisingly, the the colipase allows the lipase reactivation by anchoring the SmDL was not activated either by its own pH and/or enzyme to the bile salt/lipid interface [44]. The colipase pro- temperature-treated homogenate (own colipase preparation) tein was characterized by an amphipathic overall that con- or by the turkey pancreatic colipase (Figure 5). However, in tains hydrophobic residues which are located at the tips of the same conditions, the TPL was activated. This suggests the fingers and interact with the lipidic substrate and BioMed Research International 7 Pancreatic colipase HPL/SmDL Superposition (a) R65 colipase Y404 HPL N404 SmDL (b) Figure 6: Lipase-colipase interaction. (a) Ribbon representation of SmDL and HPL interaction with colipase. The HPL structure is in complex with colipase (PDB code 1LPB). HPL and SmDL structures are well superposed. Residues involved in the lipase-colipase interaction are shown as sticks. (b) Zoom in from (a) showing the interaction interface between superimposed lipases (HPL and SmDL) and colipase. Residues conserved between the two lipases are shown only once. Residue Y404 in HPL and the corresponding residue in SmDL (N404) are both shown, and each of them is in interaction with colipase residue R65, shown in red. The hydrogen bond between R65 of the colipase and Y404 of HPL is shown as a dotted line. hydrophilic residues that bind to the C-terminal domain of classical bile salt-colipase system [24]. In fact, HPL Tyr404 the enzyme [11]. The colipase was found to interact also with is stabilized through hydrophobic contacts with the bulky the open lid of pancreatic lipases [45]. It is well established chain of Arg65 and also hydrogen bonding (by its OH group) that all lipase residues involved in colipase interaction are to the Arg65 NH2 group (Figure 6). Tyr404/Asn substitution well conserved throughout the pancreatic lipase subfamily. might disrupt the Tyr-Arg interaction and lead to uncom- Table 2 shows that residues Asn241, Asp248, and Lys400 fortable contacts between the colipase arginine bulky chain involved in HPL binding to colipase are conserved in the and the polar lipase Asn groups which prevent the colipase smooth-hound digestive lipase. Nevertheless, Ile402 which binding to the SmDL. This might explain why SmDL is not belongs to the C-terminal domain of HPL and interacts with reactivated by colipase. The Tyr404 of HPL and SmDL was Arg65 of colipase is substituted by Leu400 in the SmDL. Res- replaced by Asn406 in the HPLRP2 (Table 2). As described idues Asn241, Ser244, and Val247 of the lid are involved in by De Caro et al., this substitution might be responsible for interaction with Arg38, Leu16, and Glu15 of the colipase weaker HPLRP2-colipase interactions, since HPLRP2 is [45]. Val247 is changed to Ile247 in the SmDL. Interestingly, inhibited by NaDC and partially reactivated by the pancreatic Tyr404 known to be a key residue for HPL binding to coli- colipase [6, 46]. Furthermore, Xiao et al. [47] have shown pase [12] is replaced by Asn in the smooth-hound lipase that, unlike HPLRP2, porcine PLRP2 activity increases with (Table 2 and Figure 6). This Tyr404/Asn substitution was increasing colipase concentration due to the conservation of previously observed in the annular and red seabream diges- the lid and C-terminal residues in the porcine PLRP2 tive lipases (AsDL and RsDL) which are independent of the sequence (including Tyr404) which is involved in the 8 BioMed Research International interactions of classical HPL with colipase. Altogether, these RsDL: Red seabream lipase observations might give more insights on the absence of the PPLRP2: Porcine pancreatic lipase-related protein 2 classical system in the smooth-hound fish when assessing TPL: Turkey pancreatic lipase SmDL activity (Figure 6). The comparative studies of the RT-PCR: Reverse transcription-polymerase chain reaction lipolytic systems can be of great importance to understand THL: Tetrahydrolipstatin the evolutionary relationship between animals, such as bony NaDC: Sodium deoxycholate. fish and cartilaginous fish. It has been shown that lower animals such as scorpions [18] and crabs [19] possess a Data Availability combined hepatopancreas, whereas higher animals have well-differentiated separate livers and pancreases. This The data used to support the findings of this study are avail- was thought to be the main evolutionary aspect of lipolytic able from the corresponding author upon request. systems. This work shows that another evolutionary finger- print to consider is that of the colipase/bile salt/lipase system. Ethical Approval Although they seem to be a homogenous digestive enzyme group, pancreatic lipases can be thereafter classified in two The experimental protocols and procedures used in the subgroups: colipase-dependent and colipase-independent present work were approved by the Ethics Committee of enzymes. This works shows for the first time that a lipase the University of Sfax (Sfax, Tunisia) for the care and use of from a cartilaginous fish (chondrichthyans) is interacting laboratory animals. poorly with colipase. This was established for other fish belonging to bony fish. Based on this discovery, one can Conflicts of Interest notice that the colipase/bile salt/pancreatic lipase system is The authors have declared no conflict of interest. evolving independently with the animal phylogeny. More studies on the composition and the evolution of bile salt com- Acknowledgments position and colipase sequence might give more insights on the discrepancies observed between animals and fish lipolytic We thank Pr. Youssef Gargouri for his support to achieve this systems. Moreover, this can open new perspectives in opti- work, which was supported by the Tunisian Ministry of mizing the application of lipolytic enzymes in bioconversion Higher Education and Scientific Research. We thank also processes. A bile salt/colipase independent pancreatic lipase the Spanish team which was also partially supported by the might be more easily applied as a biocatalyst as compared Spanish project CTQ2016-74959-R (MINECO/EU FEDER). to a classical bile salt/colipase/lipase system. Supplementary Materials 4. Concluding Remarks Figure S1: (a) agarose gel electrophoresis (1.5%) of the RNA The genes encoding SmDL and EeDL were isolated for the extraction. Lane 1 contains 1 kb DNA molecular weight first time from smooth-hound and European eel, respec- markers; lanes 2 and 3 contain the RNA of smooth-hound tively. The nucleotide BLAST analysis for SmDL indicated and European eel pancreases. (b) Agarose gel electrophoresis that it shared a high identity (77%–87%) with pancreatic (1%) of the PCR products of fish digestive lipases. Lane 1 lipase genes from some cartilaginous fish. In accordance with contains 1 kb DNA molecular weight markers; lanes 2 and the established phylogeny, sequences of SmDL and EeDL 3 contain the PCR products of smooth-hound and European genes were gathered together with those of tuna and Sparidae eel digestive lipases, respectively. Figure S2: nucleotide lipases in one fish digestive lipase cluster. The predicted 3-D sequences of the gene encoding the mature SmDL (a) and structure of SmDL indicated the conservation of the signa- EeDL (b) and the deduced amino acid sequences of corre- ture features, such as the oxyanion hole, the lid, and the spondent lipases. (Supplementary Materials) catalytic triad, shared among mammal pancreatic lipases. The substitutions of SmDL residues involved in the bind- References ing with colipase may destabilize the lipase-colipase com- plex in the open conformation. These contradictions may [1] Y. Ben Ali, R. Verger, and A. Abousalham, “Lipases or ester- elucidate the fact that the classical pancreatic colipase is ases: does it really matter? Toward a new bio-physico- not efficient at activating the isolated lipase from the chemical classification,” in Lipases and phospholipases, pp. 31–51, Springer Protocols, 2012. smooth-hound. [2] J. De Caro, C. Eydoux, S. Chérif et al., “Occurrence of pancre- atic lipase-related protein-2 in various species and its relation- Abbreviations ship with herbivore diet,” Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, SmDL: Smooth-hound digestive lipase vol. 150, no. 1, pp. 1–9, 2008. EeDL: European eel digestive lipase [3] S. López-López, H. Nolasco, and F. Vega-Villasante, “Charac- HPL: Human pancreatic lipase terization of digestive gland esterase-lipase activity of juvenile HPLRP1: Human pancreatic lipase-related protein 1 redclaw crayfish C herax quadricarinatus,” Comparative Bio- HPLRP2: Human pancreatic lipase-related protein 2 chemistry and Physiology Part B: Biochemistry and Molecular AsDL: Annular seabream digestive lipase Biology, vol. 135, no. 2, pp. 337–347, 2003. BioMed Research International 9 [4] K. Sato, H. S. Seol, and T. Kamada, “Tissue distribution of [20] F. Shahidi and Y. V. A. Janak Kamil, “Enzymes from fish and lipase genes related to triglyceride metabolism in laying hens aquatic invertebrates and their application in the food indus- (Gallus gallus),” Comparative Biochemistry and Physiology try,” Trends in Food Science & Technology, vol. 12, no. 12, Part B: Biochemistry and Molecular Biology, vol. 155, no. 1, pp. 435–464, 2001. pp. 62–66, 2010. [21] Z. Zarai, M. B. Ali, A. Fendri, H. Louati, H. Mejdoub, and [5] R. Verger, “'Interfacial activation' of lipases: facts and arti- Y. Gargouri, “Purification and biochemical properties of Hex- facts,” Trends in Biotechnology, vol. 15, no. 1, pp. 32–38, 1997. aplex trunculus digestive lipase,” Process Biochemistry, vol. 47, [6] J. De Caro, B. Sias, P. Grandval et al., “Characterization of pan- no. 12, pp. 2434–2439, 2012. creatic lipase-related protein 2 isolated from human pancreatic [22] J. Sun, H. Ji, X. X. Li, X. C. Shi, Z. Y. Du, and L. Q. Chen, juice,” Biochimica et Biophysica Acta (BBA) - Proteins and Pro- “Lipolytic enzymes involving lipolysis in Teleost: Synteny, teomics, vol. 1701, no. 1-2, pp. 89–99, 2004. structure, tissue distribution, and expression in grass carp [7] F. Carrière, K. Thirstrup, S. Hjorth, and E. Boel, “Cloning of (Ctenopharyngodon idella),” Comparative Biochemistry and the classical guinea pig pancreatic lipase and comparison with Physiology Part B: Biochemistry and Molecular Biology, the lipase related protein 2,” FEBS Letters, vol. 338, no. 1, vol. 198, pp. 110–118, 2016. pp. 63–68, 1994. [23] H. L. Cheng, X. Wang, Y. X. Peng, X. P. Meng, S. P. Sun, and [8] A. Fendri, F. Frikha, H. Mosbah et al., “Biochemical character- X. Y. Shi, “Molecular cloning and tissue distribution of lipo- ization, cloning, and molecular modelling of chicken pancre- protein lipase full-length cDNA from Pengze crucian carp atic lipase,” Archives of Biochemistry and Biophysics, vol. 451, (Carassius auratus var. Pengze),” Comparative Biochemistry no. 2, pp. 149–159, 2006. and Physiology Part B: Biochemistry and Molecular Biology, [9] K. Thirstrup, F. Carriere, S. A. Hjorth et al., “Cloning and vol. 153, no. 1, pp. 109–115, 2009. expression in insect cells of two pancreatic lipases and a proco- [24] N. Smichi, N. Miled, Y. Gargouri, and A. Fendri, “A newly lipase from Myocastor coypus,” European Journal of Biochem- thermoactive and detergent-stable lipase from annular sea istry, vol. 227, no. 1-2, pp. 186–193, 1995. bream (Diplodus annularis): biochemical properties,” Biotech- [10] F. K. Winkler, A. D'Arcy, and W. Hunziker, “Structure of nology and Applied Biochemistry, vol. 64, no. 1, pp. 79–86, human pancreatic lipase,” Nature, vol. 343, no. 6260, pp. 771– 2017. 774, 1990. [25] N. Smichi, A. Fendri, S. Triki et al., “Biochemical characteriza- [11] H. Van Tilbeurgh, M. P. Egloff, C. Martinez, N. Rugani, tion, cloning and molecular modeling of a digestive lipase from R. Verger, and C. Cambillau, “Interfacial activation of the red seabream (Pagrus major): structural explanation of the lipase-procolipase complex by mixed micelles revealed by X- interaction deficiency with colipase and lipidic interface,” ray crystallography,” Nature, vol. 362, no. 6423, pp. 814–820, Engineering in Life Sciences, vol. 17, no. 6, pp. 664–677, 2017. 1993. [26] A. Bougatef, M. Hajji, R. Balti, I. Lassoued, Y. Triki-Ellouz, and [12] H. Van Tilbeurgh, L. Sarda, R. Verger, and C. Cambillau, M. Nasri, “Antioxidant and free radical-scavenging activities “Structure of the pancreatic lipase-procolipase complex,” of smooth hound (Mustelus mustelus) muscle protein hydroly- Nature, vol. 359, no. 6391, pp. 159–162, 1992. sates obtained by gastrointestinal proteases,” Food Chemistry, vol. 114, no. 4, pp. 1198–1205, 2009. [13] D. L. Ollis, E. Cheah, M. Cygler et al., “Theα/βhydrolase fold,” Protein Engineering, vol. 5, no. 3, pp. 197–211, 1992. [27] N. Achouri, N. Smichi, Y. Gargouri, N. Miled, and A. Fendri, “The smooth-hound lipolytic system: Biochemical characteri- [14] C. Rivera-Pérez, M. . . Á. N. del Toro, and F. García-Carreño, zation of a purified digestive lipase, lipid profile and in vitro “Purification and characterization of an intracellular lipase oil digestibility,” International Journal of Biological Macromol- from pleopods of whiteleg shrimp (Litopenaeus vannamei),” ecules, vol. 102, pp. 1120–1129, 2017. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, vol. 158, no. 1, pp. 99–105, 2011. [28] J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory [15] J. M. Steiner, B. G. Wilson, and D. A. Williams, “Purification Press, Cold Spring Harbor, NY, 1989. and partial characterization of feline classical pancreatic lipase,” Comparative Biochemistry and Physiology Part B: Biochemistry [29] T. A. Hall, “BioEdit: a user-friendly biological sequence align- and Molecular Biology, vol. 134, no. 1, pp. 151–159, 2003. ment editor and analysis program for Windows 95/98/NT,” Nucleic Acids Symposium Series, vol. 41, pp. 95–98, 1999. [16] A. Sayari, H. Mejdoub, and Y. Gargouri, “Characterization of turkey pancreatic lipase,” Biochimie, vol. 82, no. 2, pp. 153– [30] M. Nei and S. Kumar, Molecular Evolution and Phylogenetics, 159, 2000. Oxford University, Press, New York, 2000. [17] S. Cherif, N. Miled, M. Kammoun, H. Mejdoub, and [31] K. Tamura, F. U. Battistuzzi, P. Billing-Ross, O. Murillo, Y. Gargouri, “Purification and biochemical characterization A. Filipski, and S. Kumar, “Estimating divergence times in of a turkey preduodenal esterase,” Comparative Biochemistry large molecular phylogenies,” Proceedings of the National and Physiology Part B: Biochemistry and Molecular Biology, Academy of Sciences, vol. 109, no. 47, pp. 19333–19338, 2012. vol. 145, no. 3-4, pp. 358–364, 2006. [32] J. Felsenstein, “Confidence limits on phylogenies: an approach [18] N. Zouari, N. Miled, S. Cherif, H. Mejdoub, and Y. Gargouri, using the bootstrap,” Evolution, vol. 39, no. 4, pp. 783–791, “Purification and characterization of a novel lipase from the 1985. digestive glands of a primitive animal: the scorpion,” Biochi- [33] M. Biasini, S. Bienert, A. Waterhouse et al., “SWISS-MODEL: mica et Biophysica Acta, vol. 1726, no. 1, pp. 67–74, 2005. Modelling protein tertiary and quaternary structure using evo- [19] S. Cherif, A. Fendri, N. Miled, H. Trabelsi, H. Mejdoub, and lutionary information,” Nucleic Acids Research, vol. 42, Y. Gargouri, “Crab digestive lipase acting at high temperature: no. W1, pp. W252–W258, 2014. purification and biochemical characterization,” Biochimie, [34] N. Guex and M. C. Peitsch, “SWISS-MODEL and the vol. 89, no. 8, pp. 1012–1018, 2007. Swiss-Pdb Viewer: an environment for comparative protein 10 BioMed Research International modeling,” Electrophoresis, vol. 18, no. 15, pp. 2714–2723, 1997. [35] R. A. Laskowski, M. W. MacArthur, D. S. Moss, and J. M. Thornton, “PROCHECK: a program to check the stereochem- ical quality of protein structures,” Journal of Applied Crystal- lography, vol. 26, no. 2, pp. 283–291, 1993. [36] E. Bouchaâla, M. BouAli, Y. B. Ali, N. Miled, Y. Gargouri, and A. Fendri, “Biochemical characterization and molecular modeling of pancreatic lipase from a cartilaginous fish, the common stingray (Dasyatis pastinaca),” Applied Biochemistry and Biotechnology, vol. 176, no. 1, pp. 151–169, 2015. [37] A.-S. Rasmussen and U. Arnason, “Molecular studies suggest that cartilaginous fishes have a terminal position in the piscine tree,” Proceedings of the National Academy of Sciences, vol. 96, no. 5, pp. 2177–2182, 1999. [38] Y. Gargouri, H. Chahinian, H. Moreau, S. Ransac, and R. Verger, “Inactivation of pancreatic and gastric lipases by THL and C12:0-TNB: a kinetic study with emulsified tribu- tyrin,” Biochimica et Biophysica Acta, vol. 1085, no. 3, pp. 322–328, 1991. [39] C. Cudrey, H. Van Tilbeurgh, Y. Gargouri, and R. Verger, “Inactivation of pancreatic lipases by amphiphilic reagents 5- (dodecyldithio)-2-nitrobenzoic acid and tetrahydrolipstatin. Dependence upon partitioning between micellar and oil phases,” The Biochemist, vol. 32, no. 50, pp. 13800–13808, 1993. [40] N. Smichi, A. Fendri, R. Chaâbouni, F. B. Rebah, Y. Gargouri, and N. Miled, “Purification and biochemical characterization of an Acid-Stable lipase from the pyloric caeca of sardine (Sar- dinella aurita),” Applied Biochemistry and Biotechnology, vol. 162, no. 5, pp. 1483–1496, 2010. [41] A. Tiss, H. Lengsfeld, F. Carrière, and R. Verger, “Inhibition of human pancreatic lipase by tetrahydrolipstatin: further kinetic studies showing its reversibility,” Journal of Molecular Cataly- sis B: Enzymatic, vol. 58, no. 1-4, pp. 41–47, 2009. [42] Y. Gargouri, G. Piéroni, C. Rivière, L. Sarda, and R. Verger, “Inhibition of lipases by proteins: a binding study using dica- prin monolayers,” Biochemistry, vol. 25, no. 7, pp. 1733– 1738, 1986. [43] N. Smichi, Y. Gargouri, N. Miled, and A. Fendri, “A grey mullet enzyme displaying both lipase and phospholipase activities: purification and characterization,” International Journal of Biological Macromolecules, vol. 58, pp. 87–94, 2013. [44] Y. Gargouri, R. Julien, A. G. Bois, R. Verger, and L. Sarda, “Studies on the detergent inhibition of pancreatic lipase activ- ity,” Journal of Lipid Research, vol. 24, pp. 1336–1342, 1983. [45] M. P. Egloff, F. Marguet, G. Buono, R. Verger, C. Cambillau, and H. Van Tilbeurgh, “The 2.46 .ANG. resolution structure of the pancreatic lipase-colipase complex inhibited by a C11 alkyl phosphonate,” The Biochemist, vol. 34, no. 9, pp. 2751– 2762, 1995. [46] B. Borgström, C. Erlanson-Albertsson, and T. Wieloch, “Pan- creatic colipase: chemistry and physiology,” Journal of Lipid Research, vol. 20, no. 7, pp. 805–816, 1979. [47] X. Xiao, L. E. Ross, W. A. Sevilla, Y. Wang, and M. E. Lowe, “Porcine pancreatic lipase related protein 2 has high triglycer- ide lipase activity in the absence of colipase,” Biochimica et Biophysica Acta, vol. 1831, no. 9, pp. 1435–1441, 2013.