Intestinal phospholipase A2 from Sparidae species: Functional properties and cytotoxic potential evaluation

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Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights

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

Functional and Structural Characterization of a Thermostable Phospholipase A(2) from a Sparidae Fish (Diplodus annularis)

International audienceNovel phospholipase (PLA(2)) genes from the Sparidae family were cloned. The sequenced PLA(2) revealed an identity with pancreatic PLA(2) group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA(2) (DaPLA(2)) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPIA(2) was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 31) structure of DaPLA(2) was constructed to gain insights into the functional properties of sparidae PLA(2). Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA(2). Using the monolayer technique, the purified DaPLA(2) was found to be active on various phospholipids ranging from 10 to 20 mN-m(-1), which explained the absence of the hemolytic activity for DaPLA(2)

Efficient heterologous expression, functional characterization and molecular modeling of annular seabream digestive phospholipase A2.

International audienceHere we report the cDNA cloning of a phospholipase A2 (PLA2) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA2. In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA2 are closer to avian PLA2 group IB than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA2 (AsPLA2). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA2 with a molecular mass of 15kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400U.mg(-1) measured on phosphatidylcholine at pH 8.5 and 50°C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA2. By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA2 at high temperatures was explained. Using the monomolecular film technique, AsPLA2 was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25dyncm(-1). Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA2, provides new insights on structure-activity relationships of fish PLA2

Functional and Structural Characterization of a Thermostable Phospholipase A₂ from a Sparidae Fish (<i>Diplodus annularis)</i>

Novel phospholipase (PLA₂) genes from the Sparidae family were cloned. The sequenced PLA₂ revealed an identity with pancreatic PLA₂ group IB. To better understand the structure/function relationships of these enzymes and their evolution, the <i>Diplodus annularis</i> PLA₂ (DaPLA₂) was overexpressed in <i>E. coli</i>. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA₂ was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA₂ was constructed to gain insights into the functional properties of sparidae PLA₂. Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA₂. Using the monolayer technique, the purified DaPLA₂ was found to be active on various phospholipids ranging from 10 to 20 mN·m^–1, which explained the absence of the hemolytic activity for DaPLA₂