Study of galactolipid enzymatic hydrolysis by various chromatographic and spectroscopic techniques

Les galactolipides sont les principaux constituants des membranes formant les thylacoïdes des chloroplastes des plantes, des algues et des cyanobactéries. Ils sont considérés comme la plus grande réserve d’acide gras sur la planète. La lipase pancréatique apparentée de type 2 (PLRP2) est une enzyme permettant à de nombres mammifères de digérer les galactolipides et d’accéder à cette importante source d’acide gras. Cette thèse a contribué au développement de diversesméthodes d’analyse pour l’étude de l’hydrolyse enzymatique des galactolipides, en particulier le monogalactosyldiacylglycerol (MGDG), par la PLRP2 du cobaye (GPLRP2).Dans un premier temps, nous avons étudié l’hydrolyse d’un galactolipide synthétique à chainesacyles moyennes, le C8-MGDG en utilisant un outil classique de la lipidomique, lachromatographie sur couche mince (CCM) couplée à la densitométrie. Le choix du réactif authymol et à l’acide sulfurique comme révélateur nous a permis de quantifier simultanément tousles composés galactosylés dans le milieu réactionnel. Les courbes cinétiques de la disparitionde MGDG et de la production de monogalactosylmonoacylglycerol (MGMG) et de monogalactosylglycerol (MGG) ont pu ainsi être établies. Le désavantage de cette méthode estqu’elle nécessite de stopper la réaction, d’extraire et de transformer les composés à quantifier,autant d’étapes qui augmentent l’erreur dans la quantification mais aussi le risque de l’évolutiondes composés avant leur révélation.Dans un second temps, la même réaction a été étudiée mais avec des outils moins fréquents en lipidomique, permettant de suivre l’hydrolyse en temps réel sans extraction ni transformation des composés à analyser. Le premier outil utilisé a été la spectroscopie infrarouge (IR), nécessitant de réaliser la réaction dans le D2O. L’enregistrement successif de spectres IR dumélange réactionnel au cours du temps a permis d’observer des changements importants dans les vibrations des carbonyle, carboxylate et méthylène. Ces changements ont été attribués à la consommation de MGDG et à la production de MGMG et d’acide octanoïque. L’étalonnage de l’absorption du MGDG, du MGMG et de l’acide octanoïque dans la région des vibrationsprécédemment citées a ensuite permis de quantifier ces composés à partir du spectre du mélange réactionnel. Par la spectroscopie infrarouge et par la CCM, la même activité spécifique de la GPLRP2 et les mêmes courbes cinétiques de disparition du substrat et d’apparition des produits ont été obtenues en étudiant la réaction dans le D2O. La chromatographie sur couche mince et la spectroscopie infrarouge nous ont aussi permis de mettre en évidence l’action direct de la GPLRP2 sur les galactolipides membranaires des chloroplastes. Enfin, la spectroscopie de résonance magnétique nucléaire (RMN) du proton a été utilisée pour analyser la même réaction dans le D2O. Contrairement à la CCM et à la spectroscopie IR, quin’ont pas permis de quantifier directement l’acide octanoïque et le MGG respectivement, laRMN a permis de quantifier à la fois le MGDG, le MGMG, le MGG et l’acide octanoïque grâceà l’intégration des pics de résonnance de certains protons identifiés. Outre l’obtention de la même cinétique qu’avec les deux premières méthodes, avec des données plus complètes, la RMN a permis aussi de caractériser les structures supramoléculaires dans lesquelles se trouvent les différents composés par l’estimation de coefficients de diffusion et de diamètres.hydrodynamiques en utilisant la RMN DOSY (Diffusion Ordered SpectroscopY)Galactolipids are the main components of thylakoid membranes in the chloroplasts of plants, algae and cyanobacteria.Galactolipids are considered as the most abundant source of fatty acid on the planet. The pancreatic lipase related protein 2 (PLRP2) is an enzyme allowing some mammals to digest this important source of fatty acids. The present thesis contributed to the development of analytical methods for the study of galactolipids hydrolysis, in particular monogalactosyldiacylglycerol (MGDG), by guinea pig PLRP2 (GPLRP2). First, we studied the hydrolysis of a synthetic medium acyl chains galactolipid, C8-MGDG , by GPLRP2 using a classical lipidomics tool, the thin layer chromatography (TLC) coupled with densitometry. We selected the thymol and sulfuric acid reagent as derivating and staining agent, this allowed us to simultaneously quantify all galactosylated compounds in the reaction medium. Kinetics curves for MGDG consumption and monogalactosylmonoacylglycerol(MGMG) and monogalactosylglycerol (MGG) production were thus established. The drawbackof this method is the need to stop the reaction, to extract and derivatize the compounds to quantify, many steps that increase error in quantification and also the risk of evolution of the compounds before their revelation. Second, the same reaction was studied with less common tools in lipidomics, which allow to monitor the hydrolysis in real time without extraction or derivatization of the compounds. The first tool used was infrared (IR) spectroscopy, which requires to perform the reaction in D2O. By recording series of reaction mixture spectra over time, we observed significant changes in the vibrations of carbonyl, carboxylate and methylene. These changes were attributed to the consumption of MGDG and the production of MGMG and octanoic acid. Calibration of MGDG, MGMG and octanoic acid absorption in the region of the above mentioned vibrationsallowed the quantification of these compounds from the reaction mixture spectra. The same specific activity of GPLRP2 and kinetic curves were obtained by studying the reaction in D2O by infrared spectroscopy and TLC. Thin-layer chromatography and infrared spectroscopy also allowed observing the direct action of GPLRP2 on galactolipids from chloroplast membranes. Proton nuclear magnetic resonance (NMR) spectroscopy was then used to analyze the same reaction in D2O. Unlike TLC and IR pectroscopy, which could not directly quantify octanoic acid and MGG respectively, NMR allowed the quantification of MGDG, MGMG, MGG and octanoic acid from the integration of the resonance peaks of the protons attributed to these compounds. This method allowed not only to obtain the same kinetics than with the first two methods, with more complete data, but also to associate supramolecular structures such as micelles to the identified compounds, based on diffusion coefficients and hydrodynamic diameters estimated by DOSY (Diffusion Ordered SpectroscopY) NMR

IR spectroscopy analysis of pancreatic lipase-related protein 2 interaction with phospholipids: 3. Monitoring DPPC lipolysis in mixed micelles

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Monitoring galactolipid digestion and simultaneous changes in lipid-bile salt micellar organization by real-time NMR spectroscopy

International audienceThe use of Nuclear Magnetic Resonance spectroscopy for studying lipid digestion in vitro most often consists of quantifying lipolysis products after they have been extracted from the reaction medium using organic solvents. However, the current sensitivity level of NMR spectrometers makes possible to avoid the extraction step and continuously quantify the lipids directly in the reaction medium. We used real-time 1H NMR spectroscopy and guinea pig pancreatic lipase-related protein 2 (GPLRP2) as biocatalyst to monitor in situ the lipolysis of monogalactosyl diacylglycerol (MGDG) in the form of mixed micelles with the bile salt sodium taurodeoxycholate (NaTDC). Residual substrate and lipolysis products (monogalactosyl monoacylglycerol (MGMG); monogalactosylglycerol (MGG) and octanoic acid (OA) were simultaneously quantified throughout the reaction thanks to specific proton resonances. Lipolysis was complete with the release of all MGDG fatty acids. These results were confirmed by thin layer chromatography (TLC) and densitometry after lipid extraction at different reaction times. Using diffusion-ordered NMR spectroscopy (DOSY), we could also estimate the diffusion coefficients of all the reaction compounds and deduce the hydrodynamic radius of the lipid aggregates in which they were present. It was shown that MGDG-NaTDC mixed micelles with an initial hydrodynamic radius rH of 7.3 ± 0.5 nm were changed into smaller micelles of NaTDC-MGDG-MGMG of 2.3 ± 0.5 nm in the course of the lipolysis reaction, and finally into NaTDC-OA mixed micelles (rH of 2.9 ± 0.5 nm) and water soluble MGG. These results provide a better understanding of the digestion of galactolipids by PLRP2, a process that leads to the complete micellar solubilisation of their fatty acids and renders their intestinal absorption possible

IR spectroscopy analysis of pancreatic lipase-related protein 2 interaction with phospholipids: 2. Discriminative recognition of various micellar systems and characterization of PLRP2-DPPC-bile salt complexes

International audienceThe interaction of pancreatic lipase-related protein 2 (PLRP2) with various micelles containing phospholipids was investigated using pHstat enzyme activity measurements, differential light scattering, size exclusion chromatography (SEC) and transmission IR spectroscopy. Various micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and lysophosphatidylcholine were prepared with either bile salts (sodium taurodeoxycholate or glycodeoxycholate) or Triton X-100, which are substrate-dispersing agents commonly used for measuring phospholipase activities. PLRP2 displayed a high activity on all phospholipid-bile salt micelles, but was totally inactive on phospholipid-Triton X-100 micelles. These findings clearly differentiate PLRP2 from secreted pancreatic phospholipase A2 which is highly active on both types of micelles. Using an inactive variant of PLRP2, SEC experiments allowed identifying two populations of PLRP2-DPPC-bile salt complexes corresponding to a high molecular weight 1:1 PLRP2-micelle association and to a low molecular weight association of PLRP2 with few monomers of DPPC / bile salts. IR spectroscopy analysis showed how DPPC-bile salt micelles differ from DPPC-Triton X-100 micelles by a higher fluidity of acyl chains and higher hydration / H-bonding of the interfacial carbonyl region. The presence of bile salts allowed observing changes in the IR spectrum of DPPC upon addition of PLRP2 (higher rigidity of acyl chains, dehydration of the interfacial carbonyl region), while no change was observed with Triton X-100. The differences between these surfactants and their impact on substrate recognition by PLRP2 are discussed, as well as the mechanism by which high and low molecular weight PLRP2-DPPC-bile salt complexes may be involved in the overall process of DPPC hydrolysis

In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves

International audienceThe removal of intact chloroplasts from their cell wall confinement offers a novel way to obtain lipophilic nutrients from green biomass, especially carotenoids and galactolipids. These latter are the main membrane lipids in plants and they represent a major source of the essential -linolenic acid (18:3; ALA). Nevertheless, knowledge on their digestion is still limited. We have developed a physical method of recovering a chloroplast-rich fraction (CRF) from green biomass and tested its digestibility in vitro under simulated gastrointestinal conditions. Using a two-step static model, CRF from both spinach leaves and postharvest, pea vine field residue (haulm) were first exposed to enzymes from rabbit gastric extracts and then either to pancreatic enzymes from human pancreatic juice (HPJ) or to porcine pancreatic extracts (PPE). The lipolysis of monogalactosyldiacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) was monitored by thin layer chromatography and gas chromatography of fatty acid methyl esters. For both CRF preparations, MGDG and DGDG were converted to monogalactosylmonoacylglycerol (MGMG) and digalactosylmonoacylglycerol (DGMG), respectively, during the intestinal phase and ALA was the main fatty acid released. Galactolipids were more effectively hydrolysed by HPJ than by PPE, and PPE showed a higher activity on MGDG than on DGDG. These findings may be explained by the higher levels of galactolipase activity in HPJ compared to PPE, which mainly results from pancreatic lipase-related protein 2. Thus, we showed that CRF galactolipids are well digested by pancreatic enzymes and represent an interesting vehicle for ALA supplementation in human diet

Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles, monomolecular films and UV-absorbing surface-coated substrate

International audienceTalaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10 ng of enzymes, against 100 ng to 10 μg with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate

Using Enzymes to Harvest Fatty Acids from Galactosyldiacylglycerols, the Most Abundant Lipids in Plant Biomass

International audienceMono- and digalactosyldiacylglycerols (MGDG, DGDG), the main lipids of plant photosynthetic membranes, represent a large but unexploited reservoir of fatty acids on earth. They are dispersed in plant biomass (milligrams per gram of dry mass) and not as accessible as vegetable oils by simple physical means. The identification and characterization of galactolipid acyl hydrolases, or galactolipases, raise the possibility to use these biocatalysts for the bioconversion of galactolipids. Here, we show that two enzymes of mammalian and microbial origins, pancreatic lipase-related protein 2 from guinea pig (GPLRP2) and cutinase from Fusarium solani, have the capacity to directly and fully release the fatty acids of MGDG and DGDG present in various plant leaves and green wastes. This high substrate accessibility to enzymes was further explored by performing alcoholysis reactions in situ and showing the conversion of galactolipid fatty acids into fatty acid alkyl esters (FAAE) when the enzyme and leaves were incubated in the presence of 6 or 2.5 M ethanol. These findings pave the way to the recovery of fatty acids dispersed in green biomass and the exploitation of an additional and renewable source of fatty acids for oleochemistry and nutrition in a context of competition for vegetable oils

Bioaccessibility of essential lipophilic nutrients in a chloroplast-rich fraction (CRF) from agricultural green waste during simulated human gastrointestinal tract digestion

International audienceThe lipophilic nutrients in a chloroplast-rich fraction derived from pea vine postharvest field-residue are released in an in vitro digestion model; the extent of their release (bioaccessibility) is affected by heat-treatment of biomass or juice

The digestion of galactolipids and its ubiquitous function in Nature for the uptake of the essential α-linolenic acid

International audienceGalactolipids, mainly monogalactosyl diglycerides and digalactosyl diglycerides are the main lipids found in the membranes of plants, algae and photosynthetic microorganisms like microalgae and cyanobacteria. As such, they are the main lipids present at the surface of the earth. They may represent up to 80 % of the fatty acid stocks, including a large proportion of polyunsaturated fatty acids mainly -linolenic acid (ALA). Nevertheless, the interest in these lipids for nutrition and other applications remains overlooked, probably because they are dispersed in the biomass and are not as easy to extract as vegetable oils from oleaginous fruit and oil seeds. Another reason is that galactolipids only represent a small fraction of the acylglycerolipids present in modern human diet. In herbivores such as horses, fish and folivorous insects, galactolipids may however represent the main source of dietary fatty acids due to their dietary habits and digestion physiology. The development of galactolipase assays has led to the identification and characterization of the enzymes involved in the digestion of galactolipids in the gastrointestinal tract, as well as by microorganisms. Pancreatic lipase-related protein 2 (PLRP2) has been identified as an important factor of galactolipid digestion in humans, together with pancreatic carboxyl ester hydrolase (CEH). The levels of PLRP2 are particularly high in monogastric herbivores thus highlighting the peculiar role of PLRP2 in the digestion of plant lipids. Similarly, pancreatic lipase homologs are found to be expressed in the midgut of folivorous insects, in which a high galactolipase activity can be measured. In fish, however, CEH is the main galactolipase involved. This review discusses the origins and fatty acid composition of galactolipids and the physiological contribution of galactolipid digestion in various species. This overlooked aspect of lipid digestion ensures not only the intake of ALA from its main natural source, but also the main lipid source of energy for growth of some herbivorous species