The substrate specificities of sunflower and soybean phospholipases D using transphosphatidylation reaction

<p>Abstract</p> <p>Background</p> <p>Phospholipase D (PLD) belongs to a lipolytic enzyme subclass which catalyzes the hydrolysis and transesterification of glycerophospholipids at the terminal phosphodiester bond.</p> <p>Results</p> <p>In this work, we have studied the substrate specificity of PLDs from germinating sunflower seeds and cultured-soybean cells, using their capacity of transphosphatidylation. In the presence of a nucleophilic acceptor, such as [¹⁴C]ethanol, PLD catalyzes the production of phosphatidyl-[¹⁴C]-ethanol. The resulting product is easily identified since it is well separated from the other lipids by thin-layer chromatography. The main advantage of this assay is that the phospholipid used as substrate does not need to be radiolabelled and thus allow us a large choice of polar heads and fatty acids. <it>In vitro</it>, we observed that sunflower and soybean cell PLD show the following decreasing order of specificity: phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol; while phosphatidylserine and phosphatidylinositol are utilized much less efficiently.</p> <p>Conclusions</p> <p>The substrate specificity is modulated by the fatty acid composition of the phosphatidylcholine used as well as by the presence of other charged phospholipids.</p

Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

Cysteine-rich domains (Cys-domains) are ∼50–amino acid–long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-γ (Cys1–GFP). Strikingly, stimulation of G-protein or tyrosine kinase–coupled receptors induced a transient translocation of cytosolic Cys1–GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1–GFP in the membrane, whereas DiC8 left Cys1–GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1–GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-γ also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2–GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester–mediated translocation of proteins to selective lipid membranes

Development of a direct and continuous phospholipase D assay based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline

International audienceOur knowledge about mesenchymal stem cells has considerably grown in the last years. Since the proof of concept of the existence of such cells in the 70s by Friedenstein et al., a growing mass of reports were conducted for a better definition of these cells and for the reevaluation from the term “mesenchymal stem cells” to the term “mesenchymal stromal cells (MSCs).” Being more than a semantic shift, concepts behind this new terminology reveal the complexity and the heterogeneity of the cells grouped in MSC family especially as these cells are present in nearly all adult tissues. Recently, mesenchymal stromal cell antigen-1 (MSCA-1)/tissue nonspecific alkaline phosphatase (TNAP) was described as a new cell surface marker of MSCs from different tissues. The alkaline phosphatase activity of this protein could be involved in wide range of MSC features described below from cell differentiation to immunomodulatory properties, as well as occurrence of pathologies. The present review aims to decipher and summarize the role of TNAP in progenitor cells from different tissues focusing preferentially on brain, bone marrow, and adipose tissue

Lipases or esterases: does it really matter? Toward a new bio-physico-chemical classification.

International audienceCarboxylester hydrolases, commonly named esterases, consist of a large spectrum of enzymes defined by their ability to catalyze the hydrolysis of carboxylic ester bonds and are widely distributed among animals, plants, and microorganisms. Lipases are lipolytic enzymes which constitute a special class of carboxylic esterases capable of releasing long-chain fatty acids from natural water-insoluble carboxylic esters. However, up to now, several unsuccessful attempts aimed at differentiating "lipases" from "esterases" by using various criteria. These criteria were based on the first substrate used chronologically, primary sequence comparisons, some kinetic parameters, or some structural features.Lipids are biological compounds which, by definition, are insoluble in water. Taking into account this basic physico-chemical criterion, we primarily distinguish lipolytic esterases (L, acting on lipids) from nonlipolytic esterases (NL, not acting on lipids). In view of the biochemical data accumulated up to now, we proposed a new classification of esterases based on various criteria of physico-chemical, chemical, anatomical, or cellular nature. We believe that the present attempt matters scientifically for several reasons: (1) to help newcomers in the field, performing a few key experiments to figure out if a newly isolated esterase is lipolytic or not; (2) to clarify a debate between scientists in the field; and (3) to formulate questions which are relevant to the still unsolved problem of the structure-function relationships of esterases

Functional Characterization of the N-Terminal C2 Domain from Arabidopsis thaliana Phospholipase Dα and Dβ

Most of plant phospholipases D (PLD) exhibit a C2-lipid binding domain of around 130 amino acid residues at their N-terminal region, involved in their Ca2+-dependent membrane binding. In this study, we expressed and partially purified catalytically active PLDα from Arabidopsis thaliana (AtPLDα) in the yeast Pichia pastoris. The N-terminal amino acid sequence of the recombinant AtPLDα was found to be NVEETIGV and thus to lack the first 35 amino acid belonging to the C2 domain, as found in other recombinant or plant purified PLDs. To investigate the impact of such a cleavage on the functionality of C2 domains, we expressed, in E. coli, purified, and refolded the mature-like form of the C2 domain of the AtPLDα along with its equivalent C2 domain of the AtPLDβ, for the sake of comparison. Using Förster Resonance Energy Transfer and dot-blot assays, both C2 domains were shown to bind phosphatidylglycerol in a Ca2+-independent manner while phosphatidic acid and phosphatidylserine binding were found to be enhanced in the presence of Ca2+. Amino acid sequence alignment and molecular modeling of both C2 domains with known C2 domain structures revealed the presence of a novel Ca2+-binding site within the C2 domain of AtPLDα

Plant phospholipase D mining unravels new conserved residues important for catalytic activity

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Phospholipases: An Overview

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Development of a Direct and Continuous Phospholipase D Assay Based on the Chelation-Enhanced Fluorescence Property of 8‑Hydroxyquinoline

Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca²⁺ complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca²⁺/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 μM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant <i>Vigna unguiculata</i> PLD, as well as the PLD from <i>Streptomyces chromofuscus</i>, cabbage, or peanuts, and no PA production could be detected using phospholipase A₁, phospholipase A₂, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale