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M. Méthot, E. Demers, R. Breton, B. Desbat, C. Salesse; Membrane Interactions of Cytosolic Phospholipase A2 Gamma From the Human Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2942.
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The brush-shaped apical surface of the retinal pigment epithelium (RPE) enables phagocytosis of rod outer segments (ROS) and the production of phagosomes. RPE is responsible to digest those phagosomes via lysosomial enzymes such as phospholipases A2 (PLA2) enzymes, and also to recycle some components of the phagosomes such as the polyunsaturated fatty acids (PUFA), which have a unique composition in ROS membranes. As a matter of fact, more than 60% of the fatty acids of the ROS phospholipids are polyunsaturated. It is by far the most unsaturated natural membrane known to this date. Those membranes are thus particularly vulnerable to oxidation. We have recently demonstrated that RPE expresses a cytosolic PLA2 (cPLA2) gamma. The specificity and constitutive activity of this PLA2 suggests that it could participate in some key roles of RPE. The objectives of this research project were to overexpress and purify this protein, and to characterize its enzymatic and binding properties in model membranes.
The sequence corresponding to the human retina cPLA2 gamma was cloned into the expression vector pET Champion containing an N-terminal His-tag, and this vector was transformed in E. coli. Protein expression was induced by IPTG and purification was accomplished by a sequence of affinity and gel exclusion chromatographies. Enzymatic activity was measured in a calcium-depleted media by the hydrolysis of radiolabelled small unilamellar vesicles. The interaction of cPLA2-gamma with lipid monolayers at the air-water interface was assayed using surface pressure measurements along with infrared spectroscopy (PMIRRAS).
Hydrolysis experiments with radiolabelled vesicles demonstrated that the recombinant protein was active in a calcium-depleted media. The injection of the enzyme underneath phospholipid monolayers of varying chain lengths and polar head group moieties led to an increase in surface pressure. Infrared spectra obtained by PMIRRAS showed the characteristic Amide I and II bands that are typical of alpha helix secondary structure, suggesting that the integrity of the protein is maintained in this environment.
The recombinant protein cPLA2-gamma was found to be active in hydrolysis experiments in vesicles in a calcium-independent manner. It readily interacts with phospholipid monolayers and maintains a high degree of alpha helical structure at the air-water interface. Measurements will then be performed to find out the specificity of this enzyme towards PUFA.
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