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Benoît Van der Rest

Glycerophosphocholine metabolism in plant: Characterization of a new glycerophosphodiester phosphodiesterase

Published on 1 February 2002

Thesis presented February 01, 2002

Glycerophosphocholine (GPC) is a diester usually present in plant cells at a low concentration. However, it can accumulate under different physiological processes leading to phospholipid remodeling in higher plant cells. Therefor, the aim of this work was to characterize its catabolism.
31P-nuclear magnetic resonance spectroscopy and biochemical analyses performed on carrot cells (Daucus carota) fed with glycerophosphocholine revealed the existence of an extracelllar glycerophosphocholine phosphodiesterase (GPC-PDE). This enzymatic activity splits glycerophosphocholine into sn-glycerol-3-phosphate and free choline. In vivosn-glycerol-3-phosphate was further hydrolyzed in glycerol and inorganic phosphate by acid phosphatases. We visualized the incorporation and the compartmentation of choline, and observed that the major pool of choline was phosphorylated and accumulated in the cytosol whereas a minor fraction was incorporated in the vacuole as free choline. Extracellular GPC-PDE was localized in the cell wall. We also report the existence of a similar GPC-PDE, located in the vacuole sap. Both extra- and intracellular GPC-PDE activities are widespread among different plant species and are often enhanced during phosphate deprivation.
The enzyme responsible for GPC-PDE activity was purified from carrot cell wall over 2700-fold. A 55 kDa polypeptide was co-purified with GPC-PDE activity. It consists of a glycoprotein that exhibits a high affinity for GPC (Km=33 µM) and that hydrolyzes GPC and other glycerophosphodiesters (glycerophosphoethanolamine, glycerophosphoinositol, glycerophosphoserine and glycérophosphoglycerol).
Alternatively, a molecular approach was based on the existence of a bacterial GPC-PDE. Indeed carrot GPC-PDE shares common features with the Escherichia coli glycerol-phosphodiester phosphodiesterase (GLPQ). Therefore, we isolated an Arabidopsis thaliana cDNA clone homologous to GLPC. Overexpression of Arabidopsis thaliana cDNA did not yield to any soluble GPC-PDE (accumulation in inclusion bodies). However, antibodies raised against the recombinant protein hybridized specifically with the 55 kDa protein co-purified with GPC-PDE activity. The strong correlation between GPC-PDE activity and the detection of the 55 kDa protein by the antibodies and the sequences deduced from the 55 kDa protein inner fragments indicate that the 55 kDa protein, homologous to bacterial phosphodiesterase, is responsible for the hydrolysis of GPC in plant cells.

Phospholipid polar head turnover, glycerophosphocholine, phosphodiesterase, nuclear magnetic resonance, carrot cells, wall, vacuole

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