Ectonucleotide pyrophosphatase/phosphodiesterase activity in Neuro-2a neuroblastoma cells: changes in expression associated with neuronal differentiation

Authors

  • Rosa Gómez-Villafuertes,

    1. Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
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  • Jesús Pintor,

    1. Departamento de Bioquímica, Facultad de Óptica y Optometría, Universidad Complutense de Madrid, Madrid, Spain
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  • María Teresa Miras-Portugal,

    1. Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
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  • Javier Gualix

    Corresponding author
    1. Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
    • Address correspondence and reprint requests to Dr Javier Gualix, Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, UCM, Av. Puerta de Hierro s/n, 28040-Madrid, Spain. E-mail: jgualix@vet.ucm.es

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Abstract

Neuro-2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The ApnA-cleaving activity has been analysed with the use of the fluorogenic compound BODIPY® FL guanosine 5′-O-(3-thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 μM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY® FL guanosine 5′-O-(3-thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn2+ ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal-like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation.

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We described the presence of an ectoenzymatic activity able to hydrolyse diadenosine polyphosphates (ApnA) in N2a cells. This activity displays biochemical features that are typical of the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members, as demonstrated by the use of the fluorogenic compound BODIPY-FL-GTPγS. Both NPP1 and NPP3 ectoenzymes are expressed in N2a cells, their levels dramatically changing when cells differentiate into a neuronal-like phenotype. Activity assays in differentiated cells showed that the ApnA-hydrolytic activity largely depends on the NPP1 isozyme.

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