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Recombinant neutral endopeptidase-24.11 expressed in mouse neuroblastoma cells is associated with neurite membranes.

ABSTRACT: Neutral endopeptidase-24.11 (EC 3.4.24.11) (NEP) is a transmembrane metallo-endopeptidase that has been shown to be involved in the degradation of several mammalian neuropeptides, including enkephalins. The enzyme has recently been found to be specifically associated with the axonal and synaptic membranes of neurons in the globus pallidus of the pig brain. This result suggests that neurons must possess mechanisms for targeting NEP to particular membrane domains. Study of these mechanisms would greatly benefit from the existence of an established neuron-like cell line capable of expressing and targeting NEP to specific membrane domains. For this reason we have used a retroviral vector containing the cDNA for rabbit kidney NEP to express this enzyme in a mouse neuroblastoma cell line (Neuro2A). Labelling of the cell surface with an antibody coupled to colloidal gold particles and examination of the cells by electron microscopy revealed a non-uniform distribution of NEP at the surface of the cells, the protein being preferentially associated with the membrane of neurites compared with the cell body. This observation suggests that Neuro2A cells possess a mechanism for targeting NEP to specific domains of the plasma membrane. This cell line could thus constitute a good model for studying the mechanisms responsible for targeting this enzyme to specialized regions of the plasma membrane.

SUBMITTER: Lemay G

PROVIDER: S-EPMC1131309 | biostudies-other | 1990 Apr

REPOSITORIES: biostudies-other

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Project description:The human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation during growth, and expresses a number of brush-border-membrane-associated hydrolases typical of a differentiated phenotype. Among these are alkaline phosphatase, dipeptidyl peptidase IV and sucrase-isomaltase (sucrase, EC 3.2.1.48). Neutral endopeptidase 24.11 [EC 3.4.24.11, neprilysin (NEP)] is another abundant protease of normal enterocytes but its presence in Caco-2 cells has not been fully documented yet. In this paper, we show that Caco-2 cell extracts hydrolyse tritiated [D-Ala2Leu5]enkephalin with a Km of 180 microM, very close to the value obtained for the NEP present in the rabbit kidney (118 microM). Western-blot analysis of brush-border membranes purified from post-confluent cells revealed a protein with an apparent molecular mass of 94000 Da similar to that of the rabbit kidney NEP. The amount of enzyme in cell extracts increased as a function of the age of the culture, indicating that NEP expression is correlated with the degree of cell differentiation as is also the case for sucrase and dipeptidylpeptidase IV (DPP-IV). Binding of a radiolabelled antibody to Caco-2 cell monolayers grown on semi-permeable filters indicated that 95% of NEP molecules present at the cell surface are on the apical side. Immunocytochemical and flow cytometric analysis of intact and permeabilized cells were also used to investigate the presence of NEP and DPP-IV at the surface of Caco-2 cells. Whereas DPP-IV staining appeared to be homogeneous throughout the entire cell population, NEP-related fluorescence exhibited a bimodal distribution which indicates an uneven expression of the protein at the cell surface. Permeabilization of monolayers with saponin before staining restored a labelling pattern for NEP similar to the one obtained for DPP-IV. This suggests that although DPP-IV and NEP follow similar patterns of expression when enzymic activities are measured on whole-cell extracts, targeting of these brush-border proteins to the cell surface appears to be regulated in different ways.

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Recombinant neutral endopeptidase-24.11 expressed in mouse neuroblastoma cells is associated with neurite membranes.

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