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D Corbeil

Publications and source records attributed to D Corbeil.

8 recordsLinked to original sources
PubMed1995

Proteolytic processing of the alpha-subunit of rat endopeptidase-24.18 by furin.

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.

Amino Acid Sequence↗
PubMed1994

Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity.

Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.

Amino Acid Sequence↗
PubMed1994

[Update on menopause].

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Estrogen Replacement Therapy↗
PubMed1993

Rat endopeptidase-24.18 alpha subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells.

Endopeptidase-24.18 (EC 3.4.24.18, E-24.18) is an oligomeric Zn-ectoenzyme. The alpha and beta subunits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C-terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E-24.18 alpha subunit and to test the functionality of the astacin-like domain in the alpha subunit when expressed alone, COS-1 cells were transfected with a cloned cDNA for rat alpha subunit. Despite the presence of its putative transmembrane domain, the alpha subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the alpha subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu-157 in the active site by Val. Taken together our results suggest that the alpha subunit of Endopeptidase-24.18 contains a latent astacin-like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.

Animals↗
PubMed1992

Polarized distribution of neutral endopeptidase 24.11 at the cell surface of cultured human intestinal epithelial Caco-2 cells.

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.

Animals↗
PubMed1992

Molecular cloning of the alpha-subunit of rat endopeptidase-24.18 (endopeptidase-2) and co-localization with endopeptidase-24.11 in rat kidney by in situ hybridization.

Endopeptidase-24.18 (endopeptidase-2, EC 3.4.24.18, E-24.18) is a Zn-ectoenzyme of rat renal and intestinal microvillar membranes exhibiting an oligomeric structure, alpha 2-beta 2. The primary structure of the alpha-subunit of E-24.18 has been defined by molecular cloning and its expression mapped in rat kidney by in situ hybridization. A 2.9-kb cDNA coding for the alpha-subunit was isolated and sequenced. It had an open reading frame of 2,244 base pairs coding for a type I membrane protein of 748 amino acids. The deduced amino acid sequence showed 87% identity with that of meprin A, a mouse metallo-endopeptidase, sharing common properties with the rat enzyme, and 85% identity with the human intestinal enzyme, 'PABA-peptide hydrolase'. Northern blot analysis revealed the alpha-subunit to be encoded by a single mRNA species of 3.2-kb. In situ hybridization performed on rat kidney showed a co-localization of E-24.18 with endopeptidase-24.11 in proximal tubules of juxtamedullary nephrons, suggesting that the two enzymes have similar or complementary physiological functions in kidney.

Amino Acid Sequence↗
PubMed1992

Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains.

Neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) is an abundant metalloendopeptidase of the brush border membrane of kidney proximal tubules. We have recently shown that NEP is delivered directly to the apical domain of the plasma membrane when expressed in polarized Madin-Darby canine kidney (MDCK) cells in culture (Jalal, F., Lemay, G., Zollinger, M., Berteloot, A., Boileau, G., and Crine, P. (1991) J. Biol. Chem. 266, 19826-19832). Here, a soluble form of NEP consisting of the signal peptide of pro-opiomelanocortin fused in-frame with the ectodomain of NEP has been expressed in MDCK cells. Enzymatic assays performed on apical and basolateral culture media of MDCK cells grown on semi-permeable supports indicated that the recombinant enzyme was predominantly released at the apical surface. In contrast, when the chimeric protein was expressed in NIH 3T3 cells or when pro-opiomelanocortin was expressed in MDCK cells, non-polarized secretion was observed into both the apical and basolateral compartments of the culture chamber. Our results suggest that the ectodomain of NEP is sufficient for directing the targeting of this protein to the apical membrane of polarized MDCK epithelial cells.

3T3 Cells↗