Distribution of, and a putative role for, the cell-surface neutral metallo-endopeptidases during mammalian craniofacial development

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3213-3226 ◽  
Author(s):  
B. Spencer-Dene ◽  
P. Thorogood ◽  
S. Nair ◽  
A.J. Kenny ◽  
M. Harris ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Cell Surface ◽  
Choroid Plexus ◽  
Neutral Endopeptidase ◽  
Apical Surface ◽  
Endopeptidase 24.11 ◽  
Ependymal Layer ◽  
Enzymes Inhibition ◽  
Wide Range ◽  
Inhibition Studies

Endopeptidase-24.11 (neutral endopeptidase, neprilysin, ‘enkephalinase’, EC 3.4.24.11) and endopeptidase-24.18 (endopeptidase-2, meprin, EC 3.4.24.18) are cell-surface zinc-dependent metallo-endopeptidases able to cleave a variety of bioactive peptides including growth factors. We report the first study of the cellular and tissue distribution of both enzymes and of the mRNA for NEP during embryonic development in the rat. Endopeptidase-24.11 protein was first detected at E10 in the lining of the gut and, at E12, the enzyme was present on the notochord, medial and lateral nasal processes, otocyst, mesonephros, heart and neuroepithelium. In contrast, at this time endopeptidase-24.18 was present only on the apical surface of the neuroepithelial cells. By E14 and E16, NEP was also detected in a wide range of craniofacial structures, notably the palatal mesenchyme, the choroid plexus, tongue and perichondrium. The distribution of endopeptidase-24.18 at these stages was restricted to the inner ear, the nasal conchae, and ependymal layer of the brain ventricles and the choroid plexus. Although endopeptidase-24.11 had been detectable in the craniofacial vasculature at E12 and E14, this was no longer apparent at E16. Significantly, the distribution of endopeptidase-24.11 mRNA closely matched the immunolocalization of the protein at all stages investigated. In order to explore the functional role of these enzymes, inhibition studies were carried out using two selective inhibitors of endopeptidase-24.11, phosphoramidon and thiorphan. E9.5 and E10.5 embryos exposed to either inhibitor displayed a characteristic, asymmetric abnormality consisting of a spherical swelling, possibly associated with a haematoma, predominantly on the left side of the prosencephalon, and the severity of this defect appeared to be a dose-dependent phenomenon. This study suggests that these enzymes play previously unrecognized roles during mammalian embryonic development.

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

1992 ◽  
Vol 288 (3) ◽  
pp. 945-951 ◽  
Author(s):  
F Jalal ◽  
C Jumarie ◽  
W Bawab ◽  
D Corbeil ◽  
C Malo ◽  
...  
Keyword(s):  
Cell Surface ◽  
Brush Border ◽  
Flow Cytometric Analysis ◽  
Neutral Endopeptidase ◽  
Rabbit Kidney ◽  
Human Colon Cancer ◽  
Cell Extracts ◽  
Endopeptidase 24.11 ◽  
Dpp Iv ◽  
Apical Side

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.

Immunogold labelling of neutral endopeptidase 24.11 (enkephalinase), on human neutrophils and neutrophil cytoplasts

1989 ◽  
Vol 47 ◽  
pp. 920-921
Author(s):  
V. Kriho ◽  
B. Wagner ◽  
E.G. Erdos ◽  
R.P. Becker
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Intact Cell ◽  
Neutral Endopeptidase ◽  
Immunogold Labelling ◽  
Human Neutrophils ◽  
Endopeptidase 24.11 ◽  
Cell Surface Structure

We have documented the presence of neutral endopeptidase 24.11 (NEP), on the surface of human neutrophils (PMN) and PMN cytoplasts. Cytoplasts are whole cell preparations which contain cytomatrix, but lack internal membranes and organelles ,such as nuclei and lysosomal granules. These structures have been extracted mechanically, leaving the plasma membrane “outside-out” topology intact. Cytoplasts are very useful in correlative studies of cell surface structure and function. Biochemically, the membrane component of cytoplasts is predominantly plasma membrane; structurally, chemical activity may be localized to domains of the intact cell surface. NEP is a membrane-bound metalloendopeptidase present in human PMN' s. We have marked NEP on the plasma membranes of PMNs and PMN cytoplasts via pre-embedding iramunocytochemistry. We used scanning electron microscopy (SEM) with backscattered electron imaging (BEI) to visualize Au labelled anti-NEP on the surface of a large number of cells. Transmission electron microscopy (TEM) was used to confirm the presence of the enzyme on PMN's and PMN cytoplasts.Suspensions of PMN or PMN cytoplasts (2 x 106 cells/ml) were fixed for 8 min at room temp. in 0.25% glutaraldehyde in phosphate buffered saline (PBS) pH 7.2 rinsed in PBS, treated with 0.1% glycine in PBS for 10 rain and then incubated for 15 min in 5% normal goat serum (NGS) in 0.1% bovine serum albumin dissolved in PBS (BSA/PBS). Following this step, cells were incubated for 20 min in anti- NEP antibody, rinsed in BSA/PBS, incubated in goat anti-rabbit IgG coupled to 15nm colloidal Au particles (GARG15) for 1 h and again rinsed in PBS. Postfixation for 30 min in 2.5% glutaraldehyde and PBS rinsing followed. For SEM a drop of cell suspension was put on a polylysine- treated Formvar-carbon-coated Au grid and cells were allowed to settle and attach for 30 min. The grid was rinsed in water, dehydrated and critical point dried. Cells were coated with carbon before viewing by SEM. For TEM, following immunolabelling, cells were post-fixed in OsO4, rinsed, dehydrated and embedded in Epon for sectioning.

CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide- induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1834-1841 ◽  
Author(s):  
MA Shipp ◽  
GB Stefano ◽  
SN Switzer ◽  
JD Griffin ◽  
EL Reinherz
Keyword(s):  
Substance P ◽  
Cell Surface ◽  
Enzymatic Activity ◽  
Inflammatory Responses ◽  
Lymphoblastic Leukemia ◽  
Neutral Endopeptidase ◽  
Biologically Active ◽  
Zinc Metalloprotease ◽  
Endopeptidase 24.11 ◽  
Induced Changes

Abstract The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu- phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.

scholarly journals CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide- induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1834-1841 ◽  
Author(s):  
MA Shipp ◽  
GB Stefano ◽  
SN Switzer ◽  
JD Griffin ◽  
EL Reinherz
Keyword(s):  
Substance P ◽  
Cell Surface ◽  
Enzymatic Activity ◽  
Inflammatory Responses ◽  
Lymphoblastic Leukemia ◽  
Neutral Endopeptidase ◽  
Biologically Active ◽  
Zinc Metalloprotease ◽  
Endopeptidase 24.11 ◽  
Induced Changes

The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu- phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.

Organization of tight junctions in the choroid plexus epithelium

1978 ◽  
Vol 36 (2) ◽  
pp. 336-337
Author(s):  
B. Van Deurs ◽  
J. K. Koehler
Keyword(s):  
Choroid Plexus ◽  
Present Report ◽  
Freeze Fracture ◽  
Barrier Properties ◽  
Cell Junctions ◽  
Structural Basis ◽  
Apical Surface ◽  
Choroid Plexus Epithelium ◽  
Solid Nitrogen ◽  
Special Composition

The choroid plexus epithelium constitutes a blood-cerebrospinal fluid (CSF) barrier, and is involved in regulation of the special composition of the CSF. The epithelium is provided with an ouabain-sensitive Na/K-pump located at the apical surface, actively pumping ions into the CSF. The choroid plexus epithelium has been described as “leaky” with a low transepithelial resistance, and a passive transepithelial flux following a paracellular route (intercellular spaces and cell junctions) also takes place. The present report describes the structural basis for these “barrier” properties of the choroid plexus epithelium as revealed by freeze fracture.Choroid plexus from the lateral, third and fourth ventricles of rats were used. The tissue was fixed in glutaraldehyde and stored in 30% glycerol. Freezing was performed either in liquid nitrogen-cooled Freon 22, or directly in a mixture of liquid and solid nitrogen prepared in a special vacuum chamber. The latter method was always used, and considered necessary, when preparations of complementary (double) replicas were made.

scholarly journals The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase).

1990 ◽  
Vol 265 (24) ◽  
pp. 14150-14155
Author(s):  
J. Vijayaraghavan ◽  
A.G. Scicli ◽  
O.A. Carretero ◽  
C. Slaughter ◽  
C. Moomaw ◽  
...  
Keyword(s):  
Neutral Endopeptidase ◽  
Endopeptidase 24.11 ◽  
Hydrolysis Of

Non-peptidic inhibitors of neutral endopeptidase 24.11 2. Design and pharmacology of orally active phosphonate prodrugs

1995 ◽  
Vol 5 (2) ◽  
pp. 151-154 ◽  
Author(s):  
Stéphane De Lombaer ◽  
Louis Blanchard ◽  
Carol Berry ◽  
Rajendra D. Ghai ◽  
Angelo J. Trapani
Keyword(s):  
Neutral Endopeptidase ◽  
Endopeptidase 24.11 ◽  
Peptidic Inhibitors ◽  
Orally Active

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 3028-3034 ◽  
Author(s):  
Soohee Lee ◽  
Asim K. Debnath ◽  
Colvin M. Redman
Keyword(s):  
Amino Acids ◽  
Blood Group ◽  
Active Site ◽  
Neutral Endopeptidase ◽  
Site Directed Mutagenesis ◽  
Peptide Hydrolysis ◽  
Enzyme Active Site ◽  
Endopeptidase 24.11 ◽  
Outer Domain ◽  
Group Protein

Abstract In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3–converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of α-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.

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