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Direct targeting of neutral endopeptidase (EC 3.4.24.11) to the apical cell surface of transfected LLC-PK1 cells and unpolarized secretion of its soluble form.

ABSTRACT: LLC-PK1 cells were transfected with a cDNA encoding rabbit neutral endopeptidase (NEP; EC 3.4.24.11), an abundant enzyme of the kidney proximal brush border. Clones of cells expressing high levels of the protein were isolated. Selective biotinylation and radioimmunolabelling were used to determine that 85-95% of NEP was localized in the apical domain of filter-grown LLC-PK1 cells. Pulse-chase and selective biotinylation studies revealed that the majority (85%) of newly made NEP was directly targeted to the apical membrane. However, a soluble form of NEP was found to be secreted in approximately equal amounts from both sides of the monolayer when expressed in LLC-PK1 cells. Transfected pro-opiomelanocortin, a pituitary hormone precursor, was secreted almost exclusively into the basolateral medium, suggesting that the bulk flow is to the basolateral membrane. This behaviour contrasts with that observed in MDCK cells, where both the transmembrane and secreted forms of NEP are directly targeted to the apical membrane and where the secretion of pro-opiomelanocortin is unpolarized.

SUBMITTER: Lanctot C

PROVIDER: S-EPMC1136445 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

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Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells.

Project description:Neutral endopeptidase (EC 3.4.24.11, NEP) is a type-II integral membrane protein found in a wide variety of cell types. We previously produced a secreted form of the enzyme by deletion of the cytoplasmic and transmembrane domains and in-frame fusion of the cleavable signal peptide of pro-opiomelanocortin [Lemay, Waksman, Roques, Crine and Boileau (1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secreted form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-anchor attachment signal of decay-accelerating factor to produce a GPI-anchored form. Expression of this chimeric form in Cos-1 cells resulted in cell-surface activity. This activity could be released from the cell surface by phosphatidylinositol-specific phospholipase C and radiolabelling studies showed that the protein could incorporate [3H]ethanolamine, indicating that the enzyme was GPI-anchored. The Km value, using [D-Ala2,Leu5]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 microM) was comparable with that of wild-type NEP (70 +/- 4 microM), as were the sensitivities to the inhibitors phosphoramidon and thiorphan. However, pulse-chase studies showed that the biosynthesis and cell-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a lower rate of biosynthesis and/or cellular transport for GPI-anchored NEP compared with its transmembrane counterpart.

| S-EPMC1138037 | biostudies-other

Hydrolysis of thymic humoral factor gamma 2 by neutral endopeptidase (EC 3.4.24.11).

Project description:A search for the natural substrates for neutral endopeptidase (NEP; EC 3.4.24.11) in the immune system led to investigation of the enzyme's action on thymic humoral factor gamma 2 (THF). The ectoenzyme rapidly and efficiently hydrolyses the Lys6-Phe7 bond of the octapeptide. The site of cleavage was confirmed by h.p.l.c. analysis, amino acid analysis and sequence determination of the products. Phosphoramidon (3.6 microM), a potent inhibitor of the enzyme, prevents this cleavage even during prolonged incubation. The high efficiency of hydrolysis of THF by NEP is similar to that reported for [Leu5]enkephalin, and the dipeptide Phe-Leu is the C-terminal product in the hydrolysis of both peptides. The presence of NEP, reportedly identified as the common acute lymphoblastic leukaemia antigen (CALLA), in bone-marrow cells and other cells of the immune system raises the possibility that it may play a role in modulating the activity of peptides such as THF.

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Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK1) receptor expressed in mammalian cells.

Project description:Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2+ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific 125I-SP binding after 20 min incubation at 37 degrees C was 92.2 +/- 3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9 +/- 0.5% of maximal, and binding was increased to 82.7 +/- 2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca2+ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca2+ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca2+ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca2+ mobilization only when coexpressed in the same cells as the SPR.

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Replicative capacity of four porcine enteric coronaviruses in LLC-PK1 cells.

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Anterograde microtubule transport drives microtubule bending in LLC-PK1 epithelial cells.

Project description:Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array.

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Polycystin-2 (TRPP2) regulates primary cilium length in LLC-PK1 renal epithelial cells.

Project description:Polycystin-2 (PC2, TRPP2) is a Ca2+ permeable nonselective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. However, little is known as to whether PC2 contributes to the primary cilium structure. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild-type LLC-PK1 renal epithelial cells. Confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated α-tubulin antibody to identify primary cilia and measure their length. Although primary cilia length measurements did not follow a Normal distribution, the data were normalized by Box-Cox transformation rendering statistical differences under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 μM) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length compared to their respective scrambled RNA transfected cells. The data indicate that conditions that regulate PC2 function or gene expression modify the length of primary cilia in renal epithelial cells. Blocking of PC2 mitigates the effects of elevated external Ca2+ concentration on primary cilia length. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.

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Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line.

Project description:Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.

| S-EPMC1132696 | biostudies-other

Expression data from ATRA-treated LLC-PK1 cells

Project description:We have established a role for p-ERK in mediating ATRA cytoprotection against chemical-induced injury to LLC-PK1 renal proximal tubule epithelial cells. The present studies were conducted to assess downstream signaling from p-ERK in ATRA-mediated cytoprotection. Gene expression profiling via microarray and ontology analyses indicated an upregulation of genes associated with cell proliferation and differentiation shortly after (0.5 and 1 hr) ATRA (25 μM) treatment. Target genes of these transcription factors associated with ribosome biogenesis, DNA replication, DNA repair, and the cell cycle were significantly increased by 4 hr.

2020-03-01 | GSE110832 | GEO

H+-gradient-dependent active transport of tetraethylammonium cation in apical-membrane vesicles isolated from kidney epithelial cell line LLC-PK1.

Project description:Transport of [3H]tetraethylammonium (NEt4+), an organic cation, has been studied by using apical-membrane vesicles isolated from cultured kidney epithelial cell line LLC-PK1. The uptake of NEt4+ by apical-membrane vesicles was osmotically sensitive, time-dependent and saturable. The presence of an H+ gradient ([H+]i greater than [H+]o) induced a marked stimulation of NEt4+ uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was inhibited by HgCl2. These results suggest that apical membranes isolated from the LLC-PK1 cells retain the transport characteristics of NEt4+ similar to those observed in renal brush-border membranes.

| S-EPMC1144826 | biostudies-other

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