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P Crine

Publications and source records attributed to P Crine.

At least 55 records · Page 3Linked to original sources
PubMed1994

Fate of a fluorescent inhibitor of endopeptidase-24.11 using enzyme-expressing MDCK cells. Modification of its cellular processing with a monoclonal antibody.

Neutral endopeptidase-24.11 (NEP) is a membrane-bound zinc metallopeptidase which cleaves biologically active peptides such as the enkephalins and atrial natriuretic peptide. Using the specific and fluorescent thiol inhibitor of the enzyme, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl)-amino-1- hexyl]-thiocarbamide (FTI), the fate of the inhibitor-enzyme complex was investigated by videomicrofluorimetry using MDCK epithelial cells expressing the rabbit peptidase thanks to a retroviral expression vector. N-[3-(R,S)-[(hydroxyamino) carbonyl]-2-benzyl-1-oxopropyl]- glycine (HACBOGly) and the corresponding tritiated molecule were also used to measure the cellular pathway of inhibitor-NEP complexes. In the present paper, we demonstrate that, for short incubation times, the fluorescent probe preferentially labeled brush border membranes of the apical side of the MDCK cells. After more than 1 h incubation, a honeycomb pattern of fluorescence was observed in videomicrofluorimetry suggesting that part of the inhibitor was bound or localized close to the basolateral plasma membrane. Confocal experiments confirmed the transcytosis of FTI/NEP complex, from the apical to the basolateral domain. Using [3H]HACBOGly on filter-grown cells, after 2 and 4 h incubation at 37 degrees C, the percentage of basolateral membrane-bound molecules was estimated to be about 12 and 23%, respectively. The coincubation of the cells with FTI and 2B12, a monoclonal antibody raised against the rabbit enzyme, greatly modified the fluorescence pattern. A patchy fluorescence was observed for short incubation times, corresponding to cluster formation induced by antigen-antibody binding. For longer incubation times (> 1 h), in addition to the basolateral labeling, some intracellular fluorescent vesicles were observed essentially localized in the vicinity of the nucleus. The colocalization of FTI with Texas Red isothiocyanate-labeled Concanavalin A (TRITC-Con A) strongly suggests an endosomal/lysosomal internalization pathway when FTI was incubated in the presence of 2B12 mAb.

Animals↗
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↗
PubMed1993

Identification and characterization of a neutral endopeptidase activity in Aplysia californica.

Kidney plasma membranes of Aplysia californica were shown to contain an endopeptidase activity which cleaved [Leu]enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Leu]enkephalinamide (Tyr-Gly-Gly-Phe-Leu-NH2) at the Gly3-Phe4 bond, as determined by reverse-phase h.p.l.c. analysis of metabolites. The optimal pH was shown to be 6.5. The bivalent cation chelating agent, 1,10-phenanthroline protected [Leu]enkephalin from degradation, suggesting that this enzyme is a metallopeptidase. The degradation of [Leu]enkephalin was also abolished by the neutral endopeptidase-24.11 inhibitors RB104 (2-[(3-iodo-4-hydroxyl)-phenylmethyl]-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)-propyl]amino-4-oxobutanoic acid), HABCO-Gly [(3-hydroxy-aminocarbonyl-2-benzyl-1-oxypropyl)glycine], phosphoramidon and thiorphan, with IC50 values of 1 nM, 1 microM, 20 microM and 30 microM respectively. By contrast, the angiotensin-converting enzyme inhibitor captopril and the serine proteinase inhibitor phenylmethanesulphonyl fluoride were without effect. Phase separation experiments using Triton X-114 showed that about 64% of the neutral endopeptidase activity in the Aplysia kidney membrane corresponds to an integral membrane protein. A specific radioiodinated inhibitor ([125I]RB104) was shown to bind the Aplysia endopeptidase with high affinity; the KD and Bmax. values were 21 +/- 5 pM and 20.3 +/- 5 fmol/mg of proteins respectively. This inhibitor was used to determine the molecular form of the enzyme, after separation of solubilized membrane proteins on SDS/PAGE and transfer on to nitrocellulose membranes. A single protein band with an apparent molecular mass of 140 kDa was observed. The labelling was abolished by specific neutral endopeptidase inhibitors. This study provides the first biochemical characterization of an endopeptidase with catalytic properties similar to those of neutral endopeptidase-24.11 in the mollusc Aplysia californica.

Animals↗
PubMed1993

Substitution of potential metal-coordinating amino acid residues in the zinc-binding site of endopeptidase-24.11.

Neutral endopeptidase (EC 3.4.24.11; NEP) is a membrane-bound zinc-metallopeptidase. The catalytic zinc ion is coordinated to three amino acid residues (His538, His587 and Glu646) and a water molecule. Here, we have systematically substituted potential metal-coordinating amino acid residues (His, Glu, Asp, Cys, Tyr, Ser) for each of the three zinc ligands of NEP using a recombinant polymerase chain reaction procedure. NEP mutants at positions 583 and 587 were devoid of catalytic activity. However, Glu587 NEP and Cys583 NEP were able to bind partially a tritiated inhibitor, the binding of which is dependent on the presence of the zinc atom. At position 646, the aspartate and cysteine mutants exhibited activity. For both mutants Km values were unaltered but kcat values were decreased by about 20-fold. Both mutants bound the tritiated inhibitor with Kd values similar to that of the wild-type enzyme. Our data suggest that neither histidine-583 nor -587 can be replaced by any other ligands. On the other hand, the glutamic acid at position 646 can be converted to an aspartic acid or a cysteine indicating the importance of a negative charge at this position.

Binding Sites↗
PubMed1993

Characterization of neutral endopeptidase 24.11 in dog glomeruli.

Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron.

Animals↗
PubMed1993

Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state.

Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane-bound Zn-metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc-endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site-directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP-Gln711 and NEP-Phe711). Determination of kinetic parameters showed that both mutants had Km values very similar to that of the non-mutated enzyme but that their kcat values were 25-fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.

Animals↗
PubMed1993

Localization of neprilysin (EC 3.4.24.11) mRNA in rat brain by in situ hybridization.

The distribution of the messenger RNA (mRNA) coding for neprilysin (EC 3.4.24.11) has been studied by in situ hybridization in the adult rat brain. A markedly heterogenous distribution among various brain regions was found. A strong signal was observed in the glomerular layer of the olfactory bulb, the olfactory tubercle, the caudate putamen, the habenular, anterior pretectal, interpeduncular, red, dorso tegmental, pontine, and vestibular nuclei, the mammillary bodies, the Purkinje cells, and the choroid plexus of the fourth ventricle. A large number of areas such as the cortex, the dentate gyrus, the hippocampus, the medial terminal nucleus of the accessory tract, the accumbens and the arcuate nuclei, the superior and inferior colliculi, and a few regions in the thalamus at the mesencephalic level exhibited a moderate or low signal of hybridization. The majority of these regions are also known to contain the neprilysin protein. On the other hand, the globus pallidus, the substantia nigra, and the central gray matter, which show a high or a moderate amount of neprilysin, did not contain any neprilysin mRNA. Comparison of the regional distribution of neprilysin mRNA with that of its translation product provides insight into neprilysin neuronal pathways in the central nervous system.

Animals↗
PubMed1993

Transformation of the signal peptide/membrane anchor domain of a type II transmembrane protein into a cleavable signal peptide.

Rabbit neutral endopeptidase-24.11 is a type II transmembrane protein with a 27-amino acid residue positively charged NH2-terminal cytoplasmic domain, a 23-amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. In order to study the mechanism of membrane anchoring of neutral endopeptidase-24.11, we created mutants in which the cytoplasmic tail was deleted. Expression of these mutants in COS-1 cells resulted in the secretion of approximately 10-20% of the protein into the culture medium, due possibly to the cleavage of part or all of the signal peptide/membrane anchor domain by the rough endoplasmic reticulum signal peptidase. In a second set of mutants, a hydrophilic sequence (GSQNS) was inserted midway in the signal peptide/membrane anchor domain of neutral endopeptidase-24.11. When this hydrophilic sequence was introduced into the full-length neutral endopeptidase-24.11, approximately 20% of the enzyme activity was recovered in the culture medium. This proportion increased to 93% when the cytosolic tail was deleted. Sequencing of the [3H]tyrosine- or [3H]isoleucine-labeled secreted protein indicated that proteolysis, possibly by signal peptidase, occurred on the COOH-terminal side of the signal peptide/membrane anchor domain. We conclude that the efficient cleavage of the signal peptide/membrane anchor domain and secretion of the protein require both the deletion of the cytosolic domain and the presence of a hydrophilic sequence.

Amino Acid Sequence↗
PubMed1993

Targeting of pro-opiomelanocortin to the regulated secretory pathway may involve cooperation between different protein domains.

The structure of pro-opiomelanocortin (POMC) can be divided into three main domains: an NH2-terminal domain formed by the NH2-terminal glycopeptide and the joining peptide, a central domain corresponding to the adrenocorticotropin sequences and a COOH-terminal domain containing the beta-lipotropin sequences. Expression of POMC in neuroendocrine cell lines such as the mouse neuroblastoma Neuro2A cells results in its targeting to the regulated secretory pathway of these cells. Intracellular targeting of proteins along non default pathways are widely believed to involve the recognition of specific structural features by a sorting machinery. To understand the nature of the signal involved in targeting prohormone to the regulated secretory pathway, we have constructed mutants of POMC in which sequences from the NH2-terminal, the central and the COOH-terminal domains were deleted and examined the sorting of these mutant POMC molecules in Neuro2A cells by immunofluorescence and immunoelectron microscopy. Our results indicate that POMC NH2-terminal glycopeptide or beta-LPH domain do not contain sufficient information for targeting to the regulated pathway since these peptides are not sorted to secretory vesicles when expressed in Neuro2A cells: Similarly, the ACTH domain does not contain essential targeting information since POMC mutants lacking these sequences were sorted to secretory vesicles. Mutant POMCs containing the sequences of more than one of the main protein domains were, however, correctly targeted to the regulated secretory pathway. Our results indicate that POMC is not targeted to the regulated secretory pathway through recognition of a unique continuous 'molecular address'.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
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

Identification and characterization of aminopeptidases from Aplysia californica.

Aminopeptidase activities were identified in extracts of kidney, ovotestis, head ganglia, heart and haemolymph of Aplysia californica. These enzyme preparations hydrolysed [3H][Leu]enkephalin at the Try-1-Gly-2 bond as determined by h.p.l.c. analysis of cleavage products. In all these tissues, enkephalin-degrading aminopeptidase activities were present both in membrane-bound and cytosolic fractions. The bivalent-cation-chelating agent, 1,10-phenanthroline, inhibited kidney membrane aminopeptidase activity with an IC50 of 30 microM, suggesting that this enzyme is a metalloproteinase. The aminopeptidase inhibitor amastatin was the most potent inhibitor of [Leu]enkephalin degradation (IC50 25 nM) by membrane-bound aminopeptidase, and bacitracin, bestatin and puromycin were about 100-1000 times less potent. In contrast with membrane-bound aminopeptidase, the cytosolic form is sensitive to puromycin. Angiotensin-converting enzyme inhibitor had no effect on [Leu]enkephalin degradation by kidney membranes, while the neutral endopeptidase inhibitors were poor inhibitors of the enzymes in this preparation. The Km values of the aminopeptidase in the kidney membranes and cytosolic fractions for the [Leu]enkephalin substrate were 2.4 and 7.4 microM respectively. The aminopeptidase present in the kidney membranes also hydrolysed endogenous Phe-Met-Arg-Phe-amide peptide at the Phe-1-Met-2 bond as well as synthetic alanine p-nitroanilide and leucine p-nitroanilide. When used in a competition assay, these substrates inhibited hydrolysis of [3H][Leu]enkephalin, suggesting that the same enzyme degraded all these substrates. Taken together, these results suggest that Aplysia tissues contain both a membrane-bound aminopeptidase related to the mammalian aminopeptidase N and a cytosolic puromycin-sensitive aminopeptidase.

Aminopeptidases↗
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

Development of [125I]RB104, a potent inhibitor of neutral endopeptidase 24.11, and its use in detecting nanogram quantities of the enzyme by "inhibitor gel electrophoresis".

Neutral endopeptidase 24.11, also known as the common acute lymphoblastic leukemia antigen, is a zinc metallopeptidase involved in the inactivation of biologically active peptides, such as the enkephalins and atrial natriuretic peptide. The highly potent radiolabeled inhibitor 2-((3-[125I]iodo-4-hydroxy)phenylmethyl)-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)propyl]amino-4-oxobutanoic acid ([125I]RB104; Ki = 30 pM) has been developed for the enzyme. [125I]RB104 is highly specific, its Ki for another widely distributed zinc peptidase, angiotensin-converting enzyme, being 15 microM. In binding studies using rat brain slices, [125I]RB104 was shown to have a high affinity (Kd = 300 +/- 20 pM) and high specific binding at the Kd concentration (90%). With rat brain homogenates the Kd of [125I]RB104 was 26.8 +/- 0.9 pM, close to the kinetically derived Kd, 7.0 +/- 0.8 pM. Using the inhibitor, we have developed a simple, rapid, and quantitative technique to detect low nanogram quantities of the endopeptidase directly from tissue extracts after SDS/PAGE. The method has been used to show the presence of low quantities of the enzyme in rabbit bone marrow. Apart from its sensitivity, "inhibitor gel electrophoresis" using [125I]RB104 has the advantage over immunohistochemical methods of being able to label the enzyme in all tissues and species. It will therefore be of great value in determining the exact role of this important regulatory peptidase in a number of biological systems. Moreover, this one-step characterization of neutral endopeptidase 24.11 could be extended to other zinc metallopeptidases such as angiotensin-converting enzyme or collagenases, and inhibitors with affinities as high as RB104 could open the way to visualization of zinc metallopeptidases in different tissues by electron microscopy.

Animals↗
PubMed1992

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

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.

Amino Acid Sequence↗
PubMed1992

Charge polarity reversal inverses the specificity of neutral endopeptidase-24.11.

Attempts to change enzyme specificity by charge polarity reversal have so far met with little success, probably due to a destabilization of the resulting ion pair in an environment naturally optimized for the inverted pair. In the zinc metallopeptidase neutral endopeptidase-24.11 (EC 3.4.24.11), Arg102, involved in substrate binding, is probably located at the edge of the active site (Bateman, R.C., Jr., Kim, Y.-A., Slaughter, C., and Hersh, L.B. (1990) J. Biol. Chem. 265, 8365-8368; Beaumont, A., Le Moual, H., Boileau, G., Crine, P., and Roques, B.P. (1991) J. Biol. Chem. 266, 214-220). This environment may be favorable for polarity reversal, as in water the energies of reverse ion pairs would be identical. We show here that, while mutating Arg102 to Glu reduces the specificity of a C-terminally negatively charged substrate 16-fold, it increases that of a substrate with an optimally positioned positive charge 29-fold. The concept of charge polarity reversal can be extended to other zinc metallopeptidases, and the mutated enzyme could also have applications in the enantiomeric separation of unnatural amino acids.

Binding Sites↗
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↗
PubMed1992

Differential expression of neuropeptide gene mRNA within the LUQ cells of Aplysia californica.

Two neuropeptide precursor cDNAs (LUQ-1 and L5-67) have been recently isolated from the Left Upper Quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica (Shyamala, Fisher, and Scheller, 1986; Wickham and DesGroseillers, 1991). Using in situ hybridization techniques as well as dot blot and polymerase chain reaction (PCR) assays, we have studied the expression of these genes in the central nervous system (CNS) of Aplysia californica. The LUQ-1 gene was found to be expressed in neuron L5 in the abdominal ganglion, whereas the expression of the L5-67 gene was observed in the other four LUQ cells (L2-4 and L6). When in situ hybridization was performed on paraffin sections of the abdominal ganglion, clusters of smaller cells located in the left hemiganglion, were also found to express either the LUQ-1 or the L5-67 gene, never both. In many sections, the mRNAs coding for the two neuropeptides were found not only in cell bodies but also in the axon of individual LUQ neurons and even as far as the pericardial nerve. The presence of neuropeptide mRNA in axons, pericardial nerve, and kidney has been confirmed by polymerase chain reaction. A specific, although diffuse hybridization in the left upper quadrant also suggests that mRNA is present in the neuritic field. Taken together these results indicate that neuron L5 is the only giant neuron expressing the LUQ-1 gene and might therefore have a physiological function different from the other four LUQ cells. Neuropeptide mRNAs were also found in the axon and/or the neuritic field of giant neurons and could play important roles related to cell signalling in axons and nerve termini.

Animals↗