Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enzyme neutral metalloendopeptidase (E.C. 3.4.24.11), also known as the common acute lymphocytic leukemia antigen, neutral endopeptidase, or enkephalinase, functions as an inactivator of a wide variety of signaling oligopeptides such as substance P, neurokinin A, enkephalins, endothelin, atrial natriuretic factor, and formyl chemotactic peptides. A cDNA clone isolated from a human lung library encodes a fragment of neutral metalloendopeptidase containing an internal 81 base pair deletion when compared with the human placental cDNA for this enzyme. Comparison of the deleted cDNA sequence with the intron-exon structure recently determined as the common acute lymphocytic leukemia antigen reveals that the 81 base pairs corresponds precisely with exon 16. RNA analysis using splice junction oligonucleotides indicates that the 16 del form constitutes a minor but significant fraction of the RNA species present in human lung. Expression of constructs containing "wild type" and "exon 16 del" neutral endopeptidases in COS-7 cells reveals that deletion of this 27 amino acid segment reduces enzymatic activity toward the synthetic substrate glutaryl-alanyl-alanyl-phenyl-alanyl-4-methoxy-2-naphthylamide to barely detectable levels.
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PMID:Exon 16 del: a novel form of human neutral endopeptidase (CALLA). 153 84

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.
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PMID:Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line. 159 10

The depletion of an essential amino acid (aa), tryptophan, caused by interferon-gamma (IFN-gamma)-mediated induction of indoleamine 2,3-dioxygenase (IDO) in mouse allografted tumor cells, has been suggested as a reason for the allograft rejection. To elucidate the mechanism of this IDO induction, attempts were made to isolate cDNA clones encoding mouse IDO. In seven of 25 mouse cell lines, IDO was induced by IFN-gamma, and the highest IDO induction was observed in the case of rectal cancer (CMT-93) cells, which were further stimulated two- to threefold by the simultaneous addition of dibutyryl cyclic AMP (Bt2cAMP). A cDNA library was prepared from poly(A)+ RNA isolated from CMT-93 cells treated with IFN-gamma/Bt2cAMP. The cDNA clones were isolated using the cDNA encoding human IDO as a probe. The mouse IDO cDNA encodes a 407-aa protein with an Mr of 45,639. The deduced aa sequence agreed with partial aa sequences derived from endopeptidase digestion of purified mouse IDO and revealed 61% homology with that of human IDO. Transient expression of the mouse IDO cDNA in COS-7 cells yielded a high level of IDO activity in the cells. Northern hybridization analysis of RNA in CMT-93 cells indicated that IFN-gamma induced the IDO mRNA, and that the level of RNA was increased by simultaneous addition of Bt2cAMP, while Bt2cAMP itself had no effect on mRNA induction.
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PMID:Cloning and expression of a cDNA encoding mouse indoleamine 2,3-dioxygenase. 193 18

The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.
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PMID:Affinity purification, peptide analysis, and cDNA sequence of the mouse interferon gamma receptor. 213 61

We discovered that the common acute lymphoblastic leukemia antigen, CALLA (CD10), was identical to human neutral endopeptidase 3.4.24.11 (NEP), a Zn-binding glycoprotein with an extracellular active site capable of hydrolyzing several biologically active peptides. In this study we compare the expression of CALLA/NEP in terms of antigenic density and enzymatic activity at the cell surface and of messenger RNA (mRNA) levels on granulocytes, leukemic cells, and CALLA-transfected COS-1 cells. Mature granulocytes, the only readily available source of normal human CALLA, express relatively low but constant levels of antigen, NEP activity (3.5 pmol/min/10(6) cells), and mRNA. The two major CALLA-mRNA species of 6.5 kb and 3.8 kb, observed to date in a variety of cells and tissues, were also found in four independent granulocyte preparations. With leukemia cell lines, a correlation was established between the density of CALLA antigen and the level of enzymatic activity (3.4 to 21.0 pmol/min/10(6) cells). This paper constitutes the first report of NEP activity on blast cells derived from patients with non-T acute lymphoblastic leukemia (ALL); the levels of activity were variable (1.5 to 35.9 pmol/min/10(6) cells for six cases) but correlated with the level of CALLA assessed by flow cytometry. Heterogeneous levels of expression of the CALLA-mRNA species were also observed in non-T ALL cases that correlated with the level of CALLA expression at the surface of these cells. Very high levels of NEP activity were achieved by transfecting COS-1 cells with pSV-CALLA; 20% of the transfected cells were CALLA+ and expressed 550 pmol/min/10(6) cells. Extracts prepared from COS-1 cells transfected with pSV-CALLA (carrying human NEP cDNA) and pSVENK19 (carrying rabbit NEP-cDNA), respectively, gave Michaelis constant (Km) values of 50 mumol/L and similar inhibition curves with thiorphan. Thus the recombinant proteins encoded by these two genes have similar enzymatic properties, confirming the high degree of their structural relatedness. The expression of high levels of CALLA/NEP on COS-1 cells should allow the use of this system to test the effects of specific mutations on activity and might lead to the understanding of the role of CALLA in the onset and/or progression of leukemia.
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PMID:Comparative levels of CALLA/neutral endopeptidase on normal granulocytes, leukemic cells, and transfected COS-1 cells. 216 7

We have cloned a full length cDNA for human leukocyte elastase (HLE, EC 3.4.21.37)/medullasin from the cDNA library of human leukemic cell line, ML3. Recombinant plasmid for the expression of HLE cDNA in eukaryotic cells was constructed in which HLE cDNA was fused in a frame to a leader sequence of human interleukin-2 (IL-2). COS-1 cells, transfected with the plasmid, secreted fusion protein consists of N-terminal 8 amino acid (aa) residues of human IL-2 and 238 aa residues of HLE. As the fusion protein was designed to be connected through lysine residue, elastase activity was generated after digestion of the fusion protein with lysyl-endopeptidase.
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PMID:Functional expression of human leukocyte elastase (HLE)/medullasin in eukaryotic cells. 232 78

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types and is especially abundant at the apical "brush border" membrane of the kidney proximal tubules. The enzyme consists of a short amino-terminal cytosolic domain of 27 amino acids, a single hydrophobic sequence which is believed to be responsible for anchoring the enzyme in the plasma membrane, and a large extracellular domain containing the active site. This model is consistent with the proposed function of neutral endopeptidase, which is believed to play an important role in the inactivation of small regulatory peptides at the cell surface. Site-directed mutagenesis has allowed the identification of 1 glutamic acid and 2 histidine residues essential for catalysis. All are located near the COOH terminus of the protein. We do not know, however, whether other segments of the protein are involved in the structure of the active site. The exact role of the cytosolic and transmembrane domains is also unknown. In this report, we have induced the secretion of a soluble form of recombinant neutral endopeptidase in COS-1 cells by fusing in-frame, the cDNA encoding the signal peptide of a secreted protein (pro-opiomelanocortin) to the cDNA sequences of the complete ectodomain of neutral endopeptidase. Characterization of the secreted recombinant protein indicated that it has the same catalytic properties as the membrane-bound recombinant enzyme or as the enzyme extracted from kidney brush border membranes. Thus the extracellular domain alone is sufficient for conferring full catalytic activity to neutral endopeptidase.
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PMID:Fusion of a cleavable signal peptide to the ectodomain of neutral endopeptidase (EC 3.4.24.11) results in the secretion of an active enzyme in COS-1 cells. 267 Sep 43

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found in the plasma membrane of many cell types. The cDNA coding for the complete primary structure of neutral endopeptidase has recently been cloned and sequenced (Devault, A. Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322). Comparison of the sequence of neutral endopeptidase with that of thermolysin, a bacterial Zn-metalloendopeptidase, suggests that Glu-584 in neutral endopeptidase probably corresponds to Glu-143 in thermolysin, which is an essential amino acid involved in catalysis. To test directly the importance of Glu-584 in the catalytic activity of neutral endopeptidase by site-directed metagenesis, we have constructed an expression vector in which the rabbit kidney cDNA encoding the entire neutral endopeptidase sequence is introduced downstream from the SV40 virus early promotor. After transfection in COS-1 monkey kidney cells, this vector was found to promote the expression of a protein with biochemical and catalytic properties identical to kidney neutral endopeptidase. Oligonucleotide-directed mutagenesis of Glu-584 to either valine or aspartic acid completely abolished the enzymatic activity of the recombinant protein without changing its affinity for the substrate-related tritiated inhibitor [3H]N-[(2R,2S)-3-hydroxyamino-carbonyl-2-benzyl-1-oxopropyl]-glycine. This observation clearly identifies Glu-584 as one of the important residues responsible for the catalytic activity of the enzyme.
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PMID:Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site. 289 75

Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His-142 and His-146) and a third histidine (His-231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His-583, His-587 and His-637) was explored by site-directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS-1 cells. Substitution of either His-583 or His-587 of NEP for Phe completely abolished the activity and Zn-directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His-142 and His-146 of thermolysin as zinc ligands. In contrast, substitution of His-637 for a phenylalanine residue was without effect on enzyme activity.
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PMID:Exploration of the catalytic site of endopeptidase 24.11 by site-directed mutagenesis. Histidine residues 583 and 587 are essential for catalysis. 316 86

During the biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins, an N-terminal signal peptide is used to direct biosynthesis to the endoplasmic reticulum. It was previously unknown whether or not this signal must be removed during the biosynthesis of GPI-anchored proteins. Using neutral endopeptidase (EC 3.4.24.11), a well characterized type II membrane protein that is attached to the membrane via an uncleaved N-terminal signal peptide, we extended its C terminus with 33 of the 37 amino acids of the GPI anchor signal sequence of decay-accelerating factor. When expressed in COS-1 and Chinese hamster fibroblast (CHW) cells, the protein was shown to possess both transmembrane and GPI anchors, indicating that a cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of GPI-anchored proteins.
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PMID:A cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of glycosylphosphatidylinositol-anchored proteins. 751 27


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