Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The inducibility of glycosyl-phosphatidylinositol (GPI)-anchored proteins on affected paroxysmal nocturnal haemoglobinuria (PNH) neutrophils (PMN) after both in vitro and in vivo stimulation was investigated. Fc gamma R-III (CD16), decay-accelerating factor (DAF/CD55) and 20 kD homologous restriction factor (HRF20/CD59) were demonstrated to be concurrently deficient on unstimulated defective PNH PMN. Upon in vitro stimulation with either N-formyl-methionyl-leucyl-phenylalanine (fMLP), zymosan-activated serum (ZAS), or recombinant human granulocyte colony-stimulation factor (G-CSF), neither CD16 nor CD55 expression was induced on defective PNH PMN. G-CSF was administered to two patients with PNH when their conditions were complicated by bacterial infections, or to prevent infections associated with the extraction of teeth or cataract surgery. CD16 expression was induced on the defective PNH PMN in both cases during the administration of G-CSF, but the expression of CD55 and CD59 was not. CD16, induced on the defective PNH PMN during the administration of G-CSF, was phosphatidylinositol-specific phospholipase C (PIPLC)-sensitive, implying that it had GPI-linkage to the membranes. The patients treated with G-CSF recovered from infection or evaded infection. These observations suggest that a deficiency of GPI-anchored proteins is not always seen in defective PNH blood cells, at least under certain stimulation conditions.
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PMID:Induction of Fc gamma R-III (CD16) expression on neutrophils affected by paroxysmal nocturnal haemoglobinuria by administration of granulocyte colony-stimulating factor. 769 30

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.
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PMID:Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells. 816 36

Normal and neoplastic cells are protected from autologous complement (C) attack by different cell-surface C-regulatory proteins including CD59 (protectin), CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor). Indirect immunofluorescence (IIF) analysis showed a differential expression of CD59, CD46, and CD55 in nine human melanoma cell lines and that the expression of CD59 was highly heterogeneous compared with that of CD46 and CD55. Levels of cell membrane CD59 were found to regulate the differential sensitivity of melanoma cells investigated to homologous C-mediated lysis; in fact, an inverse correlation (r > 0.7, p < 0.05) was found between levels of cell membrane CD59, but not of CD46 and CD55, and extent of C-mediated lysis of melanoma cells sensitized with scalar concentrations of the anti-GD3 ganglioside mAb R24. Masking of CD59 by 2.5 micrograms/ml of the anti-CD59 mAb YTH53.1 induced or enhanced C-mediated lysis of melanoma cells sensitized with 2.5 micrograms/ml of mAb R24; the latter phenomenon was found to be directly correlated (r > 0.865, p < 0.01) with levels of cell membrane CD59. CD59 is bound to melanoma cells by a glycosylphosphatidylinositol anchor: treatment of C-resistant melanoma cells Mel 97, by increasing doses of phosphatidylinositol-specific phospholipase C (PI-PLC), progressively decreased cell-surface expression of CD59 and increased C-mediated lysis of cells sensitized with mAb R24. Staining of 38 benign and malignant lesions of melanocytic origin by mAb YTH53.1 demonstrated that CD59 is consistently expressed in vivo and confirmed the heterogeneous expression detected in vitro. Our data, altogether, demonstrate that CD59 is the main restriction factor of C-mediated lysis of melanoma cells and that levels of CD59 may account for their differential resistance to C-mediated lysis. The analysis of the levels of CD59 could represent an useful strategy in selecting melanoma patients who may benefit from immunotherapeutic treatment(s) that trigger C activation.
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PMID:Levels of cell membrane CD59 regulate the extent of complement-mediated lysis of human melanoma cells. 856 95

The enzyme hepatic lipase may play several roles in lipoprotein metabolism. Recent investigation has suggested a role for the enzyme in lipoprotein and/or lipoprotein lipid uptake. To study this, a simple isolated system that mimics the in vivo system would be desirable. The enzyme is secreted by the hepatic parenchymal cell but exists, and presumably exerts its effects, while bound to capillary endothelial cells in the liver, adrenal gland, and the ovary. We constructed a cDNA that encodes the expression of a chimeric protein composed of rat hepatic lipase and the signal sequence for the addition of the glycophosphatidylinositol (GPI) anchor from human decay-accelerating factor. When transfected into Chinese hamster ovary (CHO) cells this gave rise to a cell population that had immunoreactive hepatic lipase on the cell surface. Cloning of the transfected cells produced several cell lines that expressed the chimeric protein bound to the cell surface by a GPI anchor. This was documented by demonstrating incorporation of [3H]ethanolamine into anti-hepatic lipase immunoprecipitable material; in addition, hepatic lipase was released from the cells by phosphatidylinositol-specific phospholipase C but not by heparin. Phosphatidylinositol-phospholipase C treatment of cells expressing the anchored lipase released material that comigrated with hepatic lipase on SDS-polyacrylamide gel electrophoresis and was immunoreactive with antibody to the cross-reacting determinant of GPI anchors. Cell lysates containing the anchored protein contained salt-resistant lipase activity, a known feature of the secreted hepatic lipase; thus it appears that these cells have a surface-anchored hepatic lipase molecule. Although it was not possible to demonstrate lipolysis by the enzyme while it was on the cell surface for technical reasons, the protein produced by these cells was active when studied in cell membranes. The ability of the cells to take up lipoproteins was studied. The cells demonstrated an increased affinity for low density lipoprotein (LDL) receptor mediated uptake of LDL. They did not, however, demonstrate any enhanced binding or removal of chylomicron remnants. With respect to LDL and remnants, the cells expressing anchored lipase behaved similarly to CHO cell that expressed secreted hepatic lipase. The cells expressing anchored hepatic lipase had a marked increase in the uptake of high density lipoprotein and high density lipoprotein cholesteryl ester when compared to that seen with CHO cells secreting hepatic lipase. This increase occurred primarily via the selective pathway, and was not reduced by addition of anti-LDL receptor or anti-hepatic lipase antibodies or the receptor-associated protein. Together the results suggest that hepatic lipase, when bound to the cell surface by a GPI anchor, plays a role in enhancing lipoprotein uptake. For LDL this may involve the provision of a second foot for particle binding, thus enhancing affinity for the LDL receptor. For chylomicron remnants an additional molecule or molecules are necessary to mediate this effect. For HDL, the enzyme facilitates uptake of cholesteryl ester primarily by the selective pathway.
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PMID:Chinese hamster ovary cells expressing a cell surface-anchored form of hepatic lipase. Characterization of low density lipoprotein and chylomicron remnant uptake and selective uptake of high density lipoprotein-cholesteryl ester. 866 89

Previous studies (Sivaram, P., Choi, S. Y., Curtiss, L. K., and Goldberg, I. J.(1994) J. Biol. Chem. 269, 9409-9412) from this laboratory showed that the NH2-terminal region of apoB (NTAB) has binding domains for lipoprotein lipase (LPL). LPL binding to endothelial cells, we hypothesize, involves interaction both with heparan sulfate proteoglycans and with a protein that has homology to NTAB. To test whether cell-surface NTAB would increase the amount and affinity of LPL binding to cells, we produced stable Chinese hamster ovary cell lines that have NTAB anchored to the cell surface. A cDNA encoding the amino-terminal 17% of apoB (apoB17) was fused to a cDNA coding for the last 37 amino acids of decay-accelerating factor (DAF), which contains the signal for glycosylphosphatidylinositol anchor attachment. The fused construct was sequence-verified and cloned into expression vector pCMV5. The pCMV5-apoB17-DAF plasmid was cotransfected with a neomycin resistance gene into wild-type (WT) cells and mutant heparan sulfate proteoglycan-deficient Chinese hamster ovary cells (745 cells), and stable cell lines were established. Expression of apoB17 on the cell surface was confirmed by the release of apoB17 by phosphatidylinositol-specific phospholipase C. LPL binding to WT and apoB17-DAF-transfected cells was determined. Using 0.8-6 microg of LPL, 1.3-2.2-fold more LPL associated with apoB17-DAF WT cells compared with WT cells; apoB17-DAF also increased LPL binding to 745 cells. After heparinase treatment, LPL binding to apoB17-DAF cells was still greater than to treated WT cells. This increased binding to apoB17-DAF cells was almost abolished by treatment of cells with phosphatidylinositol-specific phospholipase C or anti-apoB monoclonal antibody. LPL dissociated from WT cells with k-1 = 2.55 x 10(-2) min-1, whereas LPL dissociated more slowly from apoB17-DAF-containing cells with k-1 = 1.08 x 10(-2) min-1. Furthermore, almost 95% of the LPL on WT cells was dissociated by 1 M NaCl, while only 65% of the LPL dissociated from apoB17-DAF cells at the same high salt concentration. Similarly, in high salt, more LPL remained associated with apoB17-DAF cells than with nontransfected 745 cells. These data show that NTAB on cell surfaces can function as a LPL-binding protein. Moreover, they demonstrate that LPL association with cells can be increased by simultaneously binding to both proteoglycan and non-proteoglycan binding sites.
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PMID:Cell-surface expression of an amino-terminal fragment of apolipoprotein B increases lipoprotein lipase binding to cells. 870 44

The endothelial cells (EC) of xenografts are the target of hyperacute rejection induced by complement activation via the classical and/or the alternative pathway. To protect these cells from the attack of human complement, decay-accelerating factor (DAF, CD55) and homologous restriction factor 20 (HRF20, CD59), which belong to human complement regulatory factors, were transfected into bovine aortic EC (BAEC) using retroviral vector. Cell surface expression of DAF and HRF20 on BAEC transfectants (BAEC/DAF, BAEC/HRF20) is comparable to that on human umbilical vein EC. Phosphatidyl inositol-phospholipase C treatment diminished or abolished cell surface expression of DAF and HRF20 on BAEC. The addition of human serum to BAEC led to complement-dependent cell lysis, whereas practically no lysis was observed after addition of human serum to BAEC/DAF and BAEC/HRF20. The addition of human serum plus rabbit complement to BAEC/DAF and BAEC/HRF20 caused complement-dependent cell lysis that was comparable to that observed for BAEC. These data demonstrate that xenograft EC transfected with DAF or HRF20 cDNA using retroviral vector are protected from complement-dependent cell lysis.
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PMID:Inhibition of human complement-dependent cell lysis by bovine aortic endothelial cells transfected with membrane-bound complement-regulatory factor (DAF and HRF20) gene using a retroviral vector. 895 21

We studied the response of a human squamous cell carcinoma cell line, SCC-12F, to human complement attack and found that the cells were completely resistant to complement lysis. In the absence of lysis, there was significant C3 deposition and C5b-9 deposition on the cells. Removal of the lipid-linked complement regulatory proteins CD59 and decay-accelerating factor (DAF) by treatment of the cells with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in increased C3b and C5b-9 deposition on the cells and a slight increase in cell death. Treatment of the cells with complement caused them to release membrane vesicles containing the terminal complement proteins. In addition, complement induced SCC-12F to produce significant amounts of prostaglandin F2alpha (PGF2alpha). We conclude that CD59 and DAF are important in the resistance of SCC-12F to complement and that these cells produce membrane vesicles and PGF2alpha in response to complement attack. These responses, in the absence of cell death, may be important in the pathogenesis of inflammatory skin disease in which complement is deposited.
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PMID:Response of SCC-12F, a human squamous cell carcinoma cell line, to complement attack. 920 53

Human lung cancer expresses cell membrane complement inhibitory proteins (CIP). We investigated whether human lung cancer cell lines also express cell-membrane CIP molecules and whether the biology of CIP molecules in these cell lines differs from that of CIP in normal human respiratory epithelium in culture. The cell lines ChaGo K-1 and NCI-H596 were compared with normal human nasal epithelium in primary cultures in respect to the level of cell membrane CIP expression of membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and CD59, in respect to the level of cell resistance to complement-mediated lysis, and in respect to the contribution of cell membrane CIP to cell resistance against complement-mediated lysis. We found, using flow cytometry, that both human lung cancer cell lines expressed MCP, DAF and CD59, as did normal nasal epithelial cells. However, normal cells showed a large subpopulation of low DAF-expressing cells (60% of all cells) and a smaller subpopulation of high DAF-expressing cells (40%), while the lung cancer cell lines showed only one cell population, of high DAF expression. In addition, both lung cancer cell lines expressed higher MCP levels, and NCI-H596 cells showed higher levels of CD59. Cell resistance to complement-mediated lysis of both lung cancer cell lines was much higher than that of normal cells. Fifty percent normal human serum, under the same concentrations of complement activators, induced lysis of less than a mean of 10% of lung cancer cells, while lysing up to a mean of 50% of nasal epithelial cells. Lung cancer cell resistance to complement was due to its ability to prevent significant activation of complement upon its cell membrane, as manifested by a failure of complement activators to increase cell membrane deposition of C3-related fragments. The exact mechanism for this resistance remains obscure. Unexpectedly, neutralizing antibodies, anti-MCP and anti-DAF were entirely ineffective and anti-CD59 was only slightly effective (18% mean cell lysis) in increasing the susceptibility of the lung cancer cell lines to complement, while the same antibodies were very effective in facilitating complement-mediated lysis of the normal nasal epithelial cells (50% mean cell lysis with CD59 MoAb). On the other hand, detachment of DAF and CD59 by phosphatidylinositol-specific phospholipase C (PIPLC) from the lung cancer cell lines abrogated their resistance to lysis. We suggest that the biology of cell membrane CIP molecules in human lung cancer cell lines is different from that of CIP in normal respiratory epithelial cells. Human lung cancer cell lines are able to prevent significant complement activation upon its cell membrane and are therefore especially resistant to complement-mediated lysis. Complement resistance may serve this common and highly lethal human cancer as an escape mechanism from the body's immunosurveillance and prevent effective immunotherapy with tumour-specific MoAbs.
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PMID:Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro, and an insight into mechanism(s) of resistance. 971 65

MCA44 is a monoclonal antibody (mAb) to guinea-pig decay-accelerating factor (DAF) and, using this mAb, tissue distribution of guinea-pig DAF was studied by immunofluorescence. Guinea-pig DAF was found to be expressed not only on the vascular endothelium but also on different types of cells, such as the tubular epithelium of the kidney, epidermal cells of the skin and synovial lining cells. As there was no significant reduction in staining intensity with MCA44 following treatment with phosphatidylinositol-specific phospholipase C, many guinea-pig DAF molecules expressed in these tissues may be of the transmembrane form.
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PMID:Tissue distribution of the guinea-pig decay-accelerating factor. 982 90

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b. DAF and CD1c. DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c. DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b. DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c. DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b. DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.
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PMID:Glycosyl-phosphatidylinositol reanchoring unmasks distinct antigen-presenting pathways for CD1b and CD1c. 1090 26


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