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C N Metz

Publications and source records attributed to C N Metz.

44 records · Page 3Linked to original sources
PubMed1994

An endogenous glycosylphosphatidylinositol-specific phospholipase D releases basic fibroblast growth factor-heparan sulfate proteoglycan complexes from human bone marrow cultures.

Basic fibroblast growth factor (bFGF) is a hematopoietic cytokine that stimulates stromal and stem cell growth. It binds to a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan on human bone marrow (BM) stromal cells. The bFGF-proteoglycan complex is biologically active and is released by addition of exogenous phosphatidylinositol-specific phospholipase C. In this study, we show the presence of an endogenous GPI-specific phospholipase D (GPI-PLD) that releases the bFGF-binding heparan sulfate proteoglycan and the variant surface glycoprotein (a model GPI-anchored protein) from BM cultures. An involvement of proteases in this process is unlikely, because released proteoglycan contained the GPI anchor component, ethanol-amine, and protease inhibitors did not diminish the release. The mechanism of release is likely to involve a GPI-PLD and not a GPI-specific phospholipase C, because the release of variant surface glycoprotein did not reveal an epitope called the cross-reacting determinant that is exposed by phospholipase C-catalyzed GPI anchor cleavage. In addition, phosphatidic acid (which is specifically a product of GPI-PLD-catalyzed anchor cleavage) was generated during the spontaneous release of the GPI-anchored variant surface glycoprotein. We also detected GPI-PLD-specific enzyme activity and mRNA in BM cells. Therefore, we conclude that an endogenous GPI-PLD releases bFGF-heparan sulfate proteoglycan complexes from human BM cultures. This mechanism of GPI anchor cleavage could be relevant for mobilizing biologically active bFGF in BM. An endogenous GPI-PLD could also release other GPI-anchored proteins important for hematopoiesis and other physiologic processes.

Adult↗
PubMed1994

Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D.

Although many glycosylphosphatidylinositol (GPI)-anchored proteins have been observed as soluble forms, the mechanisms by which they are released from the cell surface have not been demonstrated. We show here that a cell-associated GPI-specific phospholipase D (GPI-PLD) releases the GPI-anchored, complement regulatory protein decay-accelerating factor (DAF) from HeLa cells, as well as the basic fibroblast growth factor-binding heparan sulfate proteoglycan from bone marrow stromal cells. DAF found in the HeLa cell culture supernatants contained both [3H]ethanolamine and [3H]inositol, but not [3H]palmitic acid, whereas the soluble heparan sulfate proteoglycan present in bone marrow stromal cell culture supernatants contained [3H]ethanolamine. 125I-labeled GPI-DAF incorporated into the plasma membranes of these two cell types was released in a soluble form lacking the fatty acid GPI-anchor component. GPI-PLD activity was detected in lysates of both HeLa and bone marrow stromal cells. Treatment of HeLa cells with 1,10-phenanthroline, an inhibitor of GPI-PLD, reduced the release of [3H]ethanolamine-DAF by 70%. The hydrolysis of these GPI-anchored molecules is likely to be mediated by an endogenous GPI-PLD because [3H]ethanolamine DAF is constitutively released from HeLa cells maintained in serum-free medium. Furthermore, using PCR, a GPI-PLD mRNA has been identified in cDNA libraries prepared from both cell types. These studies are the first demonstration of the physiologically relevant release of GPI-anchored proteins from cells by a GPI-PLD.

Antigens, CD↗
PubMed1994

An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cellular differentiation, proliferation, migration, and protein expression. These properties have been exploited to create a variety of bioassays for detecting the mature growth factor. In this paper, we describe a highly sensitive and specific, nonradioactive quantitative bioassay for TGF-beta based on its ability to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mink lung epithelial cells (MLEC) were stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Addition of TGF-beta (0.2 to > 30 pM) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. Although responsive to TGF-beta, this promoter fragment was only minimally influenced by other known inducers of PAI-1 expression. When compared to the widely used MLEC assay, this assay demonstrated greater sensitivity and specificity, allowing quantification of TGF-beta in complex biological solutions.

Animals↗
PubMed1994

Binding of GPI-PLD-treated DAF to the surface of Schistosoma mansoni schistosomula.

Decay accelerating factor (DAF,CD55) is a 70-kDa glycosylphosphatidylinositol (GPI)-anchored protein that protects human erythrocytes (HuE) from complement-mediated damage by regulation of the C3-convertase. Purified human DAF can be incorporated into sheep red blood cell (SRBC) membrane and confer complement resistance on these DAF-deficient cells. Here, we demonstrate that normal HuE or their stroma (HuES) incubated at 37 degrees C for 24 h release soluble DAF in a biologically active form into the culture medium. This soluble DAF neither inserts into SRBC plasma membranes nor presents the cross-reacting determinant (CRD) characteristic of the hydrolysis by phosphatidylinositol-specific phospholipases C (PI-PLC) but binds to schistosomula of S. mansoni protecting them from antibody-mediated complement-dependent damage. To study the binding of DAF to schistosomula in vitro, we have used purified human DAF labeled with 125I(125I-DAF), intact or treated with either PI-PLC or GPI-PLD (glycosylphosphatidylinositol-specific phospholipase D). We have found that GPI-PLD-treated DAF binds to the surface of parasites more readily than intact or PI-PLC-treated DAF. Immunoprecipitation of the samples with a monoclonal anti-human DAF antibody (IA10) revealed that schistosomula incubated with GPI-PLD-treated 125I-DAF emit a stronger signal than their counterparts. This result indicates that the surface of schistosomula is capable of acquiring GPI-PLD-treated DAF more effectively than intact or PI-PLC-treated molecules.

Animals↗
PubMed1992

Control of transforming growth factor-beta activity: latency vs. activation.

Transforming growth factor-beta is a pluripotent regulator of cell growth and differentiation. The growth factor is expressed as a latent complex that must be converted to an active form before interacting with its ubiquitous high affinity receptors. This conversion involves the release of the mature growth factor through disruption of the non-covalent interactions with its pro-peptide or latency associated peptide. The mechanisms for this release in vivo have not been fully characterized but appear to be cell specific and might involve processes such as acidification or proteolysis. Although several factors including transcriptional regulation, receptor modulation and scavenging of the active growth factor have been implicated, the critical step controlling the biological effects of transforming growth factor-beta may be the activation of the latent molecule.

Animals↗
PubMed1992

Immunolocalization of a glycosylphosphatidylinositol-specific phospholipase D in mast cells found in normal tissue and neurofibromatosis lesions.

A large number of eukaryotic proteins have been shown to be anchored to the cell membrane by glycosylphosphatidylinositol (GPI). This glycolipid anchor can serve as a substrate for anchor-specific phospholipases that convert the GPI-anchored membrane proteins into soluble forms. Soluble forms of many GPI anchored proteins have been identified in vivo in connective tissue, plasma, and urine. The authors have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD). Because it recognizes a portion of the conserved glycan core structure, all GPI-anchored proteins are potential substrates. The authors report the development of a murine monoclonal antibody specific for one form of the human GPI-PLD and the immunohistochemical localization of this enzyme to mast cells.

Animals↗
PubMed1991

Production of the glycosylphosphatidylinositol-specific phospholipase D by the islets of Langerhans.

A large number of diverse cell surface proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. One proposed function for the GPI anchor is that it facilitates the release of the protein from the cell by acting as a target for anchor-specific phospholipases. We and others have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD) (Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem, Biophys. Res. Commun. 150, 476-482; Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984). Because the GPI-PLD recognizes a conserved portion of the anchor, all GPI-anchored proteins are potential substrates for the enzyme. We demonstrate in this communication the production of the plasma GPI-PLD by the islets of Langerhans. GPI-PLD enzymatic activity was found in dog pancreatic microsomes, but not pancreatic juice. Both the pancreatic and plasma enzymes were divalent cation-dependent and had identical substrate specificities. Purified murine islets of Langerhans, as well as alpha and beta cells, contained and released GPI-PLD activity. A GPI-PLD DNA fragment was amplified by polymerase chain reaction from a normal human islet cDNA library; the amplified fragment hybridized with the GPI-PLD cDNA clone. These findings represent the first demonstration of the production of the plasma GPI-PLD by a specific tissue site as well as cell type.

Animals↗