scholarly journals Collagen binding proteins derived from the embryonic fibroblast cell surface recognize arginine-glycine-aspartic acid

1989 ◽  
Vol 9 (3) ◽  
pp. 329-340 ◽  
Author(s):  
Roy C. Ogle ◽  
Charles D. Little
Keyword(s):  
Cell Surface ◽  
Aspartic Acid ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Fibroblast Cell ◽  
Surface Proteins ◽  
Collagen Molecule ◽  
Collagen Binding ◽  
Arginine Glycine Aspartic Acid ◽  
Embryonic Fibroblast

Several cell surface proteins (Mr=120,000, 90,000, 63,000 and 47,000) apparently integral to embryonic fibroblast plasma membranes were extracted with detergent and isolated by collagen affinity chromatography. Certain of these proteins (Mr=120,000, 90,000 and 47,000) were specifically eluted from collagen affinity columns by synthetic peptides containing the amino acid sequence arginyl-glycyl-aspartic acid (RGD). These data show that a number of collagen binding proteins exist on the embryonic fibroblast cell surface. Some of the proteins may be collagen receptors binding to RGD sequences in the collagen molecule while at least one of the proteins (Mr=63,000) recognizes features other than RGD.

scholarly journals The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus

2015 ◽  
Vol 197 (23) ◽  
pp. 3720-3730 ◽  
Author(s):  
Jessica L. Danger ◽  
Nishanth Makthal ◽  
Muthiah Kumaraswami ◽  
Paul Sumby
Keyword(s):  
Cell Surface ◽  
Virulence Factors ◽  
Binding Proteins ◽  
Mrna Translation ◽  
Regulatory Rna ◽  
Collagen Binding ◽  
Content Type ◽  
Small Regulatory Rna ◽  
Group A ◽  
Fibronectin Binding

ABSTRACTThe group AStreptococcus(GAS;Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to theprtF1andprtF2mRNAs within their 5′ untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and theprtF1andprtF2mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection.IMPORTANCEMore than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small RNA FasX. In addition to identifying that FasX reduces the abundance of the cell surface-located fibronectin-binding proteins PrtF1/2, fibronectin is present in high abundance in human tissues, and we have determined the mechanism behind this regulation. Importantly, as FasX is the only mechanistically characterized regulatory RNA in GAS, it serves as a model RNA in this and related pathogens.

scholarly journals Antiadhesive Role of Apical Decay-accelerating Factor (CD55) in Human Neutrophil Transmigration across Mucosal Epithelia

2003 ◽  
Vol 198 (7) ◽  
pp. 999-1010 ◽  
Author(s):  
Donald W. Lawrence ◽  
Walter J. Bruyninckx ◽  
Nancy A. Louis ◽  
Douglas M. Lublin ◽  
Gregory L. Stahl ◽  
...  
Keyword(s):  
Cell Surface ◽  
Plasma Membranes ◽  
Apical Cell ◽  
Neutrophil Migration ◽  
Surface Proteins ◽  
Epithelial Surface ◽  
Decay Accelerating Factor ◽  
Apical Cell Membrane ◽  
Epithelial Monolayers ◽  
Epithelial Cell Surface

Neutrophil migration across mucosal epithelium during inflammatory episodes involves the precise orchestration of a number a cell surface molecules and signaling pathways. After successful migration to the apical epithelial surface, apically localized epithelial proteins may serve to retain PMN at the lumenal surface. At present, identification of apical epithelial ligands and their PMN counter-receptors remain elusive. Therefore, to define the existence of apical epithelial cell surface proteins involved in PMN–epithelial interactions, we screened a panel of antibodies directed against epithelial plasma membranes. This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers. Microsequence analysis revealed that OE-1 recognized human decay-accelerating factor (DAF, CD55). DAF is a highly glycosylated, 70–80-kD, glycosyl-phosphatidyinositol–linked protein that functions predominantly as an inhibitor of autologous complement lysis. DAF suppression experiments using antisense oligonucleotides or RNA interference revealed that DAF may function as an antiadhesive molecule promoting the release of PMN from the lumenal surface after transmigration. Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface. The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.

scholarly journals Selective labelling of cell-surface polyamine-binding proteins on leukaemic and solid-tumour cell types using a new polyamine photoprobe

1997 ◽  
Vol 328 (3) ◽  
pp. 889-895 ◽  
Author(s):  
M. Donna FELSCHOW ◽  
Zenghui MI ◽  
J. STANEK ◽  
J. FREI ◽  
W. Carl PORTER
Keyword(s):  
Cell Surface ◽  
Tumour Cell ◽  
Solid Tumour ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Protein S ◽  
Cell Types ◽  
Biochemical Properties ◽  
Surface Proteins ◽  
Polyamine Transport

Polyamine transport is an active process which contributes to the regulation and maintenance of intracellular polyamine pools. Although the biochemical properties of polyamine transport in mammalian cells have been extensively studied, attempts to isolate and characterize the actual protein(s) have met with limited success. As one approach, photoaffinity labelling of cell surface proteins using a polyamine-conjugated photoprobe may lead to the identification of polyamine-binding proteins (pbps) associated with the transport apparatus and/or other regulatory responses. In a previous study [Felschow, MacDiarmid, Bardos, Wu, Woster and Porter (1995) J. Biol. Chem. 270, 28705-28711], we demonstrated that the photoprobes N4-ASA-spermidine and N1-ASA-norspermine [where the ASA (azidosalicylamidoethyl) group represents the photoreactive moiety] competed effectively with polyamines for transport and selectively labelled two major pbps at 118 and 50 kDa on the surface of murine and human leukaemia cells. In the present study, a new and more potent polyamine-conjugated photoprobe, N1-ASA-spermine, has been synthesized and used to develop a method based on detergent lysis for identifying putative cell-surface pbps on solid-tumour cell types. Transport kinetic assays showed that the new photoprobe competed with spermidine uptake with an apparent Ki of 1 μM, a value 20-50-fold lower than those of earlier probes. In L1210 cells, the new probe identified pbp50 and pbp118 thus reaffirming their identity as pbps. Two new bands were also detected. In A549 human lung adenocarcinoma cells, N1-ASA-spermine identified pbps at 39, 62, 73 and 130 kDa, the latter believed to be a size variant of pbp118. The presence of pbp130/118 in two very different cell types suggests the generality of the protein among mammalian cell types as well as its importance for further study. The high affinity of the photoprobe for the polyamine-transport system strongly suggests that at least some of the identified pbps may be associated with that function.

Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium

2004 ◽  
Vol 287 (3) ◽  
pp. H1344-H1352 ◽  
Author(s):  
Malgorzata Czarny ◽  
Jan E. Schnitzer
Keyword(s):  
Endothelial Cell ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Fluid Shear Stress ◽  
Experimental Models ◽  
Surface Proteins ◽  
Rat Lung ◽  
Neutral Sphingomyelinase

Recently, we showed that neutral sphingomyelinase (N-SMase) is concentrated at the endothelial cell surface in caveolae and is activated to produce ceramide in an acute and transient manner by increase in flow rate and pressure in rat lung vasculature (Czarny M, Liu J, Oh P, and Schnitzer JE, J Biol Chem 278: 4424–4430, 2003). Here, we report further on our investigations of this new acute mechanotransduction pathway. We employed three experimental models to explore the role of N-SMase and ceramides in mechanosignaling: 1) a cell-free, in vitro model using isolated luminal plasma membranes of rat lung endothelium; 2) a fluid shear stress model using monolayers of intact bovine aorta endothelial cell in culture; and 3) an in situ model using controlled perfusion of the rat lung vasculature. Scyphostatin, which specifically inhibited N-SMase but not acid SMase activity, prevented mechanoactivation of N-SMase as well as downstream tyrosine and mitogen-activated protein kinases. Cell-permeable ceramide analogs ( N-acetylsphingosine, C2-ceramide, and N-hexanoylsphingosine, C6-ceramide) but not the inactive dihydroderivatives D2-ceramide and D6-ceramide ( N-acetylsphinganine and N-hexanoylsphinganine, respectively) mimic rapid mechano-induced tyrosine phosphorylation of cell surface proteins as well as mechanoactivation of Src-like kinases and the extracellular regulated kinase pathway. The responses common to ceramide and mechanical stress were inhibited by genistein, herbamycin A, and PP2, but not PP3, which suggests an obligate role of Src-like kinases in ceramide-mediated mechanotransduction. Ceramides also induced serine/threonine phosphorylation to activate the Akt/endothelial nitric oxide synthase pathway. Thus N-SMase at the plasma membrane in caveolae may be an upstream initiating mechanosensor, which acutely triggers mechanotransduction by generation of the lipid second messenger ceramide.

scholarly journals Embryonic chicken fibroblast collagen binding proteins: distribution, role in substratum adhesion, and relationship to integrins

1989 ◽  
Vol 94 (2) ◽  
pp. 361-369
Author(s):  
R.C. Ogle ◽  
A.J. Potts ◽  
M. Yacoe ◽  
C.D. Little
Keyword(s):  
Cell Surface ◽  
Binding Proteins ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Type I ◽  
Dependent Manner ◽  
Filamentous Actin ◽  
Collagen Binding ◽  
Chicken Heart ◽  
Embryonic Chicken

Collagen binding proteins (CBP) are hydrophobic, cell surface polypeptides, isolated by collagen affinity chromatography. Antibodies to CBPs inhibit the attachment of embryonic chicken heart fibroblasts to native type I collagen fibrils in a dose-dependent manner. The CBP antibodies also induce rounding and detachment of cells adherent to a planar substratum. This process of antibody-mediated substratum detachment resulted in a clustering of CBP and cell-associated extracellular matrix at the cell surface, and the rearrangement of filamentous actin. Other functional studies showed that cells grown within a three-dimensional gel of type I collagen cannot be immunostained at the cell surface with CBP antibodies. However, treatment of cultures with purified collagenase, unmasks immunoreactive sites and permits strong cell surface immunolabeling. This result suggests that collagen sterically blocks antibody access to CBP. Finally, we show that antibodies to CBP recognize purified avian integrin beta subunits; and that antibodies to avian integrins recognize a 100,000 Mr CBP. These data demonstrate that chicken embryonic fibroblasts possess surface polypeptides that mediate adhesion to type I collagen, and suggest that two of these proteins are related to the integrin family.

scholarly journals A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis.

1990 ◽  
Vol 110 (4) ◽  
pp. 1077-1088 ◽  
Author(s):  
T K Rosiere ◽  
J A Marrs ◽  
G B Bouck
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Euglena Gracilis ◽  
Plasma Membranes ◽  
Membrane Skeleton ◽  
Surface Proteins ◽  
Plasma Membrane Protein ◽  
Octyl Glucoside ◽  
Skeletal Protein

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39. Other (62, 51, and 25 kD) quantitatively minor peripheral proteins also interact with IP39 on the nitrocellulose overlays, and the possible significance of this binding is discussed.

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