Binding ofEscherichia coliverotoxins to cell surface protein on wild-type and globotriaosylceramide-deficient Vero cells

1998 ◽  
Vol 44 (1) ◽  
pp. 28-34 ◽  
Author(s):  
John Devenish ◽  
Carlton Gyles ◽  
Jonathan LaMarre
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Surface Protein ◽  
Vero Cells ◽  
Surface Proteins ◽  
Cell Protein ◽  
Scatchard Analysis ◽  
Single Class ◽  
B Subunit ◽  
Protein Receptors

We have examined verotoxin (VT) binding to cell surface proteins. When Vero or globotriaosylceramide (Gb3) deficient Vero (VRP) cells were incubated with125I-labelled verotoxin 2 (VT2) and disuccinimidyl suberate cross-linker, SDS-PAGE of cell lysates showed radiolabelled bands at 44, 50, 60, 86, 102, and 138 kDa. When125I-labelled verotoxin 1 (VT1) was cross-linked, radioactive bands occurred at 51, 67, 101, 160, 188, and 232 kDa. In contrast,125I-labelled VT1 B subunit produced a single radioactive band migrating at 50 kDa. CHO cells did not bind labelled VT. VT2 binding to VRP cells fit a rectangular hyperbola suggesting a single class of binding sites. In contrast, VT1 and VT1 B subunit binding to VRP cells was best fit by sigmoidal curves suggesting the presence of positive cooperativity between at least two binding sites. Scatchard analysis of VT2 binding data yielded 3.5 times 109molecules bound/ µg of cell protein with an equilibrium dissociation constant (KD) of 13 nM. The apparent KDwas 9.7 nM for VT1 and 73.2 nM for VT1 B subunit. These results indicate that VT binds to a protein, or proteins, on the surface of susceptible cells and that there appear to be differences between VT1 and VT2 binding. Interactions between VT1 or VT2 and the proteins demonstrated here may be important in the biological activity of VT.Key words: verotoxin, protein receptors, hemolytic uremic syndrome, Escherichia coli.

scholarly journals Microfluidic Techniques for Single-Cell Protein Expression Analysis

2006 ◽  
Vol 52 (6) ◽  
pp. 1080-1088 ◽  
Author(s):  
Ethan Fitzpatrick ◽  
Sterling McBride ◽  
Jonathan Yavelow ◽  
Saltanat Najmi ◽  
Peter Zanzucchi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Cancer Cells ◽  
Microfluidic Device ◽  
Single Cells ◽  
Surface Protein ◽  
Surface Proteins ◽  
Cell Protein ◽  
Surface Protein Expression ◽  
Mcf 7

Abstract Background: The analysis of single cells obtained from needle aspirates of tumors is constrained by the need for processing. To this end, we investigated two microfluidic approaches to measure the expression of surface proteins in single cancer cells or in small populations (<50 cells). Methods: One approach involved indirect fluorescence labeling of cell-surface proteins and channeling of cells in a microfluidic device past a fluorescence detector for signal quantification and analysis. A second approach channeled cells in a microfluidic device over detection zones coated with ligands to surface proteins and measured rates of passage and of retardation based on transient interactions between surface proteins and ligands. Results: The fluorescence device detected expression of integrin α5 induced by basic fibroblast growth factor (FGF-2) treatment in MCF-7 cells and that of Her-2/neu in SK-BR-3 cells compared with controls. Experiments measuring passage retardation showed significant differences in passage rates between FGF-2–treated and untreated MCF-7 cells over reaction regions coated with fibronectin and antibody to integrin α5β1 compared with control regions. Blocking peptides reversed the retardation, demonstrating specificity. Conclusions: Immunofluorescence detection in a microfluidic channel demonstrates the potential for assaying surface protein expression in a few individual cells and will permit the development of future iterations not requiring cell handling. The flow retardation device represents the first application of this technology for assessing cell-surface protein expression in cancer cells and may provide a way for analyzing expression profiles of single cells without preanalytical manipulation.

scholarly journals Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition

2020 ◽  
Vol 22 (1) ◽  
pp. 85
Author(s):  
Julia L. Sanwald ◽  
Jochen Dobner ◽  
Indra M. Simons ◽  
Gereon Poschmann ◽  
Kai Stühler ◽  
...  
Keyword(s):  
Cell Surface ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Microscopic Analysis ◽  
Surface Protein ◽  
Surface Proteins ◽  
Receptor Associated Protein ◽  
Independent Functions ◽  
Protein Receptors

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome composition of TKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila

1986 ◽  
Vol 6 (9) ◽  
pp. 3240-3245
Author(s):  
G A Bannon ◽  
R Perkins-Dameron ◽  
A Allen-Nash
Keyword(s):  
Cell Surface ◽  
Specific Surface ◽  
Incubation Temperature ◽  
Tetrahymena Thermophila ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Surface Proteins ◽  
Ciliated Protozoan ◽  
Temperature Dependent ◽  
Molecular Sizes

The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.

Electrophoretic and Chemical Characterization of the Charged Groups at the Surface of Murine CL3 Ascites Leukaemia Cells

1969 ◽  
Vol 4 (2) ◽  
pp. 289-298
Author(s):  
P. D. WARD ◽  
E. J. AMBROSE
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Chemical Characterization ◽  
Surface Protein ◽  
Subsequent Treatment ◽  
Surface Proteins ◽  
Carboxyl Groups ◽  
Neuraminidase Treatment ◽  
Ionic Nature ◽  
Ph Range

The electrophoretic characteristics of the murine CL3 ascites tumour were investigated. Treatment of the cells with formaldehyde raised the electrophoretic mobility (E.P.M.) from - 1.06 to - 1.28 µ/sec/V/cm; subsequent treatment with diazomethane reduced their mobility to zero. The E.P.M. of the diazomethane-treated cells did not alter over the pH range 3.0-8.0. This proved that the only ionic groups at this cell surface were amino and carboxyl groups. The absence of phosphate groups, another possibility, was confirmed by the lack of calcium-ion binding from 10 mM Ca2+ solutions. Neuraminidase treatment reduced the E.P.M. from -1.06 to -0.55 µ/sec/V/cm and free sialic acid was identified in the enzyme supernatant. Subsequent treatment of the cells with formaldehyde raised the mobility to -1.22 µ/sec/V/cm indicating that the change in E.P.M. on neuraminidase treatment was not due solely to the removal of the carboxyl groups of sialic acid but also to a change in the ionic nature of the surface. This change is ascribed to a change in the conformation of the surface protein. The reason for this change and a suggestion for the possible role of sialic acid at the cell surface are mentioned. Treatment of the cells with trypsin did not affect the viable cells in any way, suggesting that the surface proteins lack the basic amino acids lysine and arginine. Pronase treatment served only to show that much of the sialic acid was bound to protein; the total amount was not determined.

scholarly journals Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 542-551 ◽  
Author(s):  
AA Higazi ◽  
RH Upson ◽  
RL Cohen ◽  
J Manuppello ◽  
J Bognacki ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Binding Sites ◽  
Ldl Receptor ◽  
Cell Types ◽  
Surface Proteins ◽  
Single Chain ◽  
Cell Surface Proteins ◽  
Functional Changes ◽  
And Migration

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI- 1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell- bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.

scholarly journals Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T Kitamura ◽  
A Tojo ◽  
T Kuwaki ◽  
S Chiba ◽  
K Miyazono ◽  
...  
Keyword(s):  
Cell Line ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Scatchard Analysis ◽  
Hematopoietic Growth Factors ◽  
Single Class ◽  
Gm Csf ◽  
Molecular Weights ◽  
Human Erythropoietin ◽  
Macrophage Colony

Abstract We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.

CORTICOSTERONE BINDING BY MOUSE SERA DURING FOETAL AND POST-NATAL DEVELOPMENT

1977 ◽  
Vol 84 (1) ◽  
pp. 177-190 ◽  
Author(s):  
Lia Savu ◽  
Emmanuel Nunez ◽  
Max-Fernand Jayle
Keyword(s):  
Amniotic Fluid ◽  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Normal Adult ◽  
Scatchard Analysis ◽  
Mammalian Development ◽  
Single Class ◽  
Mean Values

ABSTRACT The binding properties of corticosterone binding globulin (CBG) of mouse sera have been studied by equilibrium dialysis and electrophoretic techniques, at different stages of foetal and post-natal development. Scatchard analysis has demonstrated in all cases a single class of high affinity saturable binding sites for corticosterone. Remarkable increases of the binding capacities were observed in the foetal and pregnant sera, as compared to normal adult and immature levels. The mean values of n1M1 × g−1 of serum proteins (concentration of binding sites, n1 × moles of binding proteins M1) were 21 10−8 in 14–19 day pregnant females, 17 10−8 in the amniotic fluid, 4.2 10−8 in 14–19 day embryos, and only 0.8 10−8 in the normal adult female. Neonatal mice, aged 0–6 days exhibited no CBG activities. The association constants showed values of 2.5–4.1 108 m−1 when measured with foetal sera, and of 1.2–2.1 108 m−1 with pregnant or control adult sera and with the amniotic fluid, at 25°C. Comparative electrophoretic, thermal denaturation and competition studies with foetal and pregnant plasma CBG's are also reported. The results are discussed in relation to the origin of CBG in the foetal serum, and also with respect to similar studies in the rat, guinea pig and man. The possible biological implications of serum steroid binding proteins in mammalian development are briefly outlined.

Involvement of estrogen receptors α and β in the regulation of cervical permeability

2000 ◽  
Vol 278 (4) ◽  
pp. C689-C696 ◽  
Author(s):  
George I. Gorodeski ◽  
Dipika Pal
Keyword(s):  
Estrogen Receptor ◽  
Binding Sites ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Scatchard Analysis ◽  
Single Class ◽  
Estrogen Receptor Modulators ◽  
Transcription Inhibitor ◽  
Estrogen Binding ◽  
In Cells

Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888–C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of ∼0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time ≈ 4 h, and EC50≈ 1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor α (αER) and estrogen receptor β (βER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of αER and βER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased βER mRNA. In cells treated with estrogen, neither ICI-182780 nor tamoxifen had modulated significantly the increase in αER or βER mRNA. The transcription inhibitor actinomycin D blocked the estrogen-induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an αER-dependent increase in transcription.

288 CHARACTERIZATION OF PORCINE ADIPOSE TISSUE-DERIVED ADULT STEM CELL SURFACE PROTEINS

2009 ◽  
Vol 21 (1) ◽  
pp. 241
Author(s):  
K. J. Williams ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Tissue Engineering ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Cell Surface ◽  
Cell Lines ◽  
Adult Stem Cell ◽  
Surface Protein ◽  
Surface Proteins ◽  
Early Passage ◽  
Cell Surface Proteins

Human adipose tissue-derived adult stem (ADAS) cells are a self-renewing population of cells with a multilineage plasticity similar to bone marrow-derived mesenchymal stem cells. Human ADAS have promise for use in combination with various biomaterials for reconstructive tissue engineering. The phenotypic profile of human ADAS cell surface proteins has been partially characterized for stem cell-associated cluster differentiation molecules including CD29, CD44, and CD90. Porcine ADAS cells, an animal model for tissue engineering, also have the ability to self-renew and differentiate into multiple tissue lineages. However, the surface protein phenotype has not been described. Because porcine ADAS are isolated from fat depots likely different from human ADAS liposuction aspirates, it is important to characterize these cells. In this study, we have partially characterized the surface protein phenotype of undifferentiated porcine ADAS cells in comparison with the immunophenotype of undifferentiated human ADAS cells as reported in the literature. Flow cytometry and enhanced chemiluminescence Western blot analysis of early passage (passages 0–4) porcine ADAS cell populations demonstrated that the profiles are not similar to the human ADAS cell surface. Immunoblot detection paired with an enhanced chemiluminescence kit revealed a positive expression for CD44 and CD90 in human ADAS cells as indicated by bands present at the expected sizes and a negative expression for CD44 and CD90 in porcine ADAS cells. Flow cytometric analysis also indicated differences between human and early passage porcine ADAS cell surfaces with a relatively low expression of CD29 (5 cell lines with a mean percent positive of 4.5 ± 1.7 and a range of 2.5–7.2%) and CD44 (5 cell lines with a mean percent positive of 0.66 ± 0.67 and a range of 0.0–1.8%) compared with human ADAS values of 98 ± 1 and 60 ± 15, respectively (Gronthos et al. 2001). Other cell surface proteins analyzed at early passages include CD3 (3 cell lines; 0.07 ± 0.06% positive and 0.0–0.1 range), CD8 (3 cell lines; 0.10 ± 0.10% positive and 0–0.2 range), and CD90 [5 cell lines; 12.7 ± 11.9% positive and 2.4–33 range; human ADAS geometric mean 25.96% (Zuk et al. 2002)]. Analysis of late passage (passages 5–11) porcine ADAS cell populations revealed an increased expression of CD29 (3 cell lines; 26.4 ± 7.2% positive and 21.2–34.6 range). The expression level of CD90 at late passages were 21.3 and 26.9% positive for 2 cell lines and CD44 remained low (3 cell lines; 4.1 ± 3.5% positive and 0.2–7.0 range). Later passages were also analyzed for c-Kit (CD117), which was expressed at low levels (2 cell lines; 0.3 and 0.4% positive). The characterization of adipose tissue-derived adult stem cell surface proteins present at different stages of in vitro culture from a model animal, such as the pig, could have valuable impacts on tissue engineering research. These results suggest that care should be taken when interpreting results from animal models of somatic stem cells.

Sign in / Sign up

Export Citation Format

Share Document