scholarly journals CD19 has a potential CD77 (globotriaosyl ceramide)-binding site with sequence similarity to verotoxin B-subunits: implications of molecular mimicry for B cell adhesion and enterohemorrhagic Escherichia coli pathogenesis.

1994 ◽  
Vol 180 (1) ◽  
pp. 191-201 ◽  
Author(s):  
M D Maloney ◽  
C A Lingwood
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
B Cell ◽  
Germinal Center ◽  
Sequence Similarity ◽  
Wild Type ◽  
B Subunit ◽  
Daudi Cells ◽  
Globotriaosyl Ceramide ◽  
Mutant Cells

The glycosphingolipid globotriaosyl ceramide (CD77) and other globo-series glycolipids containing terminal galactose (Gal)alpha 1-4Gal residues function as receptors for the verotoxin (Shiga-like toxin) family of Escherichia coli-elaborated toxins. CD77 is also a marker for germinal center B lymphocytes and Burkitt's lymphoma cells. The pan B cell marker CD19 is a 95-kD membrane protein that appears early in B cell differentiation and is only lost upon terminal differentiation to plasma cells. CD19 is involved in signal transduction and has a regulatory role in B cell proliferation and differentiation in response to activation in vitro. However, an endogenous ligand for CD19 has not yet been identified. We report herein that the extracellular domain of CD19 has a potential CD77-binding site with extensive sequence similarity to the verotoxin B-subunits. These B-subunit-like sequences on CD19 are in close proximity following the organization of intervening amino acids into disulfide-linked domains. Cocapping of CD19 and CD77 on Burkitt's lymphoma-derived Daudi cells with anti-CD19 antibodies indicates that CD19 and CD77 are associated on the B cell surface. Cell surface binding of anti-CD19 antibodies is decreased on CD77-deficient mutant Daudi cells, suggesting that CD77 expression influences the surface expression of CD19. Wild-type Daudi cells, but not the CD19/CD77-deficient mutants, bind to matrices expressing the carbohydrate moiety of CD77 or other Gal alpha 1-4Gal containing glycolipids. This binding can be inhibited by anti-CD77 antibodies, the CD77-binding verotoxin B-subunit or anti-CD19 antibodies. Daudi cells exhibit a degree of spontaneous homotypic adhesion in culture while the CD77/CD19-deficient Daudi mutants grow as single cells. The stronger homotypic adhesion that occurs in B cells after antibody ligation of CD19 and that involves, to some extent, the integrin system, is also dramatically lower in the mutant cells relative to the parent cell line. However, reconstitution of mutant cells with CD77 restores the anti-CD19 mAb-induced adhesion to wild-type Daudi cell levels. These studies represent the first time that CD19-mediated signaling has been reconstituted in a low-responder B cell line. These convergent observations provide compelling evidence that CD19/CD77 interactions function in adhesion and signal transduction at a specific stage in B cell development and suggest that such interactions have a role in B lymphocyte homing and germinal center formation in vivo. By targeting CD77+ B cells, verotoxins may suppress the humoral arm of the immune response during infection.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Glycolipid modification of α2 interferon binding. Sequence similarity between the α2 interferon receptor and verotoxin (Shiga-like toxin) B-subunit

1992 ◽  
Vol 283 (1) ◽  
pp. 25-26 ◽  
Author(s):  
C A Lingwood ◽  
S K Yiu
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Sequence Similarity ◽  
Structural Basis ◽  
Toxin B ◽  
B Subunit ◽  
Interferon Receptor ◽  
Binding Sequence ◽  
Glycolipid Receptor ◽  
Binding Subunit

Previous studies have implicated the glycolipid receptor for the Escherichia coli-derived verotoxin, globotriaosylceramide (Gb3; Gal alpha 1-4Gal beta 1-4Glc-ceramide), in the mechanism of alpha 2 interferon signal transduction. Comparison of the amino acid sequence of the human alpha 2 interferon receptor with that of the B (receptor-binding)-subunit of verotoxin shows three regions of similarity which may provide a structural basis for alpha 2-interferon-receptor/Gb3 interaction.

scholarly journals Differential Biological and Adjuvant Activities of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Hybrids

2001 ◽  
Vol 69 (3) ◽  
pp. 1528-1535 ◽  
Author(s):  
Christal C. Bowman ◽  
John D. Clements
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
The Other ◽  
Toxin Gene ◽  
Wild Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit ◽  
Ifn Γ

ABSTRACT Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A–CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A–CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-γ) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A–LT-B and native CT both produced lower levels of antigen-specific IFN-γ. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

scholarly journals TolC-Dependent Exclusion of Porphyrins in Escherichia coli

2008 ◽  
Vol 190 (18) ◽  
pp. 6228-6233 ◽  
Author(s):  
Ryoko Tatsumi ◽  
Masaaki Wachi
Keyword(s):  
Escherichia Coli ◽  
Uv Irradiation ◽  
High Performance ◽  
Aminolevulinic Acid ◽  
Wild Type ◽  
E Coli ◽  
Chromatography Analysis ◽  
Increased Sensitivity ◽  
Mutant Cells ◽  
Reddish Brown Color

ABSTRACT We found that Escherichia coli tolC mutants showed increased sensitivity to 5-aminolevulinic acid (ALA), a precursor of porphyrins. The tolC mutant cells grown in the presence of ALA showed a reddish brown color under visible light and a strong red fluorescence under near-UV irradiation. Fluorescence spectrometry and high-performance liquid chromatography analysis showed that the tolC mutant cells grown in the presence of ALA accumulated a large amount of coproporphyrin(ogen) intracellularly. In contrast, the wild-type cells produced coproporphyrin extracellularly. The tolC mutant cells grown in the presence of ALA, which were capable of surviving in the dark, were killed by near-UV irradiation, suggesting that the intracellular coproporphyrin(ogen) renders these cells photosensitive. These results suggest that the TolC-dependent efflux system is involved in the exclusion of porphyrin(ogen)s in E. coli.

scholarly journals Genetic and Biochemical Analysis of Phosphatase Activity of Escherichia coli NRII (NtrB) and Its Regulation by the PII Signal Transduction Protein

2003 ◽  
Vol 185 (4) ◽  
pp. 1299-1315 ◽  
Author(s):  
Augen A. Pioszak ◽  
Alexander J. Ninfa
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Binding Site ◽  
Phosphatase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Small Surface ◽  
Central Domain ◽  
Atp Binding Site ◽  
Terminal Domain

ABSTRACT Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the “back” or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the “back” of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.

Role of FOXO1 in Germinal Center Development and Lymphomagenesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3536-3536 ◽  
Author(s):  
David Dominguez-Sola ◽  
Jennifer Kung ◽  
Victoria A Wells ◽  
Antony B Holmes ◽  
Laura Pasqualucci ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Nuclear Localization ◽  
Germinal Center ◽  
Target Genes ◽  
Cell Receptor ◽  
Missense Mutations ◽  
Wild Type ◽  
Foxo1 Gene

Abstract A significant fraction of B cell non-Hodgkin lymphomas (B-NHL) of germinal center origin carry heterozygous missense mutations in FOXO1, a member of the FOXO family of transcription factors. FOXO1 is a central component of the PI3K signaling cascade engaged by the B cell receptor and is essential for B cell homeostasis and survival (Dengler et al, Nat Immunol 2008; Srinivasan et al, Cell 2009; Lin et al, Nat Immunol 2010). In response to PI3K activation, AKT phosphorylates FOXO1 leading to its nuclear-cytoplasmic translocation and inactivation. Missense mutations of the FOXO1 gene are detectable in germinal center (GC)-derived B-NHL, including ~12% of Burkitt Lymphoma (BL) and ~9% of Diffuse Large B Cell Lymphoma (DLBCL) cases (Schmitz et al, Nature 2012; Trinh et al, Blood 2013; Pasqualucci et al, Cell Rep 2014). The role of FOXO1 in normal GC development as well as the contribution of its mutations to lymphomagenesis is unclear. We show that FOXO1 expression is restricted to the dark zone of GCs, where its nuclear localization is detectable in most B cells. Mice carrying the conditional inactivation of FOXO1 in GC B cells display normal GC in number and size. However, these GCs lack phenotypically defined (CXCR4hi/CD86lo) dark zones and are entirely composed by light zone B cells (CXCR4lo/CD86hi). FOXO1-/- GC B cells express AICDA and carry a normal number of mutations in their immunonoglobulin genes, but do not undergo affinity maturation, resulting in severely impaired antigen responses. In order to identify the biological program controlled by FOXO1 in GC B cells, we identified candidate transcriptional target genes by integrating ChIP-seq and gene expression data. These analyses showed that that the establishment of the dark zone fate relies on a FOXO1-dependent transcriptional network that is enriched for genes involved in immune signaling cascades triggered by the B cell receptor and by a variety of cytokines controlling GC polarity. Notably, a majority of these target genes are co-bound and co-regulated, in a FOXO1-dependent manner, by BCL6, a well characterized GC master regulator. To assess the role of BL- and DLBCL-associated mutations, we first investigated the subcellular localization of FOXO1 mutant proteins by transfecting wild type and mutant GFP-tagged FOXO1 alleles into HeLa cells. As previously shown (Trinh et al, Blood 2013), this analysis showed that mutant FOXO1 proteins, but not the wild-type one, readily localize in the nucleus. Analogously, immunofluorescence analysis of BL and DLBCL samples showed the presence of nuclear FOXO1 in all tumors carrying mutations in the FOXO1 gene. However, nuclear localization was also detectable in virtually all cases carrying normal FOXO1 genes. Accordingly, in vitro experiments testing the ability of normal and mutated FOXO1 proteins to respond to various signals activating the PI3K pathway in multiple BL and DLBCL cell lines, failed to display a correlation between the presence of mutations and responsiveness to these signals. Taken together, these results suggest that other mechanisms in addition to direct gene mutation are responsible for the constitutive nuclear localization of FOXO1 in tumors. We are now examining the consequences of FOXO1 missense mutations in vivo, by reconstituting FOXO1-/- GC B cells with FOXO1 mutants using bone marrow chimeras. Disclosures No relevant conflicts of interest to declare.

scholarly journals Substrate Specificity and Signal Transduction Pathways in the Glucose-Specific Enzyme II (EIIGlc) Component of the Escherichia coli Phosphotransferase System

2000 ◽  
Vol 182 (16) ◽  
pp. 4437-4442 ◽  
Author(s):  
Lucinda Notley-McRobb ◽  
Thomas Ferenci
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Substrate Specificity ◽  
Dependent Manner ◽  
Wild Type ◽  
Specific Enzyme ◽  
Phosphotransferase System ◽  
Transmembrane Segments ◽  
In The Wild ◽  
Second Category

ABSTRACT Escherichia coli adapted to glucose-limited chemostats contained mutations in ptsG resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EIIGlc) and resulting in increased transport of glucose and methyl-α-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increasedptsG transcription in the absence of glucose. Increased expression of wild-type EIIGlc was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EIIGlc. The conformation of EIIGlc elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of pts genes.

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