Association between diphtheria toxin A- and B-fragment and their fusion proteins

Stenmark, Harald; Afanasiev, Boris N.; Ariansen, Sarah; Olsnes, Sjur

doi:10.1042/bj2810619

Cited by 27 publications

(31 citation statements)

References 24 publications

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“…Fragments from paFGF(K132E) and vector from pHBGF-DT1 were ligated, creating a plasmid containing the sequence encoding aFGF(K132E) in front of DTA. pBD-23 encodes the B-fragment of DT (11).…”

Section: Plasmid Construction and Protein Purification Pafgf(k132e)-mentioning

confidence: 99%

“…After translation, when appropriate, lysates containing fusion protein were mixed with an equal volume of lysate containing unlabeled DT-B. The lysates were dialyzed against dialysis buffer (20 mM HEPES, pH 7.0, 140 mM NaCl, 2 mM CaCl 2 ) to remove free [ 35 S]methionine and reducing agent, allowing disulfide bridges to be formed (11).…”

Section: Cell Culture Polyacrylamide Gel Electrophoresis In the Presmentioning

confidence: 99%

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Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells

Klingenberg¹,

Wiedlocha

Rapak³

et al. 1998

Journal of Biological Chemistry

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Acidic fibroblast growth factor (aFGF) is a potent mitogen. It acts through activation of specific cell surface receptors leading to intracellular tyrosine phosphorylation cascades, but several reports also indicate that aFGF enters cells and that it has an intracellular function as well. The aFGF(K132E) mutant binds to and activates fibroblast growth factor receptors equally strongly as the wild-type, but it is a poor mitogen. We demonstrate that aFGF(K132E) enters NIH 3T3 cells and is transported to the nuclear fraction like wild-type aFGF. A fusion protein of aFGF(K132E) and diphtheria toxin A-fragment (aFGF(K132E)-DT-A) and a similar fusion protein containing wild-type aFGF (aFGF-DT-A) were reconstituted with diphtheria toxin B-fragment. Both fusion proteins were translocated to the cytosol by the diphtheria toxin pathway and subsequently recovered from the nuclear fraction. Whereas translocation of aFGF-DT-A stimulated DNA synthesis in U2OSDR1 cells lacking functional fibroblast growth factor receptors, aFGF(K132E)-DT-A did not. The mutation disrupts a protein kinase C phosphorylation site in the growth factor making it unable to be phosphorylated. The data indicate that a defect in the intracellular action of aFGF(K132E) is the reason for its strongly reduced mitogenicity, possibly due to inability to be phosphorylated.

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Section: Plasmid Construction and Protein Purification Pafgf(k132e)-mentioning

confidence: 99%

Section: Cell Culture Polyacrylamide Gel Electrophoresis In the Presmentioning

confidence: 99%

Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells

Klingenberg¹,

Wiedlocha

Rapak³

et al. 1998

Journal of Biological Chemistry

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“…Published May 14, 2010. Diphtheria toxin (DT) is an A-B type protein toxin produced by Corynebacterium diphtheriae (1)(2)(3)(4). The B fragment (DTB) binds to the receptor on the host cell surface and mediates the translocation of the A fragment (DTA) through the cell membrane, which inactivates the protein synthesis elongation factor 2 in some mammalian cells (5)(6)(7)(8)(9). The expression of recombinant DTB (rDTB) in other prokaryotic organisms is necessary to understand the role of DT in the development and severity of toxemic infectious processes (10)(11)(12).…”

mentioning

confidence: 99%

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

Nascimento¹,

Lemes

Queiroz

et al. 2010

Braz J Med Biol Res

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The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

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“…S]methionine, 25 M unlabelled methionine was used. The concentrations of translation products obtained were estimated as previously described (56). After translation, the lysates were dialyzed at 4ЊC first for 16 h against PBS and then for 4 h against HEPES medium to remove free [ 35 S]methionine and the reducing agent, allowing disulfide bridges to be formed.…”

mentioning

confidence: 99%

Stimulation of Proliferation of a Human Osteosarcoma Cell Line by Exogenous Acidic Fibroblast Growth Factor Requires both Activation of Receptor Tyrosine Kinase and Growth Factor Internalization

Wiedlocha

Falnes

Rapak

et al. 1996

Molecular and Cellular Biology

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102

103

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U2OSDr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.Acidic fibroblast growth factor (aFGF) induces a multitude of responses in target cells. In many cells, this growth factor is a potent mitogen (3, 9, 23, 24); in others, it inhibits proliferation (34) or induces various kinds of differentiation (6,17,27,39,41,44,50,58). The different responses may partly be due to the fact that there are four known FGF receptor genes and a number of splicing variants for three of them (11,22,26,64,65).In a previous paper, we showed by cell fractionation that externally added aFGF was recovered in the nuclear fraction of NIH 3T3 cells, which contain specific FGF receptors. We also presented evidence that targeting to this location is required for the stimulation of DNA synthesis, whereas external binding to the receptor is sufficient to induce tyrosine phosphorylation (62). In Vero Dr22 and U2OS Dr1 cells, which lack specific FGF receptors and do not respond to aFGF, we were able to stimulate DNA synthesis by translocating the growth factor into the cytosol as a fusion protein with the diphtheria toxin A fragment (aFGF-dtA). Reconstituted with the diphtheria toxin B fragment (dtB), the fusion protein was translocated by the diphtheria toxin pathway (42, 43) into the cytosol of Vero Dr22 and U2OS Dr1 cells, which are insensitive to the intracellular action of diphtheria toxin but rich in diphtheria toxin receptors, and it was subsequently found in the nuclear fraction (63). However, there was no stimulation of cell proliferation.In NIH 3T3 ...

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Association between diphtheria toxin A- and B-fragment and their fusion proteins

Cited by 27 publications

References 24 publications

Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells

Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

Stimulation of Proliferation of a Human Osteosarcoma Cell Line by Exogenous Acidic Fibroblast Growth Factor Requires both Activation of Receptor Tyrosine Kinase and Growth Factor Internalization

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