scholarly journals Involvement of Domain 3 in Oligomerization by the Protective Antigen Moiety of Anthrax Toxin

2001 ◽  
Vol 183 (6) ◽  
pp. 2111-2116 ◽  
Author(s):  
Jeremy Mogridge ◽  
Michael Mourez ◽  
R. John Collier
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Protective Antigen ◽  
Anthrax Toxin ◽  
Membrane Insertion ◽  
A Cell ◽  
Domain 3 ◽  
Active Fragment ◽  
Cell Surface Protease ◽  
Pa Domain

ABSTRACT Protective antigen (PA), a component of anthrax toxin, binds receptors on mammalian cells and is activated by a cell surface protease. The resulting active fragment, PA63, forms ring-shaped heptamers, binds the enzymic moieties of the toxin, and translocates them to the cytosol. Of the four crystallographic domains of PA, domain 1 has been implicated in binding the enzymic moieties; domain 2 is involved in membrane insertion and oligomerization; and domain 4 binds receptor. To determine the function of domain 3, we developed a screen that allowed us to isolate random mutations that cause defects in the activity of PA. We identified several mutations in domain 3 that affect monomer-monomer interactions in the PA63 heptamer, indicating that this may be the primary function of this domain.

scholarly journals Modulation of RECK levels in Xenopus A6 cells: effects on MT1-MMP, MMP-2 and pERK levels

2019 ◽  
Vol 26 (1) ◽  
Author(s):  
Jessica A. Willson ◽  
Bradley S. Bork ◽  
Carlie A. Muir ◽  
Sashko Damjanovski
Keyword(s):  
Cell Migration ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Mmp Inhibitors ◽  
Erk Activation ◽  
A6 Cells ◽  
A Cell ◽  
Plasmid Transfection

Abstract Background MT1-MMP is a cell-surface enzyme whose regulation of pro-MMP-2 and ERK activation position it as a key facilitator of ECM remodelling and cell migration. These processes are modulated by endogenous MMP inhibitors, such as RECK, a GPI-anchored protein which has been shown to inhibit both MT1-MMP and MMP-2 activity. Our previous studies have revealed a link between MT1-MMP levels, and pro-MMP-2 and ERK activation in mammalian cells, as well as MT1-MMP and RECK co-localization in Xenopus embryos. We here investigated how modulation of RECK would impact MT1-MMP and MMP-2 levels, as well as ERK signalling in Xenopus A6 cells. Results We used a Morpholino approach to knockdown RECK, plasmid transfection to overexpress RECK, and PI-PLC treatment to shed RECK from the cell surface of Xenopus A6 cells. RECK reduction did not alter pERK or MT1-MMP levels, nor MMP-2 activity as measured by zymography; thus RECK-knockdown cells maintained the ability to remodel the ECM. RECK overexpression and PI-PLC treatment both increased ECM remodelling potential through increased MT1-MMP protein and relative MMP-2 activation levels. Conclusions RECK changes that reduce the ability of the cell to remodel the ECM (overexpression and cell surface shedding) are compensated for by increases in MT1-MMP, and MMP-2 levels as seen by zymography.

scholarly journals Interaction of Chlamydia trachomatis with Mammalian Cells Is Independent of Host Cell Surface Heparan Sulfate Glycosaminoglycans

2006 ◽  
Vol 74 (3) ◽  
pp. 1795-1799 ◽  
Author(s):  
Richard S. Stephens ◽  
Jesse M. Poteralski ◽  
Lynn Olinger
Keyword(s):  
Chlamydia Trachomatis ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Host Cell ◽  
Cell Line ◽  
Mammalian Cells ◽  
Chlamydial Infection ◽  
Functional Role ◽  
A Cell ◽  
Host Cell Surface

ABSTRACT The hypothesis that host cell surface heparan sulfate is required to promote chlamydial infection was tested using a cell line (CHO-18.4) containing a single retroviral insertion and the concomitant loss of heparan sulfate biosynthesis. Tests of chlamydial infectivity of heparan sulfate-deficient CHO-18.4 cells and parental cells, CHO-22, demonstrated that both were equally sensitive to infection by Chlamydia trachomatis serovars L2 and D. These data do not support the hypothesis and demonstrate that host cell surface heparan sulfate does not serve an essential functional role in chlamydial infectivity.

Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells

1999 ◽  
Vol 67 (4) ◽  
pp. 1853-1859
Author(s):  
Yogendra Singh ◽  
Kurt R. Klimpel ◽  
Seema Goel ◽  
Prabodha K. Swain ◽  
Stephen H. Leppla
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin

scholarly journals Cytosolic Delivery and Characterization of the TcdB Glucosylating Domain by Using a Heterologous Protein Fusion

2001 ◽  
Vol 69 (1) ◽  
pp. 599-601 ◽  
Author(s):  
Lea M. Spyres ◽  
Maen Qa'Dan ◽  
Amy Meader ◽  
James J. Tomasek ◽  
Eric W. Howard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Small Gtpases ◽  
Anthrax Toxin ◽  
Coding Region ◽  
Protein Fusion ◽  
Actin Reorganization ◽  
Amino Terminal ◽  
Cell Extracts

ABSTRACT TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB1-556 coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB1-556was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB1-556 plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.

scholarly journals Cytotoxic T-Lymphocyte Epitopes Fused to Anthrax Toxin Induce Protective Antiviral Immunity

1999 ◽  
Vol 67 (7) ◽  
pp. 3290-3296 ◽  
Author(s):  
Amy M. Doling ◽  
Jimmy D. Ballard ◽  
Hao Shen ◽  
Kaja Murali Krishna ◽  
Rafi Ahmed ◽  
...  
Keyword(s):  
Fusion Protein ◽  
T Lymphocyte ◽  
Mammalian Cells ◽  
Protective Antigen ◽  
Cytotoxic T Lymphocyte ◽  
Lethal Factor ◽  
Antiviral Immunity ◽  
Anthrax Toxin ◽  
Listeriolysin O

ABSTRACT We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes. During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells. Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens. Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ld and one presented by Db. The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens.

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