Abstract
Protective antigen (PA), a component of anthrax toxin, mediates translocation of the toxin's lethal and edema factors (LF and EF, respectively) to the cytoplasm, via a pathway involving their release from an acidic intracellular compartment. PA63, a 63-kDa proteolytic fragment of PA, can be induced to form ionconductive channels in the plasma membrane of mammalian cells by acidification of the medium. These channels are believed to be comprised of dodecyl sulfate-resistant oligomers (heptameric rings) of PA63 seen by electron microscopy of the purified protein. Here we report that the PA63-mediated efflux of 86Rb+ from preloaded CHO-K1 cells under acidic conditions is strongly inhibited (> or = 70%) by LF or LFN, a PA-binding fragment of LF. Control proteins caused no inhibition. Evidence is presented that the inhibition involves partial blockage of ion conductance by the PA63 channel. Also, oligomer formation is slowed somewhat by LF at pH values near the pH threshold of channel formation (pH approximately 5.3), suggesting that channel formation may also be retarded under these conditions. The relevance of these results to the location of the LF-binding site on PA63 and the mechanism of LF and EF translocation is discussed.
Highlights
From the Department of Microbiology and Molecular Genetics and Shipley Institute of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Protective antigen (PA), a component of anthrax toxin, mediates translocation of the toxin's lethal and edema factors (LF and EF, respectively) to the cytoplasm, via a pathway involving their release from an acidic intracellular compartment
After binding to a cell-surface receptor, these toxins generally undergo receptor-mediated endocytosis and are transported to an appropriate intracellular compartment, where their enzymic moieties are transferred across the membrane and into the cytosol
Summary
Vol 270, No 31, Issue of August 4, pp. 18626-18630, 1995 Printed in U.S.A. Effect of Anthrax Toxin's Lethal Factor on Ion Channels Formed by the Protective Antigen*. The relevance of proteolytic activation and exposure to acidic pH has been demonstrated in vivo: mutation or deletion of the protease cleavage site in PA abolishes EF and LF binding [10, 17], and lysosomotropic agents protect cells from the PA-mediated toxic effect ofEF and LF [12, 13]. We found that purified PAas forms high molecular weight, dodecyl sulfate-resistant oligomers, which appear predominantly as heptameric rings by electron microscopy [18], Similar or identical oligomers are generated in a time-dependent manner in cells incubated with PA, and oligomer formation in vivo appears to require exposure of PAas to acidic pH, presumably in an acidic intracellular compartment. We report that LF inhibits the Pl\,3-induced permeabilization of the plasma membranes ofCHO-KI cells under acidic pH conditions and present data relevant to the mechanism of the inhibition
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