Stabilization of the Tertiary Structure of the Cholera Toxin A1 Subunit Inhibits Toxin Dislocation and Cellular Intoxication

    Authors

    S. Massey; T. Banerjee; A. H. Pande; M. Taylor; S. A. Tatulian;K. Teter

    Comments

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    Abbreviated Journal Title

    J. Mol. Biol.

    Keywords

    cholera toxin; circular dichroism; endoplasmic reticulum-associated; degradation; surface plasmon resonance; toxin translocation; RETICULUM-ASSOCIATED DEGRADATION; CHO-CELLS RESISTANT; ENDOPLASMIC-RETICULUM; CHEMICAL CHAPERONES; RETRO-TRANSLOCATION; PROTEIN; TOXINS; CULTURED-CELLS; POLYPEPTIDE; PATHWAY; RICIN; Biochemistry & Molecular Biology

    Abstract

    Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic subunit of CT (CTA1) then crosses the ER membrane and enters the cytosol in a process that involves the quality control mechanism of ER-associated degradation. The molecular details of this dislocation event have not been fully characterized. Here, we report that thermal instability in the CTA1 subunit-specifically, the loss of CTA1 tertiary structure at 37 degrees C-triggers toxin dislocation. Biophysical studies found that glycerol preferentially stabilized the tertiary structure of CTA1 without having any noticeable effect on the thermal stability of its secondary structure. The thermal disordering of CTA1 tertiary structure normally preceded the perturbation of its secondary structure, but in the presence of 10% glycerol the temperature-induced loss of CTA1 tertiary structure occurred at higher temperatures in tandem with the loss of CTA1 secondary structure. The glycerol-induced stabilization of CTA1 tertiary structure blocked CTA1 dislocation from the ER and instead promoted CTA1 secretion into the extracellular medium. This, in turn, inhibited CT intoxication. Glycerol treatment also inhibited the in vitro degradation of CTA1 by the core 20S proteasome. Collectively, these findings indicate that toxin thermal instability plays a key role in the intoxication process. They also suggest the stabilization of CTA1 tertiary structure is a potential goal for novel antitoxin therapeutic agents. (C) 2009 Elsevier Ltd. All rights reserved.

    Journal Title

    Journal of Molecular Biology

    Volume

    393

    Issue/Number

    5

    Publication Date

    1-1-2009

    Document Type

    Article

    Language

    English

    First Page

    1083

    Last Page

    1096

    WOS Identifier

    WOS:000271596500008

    ISSN

    0022-2836

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