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Glucosylating and Deamidating Bacterial Protein Toxins, Page 1 of 2
< Previous page Next page > /docserver/preview/fulltext/10.1128/9781555817893/9781555812454_Chap16-1.gif /docserver/preview/fulltext/10.1128/9781555817893/9781555812454_Chap16-2.gifAbstract:
The family of large clostridial cytotoxins comprises Clostridium difficile toxins A and B, the lethal and the hemorrhagic toxins from Clostridium sordellii, the α-toxin from Clostridium novyi, and various toxin isoforms mainly produced by C. difficile. Differential glucosylation is used to measure the modification of Rho GTPases in intact cells. Toxin-modified Rho GTPase in intact cells blocks subsequent toxin-catalyzed labeling of Rho proteins in the cell lysate upon addition of UDP[14C]glucose. Covalent modification by bacterial protein toxins not only inhibits but also activates Rho GTPases. Measurement of the GTP hydrolysis by cytotoxic necrotizing factor (CNF)1-treated RhoA reveals an inhibition of the intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activity, indicating that deamidation of Gln-63 forms a constitutively active Rho protein. The modification of Rho GTPases by dermonecrotizing toxin (DNT) occurs predominantly with the GDP-bound form of Rho, while the modification of Rho by CNFs is not nucleotide dependent. Like deamidation of Gln-63, transglutamination of Gln-63 inhibits GTP hydrolase activity of Rho proteins, although the precise function of the addition of primary amines onto Rho GTPases is not completely understood. Deamidation and transglutamination by CNFs or DNT change the migration of GTPases by SDS-PAGE. This change in migration occurs with RhoA but not Rac or Cdc42 and depends on the deamidation of Glu-63 and, therefore, is also observed with the recombinant RhoA-Q63E. Mass spectrometric analysis of proteolytic peptides of Rho GTPases allows the detection of the 1-Da shift caused by CNF1-induced deamidation of Gln-63 of RhoA (Gln-61 of Rac and Cdc42).