Association of Pasteurella multocida toxin with vimentin

Abstract

To help understand the molecular mechanisms of Pasteurella multocida toxin (PMT) action, we searched for a cellular protein interacting with PMT. The ligand overlay assay revealed a 60-kDa cellular protein that binds to a region from the 840th to 985th amino acids of the toxin. This protein was identified as vimentin by peptide mass fingerprinting. The N-terminal head domain of vimentin was further found to be responsible for the binding to the toxin.

FIG. 1.

Ligand overlay assay with PMT and its deletion mutants. The lysates of Swiss 3T3 cells were subjected to SDS-PAGE, transferred to a membrane, and incubated with full-length PMT (A) or the deletion mutants of the toxin (C) for 1 h. The deletion mutants of PMT used in this experiment are schematically represented in panel B. The numbers with the mutant names indicate the positions of the N- and C-terminal amino acids. Crosshatched bars indicate FLAG peptides. After stringent washing, the toxin that remained on the membrane was detected by Western blot analysis with anti-PMT polyclonal antibody or anti-FLAG M2 antibody. The positions of the molecular mass standards are shown on the left.

FIG. 2.

Association of PMT with vimentin. (A) Purified mouse vimentin (1.5 μg) was subjected to the ligand overlay assay with PMT. (B) Pull-down assay with GST-PMT_840-985. Vimentin (2 μg) was mixed with GST-PMT_840-985- or GST-coupled glutathione Sepharose 4B and incubated at 4°C for 3 h. Vimentin associated with the beads was extracted by boiling in SDS-PAGE buffer and subjected to Western blot analysis with anti-vimentin antibody as described in the text. The positions of the molecular mass standards are shown on the left.

FIG. 3.

Association of the head domain of vimentin with PMT. The lysates of E. coli expressing the head (H; amino acids 1 to 94), rod (R; amino acids 95 to 406), and tail (T; amino acids 407 to 465) domains of vimentin in the GST-tagged forms were subjected to the ligand overlay assay with PMT_840-1285. Arrows indicate the predicted position of each domain. (A) The expression of the fusion proteins was estimated by Western blot analysis with anti-GST polyclonal antibody. The amount of each fragment was estimated from the intensity of the blotted protein band. (B) Ligand overlay assay. An equal amount of each fragment was applied to each lane. Note that the head domain at the predicted position was reacted with PMT_840-1285, whereas the rod and the tail domains were not. The bands that appeared at the position below 25 kDa should be nonspecifically reactive proteins, because they were observed even in the lysate from naive E. coli. The positions of the molecular mass standards are shown on the left.