Localization of functional domains of the mitogenic toxin of Pasteurella multocida.
The locations of the catalytic and receptor-binding domains of the Pasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed alpha/beta domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.
Pullinger
G D
GD
Department of Oral Microbiology, Kings College London, Guy's Hospital, London SE1 9RT, United Kingdom. gillian.pullinger@kcl.ac.uk
Sowdhamini
R
R
Lax
A J
AJ
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003603:Cytotoxins / Q000737:chemistry; D051379:Mice; D008934:Mitogens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016979:Pasteurella multocida / Q000472:pathogenicity*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D011503:Transglutaminases; D010566:Virulence Factors, Bordetella
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D008934:Mitogens; C062258:Pasteurella multocida toxin; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; C060969:dermonecrotic toxin, Bordetella; D011503:Transglutaminases