Localization of the intracellular activity domain of Pasteurella multocida toxin to the N terminus.
We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqalpha protein that is coupled to phosphatidylinositol-specific phospholipase Cbeta1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268-1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells.
Wilson
B A
BA
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA. bwilson@wright.edu
Ponferrada
V G
VG
Vallance
J E
JE
Ho
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism* / Q000382:microbiology*; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism / Q000382:microbiology; D016979:Pasteurella multocida / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity; D017384:Sequence Deletion; D014709:Vero Cells; D014982:Xenopus laevis
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C062258:Pasteurella multocida toxin; D010446:Peptide Fragments; D011994:Recombinant Proteins