Identification of a receptor-binding domain of Bordetella dermonecrotic toxin.
Bordetella dermonecrotic toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating or polyaminating Gln63 of the small GTPase Rho. DNT is an A-B toxin which is composed of an N-terminal receptor-binding (B) domain and a C-terminal enzymatically active (A) domain. In this study, to analyze the functional and structural organization of DNT, we prepared 10 clones of hybridoma producing anti-DNT monoclonal antibodies. One of these antibodies, 2B3, neutralized the effects of DNT on target cells when mixed with the toxin. When microinjected into cells, however, 2B3 did not inhibit the intoxication by DNT. Western blot analysis revealed that 2B3 recognized the N-terminal region of DNT. To delineate the DNT-binding domain, we examined a series of truncated DNT mutants for the ability to competitively inhibit the intoxication of cells by the full-length DNT and found that a fragment consisting of the N-terminal 54 amino acids (DNT(1-54)) was the smallest inhibitory fragment. The radioiodinated DNT(1-54) actually bound to target cells, which was inhibited by 2B3. These results suggest that the N-terminal 54 amino acids of DNT are responsible for the binding to target cells. DNT(1-54) bound to none of the DNT-resistant cells, implying the presence of a cell surface receptor specific to DNT-sensitive cells.
Matsuzawa
Takeshi
T
Department of Bacterial Toxinology, Research Institute for Microbial Diseases, Osaka University, Yamada-oka 3-1, Suita, Osaka 565-0871, Japan.
Kashimoto
Takashige
T
Katahira
Jun
J
Horiguchi
Yasuhiko
Y
D000911:Antibodies, Monoclonal / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D016950:Bordetella bronchiseptica / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D009500:Neutralization Tests; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011503:Transglutaminases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D010566:Virulence Factors, Bordetella
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D008024:Ligands; D011956:Receptors, Cell Surface; D010566:Virulence Factors, Bordetella; C060969:dermonecrotic toxin, Bordetella; D011503:Transglutaminases