Identification of a Receptor-Binding Domain of Bordetella Dermonecrotic Toxin

Abstract

Dermonecrotic toxin (DNT), commonly produced by Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis, exerts lethal, dermonecrotic, and splenoatrophic activities in a variety of experimental animals (2,3,7,8,12,19). B. bronchiseptica DNT is considered to be responsible for turbinate atrophy in swine atrophic rhinitis (4, 6, 9). The turbinate atrophy caused by DNT likely results from a deficiency of osteoblastic differentiation in bone tissues (9, 11). DNT also alters cell morphology, stimulating the anomalous reorganization of actin stress fibers and focal adhesions, which is elaborately regulated by the small GTPase Rho (5, 10). The small GTPases function as a molecular switch regulating various cell functions besides the reorganization of actin cytoskeletons by changing between the GDP-bound inactive and the GTPbound active forms. The GDP-bound GTPases in resting cells exchange GDP for GTP in response to various stimulations, transduce the signals downstream by interacting with effector proteins, and thereafter revert to the GDP-bound inactive form by hydrolyzing the bound GTP. Our research group recently demonstrated that DNT was a transglutaminase catalyzing deamidation or polyamination at Gln63 of Rho and the corresponding Gln residues of the other members of the Rho family, Rac and Cdc42 (5, 18). The deamidation and polyamination result in a reduction of the GTP hydrolyzing activity. In addition, the polyaminated Rho comes to interact with a downstream effector, ROCK, in a GTP-independent manner (17). Thus, these modifications render the intracellular GTPases constitutively active, which probably mediates various effects of DNT on target cells.DNT is a single-chain polypeptide which consists of 1,464 amino acids with a calculated molecular mass of 160,602 (13). Previously, we localized the catalytic domain of DNT to the C-terminal region from Ile 1176 to the C-terminal end. We also found that the N-terminal fragment spanning Met 1 to Pro 531 of DNT competitively blocked the intoxication of cells by the full-length DNT (13), implying that this fragment retains the receptor-binding or internalizing property. In the present study, we attempted to define the N-terminal receptor-binding region of DNT by using a series of toxin mutants with various lengths and a monoclonal antibody (MAb) that neutralizes the toxin. The results presented here indicate that DNT binds to the cells through the N-terminal region consisting of 54 amino acids, in which the MAb recognized the region including Arg 44 . MATERIALS AND METHODSMaterials. DNT was purified from B. bronchiseptica S798 as described previously (8). The numbering of the amino acids of DNT was based on the sequence available from the DDBJ/EMBL/GenBank databases under accession no. AB020025. C3 exoenzyme was provided by S. Kozaki, University of Osaka Prefecture, Osaka, Japan. MC3T3-E1 cells were cultured at 37°C in ␣-minimum essential medium (␣-MEM; Gibco Laboratories, Grand Island, N.Y.) supplemented with 10% fetal calf serum under 5% CO 2 ...

Bordetella Dermonecrotic Toxin Undergoes Proteolytic Processing to Be Translocated from a Dynamin-related Endosome into the Cytoplasm in an Acidification-independent Manner

“…5, B and D). The time course of the modification of intracellular Rho by nicked DNT apparently corresponded to that of binding of DNT (binding domain) to cells (13). These results indicate that proteolytic cleavage at the furin motif is a prerequisite and ratelimiting step for DNT to affect cells.…”

“…⌬B Is Translocated across the Membrane after Dissociating from the Binding Domain-Our previous findings demonstrated that DNT binds to target cells via the N-terminal domain and modifies intracellular Rho by the enzymatic action of its C-terminal domain (13,14). The intervening region between the binding domain and the catalytic domain, therefore, should be involved in translocation of the catalytic domain into cytoplasm through the lipid membrane.…”

“…These results prompted us to examine whether ⌬B interacts not only with artificial liposomes but also with the cell membrane. For this purpose, we applied ⌬B to Balb/3T3 cells, which were resistant to the full-length DNT because of a lack of the cell surface receptor (13). If ⌬B interacts with the cell membrane as well as liposomes and translocates the catalytic domain into the cytosol, it should exert toxicity in the cells independently of the cell surface receptors.…”

“…2A) (13). To elucidate whether the binding domain dissociates from the rest of the toxin molecule (⌬B) after the cleavage, we treated the N-terminal His 6 -tagged DNT with furin and compared the elution profile with that of intact DNT from gel filtration chromatography.…”

“…As a result of the polyamination or deamidation, the GTPases become constitutively active and thereby cause the intoxicated cells to aber-rantly express the Rho-dependent phenotypes. Recently, we found that the binding and enzymatic domains of DNT were ascribed to the N-terminal 54 amino acids (13) and the Cterminal 300 amino acids (14), respectively. However, the internalization or translocation mechanism of DNT has not been dissected.…”