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J. Biol. Chem., Vol. 279, Issue 4, 2866-2872, January 23, 2004

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

Takeshi Matsuzawa, Aya Fukui, Takashige Kashimoto¶, Kaori Nagao, Kiyomasa Oka, Masami Miyake, and Yasuhiko Horiguchi||

From the Department of Bacterial Toxinology, Research Institute for Microbial Diseases, Osaka University, Yamada-oka 3-1, Suita, Osaka 565-0871, Japan

Bordetella pertussis dermonecrotic toxin (DNT), which activates intracellular Rho GTPases, is a single chain polypeptide composed of an N-terminal receptor-binding domain and a C-terminal enzymatic domain. We found that DNT was cleaved by furin, a mammalian endoprotease, on the C-terminal side of Arg⁴⁴, which generates an N-terminal fragment almost corresponding to the receptor-binding domain and a C-terminal remainder (B) containing the enzymatic domain. These two fragments remained associated even after the cleavage and made a nicked form. DNT mutants insensitive to furin had no cellular effect, whereas the nicked toxin was much more potent than the intact form, indicating that the nicking by furin was a prerequisite for action. B, but not the nicked toxin, associated with artificial liposomes and activated Rho in cells resistant to DNT because of a lack of surface receptor. These results imply that B, dissociated from the binding domain, fully possesses the ability to enter the cytoplasm across the lipid bilayer membrane. The translocation ability of B was found to be attributable to the N-terminal region encompassing amino acids 45-166, including a putative transmembrane domain. Pharmacological analyses with various reagents disturbing vesicular trafficking revealed that the translocation requires neither the acidification of the endosomes nor retrograde vesicular transport to deeper organelles, although DNT appeared to be internalized via a dynamin-dependent endocytosis. We conclude that DNT binds to its receptor and is internalized into endosomes where the proteolytic processing occurs. B, liberated from the binding domain after the processing, begins to translocate the enzymatic domain into the cytoplasm.

Received for publication, September 17, 2003 , and in revised form, October 28, 2003.

^* This work was supported in part by Grants-in-aid for Scientific Research 11670264, 13470059, and 15390142 from the Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Kitasato Institute for Life Sciences, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan.

Both authors contributed equally to this work.

^¶ Present address: School of Veterinary Medicine and Animal Sciences, Kitasato University, Higashi 23-35-1, Towada, Aomori 034-8628, Japan.

^|| To whom correspondence should be addressed. Tel.: 81-6-6879-8284; Fax: 81-6-6879-8283; E-mail: horiguti{at}biken.osaka-u.ac.jp.

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