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Journal of Biochemistry Advance Access originally published online on May 23, 2007
Journal of Biochemistry 2007 142(1):105-112; doi:10.1093/jb/mvm115
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© 2007 The Japanese Biochemical Society.

Transgenic Mice Expressing a Fully Nontoxic Diphtheria Toxin Mutant, not CRM197 Mutant, Acquire Immune Tolerance against Diphtheria Toxin

Yasuko Kimura1, Michiko Saito1,2, Yukio Kimata1 and Kenji Kohno1,2,*

1Laboratory of Molecular and Cell Genetics, Division of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0192; and 2Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Minato-ku, Tokyo 105-0001, Japan

*To whom correspondence should be addressed. Tel: +81-743-72-5640, Fax: +81-743-72-5649, E-mail: kkouno{at}bs.naist.jp

Received April 3, 2007; Accepted May 6, 2007


  Abstract

We previously developed a method termed "toxin receptor-mediated cell knockout" (TRECK). By the TRECK method, a single or repeated shot(s) of diphtheria toxin (DT) conditionally ablates a specific cell population from transgenic mice expressing the DT receptor transgene under the control of a cell type-specific promoter. In some cases of TRECK, frequent and high-dose administration of DT is required, raising the concern that these frequent injections of DT could cause production of anti-DT antibody, which would neutralize further DT administration. To solve this problem, we aimed to generate transgenic mice genetically expressing a nontoxic DT mutant, with the expectation that they may naturally acquire immune tolerance to DT. Unexpectedly, the G52E DT mutant, which is well known as the nontoxic DT variant cross reacting material 197 (CRM197), exhibited cytotoxicity in yeast and mammalian cells. Cytotoxicity of CRM197 was abrogated in cells mutated for elongation factor 2 (EF-2), indicating that CRM197 exerts its toxic effects through EF-2, similar to wild-type DT. On the other hand, the K51E/E148K DT mutant exhibited no detectable cytotoxicity. This led us to successfully obtain DT gene transgenic mice, which exhibited no histological abnormalities, and indeed acquired immune tolerance to DT.

Key Words: ADP-ribosylation, diphtheria toxin, elongation factor 2, HB-EGF, immune tolerance

Abbreviations: ADP, adenosine diphosphate; CRM, cross reacting material; DT, diphtheria toxin; DTA, diphtheria toxin fragment A; DTB, diphtheria toxin fragment B; EF-2, eukaryotic elongation factor 2; ELISA, enzyme-linked immunosorbent assay; HB-EGF, heparin-binding epidermal growth factor-like growth factor; TRECK, toxin receptor-mediated cell knockout


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T. Kageyama, M. Ohishi, S. Miyamoto, H. Mizushima, R. Iwamoto, and E. Mekada
Diphtheria Toxin Mutant CRM197 Possesses Weak EF2-ADP-ribosyl Activity that Potentiates its Anti-tumorigenic Activity
J. Biochem., July 1, 2007; 142(1): 95 - 104.
[Abstract] [Full Text] [PDF]


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