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Journal of Biochemistry Advance Access originally published online on May 24, 2007
Journal of Biochemistry 2007 142(1):95-104; doi:10.1093/jb/mvm116
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© 2007 The Japanese Biochemical Society.

Diphtheria Toxin Mutant CRM197 Possesses Weak EF2-ADP-ribosyl Activity that Potentiates its Anti-tumorigenic Activity

Takuya Kageyama1,*,{dagger}, Minako Ohishi1,*, Shingo Miyamoto2, Hiroto Mizushima1, Ryo Iwamoto1 and Eisuke Mekada1,{ddagger}

1Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871; and 2Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Fukuoka, 814-0180, Japan

{ddagger}To whom correspondence should be addressed. Tel: +81-6-6879-8286, Fax: +81-6-6879-8289, E-mail: emekada{at}biken.osaka-u.ac.jp

Received April 16, 2007; Accepted May 2, 2007


  Abstract

CRM197, a mutated diphtheria toxin (DT), has long been recognized to be a non-toxic protein. Based on its non-toxic feature, this protein has been utilized for various purposes, including as an inhibitor of heparin-binding EGF-like growth factor (HB-EGF) and as an immunological adjuvant for vaccination. Here we show evidence that CRM197 has a weak toxicity. This toxicity was observed in cells over-expressing the DT receptor/proHB-EGF, but not in parental cells, indicating that the toxicity was mediated through DT receptor. CRM197 did not show any toxicity toward DT-resistant cells, which have a mutation in elongation factor 2, and a cell-free assay revealed the existence of weak EF-2-ADP ribosylation activity in fragment A of CRM197. Thus, the present study indicates a requirement for specific care in the use of CRM197 at a high dosage, although the toxicity of CRM197 is about 106 times less than that of wild-type DT. We found that a monoclonal antibody to DT inhibited CRM197 toxicity, but did not affect the inhibitory activity of CRM197 toward HB-EGF-induced mitogenic activity. CRM197 strongly inhibits tumour growth in nude mice. The anti-DT monoclonal antibody administered with CRM197 reduced the anti- tumourigenic effect of CRM197, indicating that the toxicity of CRM197 potentiates its anti- tumourigenic effect.

Key Words: ADP ribosylation, CRM197, diphtheria toxin, EF-2, HB-EGF

Abbreviations: ADPR, ADP-ribosylation; DT, diphtheria toxin; DTR, diphtheria toxin receptor; EGF, epidermal growth factor; EGFR, EGF receptor; HB-EGF, heparin-binding EGF-like growth factor; proHB-EGF, the membrane-anchored form of HB-EGF; TCA, trichloracetic acid

*The authors wish it to be known that, in their opinion the first two authors contributed equally to this work.

{dagger}Present address: Division of Molecular Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.


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