Combined Program on Microbiology and Immunology. Osaka University.

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Research Theme :
Analysis of the molecules and receptors involved in bacterial infection

Principal Research Scientist
Eisuke Mekada
Profile:

1974 Research Fellow, Research Institute for Microbial Diseases, Osaka University
1979 Research Associate, Research Institute for Microbial Diseases, Osaka University
1982 Research Associate, Institute of Molecular and Cellular Biology, Osaka University
1988 Associate Professor, Institute of Molecular and Cellular Biology, Osaka University
1989 Professor, Institute of Life Science, Kurume University
January, 2000 Professor, Research Institute of Microbial Diseases, Osaka University

Collaborators

Ryo Iwamoto, Hiroto Mizushima

Yukako Fujinaga(Research Associate of COE)

Research Summary

Many bacterial toxins, which play important roles in bacterial infection, bind to specific proteins (toxin receptors) of host cell membrane, enter the host cells, and then exert toxic effects. In the case of diphtheria toxin, the diphtheria toxin receptor (proHB-EGF) plays a critical role in the generation of toxic effects. Diphtheria toxin binds to the EGF domain of proHB-EGF, which facilitates entry of the toxin into cells by endocytosis. The diphtheria toxin receptor forms a complex with membrane proteins such as CD9, integrin ?3?1, and heparan sulfate proteoglycan. It is also known that CD9 and heparan sulfate proteoglycan play roles that aid toxin entry into cells. Using cultured cells, we have been conducting comprehensive analyses of the mechanisms of toxin entry through activation of the diphtheria toxin receptor complex. Interestingly, mice and rats are resistant to diphtheria toxin. This is because diphtheria toxin cannot bind to its receptor in these animals due to an amino acid substitution in the EGF domain of proHB-EGF. To generate a mouse model of diphtheria, we are currently producing knock-in mice expressing human proHB-EGF.
The diphtheria toxin receptor also functions as a growth factor. proHB-EGF is a membrane-anchored growth factor belonging to the EGF family, and also serves as a growth and differentiation factor at various tissues. This molecule plays a critical role in cardiac formation, myocardial maintenance, and wound healing of skin. proHB-EGF exists on the cell membrane as a membrane-anchored form, is cleaved by proteases on the cell surface, and secreted as a soluble form (sHB-EGF). To function as a growth factor, conversion from the membrane-anchored form (proHB-EGF) to the soluble form (sHB-EGF) is crucial both physiologically and pathologically. Aberrant regulation upon cleavage results in serious anomalies including fetal tissue hyperplasia. We conduct analyses of the mechanisms regulating the conversion from proHB-EGF to sHB-EGF, the physiological significance of this transformation, and oncogenesis caused by excess cleavage.

Diphtheria toxin consists of fragment A (shown in red) and fragment B (shown in green).
Diphtheria toxin binds to diphtheria toxin receptor via fragment B. Diphtheria toxin receptor forms a complex with membrane proteins such as CD 9. CD 9 facilitates diphtheria toxin binding to diphtheria toxin receptor. Diphtheria toxin binds to its receptor and is endocytosed into the endosome. Fragment A then passes through the endosomal membrane into the cytosol and inactivates elongation factor2, (EF2), resulting in the inhibition of protein synthesis in the host cell.

proHB-EGF, a diphtheria toxin receptor, is cleaved by a protease on the cell surface to generate a soluble form, sHB-EGF. sHB-EGFs bind to EGF receptors on same or neighboring cells and activate them. Activated EGF receptors activate the Ras/ERK pathway, which elicits the synthesis of proHB-EGF. Ras/ERK activation also induces cleavage of proHB-EGF.
Various types of GPCR ligands and stress-inducing stimuli also induce cleavage of proHB-EGF.
Therefore, under conditions where GPCR ligands or stress-inducing stimuli constantly exist, excessive levels of sHB-EFG are produced due to the positive- feedback loop; this results in tissue hyperplasia and the proliferation of cancer cells.

Publications

1. Yamazaki, S., Iwamoto, R., Saeki, K., Asakura, M., Takashima, S., Yamazaki, A., Kimura, R., Mizushima, H., Moribe, H., Higashiyama, S., Endoh, M., Kaneda, Y., Takagi, S., Itami, S., Takeda, N., Yamada, G. and Mekada. E. Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities. (2003) J. Cell Biol. in press.

2. Takeda, Y., Tachibana, I., Miyado, K., Kobayashi, M., Miyazaki, T., Funakoshi, T.,Kimura, H., Yamane, H., Saito, Y., Goto, H., Yoneda, T., Yoshida, M., Kumagai, T., Osaki, T., Hayashi, S., Kawase, I., and Mekada, E. Tetraspanins CD9 and CD81 function to prevent the fusion of mononuclear phagocytes. (2003) J. Cell Biol. 161, 945-956

3. Takenobu, H., Yamazaki, A., Hirata, M., Umata, T. and Mekada, E. The stress- and the inflammatory cytokine-induced ectodomain shedding of heparin-binding EGF-like growth factor is mediated by p38 MAPK, distinct from TPA-induced and LPA-induced signaling cascades. (2003) J. Biol. Chem. 278, 17255-17262.

4. Iwamoto, R., Yamazaki, S., Asakura, M., Takashima, S., Hasuwa, H., Miyado, K., Adachi, S., Kitakaze, M., Hashimoto, K., Raab, G., Nanba, D., Higashiyama, S., Hori, M., Klagsbrun, M. and Mekada, E. HB-EGF and ErbB signaling is essential for heart function (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 3221-3226.

5. Umata, T., Hirata, M., Takahashi, T., Ryu, F., Shida, S., Takahashi, Y., Tsuneoka, M., Miura, Y., Masuda, M., Horiguchi, Y. and Mekada, E. (2001) A dual signaling cascade that regulates the ectodomain shedding of HB-EGF. J. Biol. Chem. 276, 30475-30482.