Pseudomonas exotoxin A-epidermal growth factor (EGF) mutant chimeric protein as an indicator for identifying amino acid residues important in EGF-receptor interaction.
Epidermal growth factor (EGF) was fused to the carboxyl end of a modified pseudomonas exotoxin A that has its toxin binding domain deleted. This chimeric toxin designated as PE(delta Ia)-EGF kills A431 cells through the EGF receptor-mediated pathway. In this study, we used a random mutagenesis approach to make point mutations on EGF, followed by replacing the wild type EGF in PE(delta Ia)-EGF with these EGF mutants. We have constructed 14 different PE(delta Ia)-EGFmutants, and examined their EGF receptor binding activity as well as their cytotoxicity to A431 cells. Our results showed that individual mutations of Val19 to Glu and Val34 to Asp in the EGF domain of PE(delta Ia)-EGFmutants resulted in an increase in the binding affinity to EGF receptor and cytotoxicity to A431 cells. On the other hand, individual mutations of His16 to Asp and Gly18 to Ala in the EGF domain of PE(delta Ia)-EGFmutants lead to a decrease in the binding affinity to EGF receptor and cytotoxicity to A431 cells. In addition, mutations of any of the cysteine residues of EGF in PE(delta Ia)-EGFmutants resulted in the loss of their binding activity to EGF receptor and a corresponding loss of their cytotoxicity. This study indicates that the cytotoxicity of PE(delta Ia)-EGFmutant to EGF receptor-bearing cells may be used as an indicator to screen mutations of EGF important in EGF-receptor interactions.
Shiah
H S
HS
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Chen
T Y
TY
Chang
C M
CM
Chow
J T
JT
Kung
H J
HJ
Hwang
J
J
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000494:pharmacology; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D005098:Exotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A