Substantial increase of the inhibitory activity of Mirabilis antiviral protein by an elimination of the disulfide bond with genetic engineering.
Mirabilis antiviral protein (MAP) is a rigid, heat-stable protein composed of 250 amino acids with an intramolecular disulfide bond. MAP inhibits the in vitro protein synthesis of rabbit reticulocyte with approximately one-thirtieth the activity of the ricin A chain, a homologous protein with no such bond (Habuka, N., Murakami, Y., Noma, M., Kudo, T., and Horikoshi, K. (1989) J. Biol. Chem. 264, 6629-6637; Habuka, N., Akiyama, K., Tsuge, H., Miyano, M., Matsumoto, T., and Noma, M. (1990) J. Biol. Chem. 265, 10988-10992). The bond is presumed to induce some structural perturbation that alters the mode of interaction with the substrate ribosome and thus lowers the activity. To confirm this hypothesis, a mutant MAP gene in which the codons of both cysteines were replaced by those of serines was constructed and expressed in Escherichia coli, and its product (C36/22OS) was purified. In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, C36/220S showed the same mobility as that of MAP reduced by 2-mercaptoethanol, whereas nonreduced MAP showed faster migration. The inhibitory activity of C36/220S was approximately 22 times higher than that of native MAP, that is the mutant had an IC50 of 0.16 nM for the protein synthesis of the rabbit reticulocyte system, whereas the native MAP had an IC50 of 3.5 nM. The results indicate that the activity of MAP is increased by the elimination of the disulfide bond, and this supports the hypothesis.
Habuka
N
N
Life Science Research Laboratory, Japan Tobacco, Inc., Kanagawa.
Miyano
M
M
Kataoka
J
J
Tsuge
H
H
Ago
H
H
Noma
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D003545:Cysteine; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics* / Q000494:pharmacology*; D010944:Plants / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011817:Rabbits; D015183:Restriction Mapping; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1
D003062:Codon; D004220:Disulfides; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C059351:MAP protein, Mirabilis jalapa; D003545:Cysteine