Loss of biological activity due to Glu-->Arg mutation at residue 11 of the B subunit of cholera toxin.
Since it has been reported that a single amino acid mutation of Gly-->Arg in the CAGYC region of the beta chain of human thyroid stimulating hormone (hTSH) was responsible for congenital isolated TSH deficiency, and that the same amino acid substitution in this site of hTSH and human chorionic gonadotropin (hCG) introduced by site-directed mutagenesis resulted in loss of activity, the authors studied the role of glutamic acid at position 11 (Glu-11) from the N-terminus of the B subunit of cholera toxin (CT), which corresponds to the glycine in the CAGYC region of the beta chain of hTSH and hCG. A mutant CT constructed by site-directed mutagenesis in which Glu-11 was replaced by Arg (CT-E11R) did not induce either morphological changes or accumulation of cytosolic cyclic AMP in Chinese hamster ovary cells, although it formed the holotoxin AB5, retained the ability to bind to GM1-ganglioside and showed ADP-ribosyltransferase activity. Weak assembly of the B subunits in mutant CT-E11R demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-heating conditions might explain the loss of biological activity.
Yamaoka
J
J
Faculty of Medicine, Kyoto University, Kyoto, Sakyo-ku, 606-01, Japan.
Yamasaki
S
S
Kurazono
H
H
Imamura
S
S
Noda
M
M
Miyai
K
K
Takeda
Y
Y
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D016466:CHO Cells; D002772:Cholera Toxin / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D006063:Chorionic Gonadotropin / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli; D005677:G(M1) Ganglioside / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000633:toxicity; D013972:Thyrotropin / Q000235:genetics; D014734:Vibrio cholerae / Q000302:isolation & purification / Q000378:metabolism
D006063:Chorionic Gonadotropin; D011994:Recombinant Proteins; D005677:G(M1) Ganglioside; D018698:Glutamic Acid; D013972:Thyrotropin; D002772:Cholera Toxin; D001120:Arginine; D000242:Cyclic AMP; D011065:Poly(ADP-ribose) Polymerases