M. Nagahama et al., Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity, BBA-MOL BAS, 1454(1), 1999, pp. 97-105
Citations number
29
Categorie Soggetti
Medical Research General Topics
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE
The beta-toxin gene isolated from Clostridium perfringens type B was expres
sed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. T
he purified GST-beta-toxin fusion protein from the E. coli transformant cel
ls was not lethal. The N-terminal amino acid sequence of the recombinant be
ta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was
G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin we
re indistinguishable from those of native beta-toxin (n toxin) purified fro
m C. perfringens type C. Replacement of Cys-265 with alanine or serine by s
ite-directed mutagenesis resulted in little loss of the activity. Treatment
of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of
r toxin and n toxin, led to no loss of the activity. The substitution of t
yrosine or histidine for Cys-265 significantly diminished lethal activity.
In addition, treatment of C265H with ethoxyformic anhydride which specifica
lly modifies histidyl residue resulted in significant decrease in lethal ac
tivity, but that of r toxin with the agent did not. These results showed th
at replacement of the cysteine residue at position 265 with amino acids wit
h large size of side chain or introduction of functional groups in the posi
tion resulted in loss of lethal activity of the toxin. Replacement of Tyr-2
66, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simult
aneous administration of r toxin and W275A led to a decrease in lethal acti
vity of beta-toxin. These observations suggest that the site essential for
the activity is close to the cysteine residue. (C) 1999 Elsevier Science B.
V. All rights reserved.