The antigenic sites of trichosanthin, a ribosome-inactivating protein with multiple pharmacological properties

Abstract

Trichosanthin (TCS) fragments were produced by Tn1000 deletion mutagenesis and by cyanogen bromide (CNBr) cleavage and their immunoreactivity was examined by incubating with various antibodies. Twelve C-terminally truncated TCS variants were successfully synthesized under the control of a T7 RNA driven promoter. The smallest antigenic fragment mapped corresponded to the N-terminal 20 amino acids (aa). Six CNBr fragments of TCS were created and identified by electrospray mass spectrometry and N-terminal sequencing. Three antigenic fragments corresponding to aa 1-72, 101-152 and 153-246, respectively were mapped. Fragments corresponding to aa 1-72 and 153-246 were immunoreactive to the same monoclonal antibody showing they are components of a discontinuous epitope. On the other hand, the fragment containing aa 73-100 was not detected by any of the antibodies used.