Mohamed Badr

• Due to their N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in medical and biological research. In the present study, we have successfully cloned two different truncated gelonins into pET-28a(+) vectors and expressed intact recombinant gelonin (rGel), recombinant C-terminally truncated gelonin (rC3-gelonin) and recombinant N- and C-terminally truncated gelonin (rN34C3-gelonin). Biological experiments showed that all these recombinant gelonins have no inhibiting effect on MCF-7 cell lines. These data suggest that the truncated-gelonins are still having a specific structure that does not allow for internalization into cells. Further, truncation of gelonin leads to partial or complete loss of N-glycosidase as well as DNase activity compared to intact rGel. Our data suggest that C-and N-terminal amino acid residues are involved in the catalytic and cytotoxic activities of rGel. In addition, the intact gelonin should be selected as a toxin in the immunoconjugate rather than truncated gelonin. In the second part, an immunotoxin composed of gelonin, a basic protein of 30 kDa isolated from the Indian plant Gelonium multiflorum and the cytotoxic drug MTX has been studied as a potential tool of gelonin delivery into the cytoplasm of cells. Results of many experiments showed that, on the average, about 5 molecules of MTX were coupled to one molecule of gelonin. The MTX-gelonin conjugate is able to reduce the viability of MCF-7 cell in a dose-dependent manner (ID50, 10 nM) as shown by MTT assay and significantly induce direct and oxidative DNA damage as shown by the alkaline comet assay. However, in-vitro translation toxicity MTX-gelonin conjugates have IC50, 50.5 ng/ml which is less toxic than that of gelonin alone IC50, 4.6 ng/ml. It can be concluded that the positive charge plays an important role in the N-glycosidase activity of gelonin. Furthermore, conjugation of MTX with gelonin through α- and γ- carboxyl groups leads to the partial loss of its anti-folate activity compared to free MTX. These results, taken together, indicate that conjugation of MTX to gelonin permits delivery of the gelonin into the cytoplasm of cancer cells and exerts a measurable toxic effect. In the third part, we have isolated and characterized two ribosome-inactivating proteins (RIPs) type I, gelonin and GAP31, from seeds of Gelonium multiflorum. Both proteins exhibit RNA-N-glycosidase activity. The amino acid sequences of gelonin and GAP31 were identified by MALDI and ESI mass spectrometry. Gelonin and GAP31 peptides - obtained by proteolytic digestion (trypsin and Arg-C) - are consistent with the amino acid sequence published by Rosenblum and Huang, respectively. Further structural characterization of gelonin and GAP31 (tryptic and Arg-C peptide mapping) showed that the two RIPs have 96% similarity in their sequence. Thus, these two proteins are most probably isoforms arisen from the same gene by alternative splicing. The ESI-MS analysis of gelonin and GAP31 exhibited at least three different post-translational modified forms. A standard plant paucidomannosidic N-glycosylation pattern (GlcNAc2Man2-5Xyl0-1 and GlcNAc2Man6-12Fuc1-2Xyl0-2) was identified using electrospray ionization MS for gelonin on N196 and GAP31 on N189, respectively. Based on these results, both proteins are located in the vacuoles of Gelonium multiflorum seeds.