Download Molecular cloning, cellular targeting and substrate interaction

MOLECULAR
CLONING,
CELLULAR
TARGETING
AND
SUBSTRATE
INTERACTION OF DIFFERENT FORMS OF SAPORIN, A TYPE 1RIP FROM
Saponaria officinalis
Pittaluga E., Perconti S., Tucci A., Poma A., Spanò L.
Department of Basic and Applied Biology, University of L'Aquila, 67010 Coppito L'Aquila, Italy
A great variety of plant species contains toxins, known as ribosome-inactivating proteins
(RIPs), which inhibit protein synthesis through the catalytic inactivation of eukaryotic
ribosomes. Recently it has been demonstrated that in vitro the action of RIPs is not limited
to the ribosomal substrate since thay are active also on viral RNAs as well as on
deoxyribonucletide substrates of different complexity (Peumans et al. 2001, Faseb J 15:
1493-1506.) RIPs are classified in two groups based on their subunit composition; type 1
RIPs are single chain polypeptides with a molecular mass of about 30 kDa and highly
basic isoelectric points.
Saporins are type 1 RIPs present in different organs of the plant Saponaria officinalis.
Several iimmunologically related isoforms have been isoleted from the seeds and one of
these, saporin SO6, constitutes about 7% of the total seed protein content (Stirpe et al.1983,
Biochem. J. 216: 617-625.; Lappi et al. 1985, Biochem. Biophys. Res. Comm. 129: 934-942.).
In the present study we report the characterization of different saporin isoforms, as well as
of mutagenized enzymes, in view of a more direct evaluation of the biological function of
these toxins in the plant of origin.
Two different leaf isoform of saporin have been purified by means of CM-Sepharose Fast
Flow column, followed by an HPLC protein ion-exchange column Vydac VHP 400. Their
sub-cellular localization, N-terminal sequence as well as data on their enzymatic activity,
in vitro and in vivo, point to their probable different function in planta. In this respect it is
interesting to note that confocal microscopic analysis of fluorescent conjugates of the
toxins has evidenced their different localization within the treated cells (Ippoliti R. et al.
2002, The Italian Journal of Biochemistry 51; 1-2: 37.).
Similar analysis were extended to mutagenized forms of the toxin, in order to clarify the
relative role played by different protein domains in determining their cytotoxic properties.
Results indicate that, in analogy to what reported already for ricin (Frankel et al., 1990
Mol. and Cell. Biol. 10, 12: 6257-6263) and PAP (Hur et al., 1995 Proc. Natl. Acad. Sci. USA
92: 8448-8452), the Glu residue at position 176 is essential for the activity on both
ribosomal and DNA substrates; the C-terminal region contributes to the catalytic activity,
most probably by stabilizing the complex between the substrate and the catalytic site, as
envisaged by Savino et al.(Savino et al. 2000, FEBS Letters 470: 239-243).