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Development of novel chimeric toxins for targeted therapy by genetic engineering |
| Principal Investigator : Janendra K Batra
Ph D Students Collaborators A.
Studies on the molecular mechanism of action of ribonucleolytic and
ribosome-inactivating protein toxins The
objective of the project is to investigate structure-function relationship of
Aspergillus ribotoxin, restrictocin; ribosome inactivating protein, saporin;
human pancreatic ribonuclease, and eosinophil-derived neurotoxin to understand
their mechanism of catalysis and cytotoxicity. Saporin
has been shown to bind to a2-macroglobulin
receptor, also called as low-density-lipoprotein-receptor-related protein (LRP),
expressed particularly in fibroblasts, monocytes and hepatocytes. LRP is
responsible for the uptake and clearance of macromolecular complexes between
proteinases and a2-macroglobulin.
We showed earlier that the cytotoxic activity of saporin-6 results due to the
combined manifestation of its N-glycosidase and internucleosomal
DNA-fragmentation activities. The mechanism of saporin cytotoxicity was
further investigated using recombinant saporin-5 and saporin-6 isoforms,
expressed in E. coli and purified from the inclusion bodies. Saporin-6
was found to be about ten-fold more active than saporin-5 in inhibiting in
vitro protein synthesis. Both the isoforms specifically depurinated the
target rRNA, however, saporin-5 had a lower activity compared to saporin-6. In
U937 cells compared to saporin-6, saporin-5 appeared to be significantly less
active in causing genomic DNA fragmentation. The cytotoxic activity of saporin
isoforms was studied on LRP-positive cell lines, J774A.1 and U937, and LRP-negative
cell lines HUT 102 and HeLa. Saporin-6 was more active than saporin-5 on all
cell lines tested; the difference of activity between the two isoforms varied
between 5-20-fold. However, we found LRP-negative cell lines to have
sensitivities similar to LRP-positive cell lines towards saporin toxicity. To
find out if the difference in activities of saporin isoforms on various cell
lines was due to a difference in their internalization efficiencies, the
cytotoxicity of isoforms was tested on HeLa cells permeabilised by adenoviral
infection. Saporin-6 was 8-fold more active than saporin-5 in adenovirus
infected cells indicating the difference in the cytotoxic activity to be a
reflection of the difference in the catalytic activity of the two isoforms. To
further investigate the involvement of LRP in the cytotoxic activity of
saporin, the toxicity of saporin-6 was assayed on LRP-positive chinese hamster
ovary cell line, CHO-K1, and its mutant CHO-13-5-1, which has been shown to
have no detectable LRP mRNA or protein. Both CHO-K1 and CHO-13-5-1 cells were
found to have similar sensitivities towards saporin-6 toxicity suggesting that
binding and internalization of saporin are not mediated through LRP. In
addition, the similar sensitivities of LRP-positive and LRP-negative cells
towards saporin toxicity indicate the entry of toxin into LRP-negative cells
through receptors or mechanisms other than LRP-mediated endocytosis. To
investigate if the intracellular translocation of saporin-6 requires acidic
endosomal pH and the involvement of Golgi, cytotoxicity of saporin-6 was
assayed in the presence of ammonium chloride or brefeldin-A on J774A.1 cells.
Brefeldin-A did not affect the toxicity of saporin-6, whereas ammonium
chloride protected the cells from saporin-6 toxicity indicating that saporin-6
requires acidic endosomal pH for the manifestation of its cytotoxicity,
however, intact Golgi is not required. To investigate if saporin translocates
to nucleus to degrade DNA, J774A.1 cells were treated with iodinated saporin-6
for different time periods and presence of radio-labelled protein was checked
in the nuclear, cytosolic and membrane fractions. The concentration of
saporin-6 increased in cytosol up to 6 hours, decreased gradually and became
negligible by 16 hours. The membrane fraction did not show any significant
amount of protein at any time point. The concentration of the protein in the
nuclear fraction was found to be comparable to that in the cytosolic fraction
up to 9 hours, however, by 16 hours concomitant with the decrease in cytosol,
most of the labeled saporin-6 localized in the nucleus. The study shows that
after internalization, initially the protein stays in the cytosol and
subsequently migrates to the nucleus. Eosinophil-derived
neurotoxin (EDN) and eosinophil cationic protein (ECP) perform various
biological functions that are dependent on their RNase activity, however, they
are much weaker enzymes compared to RNase A, which does not have any special
bioaction. The loop regions of EDN and ECP, which differ from RNase A loops in
structure and sequence, have been proposed to play a role in the physiological
function of EDN and ECP. One of these loops, loop l7, contains a nine residue
insertion, AspGlnArgArgAspProProGlnTyr in EDN and AspProArgAspSerProArgTyr in
ECP as compared to the amino acid sequence of RNase A. Residues Arg117, Pro120
and Gln122 in loop l7 of EDN were mutated to alanine by site-directed
mutagenesis. The mutants were expressed in E.coli and purified from the
inclusion bodies. In a Western blot the mutants reacted with anti–EDN
antibody as well as EDN did. The CD-spectral analysis revealed that the
overall structure of the mutants was moderately altered as compared to EDN.
The mutants showed ribonucleolytic activity comparable to that of EDN on yeast
tRNA, Poly (U) and Poly (C). All the three mutants inhibited protein synthesis
in a manner similar to that of EDN in a cell-free assay indicating that
these residues per se are not critical for the catalytic activity of
EDN. However, further analysis is required to delineate the overall
contribution of this unique insertion in EDN function. B.
Construction and evaluation of ribonuclease and ribosome-inactivating
protein-based chimeric toxins The
objective of the project is to develop chimeric toxins with
ribosome-inactivating and ribonucleolytic toxins, and to characterize their in
vitro and in vivo cytotoxic activity. The project also aims to
rationally design and engineer the chimeric toxins based on the knowledge from
the structure-function analysis to improve their biological activity. We
have shown earlier that after internalization the intracellular translocation
of restrictocin takes place through the retrograde pathway. It has been shown
that the cytotoxicity of many protein toxins, translocated via the retrograde
pathway, increases remarkably if a terapeptide tail, LysAspGluLeu (KDEL), is
attached at their carboxy terminus. To enhance the cytotoxic activity of IL13
containing chimeric toxins we have made two constructs, IL13-restrictocin-KDEL
and IL13-spacer-restrictocin-KDEL, in which the C-terminal end of restrictocin
is extended by incorporating the KDEL tetrapeptide. The characterization of
these chimeric toxins is ongoing. We
have also developed two saporin-based immunotoxins namely IL13-saporin, and
saporin-IL13, targeted at the human IL13 receptor. The fusion proteins contain
IL13 fused either at the amino terminus or the carboxy terminus of saporin.
The fusion proteins are expressed in E. coli and localized in the
inclusion bodies. The purification and characterization of saporin constructs
is underway. Publications Original
peer-reviewed articles 1.
Bagga S, Seth D and Batra JK (2003) The cytotoxic activity of
ribosome-inactivating protein saporin-6 is attributed to its rRNA
N-glycosidase and internucleosomal DNA fragmentation activities. J Biol
Chem 278:4813-4820. 2.
Gaur D, Seth D and Batra JK (2002) Glycine 38 is crucial for the
ribonucleolytic activity of human pancreatic ribonuclease on double stranded
RNA. Biochem Biophys Res Commun 297:390-395. 3.
Goyal A, Seth D and Batra JK (2002) Role of cis prolines 112 and 126 in
the functional activity of ribonucleolytic toxin restrictocin. Biochem
Biophys Res Commun 295:812-817. |