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Development of novel chimeric toxins for targeted therapy by genetic engineering |
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Principal Investigator : Janendra K Batra PhD
Students Collaborators Design
and development of recombinant protein toxins for targeted therapy is the main
theme of research of this project. Fungal ribotoxins, plant ribosome
inactivating proteins (RIP), and human ribonucleases are being analysed for
structure-function relationships to understand their molecular mechanism of
action with an aim to design knowledge-based chimeric toxins. A.
Studies on the molecular mechanism of action of ribonucleolytic protein
toxins The
objective of the study is to investigate structure-function relationship of Aspergillus
ribotoxin restrictocin, human pancreatic ribonuclease (HPR), ribosome
inactivating protein saporin, eosinophil cationic protein (ECP), and
eosinophil-derived neurotoxin (EDN) to understand their mechanism of action. The
structural and functional characterization of saporin mutants was continued to
understand its mechanism of cytotoxicity. From the earlier year’s study no
direct correlation was found in the catalytic activity and cytotoxic activity
of a few saporin mutants. The mutant Y16A showed a 20-fold reduced protein
synthesis inhibitory activity whereas the mutant W208A had a similar protein
synthesis inhibitory activity as that of the native toxin. Mutants Y16A and
W208A showed similar cytotoxic activities on all the cell lines studied
despite the two mutants showing very different protein synthesis inhibitory
activity. The activity of native saporin was investigated on the genomic DNA
of cell lines treated with the toxin for different periods of time. It was
seen that in the presence of toxin the DNA of these cells was fragmented and
the appearance of DNA was similar to that of apoptotic cells. The DNA
fragmentation did not appear to be a consequence of protein synthesis
inhibition by saporin as the laddering was seen at doses of toxin where
protein synthesis was only partially inhibited. Among various mutants of
saporin only Y16A and R24A showed genomic DNA fragmentation activity. W208A
failed to affect DNA inspite of having full N-glycosidase activity.
Individually Y16A and W208A were found to be six-fold less active on U937
cells compared to saporin. Interestingly, an equimolar mixture of the two
proteins resulted in cytotoxic activity very similar to that of saporin. The
study now clearly establishes that the cytotoxic activity of saporin is a
cumulative effect of its N-glycosidase and DNA fragmentation activity. The
intracellular trafficking and processing of saporin was also investigated in
J774A.1 cells using iodinated saporin and/or various metabolic and protease
inhibitors. The results demonstrate that i) an acidic endosomal pH is required
as 10 mM ammonium chloride protected cells from saporin cytotoxicity, ii)
intracellular routing of saporin is not through Golgi as BFA did not affect
its activity, iii) upon internalization no proteolytic processing of saporin
takes place as none of the protease inhibitors used affected the cytotoxic
activity, and iv) saporin remains intact in the cell upto 24 hrs and
translocates to nucelus and cytosol. The
characterization of EDN mutants was extended by investigating their activity
on substrates, cCMP, UpA and CpA, and also in the in vitro protein
synthesis assay. Except for the mutants H82A and D112A all other mutants of
EDN namely W7A, Q14A, R36A, N39A, and Q40A had reduced activity on cCMP
compared to the native EDN. On UpA all mutants had reduced activity, whereas
on CpA, W7A, N39A, H82A and D112A had an increased catalytic activity. In the in
vitro cell-free translation mutants W7A, N39A, Q40A, H82A and D112A had an
increased inhibitory activity whereas mutants Q14A and R36A had a reduced
activity. The study indicates crucial involvement of the studied residues in
the catalytic activity and specificity of EDN. Further characterization is
required to assign specific roles to these residues in the EDN activity. We
have also initiated studies to investigate the role of unique loops and
insertions in the EDN protein in its function. Specific mutations have been
made to study the role of residues R117, P120 and Q122. Mutants have been
generated in which amino acids 1-3, 68-69, and 91-92 have been separately
deleted. Characterization of these mutants is currently being carried out. B.
Construction and evaluation of ribonuclease-based chimeric toxins The
objectives of this study are (i) development of chimeric toxins with
restrictocin and human pancreatic ribonuclease, and characterization of their in
vitro and in vivo cytotoxic activity and (ii) to design and
engineer the chimeric toxins based on the knowledge from the
structure-function analysis to improve their biological activity. IL13
receptors are overexpressed on a variety of human tumors including renal cell
carcinoma, AIDS-associated Kaposi’s sarcoma, ovarian carcinoma, prostate
cancer and malignant glioma. IL13 has been shown to be a good target for
immunotoxin therapy in these carcinomas. We developed four restrictocin-based
immunotoxins namely IL13-restrictocin, IL13-spacer-restrictocin,
restrictocin-IL13 and restrictocin-spacer-IL13, targeted at the human IL13
receptor. The fusion proteins contain IL13 fused either directly or through a
proteolytically cleavable spacer at the amino or carboxyl terminus of
restrictocin. The fusion proteins were expressed in E. coli and
purified to homogeneity. The cytotoxicity of the immunotoxins was checked on
target cell lines U373, T98G, A172, and U251, and non-target Colo205 and A431
cell lines. U251 and A172 were found to be the most sensitive cell lines.
Restrictocin-spacer-IL13 was found to be the most active immunotoxin
indicating that the intracellular proteolytic processing is crucial for the
activity and blocking the carboxyl terminus of IL13 hampers its receptor
binding activity. The
cytotoxic activity of recombinant dimeric HPR was studied on a variety of cell
lines including IMR32, SJRH30, SKNAS, U373MG, J774A.1, K562, A431 and A549.
Human neuroblastoma, IMR32 and SKNAS, human rhabdomysarcoma, SJRH30 and human
glioblastoma U373MG were found be sensitive to HPR dimer toxicity compared to
monomeric native HPR. We are currently investigating the mechanism of HPR
dimer cytotoxicity. Publications Original
peer-reviewed articles 1. Gaur D, Swaminathan S and Batra JK (2001) Interaction of human
pancreatic ribonuclease with human ribonuclease inhibitor: Generation of
inhibitor-resistant cytotoxic variants.
J Biol Chem 276:24978-24984. 2.
Nayak SK, Bagga S, Gaur D, Nair DT, Salunke DM and Batra JK (2001)
Mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic
toxin restrictocin. Biochemistry 40:9115-9124. |