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Development of novel chimeric toxins for
targeted therapy by genetic engineering |
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Principal Investigator : Janendra K Batra Project Assistants Ph D Students Design and development of recombinant protein toxins
for targeted therapy is the theme of research. Members of two families of
ribonucleases namely (i) Aspergillus ribotoxins and (ii) human ribonucleases
are being analyzed 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 this study was to investigate
structure-function relationship of Aspergillus ribotoxin restrictocin, human
pancreatic ribonuclease (HPR), eosinophil cationic protein (ECP), and
eosinophil-derived neurotoxin (EDN) to understand their mechanism of action. The HPR mutants were further characterized for their
interaction with the ribonuclease inhibitor (RI). The ribonuclease activity of
the mutants, in the presence of RI, was quantitatively studied by assaying
their activity on poly(C). At an enzyme concentration of 0.8 ng, and a RI
concentration of 0.25 U, wt HPR, K7A and N71A showed 30% activity, whereas the
mutants Q11A and E111A exhibited 60-80% activity. However, when the enzyme
concentration was increased to 1.6 ng, all the four mutants showed full
enzymatic activity in the presence of RI, whereas HPR exhibited only 50%
activity. The double mutants Q11A:E111A and N71A:E111A, and the triple mutant
K7A:N71A:E111A exhibited 60-95% activity at a concentration of 0.8 ng in the
presence of 0.25U RI. However, the behavior of mutants K7:E111A,
Q11A:N71A:E111A and K7A:Q11A:N71A:E111A in the presence of RI was found to be
similar to that of wt HPR. These results suggest that the mutants – K7A,
Q11A, N71A, E111A, Q11A:E111A, N71A:E111A and K7A:N71A:E111A are less
sensitive to inhibition by RI than wt HPR. The inhibition constants (Ki) for
the RI-HPR/mutant interactions were determined by measuring the steady state
rate of poly(C) cleavage in the presence of RI. The Ki values for the seven
mutants – K7A, Q11A, N71A, E111A, Q11A:E111A, N71A:E111A and K7A:N71A:E111A
were 5-25 fold higher than that of wt HPR, indicating that the binding
affinity of the mutants with RI had been reduced. In order to study the effect
of these mutations on the interaction of HPR with the intracellular RI, their
cytotoxic activity was assayed on U373 MG, J774.A1, K562, A431, and A549 cell
lines. The HPR triple mutant K7A:N71A:E111A, which exhibited maximum
resistance against RI, was found to be most cytotoxic. It displayed at least a
10-fold higher cytotoxic activity than HPR on the most sensitive cell line
U373MG. The mutants Q11A and E111A were 2-3 fold more cytotoxic than wt HPR on
all the cell lines studied. Similarly, the mutants N71A:E111A, Q11A:E111A, K7A
and N71A exhibited higher cytotoxicity than HPR; however, they were not as
potent as the mutants Q11A and E111A. HPR exhibits significantly high ribonuclease activity on
double stranded RNA, compared to RNase A. In the dimeric bovine seminal
ribonuclease, that exhibits a 46- fold higher activity on dsRNA compared to
RNase A, two glycine residues at positions 38 and 111 have been found to play
an important role in the activity on dsRNA. In HPR, a glycine residue is
present at position 38 only, whereas RNase A is devoid of any glycine residue
at these positions. The present study was conducted to investigate the role of
the glycine residue at position 38 and the effect of incorporating a glycine
residue at position 111 on the activity of HPR on dsRNA. Five mutants of
HPR-G38A, G38D, E111A, E111G and G38D; E111G were prepared by site directed
mutagenesis. The mutants were expressed in E.coli and the proteins were
purified to homogeneity. The CD spectra of the mutants G38A, G38D, E111A,
E111G showed structural similarity with wt HPR; however, the secondary
structure of the mutant G38D:E111G appeared to be altered. All the mutants
exhibited RNase activity on the substrates Poly(C), yeast tRNA and cCMP
similar to that of wt HPR. The activity of the mutants on double stranded RNA
was studied by assaying their activity on poly(A).poly(U). The mutants G38D
and G38D:E111G displayed a lower activity than wt HPR. The mutant E111G showed
a higher activity on Poly(A).Poly(U). The mutants G38A and E111A exhibited a
similar activity on dsRNA as wt HPR. The substitution of Gly38 to Ala (G38A)
is a conservative mutation in which there is no significant change in the
nature or charge of the side chain and thus produces no change in activity of
HPR. However, the mutation of Gly38 to Asp (G38D), a residue present in RNase
A, results in the loss of activity on duplex RNA. The substitution of Glu111
to Gly (E111G) results in an enhancement in the RNase activity on dsRNA. These
results suggest that glycine at positions 38 plays an important role in the
activity of HPR on dsRNA. To study the mechanism of action of saporin, the putative
active site residues, Y72, Y120, E176, R179 and W208 were mutated to alanine.
Two additional invariant residues Y16 and R24, proposed to be playing a role
in normal a-helical pattern of saporin were also mutated to alanine. The
mutants were expressed in E.coli, and the proteins were purified to
homogeneity. The CD-spectral analysis of mutants showed that except for Y16A,
all the mutants retained the structure similar to saporin. The protein
synthesis inhibitory activity of mutants was assessed in an in vitro
translation assay. R24A and W208A showed similar activity as saporin, whereas
mutation of Y16, Y72, Y120, E176 and R179 resulted in the loss of protein
synthesis inhibitory activity. Mutants were also assessed for their specific
28S rRNA glycosylase activity. Saporin, R24A and W208A released the a-fragment
from the 28S rRNA, on aniline treatment, at a concentration as low as
0.04mg/ml, whereas Y16A, Y72A, Y120A, E176A and R179A started showing a faint
band only at 1mg/ml, indicating at least a 25-fold reduction in the activity
due to these mutations. Y72A released a a-fragment after 5mg/ml The DNase-like
activity of saporin and its mutants was studied on plasmid pBR322. At lower
concentrations (0.25-1.0mg/ml of saporin/2mg of DNA) saporin changes the
conformation of DNA, whereas at higher concentrations (2-3mg and more) it
completely digests the DNA. Among the mutants R24A, Y120A, E176A and R179A
were found to be as active as saporin. Y16A and W208A (upto 4mg did not
completely digest the DNA, however these mutants converted the plasmid to
relaxed circular/linear form. Y72A did not act on DNA at all upto 4mg The
cytotoxic activity of saporin and its mutants was studied on J774A.1, HUT102,
L929 and U937 cell lines. The mutants having both the glycosidase and the
DNase-like activity were found to be cytotoxic. In order to study the
mechanism of toxicity, cytotoxicity experiments were done in presence of
Brefeldin A and ammonium chloride. BFA apparently does not affect the
cytotoxicity of saporin on L929 and J774A.1 cells. The study demonstrates that
(a) the residues Y72, Y120, E176 and R179 are crucially involved in the
activity of saporin, (b) both the N-glycosidase and DNase activities are
required for cytotoxicity and (c) these activities may be residing in
different locations in the saporin molecule. The genes for EDN and ECP were cloned in pVEX11, expressed
in E.coli, and the proteins purified to homogeneity. On yeast tRNA, EDN was
200-fold more active than ECP. On poly(U), EDN was 50-100 fold more active
than ECP, and on poly(C) ECP did not show any detectable activity. The protein
synthesis inhibitory activity was assessed in an in vitro translation system.
EDN was 30-fold more active than ECP. To study the mechanism of action of EDN,
putative active site and substrate binding residues, based on sequence and
structural homology to RNase A, were mutated to alanine by site-directed
mutagenesis. These residues include W7, Q14, R36, N39, Q40, H82 and D112. The
mutants were expressed in E.coli and purified to homogeneity. The mutants were
assayed for ribonuclease activity on various substrates and for in vitro
protein synthesis inhibitory activity. On the basis of various functional
assays, residues Q14 and R36 appear to be crucially involved in the
ribonucleolytic activity of EDN. B. Construction and evaluation of ribonuclease-based chimeric toxins The objective in this project was the development of
chimeric toxins with restrictocin and human pancreatic ribonuclease, and
characterization of their in vitro and in vivo cytotoxic activity. Also, to
design and engineer the chimeric toxins based on the knowledge from the
structure-function analysis to improve their biological activity. Our studies have shown that HPR can be engineered to
generate variants with reduced sensitivity to the intracellular inhibitor,
resulting in a significant enhancement in its cytotoxic activity. Bovine
seminal ribonuclease, which is a dimeric RNase with a very low sensitivity
towards the intracellular ribonuclease inhibitor, has been shown earlier to
have a potent antitumour activity. We have employed recombinant techniques to
generate a dimeric form of HPR in which two monomers of HPR are joined by a
flexible peptide linker. The gene fusion was expressed in E.coli, and the
recombinant protein was purified to homogeneity. The dimeric HPR was found to
contain similar ribonucleolytic activity as that of HPR. However, compared to
HPR the dimeric HPR was found to have a very potent cytotoxic activity on a
variety of cell lines. It is currently being evaluated on cancers of neuronal
origin, on which bovine seminal ribonuclease has been shown to be very highly
active. The dimeric human pancreatic ribonuclease should be a useful toxin for
the construction of immunotoxins with low immunogenicity. To target human IL4 receptor, DNA coding for human IL4 was
used to generate chimeric toxins IL4-restrictocin, IL4-spacer-restrictocin,
restrictocin-IL4 and restrictocin-spacer-IL4. Chimeric toxins with saporin
targeted at the IL4 receptor were also made containing the ligand at the amino
and carboxy terminus of saporin. The chimeric toxins have been produced in
E.coli and proteins have been purified to homogeneity. Various proteins are
now being characterized for their cytotoxic activity on the target cell lines. |