|
Development
of novel chimeric toxins for targeted therapy by genetic engineering |
| Principal Investigator : Janendra K Batra
Ph
D Students Collaborator The
theme of research is the design and development of recombinant protein
toxins for targeted therapy. Members of fungal ribotoxin, plant
ribosome-inactivating proteins, and ribonuclease-A families 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 and
ribosome-inactivating protein toxins The
objective of the study 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. We
had earlier demonstrated that for the catalytic activity of EDN the putative
substrate binding residue Gln14
is indispensable; Arg36
and Gln40
could be partially dispensed with, and
Trp7,
Asn39,
His82
and Asp112
individually are completely dispensable. EDN and its several mutants were
assayed for their ability to inhibit the infectivity of extracellular virions
of the paramyxovirus, RSV-B. The mutants Q14A, R36A, H82A and D112A were taken
with the aim of studying the correlation between RNase activity and antiviral
activity. While Q14A showed an almost complete loss of activity on yeast tRNA,
R36A, H82A and D112A had enzymatic activity comparable to that of EDN. RNase A
was used as a control as it is not known to have antiviral activity on RSV-B
inspite of being a much more active ribonuclease. EDN inhibited the
infectivity of RSV-B by 50% at a concentration of 0.2 mM.
While mutant R36A had antiviral activity very similar to that of EDN, the
mutant Q14A did not have any antiviral activity and did not cause any loss of
infectivity of RSV-B virions. The mutant H82A had slightly higher activity
than EDN, and D112A had about 65% of the activity shown by EDN. The three
mutants in loop L7, R117A, P120A and Q122A showed a loss of activity and their
ID50
values were 2.5-3.5-fold higher than EDN. The maximum loss of activity was
observed in R117A mutant. RNase A was inactive even at concentrations as high
as 10 mM.
The current study shows that although the antiviral activity of EDN is
dependent on its ribonucleolytic activity, loop L7 is crucial for the
antiviral activity. While Pro120
and Gln122
are required structurally for full antiviral activity, Arg117
is critical for EDN’s interaction of with RSV-B. The
saporin isoforms show a very high sequence similarity, with all catalytically
crucial residues conserved. We have earlier shown saporin-6 to have higher
N-glycosidase and DNA-fragmentation activities than saporin-5 that reflect in
a similar difference in their cytotoxic activities. The difference in the
activity of the two isoforms could be attributed to amino acid differences,
which lie outside the proposed active site of the toxin. Of the twelve
differences between saporin-5 and saporin-6, substitutions at positions
134(Q/K), 147(S/L), 149(S/F), 162(D/N), 188(I/T) and 196(N/D) result in change
in polarity or charge of amino acid residue. To understand the mechanism of
saporin toxicity and specific contribution of these residues in the activity
of toxin, we substituted theses residues singly in saporin-6 to those in
saporin-5. The preliminary results show that individually these residues are
not critical for the catalytic as well as cytotoxic activity of saporin. We
earlier analyzed a model of restrictocin-29-mer RNA substrate complex to
understand the mechanism of restrictocin catalysis, particularly to identify
residues involved in stabilizing the substrate onto the enzyme. The base G10,
analogous to G4319 of 28S rRNA, considered to be an identity element for
ribotoxin recognition, forms a large number of contacts, both van der Waal and
hydrogen bonding most of which are with Lys42, Ser46, Lys110 and Lys111
residues of restrictocin. In addition Pro48 was also predicated to be
important for restrictocin catalysis. We made four single amino acid mutants,
K42A, S46A, P48A and K111A, to investigate the roles of Lys42, Ser46, Pro48,
and Lys111 respectively in the ribonucleolytic activity of restrictocin. While
Lys111 appears to be absolutely essential for the catalytic activity of
restrictocin, mutation of Lys42, Ser46 and Pro48 rendered the toxin partially
inactive. B.
Construction and evaluation of ribonuclease and ribosome-inactivating
protein-based chimeric toxins The
objective of the study is to develop chimeric toxins with
ribosome-inactivating and ribonucleolytic toxins, and to characterize their in
vitro and in vivo cytotoxic activity and also, to rationally design
and engineer the chimeric toxins based on the knowledge from the
structure-function analysis to improve their biological activity. The
cytotoxic activity of IL13-restrictocin-KDEL and
IL13-spacer-restrictocin-KDEL, in which the carboxyl terminus of restrictocin
was extended by incorporating the KDEL tetrapeptide, was assayed on IL13
expressing cell lines U251 and A172. Though both the chimeric toxins displayed
cytotoxicity there was no improvement in the activity compared to that of
IL13-restrictocin and IL13-spacer-restrictocin respectively. We
have developed saporin, and human pancreatic ribonuclease (HPR)-based
immunotoxins 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 saporin, and a cytotoxic variant of HPR. The
fusion proteins were expressed in E.coli and purified to homogeneity.
The cytotoxicity of the immunotoxins was checked on target cell lines A172 and
U251. However, these chimeric toxins were not found to be as potent as
restrictocin-containing chimeric toxins directed at the IL-13 receptor.
Publications Original peer-reviewed articles 1.
Bagga S, Hosur MV and Batra JK (2003) Cytotoxicity of
ribosome-inactivating protein saporin is not mediated through a2-macroglobulin
receptor. FEBS Lett 541:16-20.
Review/Proceedings 1. Batra JK (2003) The Aspergillus ribotoxins. Appl Genomics Proteomics 2:145-159. 2. Basu SK, Batra JK and Salunke DM (2004) (Editors) Deep roots, open skies: New biolgy in India. Narosa Publishing House, New Delhi, 200p. |