Download Reactivity of Interleukin 13.E13C Mutant toward Interleukin 13

Reactivity of Interleukin 13.E13C Mutant toward Interleukin 13 Receptors
Shana Kondor and Dr. Jeffrey Thompson, Biology Department, York College of PA
RESEARCH DESIGN
PROJECT SUMMARY
Create IL13.E13C mutant through point mutation of WT IL13
Although fairly extensive and ongoing research is being
conducted regarding human Interleukin-13 (hIL-13) and its
various mutants, the region of uncharted territory is vast.
Understanding completely how hIL-13 interacts with its
receptors on both normal and malignant cells could be a key
component to developing a cancer-targeting drug. Only by
continuing in the efforts of understanding the functionality of
the protein can any knowledge be gained towards the subject.
The proposed research involves a mutant of hIL-13 not yet
studied, that being hIL13.E13C. This mutation resides on a
protein domain that is involved with normal cell binding but not
cancer cell binding. With this in mind, mutating at this position
will hopefully eliminate binding affinity towards the normal cells
while retaining its ability to bind with cancer cells, therefore
making it able to target cancer cells only.
RESEARCH OBJECTIVES
1) Determine ability of IL13.E13C to target cancer
cells
2) Determine effectiveness of IL13.E13C as
cytotoxin delivery complex
3) Determine the difference, if any, between
eukaryotic and prokaryotic protein expression
4) Gain insight into IL13 structure and function to
be related to other applications of IL13
Express Samples 1 and 2 by plating transformed E. coli on Ampicillin agar plates
Solution Structure of Human IL13
www.cytok.com/structure.php?start=80
Express WT (Sample 3) and mutant (Sample 4) with COS7 cell cultures for
eukaryotic assay
LITERATURE REVIEW
Thompson and Debinski, 1997
•
-helix A mutants both caused and failed to cause TF-1 cell
proliferation
•
All position 13 mutants except E13R and E13D failed to cause TF-1
proliferation
•
Position 13 mutants that failed to cause TF-1 proliferation were most
effective at blocking hIL13-CTX action on U-251 MG and SNB-19
•
Interaction with the hIL13 shared receptor depends on multiple factors
including amino acid size, electrostatics, and polarity
Purify all samples
Test TF-1
proliferation
(all samples)
Cytotoxicity
assay
(all samples)
Competition
assay
(samples 2 + 4)
EXPECTED RESULTS
Figure 2
Figure 1
Cytotoxicity Blocking Assay
with hIL13-CTX
TF-1 Cell Proliferation
IL13.E13C
hIL13 (WT)
IL13.E13C eukaryote
100
80
60
40
20
0
0.1
1
10
Interleukin, (ng/ml)
A490, percent of control
Mintz et al., 2002
•
High-grade astrocytomas bind IL13 and are sensitive to IL13
cytotoxins
•
IL13.E13K binds to HGA IL13 site 50X better than wild type IL13
•
Identified transcript for hIL13R 2 in HGA’s
•
HGA’s bind IL13 but not IL4
•
IL13.E13K completely blocked killing of HGA’s by cytotoxin
120
120
IL13.E13C
hIL13 (WT)
IL13.E13C (eukaryote)
100
80
60
40
20
0
0.1
100
1
10
100
hIL13-CTX, (ng/ml)
Figure 3
Competition Assay with
hIL13.E13C-CTX
120
Kruse et al., 2002
•
IL4 and IL13 have individual signaling processes
•
Substrate binding and signaling strength vary depending on the
variant of IL4R  protein
•
IL13 acts independently of IL4 in inducing pathophysiological features
of asthma
Wäldele et al., 2004
•
IL13 stimulates human T-cell leukemia virus type 1 (HTLV-1) through
up-regulation of IL13 by Tax, a viral transactivator
•
IL13 inhibits apoptosis and stimulates the cell cycle
•
Various cytokines that induce cell death are inhibited by IL13
•
IL13 linked to leukemogenesis as found through Hodgkin lymphoma
cell stimulation
Kuznetsov and Puri, 1999
•
IL13 inhibits B cell proliferation
•
Kinetic studies show heterogeneity in IL13 binding, possibly
explaining cytotoxicity of IL13 toxin in renal cell carcinomas
Aversa et al., 1993
•
IL13 and IL4 work independently to induce IgG4 and IgE synthesis
•
IL4 receptor complex and IL13 receptor complex are distinct receptors
that may have common components
•
B cell proliferation studies suggest that IL4 and IL13 bind the same
receptor
•
IL13 failed to bind the IL4 receptor on mouse BaF3 cells or monkey
COS-7 cells, but blocked IL4 binding on TF-1 cells
Vladich et al., 2005
•
Demonstrated that eukaryotic IL13 was much more active than
prokaryotic IL13
•
Used COS-7 cell lines as eukaryotic model to compare with E. coli
prokaryotic model
•
COS-7 cells were transfected and assessed for IL13 concentrations
by ELISA, with correction factors to detect WT versus mutant
A490, percent of control
INTRODUCTION
Transform E. coli with WT (Sample 1) and mutant (Sample 2) by transforming E. coli
with respective samples + AmpR
A490, percent of control
Interleukin 13 (IL-13) is a pleiotropic cytokine comprised of
four -helical regions (A-D) expressed in Th1 and Th2
lymphocytes, stimulated keratinocytes, activated mast cells,
and transformed B lymphocytes (Kuznetsov and Puri 1999).
Helices A and C bind to the shared receptor, while B and D
bind to IL13 exclusive sites on cancer cells. Previous studies
have shown success in decreasing normal cell affinity while
maintaining cancer cell affinity (Thompson and Debinski
1999). The objectives are to create an IL13.E13C mutant,
express the protein in Escherichia coli and human monocytes,
and assess binding affinity and cytotoxicity through TF-1
proliferation and a cytotoxicity blocking assay with
Glioblastoma cells. By mutating at position 13 on -helix A,
affinity for normal cells should decrease while maintaining
affinity for cancer cells. Replacing glutamic acid with cysteine
creates a free sulfhydryl group for use as a cytotoxin
attachment point. This mutated form of IL13 could be used as
a chemotherapy aid that will allow it to target only cancer cells,
leaving normal cells unharmed.
100
80
60
40
20
0
0.1
1
10
100
hIL13.E13C-CTX, (ng/ml)
• Figure 1 shows hypothetical TF-1 cell proliferation of hIL13 (WT), IL13.E13C
(prokaryotic expression) and IL13.E13C (eukaryotic expression). It is suspected that
both mutants of IL13 will have decreased TF-1 cell proliferation due to lack of binding on
the shared IL13/4 receptor. With large enough concentrations, however, cell proliferation
will occur.
• Figure 2 shows a hypothetical cytotoxicity blocking assay using hIL13-CTX. When
competing with WT hIL13, blocking will occur approximately 50% because the cytotoxin
bound hIL13 and the WT hIL13 should have similar binding affinities. When in
competition with the mutants, however, blocking should occur, because the mutants are
hoped to have better binding affinity for the IL13 specific receptor. It is hypothesized that
the eukaryote expressed mutant will block slightly better because it has been found that
eukaryote expressed IL13 is more active than the prokaryote expression.
• Figure 3 shows a cytotoxicity blocking assay using IL13.E13C-CTX. It is suspected
that the eukaryote expressed protein will effectively block the prokaryote expressed
protein linked with the cytotoxin, because of findings that eukaryote expression is more
effective.
LITERATURE CITED
Aversa, G., Punnonen, J., Cocks, B., de Waal Malefyt, R., Vega, F., Zurawski, S., Zurawski,
G. and deVries, J. 1993. An Interleukin 4 (IL-4) Mutant Protein Inhibits both IL-4 and IL13-induced Human Immunoglobulin G4 (IgG4) and IgE Synthesis and B Cell
Proliferation: Support for a Common Component Shared by IL-4 and IL-13 Receptors.
Journal of Experimental Medicine 178:2213-2218.
Kruse, S., Braun, S. and Deichmann, K. 2002. Distinct signal transduction processes by IL4 and IL-13 and influences from the Q551R variant of the human IL-4 receptor alpha
chain. Respiratory Research 3:24.
Kuznetsov, V. and Puri, R. 1999. Kinetic Analysis of High Affinity Forms of Interleukin (IL)13 Receptors: Suppression of IL-13 Binding by IL-2 Receptor  Chain. Biophysical
Journal 77: 154-172.
Mintz, A., Gibo, D., Slagel-Webb, B., Christensen, N. and Debinski, W. 2002. IL-13R2 is a
Glioma-Restricted Receptor for Interleukin-13. Neoplasia 4:388-399.
Thompson, J. and Debinski, W. 1999. Mutants of Interleukin 13 with Altered Reactivity
toward Interleukin 13 Receptors. Journal of Biological Chemistry 247:29944-29950.
Vladich, F., Brazille, S., Stern, D., Peck, M., Ghittoni, R. and Vercelli, D. 2005. IL-13
R130Q, a common variant associated with allergy and asthma, enhances effector
mechanisms essential for human allergic inflammation. Journal of Clinical Investigation
115:747-754.
Wäldele, K., Schneider, G., Ruckes, T. and Grassmann, R. 2004. Interleukin-13
Overexpression by Tax Transactivation: a Potential Autocrine Stimulus in Human T-Cell
Leukemia Virus-Infected Lymphocytes. Journal of Virology 78:6081-6090.