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Figure 1. Christopher R. Pope and Jeffrey P. Thompson, Ph.D. Department of Biology, York College of Pennsylvania, York, PA 17405. hIL13R 2(his) 6 EC Expression in U-87MG Cell line. #5 Total Cell Lysate. S. A. Eluted. http://www.sciencemag.org/content/vol291/issue5512/index.dtl. Control. - PowerPoint PPT Presentation
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Investigating the Glycosylation of Interleukin 13 Receptor Alpha 2 Protein Expressed in Cancerous and Non-cancerous Cell Lines
Christopher R. Pope and Jeffrey P. Thompson, Ph.D.Department of Biology, York College of Pennsylvania, York, PA 17405.
4465 bp
Blasticidin resistance
hIL13Ra2(his)6 Intact/E.C.
SV40 enhancer
FMDV IRES
SV40 polyadenylation site
Ferritin heavy chain core promoter
EM7 prokaryotic promoter
mEF1 5'UTR
Avr II (2496)
BspHI (1441)
Pst I (7)
Figure 2.
http://www.sciencemag.org/content/vol291/issue5512/index.dtl
hIL13R2(his)6EC Expression in HUVEC and COS-7L CellsGlycoprotein Purification and N-Glycosidase Assay of
HUVEC and COS-7L Cell Lines
Clone
10
U-8
7MG
Cos-7
L Man
nose
Elu
ted
Cos-7
L Sia
lic A
cid
Elute
d
Cos-7
L Gly
cosid
ase
HU
VEC M
anno
se E
lute
dH
UV
EC Gly
cosid
ase
HU
VEC S
ialic
Aci
d E
lute
d
Posit
ive C
ontro
l
***
* **N-Glycosylated Receptor De-glycosylated Receptor
hIL13R2(his)6EC Expression in U-87MG Cell lineScreening of U-87MG Clones Expressing the
hIL132(his)6EC Protein
Posit
ive C
ontro
lU
-87M
G P
aren
tal
Clone
7Clo
ne 9
Clone
12
Clone
10
Clone
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***
* Endogenous Receptor ** hIL13R2(his)6EC
Glycoprotein purification, Ni-NTA Purification, and Glycosidase Treatment of hIL13R2(his)6
Posit
ive C
ontro
lClo
ne 1
0 N
i-NTA
Pur
ified
Clone
10
Und
iges
ted
Clone
10
Gly
cosid
ase
Clone
10
Man
nose
Elu
ted
Clone
10
Sial
ic A
cid
Elute
d
N-Glycosylated Receptor De-glycosylated Receptor
***
* **
Figure 3.
Experimental Procedures
Stable Transfection of U-87MG Glioma Cell Line (Figure 3)
with the hIL13R2(his)6EC Vector
Bacterial Amplification of Extra Cellular Human Interleukin 13 Receptor Alpha 2 Containing a Poly Histidine Tag (hIL13R2(his)6EC) p-Mono-blasti Plasmid Vector
(Figure 2)
Transient Transfection of HUVECCell Line (Figure 3)
with hIL13R2(his)6EC Vector
Screen for Over-expressing Clones
Perform Western Blot Analysis of hIL13R2(his)6 for Possible Glycosylation
Conduct a Lectin Binding Assay on the Receptor to Determine the Type of N-
linked Oligosaccharides
Verify N-Linked glycosylation of the Receptor
Stable Transfection of U-87MGCell Line with the hIL13R2(his)6
Vector
Perform a receptor estimation Assay on the Parental U-87MG and Clone 5 Intact
Receptor Cell Lines
Conduct a Ligand Binding Assay on Glycosylated and
Deglycosylated Forms of the Receptor
hIL13R-2 Binding IL-13 Expected Results Binding IL-13
0.00
0.05
0.10
0.15
Receptor Boiled +IL13Receptor -IL13Receptor +IL13CRP10 + IL13CRP10 Boiled + IL13U-87mg +IL13
Means represent absorbance due to IL-13 induced STAT6 response.hIL-13R2 and IL-13 binding efficiencies were measured using Invivogen'sHEK cell STAT6 response. Absorbances were measured at 635nmwavelength. Significance was determined using a 1 way ANOVA with aKruskal-Wallis post test. A P value of less then 0.05 was considered significant.Means determined to be not significant are signified by ns. The error barsrepresent standard error of the mean.
ns ns
nsnsns
ns
Ab
so
rba
nc
e a
t 6
35
nm
0.00
0.05
0.10
0.15
0.20
0.25
Receptor -IL13Receptor +IL13Receptor Boiled +IL13U-87mg + IL13CRP10 + IL13CRP10 Boiled + IL13
Means represent expected absorbance values due to IL-13 induced STAT6response. hIL-13R2 and IL-13 binding efficiencies were measured usingInvivogen's HEK cell STAT6 response. Absorbances were measured at635nm wavelength.
Ab
so
rba
nc
e 6
35
nmFigure 4.
IntroductionMalignant Gliomas are a highly proliferative and aggressive type of cancer, which arise from the neuralgia cells in the brain. As with many cancers, glioma cells are different from normal cells by expressing unique molecular phenotypes and morphologies.
Cancerous cell lines are known to alter post-translational modifications, such as the glycosylation patterns (1). These modifications are known to support cancer cells highly mitogenic nature by regulating mechanisms active in cell proliferation (2).
Glycoproteins on the surface of cells convey molecular information identifying cells and influencing proper cellular behavior.
Human Interleukin 13 Receptor Alpha 2 is a mutated transmembrane receptor, over expressed on glioma cells. Studies have shown that hIL13R2 is glycosylated (Figure 1), and that its glycosylation is required for the proper binding to its ligand, Interleukin-13 (IL-13) (3).
Recognizing hIL13Rα-2 as a tumor-specific plasma membrane receptor, there is interest on using this receptor to deliver cytotoxic payloads directly to tumor cells (4). Other forms of cancer also express the same surface receptor as well as healthy cells locates in the testis (5).
Specific AimsThe purpose of the current study is to confirm that hIL13R2 is glycosylated, to determine whether the glycosylation patterning of this receptor varies between cancerous and non-cancerous cell lines, and to determine the role of glycosylation in ligand binding.
Cytosolic Membrane0
10000
20000
30000
40000
50000
60000
70000
80000
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110000
120000
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U-87 MG ParentalU-87 MG Clone 5
*
*
Mean data for receptor concentration on U-87 MG parentals and Clone 5.Significance according to an unpaired T-test is represented by *, (p<0.05). Clone 5is over-expressing IL-13R2 in the cytosol by a factor of 26.8, and in themembrane by a factor of 25.7.
Num
ber
of IL
-13R
2
Mol
ecul
es
Receptor Estimation Assay
Contro
l
#5 T
otal
Cell L
ysat
e
S. A. E
luted
S.A. E
luted
Dige
st
BlankM
an. E
luted
Man
. Elut
ed D
igest
Lectin Binding AssayResults
Figure 1.
Conclusions• Results from the lectin binding assay detected a sialic acid component suggesting the oligosaccharides are either of the complex or hybrid type (Figure 5).• The receptor estimation assay found the U-87MG cell line contains approximately 5,000 receptors per cell.• Further analysis, may be able to accurately determine the carbohydrate moieties that are responsible for ligand receptor interactions.
http://www.cryst.bbk.ac.uk/pps97/assignments/projects/emilia/typ.GIF
Figure 5.
References 1. Przybyto, M., Hoja-Lukowicz, D., Litynska, A., and Laidler, P. 2002. Different glycosylation of cadherins from bladder non
malignantand cancer cell lines.Cancer Cell International. 2:1475-2867.2. Dennis, J., Laferte, S., Waghorne, C., Breitman, M., and Kerbel, R. 1987. Beta 1-6 branching of asn-linked oligosacchrides is
directly associated with metastasis.Science. 236:582-585.3. Kioi, M., Seetharam, S., and Puri, R. 2006. N-linked glycosylation of IL-13Ra2 is essential for optimal IL-13 inhibitory activity. FASEB
Journal. 20: 892-6638.4. Debinski, W., Gibo, D. and Puri, R. 1998. Novel way to increase targeting specificity to a human glioblastoma-associated receptor
for interleukin 13. International Journal of Cancer 76:547-5515. Moscatello, D., Holgado-Madruga, M., Godwin, A., Ramirez, G., Gunn, G., Zoltick, P., Biegel, J., Hayes, R., Wong, A. 1995.
Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Research 55:5563-5539.