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

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cosid

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dH

UV

EC Gly

cosid

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HU

VEC S

ialic

Aci

d E

lute

d

Posit

ive C

ontro

l

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* **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

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Clone

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ne 9

Clone

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Clone

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Clone

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* Endogenous Receptor ** hIL13R2(his)6EC

Glycoprotein purification, Ni-NTA Purification, and Glycosidase Treatment of hIL13R2(his)6

Posit

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ontro

lClo

ne 1

0 N

i-NTA

Pur

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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

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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

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otal

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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.