Christopher R. Pope and Jeffrey P. Thompson, Ph.D.

1. Figure 1. Christopher R. Pope and Jeffrey P. Thompson, Ph.D. Department of Biology, York College of Pennsylvania, York, PA 17405. hIL13R2(his)6EC Expression in U-87MG Cell line #5 Total Cell Lysate S. A. Eluted http://www.sciencemag.org/content/vol291/issue5512/index.dtl Control Man. Eluted Man. Eluted Digest S.A. Eluted Digest Blank Screening of U-87MG Clones Expressing the hIL132(his)6EC Protein Glycoprotein purification, Ni-NTA Purification, and Glycosidase Treatment of hIL13R2(his)6 Clone 10 Mannose Eluted Clone 10 Sialic Acid Eluted Clone 10 Ni-NTA Purified Clone 10 Glycosidase U-87MG Parental Clone 10 Undigested Positive Control Clone 12 Positive Control Clone 11 Clone 10 Clone 7 Clone 9 * ** * ** hIL13R2(his)6EC Expression in HUVEC and COS-7L Cells ** * ** * hIL13Ra2(his)6EC N-Glycosylated Receptor De-glycosylated Receptor Endogenous Receptor Glycoprotein Purification and N-Glycosidase Assay of HUVEC and COS-7L Cell Lines HUVEC Sialic Acid Eluted Cos-7L Sialic Acid Eluted HUVEC Mannose Eluted Cos-7L Mannose Eluted HUVEC Glycosidase Cos-7L Glycosidase Clone 10 U-87MG Positive Control Expected Results Binding IL-13 hIL13R-a2 Binding IL-13 * ** ** * N-Glycosylated Receptor De-glycosylated Receptor Figure 4. Figure 2. Figure 5. Pst I (7) SV40 enhancer Ferritin heavy chain core promoter Figure 3. SV40 polyadenylation site Conclusions mEF1 5'UTR 4465 bp Blasticidin resistance • Results from the lectin binding assay detected a sialic acid component suggesting the oligosaccharides are either of the complex or hybridtype (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. EM7 prokaryotic promoter Bsp HI (1441) Specific Aims FMDV IRES Results hIL13Ra2(his)6 Intact/E.C. Avr II (2496) http://www.cryst.bbk.ac.uk/pps97/assignments/projects/emilia/typ.GIF The purpose of the current study is to confirm that hIL13Ra2 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. Lectin Binding Assay Receptor Estimation Assay References • 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. • 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. • 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. • 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 76:547-551 • 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. Investigating the Glycosylation of Interleukin 13 Receptor Alpha 2 Protein Expressed in Cancerous and Non-cancerous Cell Lines Experimental Procedures Introduction Bacterial Amplification of Extra Cellular Human Interleukin 13 Receptor Alpha 2 Containing a Poly Histidine Tag (hIL13R2(his)6EC) p-Mono-blasti Plasmid Vector (Figure 2) • Malignant 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 hIL13R2 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). Stable Transfection of U-87MG Glioma Cell Line (Figure 3) with the hIL13R2(his)6EC Vector Transient Transfection of HUVEC Cell Line (Figure 3) with hIL13R2(his)6EC Vector Stable Transfection of U-87MG Cell Line with the hIL13R2(his)6 Vector Screen for Over-expressing Clones Conduct a Ligand Binding Assay on Glycosylated and Deglycosylated Forms of the Receptor Perform Western Blot Analysis of hIL13R2(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 Perform a receptor estimation Assay on the Parental U-87MG and Clone 5 Intact Receptor Cell Lines 1.). 2.). 3.) 4.). 5.)