1. 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 hIL13R  2(his) 6 EC Expression in HUVEC and COS-7L Cells Glycoprotein Purification and N-Glycosidase Assay of HUVEC and COS-7L Cell Lines Clone 10 U-87MG Cos-7L Mannose Eluted Cos-7L Sialic Acid Eluted Cos-7L Glycosidase HUVEC Mannose Eluted HUVEC Glycosidase HUVEC Sialic Acid Eluted Positive Control * ** * N-Glycosylated ReceptorDe-glycosylated Receptor hIL13R  2(his) 6 EC Expression in U-87MG Cell line Screening of U-87MG Clones Expressing the hIL13  2(his) 6 EC Protein Positive Control U-87MG Parental Clone 7Clone 9 Clone 12 Clone 10 Clone 11 * ** * Endogenous Receptor ** hIL13R  2(his) 6 EC Glycoprotein purification, Ni-NTA Purification, and Glycosidase Treatment of hIL13R  2(his) 6 Positive Control Clone 10 Ni-NTA Purified Clone 10 Undigested Clone 10 Glycosidase Clone 10 Mannose Eluted Clone 10 Sialic Acid Eluted N-Glycosylated ReceptorDe-glycosylated Receptor ** * * Figure 3. Experimental Procedures Stable Transfection of U-87MG Glioma Cell Line (Figure 3) with the hIL13R  2(his) 6 EC Vector Bacterial Amplification of Extra Cellular Human Interleukin 13 Receptor Alpha 2 Containing a Poly Histidine Tag (hIL13R  2(his) 6 EC) p-Mono-blasti Plasmid Vector (Figure 2) Transient Transfection of HUVEC Cell Line (Figure 3) with hIL13R  2(his) 6 EC Vector Screen for Over-expressing Clones 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 Stable Transfection of U-87MG Cell Line with the hIL13R  2(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 Figure 4. Introduction  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). Specific Aims The purpose of the current study is to confirm that hIL13R  2 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. Receptor Estimation Assay Control #5 Total Cell Lysate S. A. Eluted S.A. Eluted Digest Blank Man. Eluted Man. Eluted Digest Lectin Binding Assay Results 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. 1.). 2.). 3.) 4.). 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 76:547-551 5. 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.