Determining the glycan profile of reelin glycoprotein in neural cell line

Reelin is a highly conserved extracellular glycoprotein that have an importance in neural development. There is many research related with neural disease originate from dysfunction of Reelin protein and mutation of RELN gene. However, the information about the glycans in the structure of reelin is very limited as well as no information about the possible roles of glycans in the disease. Different units of glycan in the structure of glycoproteins can control their function by altering the microenvironment of proteins. Thus, they act to function directly or indirectly in almost all functions of the cell. It is highly probable that the glycan chains of the Reelin glycoprotein also have an effect on its function since the glycosylation changes in the two proteins with the same amino acid sequences can directly alter the protein's function. In this thesis, it is aim to isolate of the Reelin glycoprotein and its important fragments from SH-SY5Y neuroblastoma cell line and to characterize of glycans profiles of these isolates by lectin blotting and structure (monosaccharide) analysis of these by CapLC-ESI-MS/MS. For this aim, SH-SY5Y neuroblastoma cell line was used. Total and cytoplasmic protein extractions were carried out after cells were grown in appropriate conditions. Band / bands belong to Reelin were determined using Western blotting. Lectin blotting was carried out using SNA, MAA, GNA, PNA and DSA lectins to determine the terminal units of glycan on the Reelin bands. Reelin glycoprotein was isolated from the cell line using an immunoprecipitation technique. Monosaccharide analysis of the isolated Reelin fragment was performed using the CapLC-ESI-MS/MS system. The total protein profile of the SH-SY5Y neuroblastoma cell line was demonstrated by SDS-PAGE using three different lysis buffers (Ripa, NucBuster, IP-lysis) and very similar results were obtained in all three cases. Reelin was obtained in the form of four fragments with weight of 400, 310, 250 and 85 kDa. α2,3 and α2,6 bound sialic acids, β-Galactose, α-Mannose, β-GlcNAc were determined in the fragments of Reelin glycoprotein as terminal unit of glycan. The highest value in terms of band area density was found to be 601 units in the α2,3 linked sialic acid band of the 400 kDa fragment of Reelin. Total value of band areas in the four fragments of Reelin were α2,3 linked sialic acid (1224 unit), β-Galactose (1165 unit), α2,6 bound sialic acids (764 unit), β-GlcNAc (672 unit), α-Mannose (205 unit) respectively. Purification of the Reelin glycoprotein was performed by immunoprecipitation from not transfected cells and Reelin fragment with weight of 250 kDa was isolated in pure form. In the result of the monosaccharide analysis of this isolate, N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose and mannose were determined from high amount to low respectively. As a result, there are many diseases associated with alteration of glycosylation and/or recognition dysfunctions of glycans. Although Reelin is a glycoprotein, literature on glycan profile of Reelin is very limited. Glycan analysis of Reelin with high analytical techniques, LC-MS/MS, was performed for the first time. In addition, purification of the natural form without the use of transfection system to produce reelin is important for preventing possible glycan changes and not been found in the literature. The 85 kDa from the determined Reelin fragment was also shown for the first time. Determination of high amounts of aminoglycans (GlcNAc and GalNAc) demonstrate that N-glycans as well as O-glycans may be an important on Reelin. It is expected that these results and future studies will contribute to detailed understanding of the mechanism of neuropsychiatric diseases and to the research done for diagnosis and treatment of the diseases from the viewpoint of glycobiology.