Isolation of egg jelly coat glycoproteins of sea urchin paracentratus lividus and identification of glycan profile by MALDI-TOF/MS and LC-MS/MS system using the glycomics approach

The surface carbohydrate content is crucial in the sperm and egg interactions. There are carbohydrate-rich glycoproteins in the jelly coat which surrounding sea urchin eggs. Although there are no studies yet they are considered to be homologous to the vertebrate recognition molecules. Although some glycoproteins involved in this layer have been characterized, glycan profiles are not determined. Therefore, in this thesis;, it is aimed to identificate various glycoproteins known importance in fertilization and to clarify the glycan profile by glycomics approach in the egg jelly coat of the sea urchin Paracentrotus lividus which is selected as a model organisms due to having alecithal type egg like mammals. Hence, isolated egg jelly coat by acidic method was lyophilized and dialysed and protein content was measured by the Bradford method. Jelly coat proteins determined by SDS-PAGE, glycosylation of proteins identified by periodic acid-Schiff (PAS) staining, glycoproteins were defined by lectin blotting using LFA which is specific for the terminal sugar in the glycoprotein called sialic acid. Analysis of monosaccharides that is forming the glycan chains of glycoproteins is performed using acid hydrolysis to the egg jelly coat. PMP was used for the derivatization of the monosaccharides except sialic acids, DMB flourescent reagent used for the sialic acids. Samples were analysed using CapLC-ESI-MS/MS system. To determine glycans with glycomics approach; N-glycans were released by PNGase F from protein chain, derivatized with permethylation and analysis were performed by MALDI-TOF-MS in the presence of N2 with laser. The same samples were characterized using CapLC-ESI-MS/MS system with fragment ions. Ammonia solution was directly added on to the egg jelly coat for O-glycan analysis. Then derivatized with PMP and analysed by LC-MS/MS system. After the characterisation of protein/glycoprotein of egg jelly; by SDS-PAGE four protein band were observed near 250, 55, 50 ve 15 kDa. It is understood using PAS staining that near 250, 35 ve 10 kDa proteins are glycosylated, those of only near 250 kDa protein is a large glycoprotein. Probably near 35 and 10 kDa bands are proteoglycans. As a result of the monosaccharide analysis of jelly coat glycoproteins; fucose was found at high level and the mannose, arabinose+xylose, galactose, glucose, N-acetylgalactosamine ve N-acetylglucosamine found respectively. Ribose is derived from genetic material. Neu5Gc was found in highest level among sialic acid types. Then Neu5Ac, Neu9Ac5Gc, Neu5,9Ac2, Neu8Ac5Gc, Neu7Ac5Gc, Neu5,7Ac2, Neu5,8Ac2 was found respectively. Neu5GcS and Neu5AcS which substituted with sulphate were determined in trace level. High mannose N-glycan and O-hexose type O-Glycans were determined in the egg jelly coat glycoproteins using glycomics approach. Furthermore, core 1-8 like O-glycans were identified using LC-MSn system. Core 1-8 O-glycans determined in MALDI-TOF-MS, too. In conclusion; in this thesis, glycan profile of the egg jelly of P. lividus revealed for the first time using MALDI-TOF/MS, LC-ESI-MS/MS, lectin blotting and also software which are new and modern glycomics techniques. P.lividus is used as a model organism in this study which survives until today and has been unchanged during the evolution process compared to the other species, reveals the special nature of this species. Therefore, not only fertilization, but also it is interesting to get answers of the molecular explanation of speciation especially from glycobiological view. After the details identified in the model organism is understood, the perspective to the homologous molecules could be affected by other species. It may also contribute to the investigation and deciphering of infertility in humans by knowing these relations well.