IDENTIFYING GLYCAN UNITS OF LAMIN A/C PROTEIN IN A549 LUNG CARCINOMA CELLS SYNCHRONIZED AT SYNTHESIS AND MITOSIS PHASES
Project no. 432 · Status: Kapanmış · 2017
Summary
Nuclear lamins are type V intermediate filament proteins that play several roles in many cellular events such as chromatin organization, gene regulation, DNA damage and repair, DNA replication and signal transduction besides providing mechanical support to the nucleus. The most striking feature of the lamins is that their reversible assembly and disassembly during mitosis. In prophase, phosphorylation of lamins triggers the nuclear envelope disassembly and in late telophase, dephosphorylation of lamins reverses the process. However, it hasn’t been known whether lamin proteins undergo a modification other than phosphorylation throughout the cell cycle. The post-translational modification, glycosylation, regulates the interaction of proteins with their environment by altering the thermodynamic, kinetic, and structural properties of proteins. It has been known that especially in nucleocytoplasmic proteins, O-GlcNAcylation and phosphorylation are competing for the same Ser/Thr residues of the protein, and the interplay between two modification regulates the activation/inactivation of the protein. Based on this information, in this thesis, we hypothesized that these two modifications may function cooperatively or reversibly to organize the structural organization of the lamin A/C protein during cell cycle. Thus, the first aim of the study is to determine possible O-GlcNAcylated and O-GalNAcylated positions and to generate 3D glycoprotein structure of human lamin A/C protein using bioinformatics tools. The second aim of the study is to analyze experimentally the glycan units of lamin A/C protein and the glycan changes between the phases in A549 lung carcinoma cells synchronized at G2/M and S phases of the cell cycle. For this purpose, in order to determine possible glycosylation positions of human lamin A/C protein, surface area, secondary structure, hydropathy, surface and solvent accessibility analyzes were performed and the 3D glycoprotein structure was generated. Within the scope of the experimental studies, following the synchronization of the cells, the outermost end monosaccharide units of the protein was identified by lectin blotting and glycosylated lamin A/C protein localization in the cell was examined with TEM using colloidal gold conjugated antibody and lectins. In addition, lamin A/C protein was immunoprecipitated and monosaccharide units were analyzed further by Cap-LC-ESI/MS-MS in both two phases. As a result of bioinformatics analysis, lamin A/C protein showed O-?-GlcNAcylation at Ser392, O-ß-GlcNAcylation at Ser395, and O-GalNAcylation at Thr505 residues. Considering the experimental data, lamin A/C protein was found to be contain O-GlcNAc, O-GalNAc, galactose, mannose, fucose, and sialic acid in lung carcinoma cells synchronized at G2/M and S phases. Since it was determined with bioinformatics analysis that the protein does not contain N-glycosylation position, the presence of these sugars has been associated with O-glycosylation. Based on these findings, it was demonstrated that lamin A/C is a glycoprotein showing O-GlcNAc modification and Sia, Fuc, Man and O-GalNAc modifications on core 1 type O-glycan structure. TEM studies showed that glycosylated lamin A/C protein is localized in nucleoplasm at G2/M phase and in nuclear envelope and nucleoplasm at S phase. As a result, it is determined for the first time with this thesis that the lamin A/C protein shows a modification other than phosphorylation in interphase and mitosis phases of the cell cycle. The determination of glycosylation modification in both interphase and mitosis suggests that glycosylation plays a role in the regulation of the cell cycle by forming a post-translational code with phosphorylation in assembly and disassembly of the lamin A/C protein during the cell cycle. Keywords: Lamin A/C, Glycan, Monosaccharide, A549 cells, Immunoprecipitation, CapLC-ESI/MS-MS, TEM