Determination of the Glycosylation Profile of Histone Types in Embryonic Stem Cells
Project no. 564 · Status: Kapanmış · 2014
Summary
Embryonic stem cells in the early stages of development are highly active in terms of gene expression. The chromatin structure must be arranged so that all gene regions are open and gene expression is on. Glycosylation is the one of the histone modification of control mechanisms in the gene regulation and there is a paucity of information on this topic. Thus, the aim of the project is to predict the glycosylation positions of histone types in embryonic stem cells by using bioinformatics and to reveal the glycosylation profile of histones in the embryonic stem cells of the sea urchin Paracentrotus lividus, experimentally. The amino acid sequences of the histone proteins used in the bioinformatics study were obtained from the NCBI (National Center for Biotechnology Information) (https://www.ncbi.nlm.nih.gov) and UniProt (http://www.uniprot.org) databases. NetNGlyc 1.0; NetOGlyc 4.0 and YinOYang 1.2 servers were used in order to predict potential glycosylation regions/positions. ISOGlyP, NetSurfP v.1.1 and Polyview-2D SABLE were used to determine more specific features. For experimental studies, egg and sperm obtained from adult sea urchins were fertilized in filtered artificial sea water. Types of glycans in the structure of histones in 64-128 cell stage embryos were investigated by lectin blotting. In terms of bioinformatics; only H1 was found to be shown N-glycosylation sites in the sea urchin. While the mucin type O-glycosylation was determined in all histone types, O-ß-GlcNAc modification was determined only in H1 and H2A. The surface accessibility of all potential glycosylation positions in all histone types were found as accessible and secondary structure motifs were found as helical structure. H1 and H2B were high, H2A and H3 were both high and low, and H4 was low in terms of solvent accessibility. Glycosylation positions were found to be located predominantly in the N-terminal domains of proteins. In terms of experimental study, the protein bands of histones, which are small molecular weight proteins (~ 11- 22 kDa), were significantly observed between 11-22 kDa by SDS-PAGE. The best reaction in histone protein bands transferred to the membrane was observed with terminal mannose-specific GNA lectin. A reaction was also observed with ?2,3 linked sialic acid-specific MAA lectin, but no reaction were observed with SNA, PNA and DSA lectins. As a result, histone modifications are known to play an important role in the differentiation of cells. It is thought that glycosylation, as an alternative, may uncover some of the epigenetically missing points at the level of gene expression. Key words: Histone, Glycosylation, Bioinformatics, Lectin Blotting, Stem cell, Paracentrotus lividus.