IDENTIFICATION AND GENE EXPRESSION OF SIA-9-P SYNTHASE (SPS) THAT SYNTHESIZES KDN AND NEU5AC IN SEA URCHIN (Paracentrotus lividus)
Project no. 497 · Status: Kapanmış · 2018
Summary
Echinoderms (spiny-skinned animals) have a complex immune system to survive. The cellular defense systems of echinoderms are based on free-roaming blood cells (coelomocytes) as well as hematopoietic (axial) organ, in which these cells are produced, differentiated and stored. Their immune system is mainly based on recognize self from non-self. In this study, a local BLAST database was formed using the total transcriptome data of Paracentratus lividus, whose genome studies have not been completed. Then, using bioinformatics tools, the Cmah and Nans (Sas) gene sequences, previously defined in S. purpuratus, were aligned to this database in order to search for the potential homologues of these genes in P. lividus. These genes are responsible from the synthesis of the enzymes CMAH and NANS (SAS), which are in turn responsible from the biosynthesis of the sialic acids Neu5Ac and KDN, respectively. The sialic acids located as the terminal glycan found in glycoproteins and glycolipids present on the cell surfaces of vertebrates and higher invertebrates, and they posses important roles in differentiation and recognition. Afterwards, the expression levels of Nans and Cmah genes in total RNA isolates obtained from P. lividus samples (solomocyte and axial organ) were assessed by RT-PCR using the primers designed based on the bioinformatics results. Furthermore, the amount and type of sialic acids present in coelomocytes and axial organ were characterized by CapLC-ESI/MS-MS analysis. The gene expression levels were found higher in axial organ as determined by RT-PCR. This result was parallel to the results obtained by CapLC-ESI/MS-MS analysis such that the sialic acid quantity was 1.5x higher in axial organ. Finally, the transcriptome database of P. lividus was searched for the isoforms of Nans and Cmah genes to determine the expression levels of the Nans and Cmah genes at different embriyonic stages. In conclusion, KDN was detected for the first time in P. lividus and it was found that the NANS enzyme functions bifunctionally for the biosynthesis of KDN and Neu5Ac. AS Neu5Gc and its derivatives were found in higher quantities in coelomocytes, it was proposed that they play a more active role in the immune system. Most importantly, Nans and Cmah gene isoforms for P. lividus were detected using bioinformatics tools fir the first time. Keywords: Sea Urchin, Paracentrotus lividus, Sialic Acids, Neu5Ac, Neu5Gc, Sialic Acid Synthase (Sas), KDN, RT-PCR, LC-MS