Determining the monosaccharides of the sea urchin (Paracentrotus lividus) coelomocytes via the CapLC-ESI-MS/MS system and the lectin histochemistry

Abstract

The essential mechanism within immune systems is the recognition of pathogens and parasites by the immune system cells, which attach to their targets and destroy them. Glycans are fundamental macromolecular components of all cells, and are important in the vertebrate immunity. But, glycans have been investigated rarely in coelomocytes of echinoids. Hence, the aim of this study is to determine the monosaccharides which form glycan chains on the sea urchin immune system cells, coelomocytes, via analytical and lectin histochemistry methods. The study material is the coelomocytes obtained from adult sea urchin Paracentrotus lividus. In order to analyze the monosaccharides with the Capillary Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (CapLC-ESI-MS/MS) system, the samples underwent hydrolysation, reacetylation and derivatization steps. In order to determine the monosaccharides with the lectin histochemistry, the cells were incubated with fluorescein isothiocyanate (FITC) conjugated PNA, HPA, WGA-suc, WGA, and PSL lectins and then photographed with the fluorescence microscope. As a result of the CapLC-ESI-MS/MS analysis; mannose, ribose, N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, arabinose, xylose and fucose monosaccharides were detected. A peak area calculation analysis revealed the most prevalent saccharides as glucose, galactose and fucose, respectively. Lectin histochemistry came out with higher intensity emission signals obtained from the FITC-conjugated lectin WGA, which is specific to N-acetylglucosamine and sialic acid in comparison to the emission obtained from the sialic acid unspecific WGA-suc lectin. This finding indicates the existence of sialic acid within coelomocytes. Fluorescent emissions from other lectins were detected at lower levels. Determination of the monosaccharides which form glycan chains of the sea urchin coelomocytes and elucidating their similarities among other invertebrate and vertebrate systems is vital in terms of understanding the uncovered complex features of the immune systems of higher vertebrates.