Computational analysis of the structure, glycosylation and CMP binding of human ST3GAL sialyltransferases.

Sialyltransferases (STs) are the fundamental enzymes which are related to many biological processes such as cell signalling, cellular recognition, cell-cell and host-pathogen interactions and metastasis of cancer. All STs catalyse the terminal sialic acid addition from CMP donor to the glycan units. ST3GAL family is one of the most important STs and divided into the six subfamily in mouse and humans which are ST3Gal I, ST3Gal II, ST3Gal III, ST3Gal IV, ST3Gal V, and ST3Gal VI. The members of the ST3GAL family transfer sialic acid to the terminal galactose residues of glycochains through an α2,3-linkage. There are many reports on the ST3GAL function in mammals but, there is a paucity of information about structure of human ST3GAL family. Herein, we investigated the structure, glycosylation and CMP binding site of human ST3GAL family using computational methods. We found for the first time N-glycosylation positions in ST3Gal IV and VI, mucin type glycosylation in ST3Gal III and O-GlcNAcylation in ST3Gal V and their relation with sialylmotifs. In addition, we predicted CMP binding positions of human ST3GAL enzyme family on three-dimensional structure using molecular docking and first demonstrated the sialylmotifs relation with the CMP binding positions in ST3Gal III-VI subfamilies.

In silico analysis of Pax6 protein glycosylation in vertebrates.

Pax6 is a transcription factor that involves in the formation of the eye, brain, and central nervous system in vertebrates. Due to various roles in the eye morphogenesis, Pax6 interacts with DNA and various transcription factors via post-translational modifications. Post-translational modifications of Pax6 have been studied extensively but there is a paucity of information about the glycosylation. Here, we focused on predicting the glycosylation positions of Pax6 protein in vertebrates. Also, 3D protein and glycoprotein models were generated using I-TASSER and GLYCAM servers in order to understand the effect of glycosylation on the Pax6 protein structure. We predicted N-glycosylation, mucin-type O-glycosylation, O-α-GlcNAcylation, and O-ß-GlcNAcylation positions on Pax6 protein besides O-GlcNAc modification. Moreover, we found ying-yang positions suggesting the presence of O-GlcNAcylation/phosphorylation competition in Pax6. As to 3D glycoprotein models of Pax6, Ser24, Ser65, and Ser74 residues at the PD domain of Pax6 protein was evaluated as a strong candidate for the DNA binding site. We suggest that determination of the glycosylation positions on 3D glycoprotein model will facilitate the understanding of glycosylation role on Pax6 protein interactions in transcription and intracellular activities.

Morphological and ultrastructural characterization of sea urchin immune cells.

The free circulating coelomocytes in the coelomic cavity of echinoderms are considered to be immune effectors by phagocytosis, encapsulation, cytotoxicity, and by the production of antimicrobial agents. Although echinoderms (especially sea urchin embryo) have been used as a model organisms in biology, no uniform criteria exist for classification of coelomocytes in echinoderms, and few studies have reported about the biological functions of their coelomocytes. Hence, we study the coelomocytes in the echinoid sea urchin, Paracentrotus lividus, and describe their morphological and ultrastructural features using light and transmission electron microscopes. We classify the coelomocytes of P. lividus into red spherule and colorless spherule cells, small cells, vibratile cells, and phagocytic cells; petaloid and filopodial cells. To our knowledge, this is the first report describing ultrastructural details of the coelomocytes of P. lividus.

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

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.

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin, Paracentrotus lividus, using capillary LC-ESI-MS/MS.

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac2, Neu5,7Ac2, Neu5,8Ac2, Neu5,7,9Ac3, Neu5Gc7,9Ac2, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).