**3. Sialoglycoconjugates of leukocytes as the main regulators of molecular and cellular interactions**

Glycoconjugates of leukocytes contain sialic acid as the terminal sugar and play important roles in many physiological processes. Sialic acid normally exists in the periphery of non-reducing end of the oligosaccharide chains of many glycoproteins and glycolipids. They are involved in carbohydrate-protein interactions during cell recognition, in cell–cell interactions involving functional receptors, in the binding of pathogens such as viruses, bacteria or parasites [19, 23]. Sialic acids are also implicated in the processes of activation, differentiation, survival and apoptosis of leukocytes. Sialoglycoconjugates affects cellular adhesiveness, antigenicity, action of some hormones, catalytic properties of enzymes, modulating the affinity of cell surface receptors and transmembrane signaling [19, 20]. It is obvious that sialic acids are important molecular determinants of many immune processes. To implement these functions, organisms have a range of proteins (sialospecific lectins) that recognize surface-exposed sialic acids in glycoconjugates [19].

The variety of functions indicates the importance of sialic acid in cell biology. The biology of sialic acids should be considered from the point of view of their dual function. On the one hand, sialic acid acts as biological mask agent by masking recognition sites such as receptor molecules of cell membranes. On the other hand, sialic acid plays a role as recognizable cell patterns. Sialic acids as ligands are recognized by lectins, antibodies, hormones or as receptors recognize extracellular markers in the molecular processes of cell interactions [23–25]. Activation of cells can lead to the opening of ligand-binding sites with a subsequent increase in binding affinity, lowering the cellular activation threshold, or removal of inhibitory signals [26, 27].

Sialic acids can participate directly or indirectly in multiple cellular events and overall immune response [28]. Sialic acids contribute to cells being "self" and, thus, weakens immunoreactivity. That is why they are not recognized by immune system cells or macrophage lectins. The loss of these masking monosaccharides makes the cell "foreign", activating the body's immunoreactive response. Therefore, sialic acids can be considered components of innate immune protection [29]. These acids have recently been recognized as being involved in most important phenomena of molecular and cellular interactions in immune regulation [30, 31]. In this respect, sialic acids have been associated with inflammatory diseases, malignancies, cardiovascular disease and diabetes [32].

Sialic acids are group of monosaccharides with high structural diversity, which are chemically derived from nine carbon acidic sugars – neuraminic acids. The most abundant member of the family carries an acetyl moiety linked to the amino group of fifth carbon (C5) giving the Neu5Ac. A feature of its structure is the presence of a carboxyl group near C1, which determines the negative charge of the molecule at physiological pH and characterizes it as a strong organic acid (pK 2.2). More than fifty derivatives of neuraminic acids have been found in nature. The most common sialic acid derivatives found in mammals are Neu5Ac and N-glycolylneuraminic acid (Neu5Gc), whereas in humans Neu5Ac is the dominant sialic acid [19, 23, 33].

Due to their negative charge at physiological pH and hydrophilic property sialic acids stabilize conformation of molecules, can impact protein oligomerization, the

#### *The Structure of Leukocyte Sialic Acid-Containing Membrane Glycoconjugates is a Differential… DOI: http://dx.doi.org/10.5772/intechopen.97199*

interactions of proteins with other proteins and the extracellular matrix. Sialic acids as an essential compound of all cell membranes play an important role in maintaining the structure, permeability and integrity of the cell membrane [28, 34]. Not surprisingly, sialic acid exponation is dynamic, changes during development and is altered in numerous diseases [35]. Changes in sialylation are associated with oxidative stress induced by several disorders including diabetes. It has been proven that level of sialic acid increased in plasma in condition of inflammatory processes and DM [36]. The relation between sialic acid and diabetes duration most likely follows from the association sialic acid with microvascular complications, which are well established to be related to glycemic control [37]. Therefore, sialic acid concentrations in the blood may be a useful marker of the development of diabetic complications, but there have been no many studies examining the link between sialoglycoconjugates and complications in type 1 DM.

The structural diversity of sialoglycoconjugates is due not only to the diversity of derivatives of sialic acids in their composition, but also depends on the type of glycosidic linkages (2,3, 2,6, 2,8, and 2,9) with subterminal sugars. The sialylation of oligosaccharide chains of glycoconjugates is carried out with the participation of the family of enzymes sialyltransferases (STs). About 20 STs have been characterized [19]. STs are divided in four main subfamilies, namely the ST3Gal, ST6Gal, ST6GalNAc and ST8Sia, depending on the glycosidic linkage formed and the monosaccharide acceptor recognized [35, 38]. ST3Gal, ST6Gal, ST6GalNAc and ST8Sia link Neu5Ac via its C2 to the C3, C6 positions of other carbohydrates or the C8, C9 positions of another sialic acids, generating α2,3-, α2,6-, α2,8, or α2,9-linked sialic acids, respectively [19, 39]. Sialyltransferase-mediated addition of sialic acid on glycans usually stops their further growth and modifies charge, steric hindrance, conformation and flexibility, underlying the importance of STs in shaping the structures and functions of sialoglycans [35, 40].

In the structure of leukocytes' glycans sialic acids are frequently the terminal residues of glycans and are mostly attached either by a 2,3- or 2,6-glycosidic bond to Gal or GalNAc of oligosaccharide chains [19]. The ST6Gal and ST6GalNAc, which are present in leukocytes, catalyze the transfer of Neu5Ac from CMP-Sia (cytidine-5′-monophospho-N-acetylneuraminic acid) to the C6 hydroxyl group of a terminal Gal or GalNAc residues, respectively, with the formation of α2,6-linkaged sialic acids in the oligosaccharide chains of glycans [19, 20, 38]. The ST3Gal comprises family with six members (ST3Gal I–VI). The expression of ST6Gal-I is tissue specific and regulated by multiple transcriptional promoters [41, 42]. An inducible and liver-specific promoter drive high ST6Gal-I expression during inflammation with increase in secreted ST6Gal-I in blood [43]. Activated platelets release the CMP-Sia that serves as the donor for circulating ST6Gal-I, allowing for the remodeling of the glycans of hematopoietic stem cells and multipotent progenitors (HSC/MPPs) [44]. Thus, inducible promoter is important for regulation of hematopoiesis [45]. The ST3Gal-V add a sialic acid to terminal Gal residues with the formation of α2,3 glycosidic linkage, while ST3Gal-IV sialylates Galβ1,3GalNAc terminated structures in glycoconjugates and Galβ1,4(3)GlcNAc structures found on N- and O-glycans [46]. The ST3Gal-IV and ST3Gal-VI involved in the synthesis of the sialyl Lewis<sup>X</sup> (sLeX) determinant on leukocyte E-, L- and P-selectin ligands [19, 46]. Leukocytes express a number of different selectin ligands, including E-selectin ligand-1, P-selectin glycoprotein ligand-1, CD43, CD44, β2-integrins, ets. [35, 47].

Glycosylation of cell-surface structures of leukocytes is important in the accomplishment of the immune function by these cells in organism. The membrane structures of leukocytes are decisive in the processes of extravasation, the migration of leukocytes from blood vessels into the extracellular space. In order to penetrate the vascular wall, leukocytes initially interact with the endothelium, roll over its surface, undergo dense adhesion, dissolve, and finally move through or between endothelial cells of the blood vessel [48–50]. Leukocyte chemotaxis depends on the surface sLeX and E-selectin of vascular endothelial cells. E-, L- and P-selectins are exposed by endothelial cells, leukocytes and platelets, respectively. Selectins are carbohydrate-binding proteins that recognize the sLeX structure (Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAcβ-R), capping N- and O-glycans as specific ligands [51–53]. These sialic acid-containing moieties are required for leukocyte binding to selectins on endothelial cells and their rolling [54, 55]. Combinatorial knockout of ST3Gal-IV and ST3Gal-VI that are the involved in sLeX synthesis leds to a decrease in neutrophil binding to E- and P-selectins, selectin-dependent rolling, and lymphocyte homing [46]. The selectin profile of cells can change under the influence of cytokines in case of development of inflammatory process, infection or under the influence of ROS. In tissue inflammation, cytokines stimulated endothelial cell production of E-selectin, which could recognize sLeX on the leukocyte surface and bind it, promoting leukocyte adhesion to the vascular endothelium and, subsequently, to the inflammatory tissue or locations of injury [32, 56]. Therefore, the recognition of all types of selectins is mediated with sialic acid residues [19].

In the catabolism of sialoglycans of glycoconjugate involved extracellular and intracellular sialidases, a glycoside hydrolase, that specifically hydrolyze release α-linked sialic acid residues through hydrolysis of the glycosidic bond between the acidic sugar(s) and the internal acceptor. Four different sialidases (also termed as neuraminidases – NEUs) in mammalian cells, NEU1, NEU2, NEU3 and NEU4, have been described [57]. These NEUs exhibit differences in cellular localization, substrate specificities, physiological functions and expression patterns in different tissues and physio/pathological conditions [35, 57, 58]. The NEU1 is found in the lysosome and on the cell surface and is the most highly expressed of this sialidase family [35]. The level of NEU2 is extremely low and the content of NEU3 and NEU4 are about 10% of NEU1 in tissue separately [57]. The lysosomal sialidase NEU1 initiates the degradation of sialoglycoconjugates [59]. The NEU1 is capable of hydrolyzing a wide range of glycoproteins, oligosaccharides and ganglioside near neutral pH. It exclusively acts on glycoproteins and preferentially cleaves α2,3 linkages over α2,6- or α2,8-linkages [19, 35]. In addition, NEU1 may have extralysosomal localization and focus on the periphery of activated lymphocytes. The NEU1 controls several aspects of the immune response by the desialylation of molecules, such as Toll-like receptor 4 and adhesion molecules involved in the recruitment of leukocytes to inflammatory sites [35, 57]. Desialylation of sialyl α2,3-linked Gal residues of Toll-like receptor 4 is essential for receptor activation and cellular signaling [60]. The cytosolic sialidase (NEU2) can hydrolyze sialic acids from glycoproteins and gangliosides [61]. The plasma membrane-associated sialidase (NEU3) is a key enzyme for ganglioside hydrolysis [57]. The NEU4 is localizing in the lysosomal lumen or bound to the outer mitochondrial membranes via protein–protein interactions or the ER membrane-associated. Its exhibits the highest activity with gangliosides as well as sLeX and sLea antigens [35, 57, 58]. Sialic acid is actively exfoliated from the cell surface by extracellular sialidases during leukocyte activation. This process plays an important regulatory role in cell activation and differentiation [62].

Metabolism of sialic acids includes the cooperation of enzymes that catalyze the biosynthesis, activation, transfer of sialic acids to glycoconjugates, as well as the removal and degradation of sialic acids [63]. The aberrant expression of STs and NEUs accelerates and sustains sialylation status on glycoconjugates [64]. Therefore, knowledge in this field of glycobiology allows to predict biological events in case of increase or decrease in the amount of sialoglycoconjugates on the cell surface or under conditions of modification or structural changes of these acids in certain

*The Structure of Leukocyte Sialic Acid-Containing Membrane Glycoconjugates is a Differential… DOI: http://dx.doi.org/10.5772/intechopen.97199*

types of cells. Thus, STs, NUEs and sialic acids itself represent important therapeutic targets for medicinal chemistry and biopharmaceutical industry [65, 66].
