**5. Сhanges of carbohydrate determinants of glycoconjugate mediate the functional state of leukocyte in type 1 DM**

Leukocytes are markers of the immune homeostasis and receive signals from the microenvironment through the glycans of receptors. Lectins of certain carbohydrate specificity are ligands that selectively activate chemokine receptors. The response of cells to lectins *in vitro* makes it possible to analyze the chemical structure of the carbohydrate determinants of glycoconjugates on the membrane of leukocytes [66, 78].

Lectins WGA, SNA and MAA, which specific to sialic acids, are used to determination sialylated glycoconjugates and differentiation various types of sialic acid links with subterminal carbohydrates of glycans (SNA recognizes α2,6-links, while МАА identifies α2,3-links, **Figure 2**) [75, 79].

Alteration of the amount of sialic acids on the surface of leukocytes is an additional level of regulation of cells affinity to signaling molecules (cytokines, hormones), pathogenic microorganisms and determines the nature of cell–cell interactions [20].

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

#### **Figure 2.**

*The structure of leukocyte sialic acid-containing membrane glycans in physiological state of cells and in type 1 diabetes. Sialic acids, depending on the type of glycosidic bond in the structure of the glycan, are recognized by WGA, SNA and MAA lectins.*

The most significant changes of increasing lectin-induced aggregation of leukocytes in type 1 diabetes have been observed using lectin WGA. An increase in the degree and rate of WGA-induced aggregation of neutrophils in diabetes is a sign of increased of N-acetyl-β,D-glucosamine-containing and sialic acid-containing glycoconjugates on surface of leukocytes [80]. This indicates that synthesis of hybrid types of N-glycans is occured by activated N-acetylglucosaminyltransferase-III (GnT-III) and incomplete glycosylation of proteins and lipids [20]. As a result, glycoconjugates with terminal β,D-GlcNAc residues are exhibited on the leukocyte surface and determined high rates of WGA-induced aggregation [81].

The structural characterization of neutrophils glycoconjugates showed that cell surface N-glycans are highly sialylated, and many of their "antenna" play an important role in selectin-mediated neutrophil circulation [82]. Glycome of neutrophils is consisted mainly of complex bi- tri- and tetra-antennary N-glycans (**Figure 2**). Their antennae are predominantly terminated with Neu5Ac and LeX (Galβ1,4(Fucα1,3)GlcNAc) epitopes [83]. The ST3Gal-IV knockout results in significant reduction in the synthesis of sLeX structures in neutrophils. These cells show significant impairment in rolling and adhesion to the endothelial cells [84]. All these structural changes in the carbohydrate chains of glycoconjugates of leukocytes induce disturbances of molecular signals perception from the microenvironment, affecting interaction of leukocytes with other circulating blood cells and vascular endothelium in condition of diabetes [7, 20, 85].

Sialic acids can mask, i.e. change the structure of carbohydrate components of various specific receptors on the cell surface [19]. There is the receptor to N-formyl-methionyl-leucil-phenylalanine, C5a component of the complement system, IL-8, the receptor of granulocyte-monocyte colony-stimulating factor and the cell receptor 3 (Mac-1) among WGA-binding glycoproteins. The interaction of neutrophils with the intercellular adhesion molecule 1 (ICAM-1, CD54), which is involved in the adhesion of leukocytes to the vascular endothelium occurs via the Maс-1 receptor. On the other hand, WGA-specific receptors are involved in the

#### *Fundamentals of Glycosylation*

stimulation of respiratory burst in neutrophils by activating NADPH oxidase and followed formation of ROS [86–88].

The content of GlcNAc and Neu5Ac residues in glycans of glycoconjugates of the plasma membrane of neutrophils increases in type 1 diabetes [72]. It may be one of the main causes of nonspecific damage of tissues and cells, which are close to stimulated neutrophils. Under such conditions, neutrophils produce ROS and cause erythrocytes, platelets, fibroblasts and endotheliocytes death, inactivate enzymes, lead to changes in the structure of proteins and lipid peroxidation [6, 20].

Interaction of glycoconjugates of polymorphonuclear leukocytes with lectin SNA changes significantly under DM [80]. The level, velocity and time required for the maximum neutrophilic granulocyte aggregation in patients with type 1 DM duration of up to 5 years have been different from these indicators in patients with diabetes lasting more than 10 years. In particular, in the early stages of the disease, the degree of neutrophils aggregation, as well as the rate of SNA-induced aggregation have been four times higher than in patients with the disease over ten years [20, 80]. It is assumed that with the disease progresses, changes in leukocytes are associated with neutrophil subactivation processes that lead to the release of granule contents into the extracellular space, especially intravascularly. Degranulation leads to the lowering of cells aggregation [88, 89]. It is known that elevated glucose levels inside the cell have an inhibitory effect on a number of enzymes that are involved in the biosynthesis of the oligosaccharide chain of glycans. One of such enzymes is STs, which catalyze the attachment of sialic acid to the terminal sugar in glycan structure [19, 39]. Hyperglycemia is probably one of the factors that mediates the glycan profile violation of leukocytes in diabetes.

Decreased aggregation of neutrophils in patients with DM under the addition of MAA lectin indicates the presence sialic acids in the structure of glycoconjugates of neutrophil membranes in a small amount. These α2,3-linked sialic acids affect both the dynamic and kinetic parameters of the neutrophil aggregation process [72, 90]. The decrease in sialic acid content in the cellular glycocalyx is most often due to the enhanced desialylation of the membrane glycoconjugate. It is worth noting that sialic acids which are linked to subterminal sugars of the glycoconjugates oligosaccharide chains by the α2,3-glycoside bond are much more likely to undergo hydrolytic cleavage by sialidases than α2,6-linked residues of these sugars [90]. Cleavage of sialylated oligosaccharide fragments from glycoconjugates or exfoliation of the whole molecules of sialoglycoconjugates can be another reason of loss of sialic acids from the cell surface. However, there is often a combination of all these factors [20, 81]. Decreased α2,3-linked sialic acid on the surface of leukocytes leads to impaired perception of signals from the extracellular space, interaction with other cells, as well as numerous bacteria, protozoa and viruses. Desialylation of surface glycoconjugates of polymorphonuclear leukocytes leads to increasing of their adhesive properties, which promotes the migration of neutrophils through the vascular endothelium [91].

The interaction of glycoconjugates of mononuclear leukocytes with lectin MAA, which reacts with Neu5Acα2,3 Gal/GalNAc terminal endings glycan, have been markedly inhibited under diabetes [90, 92]. It has been found that the decrease in sialic acid content usually occurs due to increased activity of endogenous sialidases in activated T cells and monocytes [46]. This leads to increased production of cytokines by lymphocytes and interaction of monocytes with hyaluronic acid – a component of extracellular matrix [93, 94]. The NEU1 and NEU3 are expressed in monocytes in the process of their differentiation into macrophages. Desialylation of glycans on the surface of monocytes by exogenous NEU resulted in activation of ERK1/2 and p38 MARK signaling pathways and increased production of IL-6, IL-1β, MIP-1α and MIP-1β [94, 95]. Рro-inflammatory cytokines cause endothelial

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

dysfunction by increasing capillary permeability, inducing prothrombotic properties, promoting leukocyte recruitment by synthesis of adhesion molecules and chemoattractants, and play a role in macroangiopathy by promoting dyslipidemia. Thus, it is unlikely that the increased circulation of sialic acid is the result of desialylation of glycoconjugates. However, there is evidence that sialic acid is reduced in endothelium and erythrocytes in diabetes, which may be important in the pathophysiology of vascular disease [37].

Due to fact that terminal α2,3-linked sialic acids are included, in particular, in the structure of the CD45 receptor, which mediated an increasing of T cell proliferation [96], the decrease content of sialic acids in type 1 diabetes indicates a violation of this function in immunocompetent blood cells. Studies showed that the sialylation of T cell CD45 by ST6Gal-I blocks galectin-1 clustering of CD45 and resulting cell death [97]. The α2,6-sialylation of FasR blocks binding of Fasassociated adaptor molecule to the FasR death domain, thus inhibiting the formation of the death-inducing signaling complex [98].

Lectins SNA and MAA interact with CD45<sup>+</sup> leukocytes [96]. CD45 is a transmembrane glycoprotein found on T, B, NK cells, granulocytes, and monocytes. It has a cytoplasmic tail with cytosolic phosphotyrosine phosphatase activity. CD45 is the antagonist of tyrosine kinase of insulin receptor, whereas it can show high activity towards membrane-bound molecules (receptors of insulin and epidermal growth factor) [96, 99]. The increased content of sialoglycans in CD45 may cause masking of insulin receptors on organs and tissues, preventing the effect of minimal amounts of the hormone, which can still be produced in type 1 diabetes. This effect may disimprove complications during the development of the disease [96].

The α2,6-sialylation of leukocyte glycoconjugates undergoes certain changes in type 1 DM (**Figure 2**) [100]. Therefore, the quantity of sialic acids linked by α2,6-glycosidic bonds correlate with the disease duration. The content of sialoglycoconjugants on leukocytes surfaces increases for patients with the disease up to five years, while it decreases for patients with the disease duration over ten years. The pathology is accompanied by an increase of linkage places for SNA, which indicates the replacement of α2,3-linked sialic acids by α2,6-linked acids. It is likely to as a result of quantitative changes in the cells or in the enzyme activity of ST6Gal and/or ST6GalNAc [45]. The activity of α2,6 sialyltransferase decreases during the biosynthesis of O-glycans of T lymphocyte in the process of their activation. Thus, an increase in the content of α2,6-linked sialic acids of leukocyte cell surfaces along with a decline in the number of α2,3-linked sialic acids may indicate an increased sensibilization towards B lymphocyte stimulation and the inhibition of T lymphocyte activity under type 1 DM [20, 58].
