**4. Lectins as diagnostic molecular probes for determining the glycosylation profile and structural changes of glycans**

Glycocode information is read in living organisms with the help of specific compounds – lectins. Lectins are sugar-binding proteins that can specifically recognize glycans of glycoconjugates without disrupting the structure of the recognizable carbohydrate-containing ligands.

Since surface glycoconjugates have a unique structure for each cell type, they can be identified, quantified and characterised structural changes in glycans using specific lectins. Nowadays, lectins, their properties, the importance of these proteins in the life of organisms and their applying in experimental biology and medicine are the subject of research in the world's science laboratories. Lectins excluded from living objects are valuable biochemical reagents that are used in experimental cytochemistry, in the diagnosis of some diseases, and in biotechnology for isolating certain carbohydrate-containing molecules [20, 67].

Interactions of sialic acids with lectins play a leading role in many physiological and pathological processes. Therefore, sialospecific lectins are used to recognize sialic acids with specific linkages to subterminal sugars. Wheat germ lectin (WGA) specifically binds to β,DGlcNAc and Neu5Ac. The *Maackia amurensis* lectin (MAA) and *Sambucus nigra* lectin (SNA) are commonly used to recognize the α2,3-linked (Neu5Acα2,3Gal) and α2,6-linked (Neu5Acα2,6Gal/GalNAc) sialic acid residues, respectively [20, 68, 69]. Sialospecific lectins apply in lectin microarray [70], histochemistry [71], in lectin blot [72, 73], fluorescent image and flow cytometry [74] (**Figure 1**). At the same time, the combination of lectins with monoclonal antibodies can be used to obtain complete information on the antigenic repertoire of cells both in normal and in case of pathologies [73].

Blood leukocytes are similar in structural organization, and, at the same time, they differ significantly in biochemical structure. It is very important to understand the morphofunctional state of the cell is to be able to detect these differences. Numerous methods are used for this purpose [70]. Aggregatometry is one of the assays used to evaluate the functional properties of platelets, leukocytes and erythrocytes in the dynamics, monitor antiplatelet therapy, study the mechanisms of aggregation. The aggregation capacity of cells is assessed by such parameters as the degree, rate and time of aggregation [20].

The substances of protein (lectins, proteolytic enzymes, chemoactive peptides); lipid (metabolites of arachidonic acid, liposomes); carbohydrate (heparin, dextransulfates) or other nature (phorbol esters, amphotericin B, ADP, organic dyes – alcyanine blue, ruthenium red) can be inducers of aggregation. Lectins used in aggregatometry are divided into lectins-mitogens (СonA, PHA) and polyvalent lectins (WGA, SNA). Polyvalent lectins have two or more binding centers of carbohydrate determinants (carbohydrate-recognition domains) on the cell surface. The aggregation of cells by such lectins is due to the formation of intercellular molecular bridges. The ability of each subunit of lectin to bind sugars individually leads to the formation of a cross-linked structure of the aggregate. The efficiency of lectininduced aggregation is determined by the processes of clustering of lectin receptors on the cell surface [20, 75].

The aggregation capacity of leukocytes is studied to model their pre-migratory state before leaving the bloodstream, i.e. before diapedesis, or to analyze phagocytic activity. It is consider that phagocytosis involving lectin-carbohydrate interactions

#### **Figure 1.**

*Examples of uses of lectins in glycobiology. Many plant and animal lectins are multivalent. In particular, the lectin is shown with four carbohydrate binding domains. (A) Lectins bind of surface glycoconjugates of leukocytes, causes cell aggregation. (B) Histochemical analysis of surface glycans. (C) Enzyme linked lectin assay: biotinylated lectins bind to glycoconjugates on the surface of cells immobilized to the bottom of the well of a flat-bottomed plate; bound lectins are detected by antibodies to biotin with horseradish peroxidase.*

is one of the oldest evolutionary forms of this process [76]. Phagocytosis during evolution was significantly displaced by antigen–antibody interactions, but did not lose importance in the formation of a nonspecific immune response [77].
