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α2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosomacruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. сruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for α2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.

Pseudo-α1-acid glycoprotein with carbohydrate chain ratio typical of native form was synthesized by a previously developed original technique of quantitative transfer of α1-acid glycoprotein carbohydrate chains to other polymeric carrier. Similarly to native glycoprotein, the semisynthetic analog inhibited lymphocyte proliferation and stimulated the production of antiinflammatory cytokines by peripheral blood mononuclear leukocytes. However, it possessed no antioxidant activity and did not inhibit complement activation by the alternative pathway. The role of carbohydrate and protein components of α1-acid glycoprotein molecule in the realization of its biological effects is discussed.

Effects of various glycoforms of ±1-acid glycoprotein on the production of tumor necrosis factor and interleukin-1 are studied. After fractionation of donor blood or ascitic fluid from cancer patients 4 glycoforms were obtained, differing by affine binding to concanavalin A and by the carbon chain structure. Different glycoforms of ±1-acid glycoprotein inhibit or stimulate the production of tumor necrosis factor-α and interleukin-1β by peripheral blood mononuclears. The glycoform with the highest affinity for ConA produced the strongest stimulating effect.

The acute phase protein, alpha1-acid glycoprotein (AGP), is a normal constituent of human blood (0.2-1 mg ml(-1)) and its glycosylation and concentration in the blood change during inflammation. In this review of our recent work, we discuss the immunomodulatory properties of AGP in connection with the structure of its carbohydrate chains. AGP samples prepared from normal donor serum (nAGP), serum obtained during abortion (fAGP), serum of cancer patients (cAGP), and ascitic fluid of patients with stomach cancer (sAGP) were subjected to analysis. All the samples except for fAGP had five N-linked chains of the 'complex' type, however, the numbers of bi-, tri-, and tetra-antennary chains, as well as glycan structures terminating these chains, were different. fAGP had three N-linked chains of the lactosamine and polylactosamine type and three O-chains which were not present in AGP isolated from the other sources. The glycoforms of nAGP and sAGP that were isolated using a ConA affinity column were similar in respect to their branching, but differed in their terminal oligosaccharides. sAGP was enriched in units ending in Le(x) and asialoagalacto (GlcNAc-terminating) forms. Immunomodulatory activity of different AGP preparations was tested in vitro by measuring their effect on the proliferative response of human lymphocytes stimulated by PHA, and by determining their influence on the production of IL-1, IL-2, IL-6, and TNF in the stimulated cells. nAGP was less active compared to cancer or fetal AGP in the proliferation test, but more active in affecting cytokine production. Some AGP glycoforms had opposite immunomodulatory effects. A new approach was developed in order to clarify the role of carbohydrate chains in the biological activity of AGP. A pool of N-linked oligosaccharide chains were attached to a soluble polyacrylamide matrix. This 'pseudoglycoprotein' was similar to AGP in its molecular weight; in its relative amounts of tetra-, tri-, and bi-antennary chains; and in the content of mono-, di-, tri-, and tetra-sialylated-oligosaccharides. This pseudo-AGP displayed a similar activity to its parent AGP in the biological tests. Analytical flow cytometry of leukocyte subpopulation from human peripheral blood showed that monocytes and granulocytes but not lymphocytes were the main targets for the binding of AGP and pseudo-AGP. This binding was inhibited by synthetic glycoconjugates containing mannose or sialic acid. The binding curve data suggested that there are two monocyte and granulocyte populations. These may have different carbohydrate specificities. All the evidence provided by these studies indicate that it is the carbohydrate chains on AGP that are important in modulating the immune system and not the AGP molecule itself.

Surface-enhanced Raman scattering (SERS) spectra of sialic acid (SA) benzyl, and methyl glycosides as well as natural sialylated glycoproteins from human cells of healthy donors and tumor patients have been analyzed in view of the fact that peripheral fragments of many bioactive glycoconjugates are SA residues. SA residues can be detected by SERS at concentrations as low as 10−6M. The pattern of interaction of SA with the surface of a silver hydrosol involves the C-8 and C-9 hydroxy groups of SA. SERS spectroscopy is sensitive to changes in the content and type of branching of sialylated sugar chains in sialylglycoproteins. The differences in sialylated sugar-chain organization for α1-acid glycoproteins from healthy donors and tumor patients have been detected by means of SERS spectroscopy. The first example of the detection of SA residues for a suspension of living cells has been presented.

The mechanisms of the antiproliferative effect of α1-acid glycoprotein (AGP) isolated from the blood of healthy donors (nAGP) and from the ascitic fluid of patients with stomach cancer (aAGP) are studied. Three fractions of AGP are divided into 3 groups according to their ability to bind to concanavalin A (ConA): AGP not binding to ConA (AGP-1) and AGP weakly (AGP-2) and strongly (AGP-3) binding to ConA. It is shown that native preparation of aAGP has a more potent inhibitory effect on lymphocyte proliferation than native preparation of nAGP. The most potent inhibitory effect is exerted by AGP-3. Native preparation of aAGP does not affect the secretion of interleukin-2 (IL-2) by lymphocytes, whereas AGP-1 inhibits this process. The weakly bound fraction has a stimulatory effect both on the proliferative response and on IL-2 secretion.

Zymosan from cell wall of the yeast Saccharomyces cerevisiae was found to interact with synthetic and natural glycoconjugates; mannose-rich glycoprotein N-chains demonstrated the maximal affinity. The carbohydrate-carbohydrate nature of the interaction has been confirmed by the following data: 1) periodate treatment of the zymosan impairs the binding; 2) neither protease nor alkali treatment of the zymosan weaken the binding; 3) beta-glucan from S. cerevisiae, which is the major zymosan component, interacts with glycoconjugates similarly to zymosan. The binding is reversible, Ca2+-dependent, and cooperative; it can be dose-dependently inhibited by saccharides relative to one of the partners of the carbohydrate-carbohydrate interaction.

Pseudo-alpha(1)-acid glycoprotein with carbohydrate chain ratio typical of native form was synthesized by a previously developed original technique of quantitative transfer of alpha(1)-acid glycoprotein carbohydrate chains to other polymeric carrier. Similarly to native glycoprotein, the semisynthetic analog inhibited lymphocyte proliferation and stimulated the production of antiinflammatory cytokines by peripheral blood mononuclear leukocytes. However, it possessed no antioxidant activity and did not inhibit complement activation by the alternative pathway. The role of carbohydrate and protein components of alpha(1)-acid glycoprotein molecule in the realization of its biological effects is discussed.

α1-Acid glycoprotein (AGP) belongs to a group of acute phase proteins that play a role in the modulation of the immune system in response to stress, along with many other functions [1]. AGP has five N-glycosylation sites. The variability of glycans at each site determines the existence in blood plasma of several AGP glycoforms that differ mainly by the antennary of carbohydrate chains [1, 2], (table 1).

The acute phase protein, alpha1-acid glycoprotein (AGP), is a normal constituent of human blood (0.2-1 mg ml(-1)) and its glycosylation and concentration in the blood change during inflammation. In this review of our recent work, we discuss the immunomodulatory properties of AGP in connection with the structure of its carbohydrate chains. AGP samples prepared from normal donor serum (nAGP), serum obtained during abortion (fAGP), serum of cancer patients (cAGP), and ascitic fluid of patients with stomach cancer (sAGP) were subjected to analysis. All the samples except for fAGP had five N-linked chains of the 'complex' type, however, the numbers of bi-, tri-, and tetra-antennary chains, as well as glycan structures terminating these chains, were different. fAGP had three N-linked chains of the lactosamine and polylactosamine type and three O-chains which were not present in AGP isolated from the other sources. The glycoforms of nAGP and sAGP that were isolated using a ConA affinity column were similar in respect to their branching, but differed in their terminal oligosaccharides. sAGP was enriched in units ending in Le(x) and asialoagalacto (GlcNAc-terminating) forms. Immunomodulatory activity of different AGP preparations was tested in vitro by measuring their effect on the proliferative response of human lymphocytes stimulated by PHA, and by determining their influence on the production of IL-1, IL-2, IL-6, and TNF in the stimulated cells. nAGP was less active compared to cancer or fetal AGP in the proliferation test, but more active in affecting cytokine production. Some AGP glycoforms had opposite immunomodulatory effects. A new approach was developed in order to clarify the role of carbohydrate chains in the biological activity of AGP. A pool of N-linked oligosaccharide chains were attached to a soluble polyacrylamide matrix. This 'pseudoglycoprotein' was similar to AGP in its molecular weight; in its relative amounts of tetra-, tri-, and bi-antennary chains; and in the content of mono-, di-, tri-, and tetra-sialylated-oligosaccharides. This pseudo-AGP displayed a similar activity to its parent AGP in the biological tests. Analytical flow cytometry of leukocyte subpopulation from human peripheral blood showed that monocytes and granulocytes but not lymphocytes were the main targets for the binding of AGP and pseudo-AGP. This binding was inhibited by synthetic glycoconjugates containing mannose or sialic acid. The binding curve data suggested that there are two monocyte and granulocyte populations. These may have different carbohydrate specificities. All the evidence provided by these studies indicate that it is the carbohydrate chains on AGP that are important in modulating the immune system and not the AGP molecule itself.

Biological properties of α1-acid glycoprotein (AGP) are determined by its carbohydrate chains [1], containing sialic acids (SA) as terminal residues. SA provide specific binding of AGP in biological processes. Therefore, determination of SA location relative to a glycan chain and/or protein core, estimation of whether SA are accessible to interact or screened by other parts of the molecule, is a key to the understanding of molecular mechanism of AGP interaction with the cellular membrane.

Albumin-like glycoprotein (Gp66) with a molecular mass of 66 kDa has been isolated from human fetal tissue by size-exclusion, ion-exchange chromatography and reverse-phase HPLC. Reactivity of Gp66 with antiserum raised against the major protein components fraction of human fetal serum was observed. The N-terminal 35 amino acid residues of Gp66 were identical to human serum albumin. Meanwhile Gp66 differed from albumin by a/ the presence of 3-5 Trp residues instead of 1 according to fluorescence and UV-spectra, b/ the glycosylation pattern: bi-, tri-, and tetraantennary sialooligosaccharides of a complex type were present. Isoelectric focusing revealed 4 isoforms (pI ranging within 4.8 to 5.1) of Gp66. Gp66 (but not asialo-Gp66) was able to inhibit the cytotoxic effect of TNF against the tumor cell line L929. Inhibition of WEHI-3 and L929 tumor cells proliferation by Gp66 was similar to that of albumin.

beta-D-Glucopyranosyl-(1S and 1R)-epoxyethanes (I and II), 1-(beta-D-glucopyranosyl)-(2R and 2S)-2,3-epoxypropanes (III and IV), beta-D-glucopyranosyl isothiocyanate (V) and beta-D-galactopyranosylepoxyethane (VI) are active-site-directed irreversible inhibitors of sweet-almond beta-glucosidase B (beta-D-Glucoside glucohydrolase, EC 3.2.1.21). Formation of the covalent bond is preceded by the binding of these inhibitors in the active site of the enzyme. This is testitified by the competitive character of inhibition of beta-glucosidase component B by compounds I-VI at the early period and by the protection of the enzyme from inactivation by its competitive inhibitors D-glucose and 1,5-D-gluconolactone. Epoxides I-IV are bound covalently with componet B at a molar ratio 1 : 1 as shown with the aid of 14C-labelled inhibitors. The release of the label from modified enzyme (E-I covalent) by treatment with hydroxylamine suggests the formation of an ester bond between inhibitors I-IV and the carboxyl group of the enzyme active site. The pH dependence curve of the inactivation rate of beta-glucosidase B is of a bell-shaped form for V and of a sigmoid character for I-IV and points to the involvement of the active site groups with pKa 5.6-5.9 and 4.2-4.4.

Diastereomeric epoxy-(β- D -glucopyranosyl)ethanes and 1,2-epoxy-3-(β- D -glucopyranosyl)propanes were synthesized to study the active site of β- D -glucosidases. The mixtures of the diastereomeric epoxides of β- D -glucopyranosylethene and of 2-(β- D -glucopyranosyl)-1-propene tetraacetates were prepared by reaction of 2,3,4,6-tetra- O -acetyl-α- D -glucopyranosyl bromide with the appropriate Grignard reagents, followed by oxidation with peroxyphthalic acid in dichloromethane. Chromatographic separation of these mixtures, followed by deacetylation of each epoxide resulted in two pairs of diastereomers that inactivated irreversibly sweet-almond β- D -glucosidase.

The diastereomericβ-D-galactopyranosylepoxyethanes were synthesized for the irreversible inactivation of sweet almondβ-glucosidase B.

N -Acetylneuraminic (sialic) acid which plays a key role in process of cell recognition and interaction was studied by Raman spectroscopy. It was revealed that the strong band at 873 cm −1 arising from the glycerol fragment vibrations can be used as a Raman marker of sialic acid. In the Raman spectra of oligosaccharides and glycoproteins this band is observed at 880 cm −1 . The strong dependence of the 880 cm −1 band intensity on the sialic acid ( Sia ) content in α 1 -acid glycoprotein was shown. The data demonstrate the possibility to use Raman spectroscopy approach as a simple and non-destructive assay for the rapid registration and quantification of Sia in the glycoproteins and on the membranes of the living cells.