Effect of semisynthetic analog of alpha(1)-acid glycoprotein on immunomodulatory and antiinflammatory activity of natural glycoprotein.

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.

Carbohydrate-carbohydrate interaction: zymosan and beta-glucan from Saccharomyces cerevisiae bind mannosylated glycoconjugates.

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.

Modification of the functional activity of neutrophils treated with acute phase response proteins.

The effects of alpha1-acidic glycoprotein (AGP) and its subfractions (glycoforms) with different affinity for concanavalin A on generation of H2O2 by human neutrophils exposed to stimulators of different nature, namely, galactose-specific mistletoe (Viscum album) lectin (VAA), digitonin, and N-formyl-Met-Leu-Phe, a chemotactic peptide (FMLP), were studied. Within the concentration range of 13-500 microgram/ml, AGP and its glycoforms produced dose-dependent inhibition of digitonin-induced cell responses. AGP also inhibited the VAA-induced generation of H2O2; however, this cell response was potentiated by low concentrations (50 microgram/ml) of AGP and AGP-A. FMLP induced the most consistent response of neutrophils, which changed only slightly in the presence of AGP and AGP-B; however, low concentrations of AGP-A inhibited this response. The presence of sialic acid in the terminal position of carbohydrate antennae of AGP is not necessary for its inhibitory effect on human neutrophil respiratory burst because asialo-AGP (250 microgram/ml) inhibited H2O2 generation by cells stimulated with agonists of the NADPH-oxidase system of phagocytes. In contrast to AGP, two other acute phase response proteins displaying a lectin activity (C-reactive protein and serum amyloid P component) within the concentration range of 10-100 microgram/ml produced no significant effect on H2O2 generation by stimulated neutrophils. These data suggest that AGP is an effector molecule responsible for feedback regulation of the functional activity of neutrophils.

Carbohydrate composition and immunomodulatory activity of different glycoforms of alpha1-acid glycoprotein.

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.

Albumin-like glycoprotein from human fetal tissue.

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.

Specfic irreversible inhibition of sweet-almond beta-glucosidase by some beta-glycopyranosylepoxyalkanes and beta-d-glucopyranosyl isothiocyanate.

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.