Probing sialic acid binding Ig-like lectins (siglecs) with sulfated oligosaccharides.

Soluble siglecs-1, -4, -5, -6, -7, -8, -9, and -10 were probed with polyacrylamide glycoconjugates in which: 1) the Neu5Ac residue was substituted by a sulfate group (Su); 2) glycoconjugates contained both Su and Neu5Ac; 3) sialoglycoconjugates contained a tyrosine-O-sulfate residue. It was shown that sulfate derivatives of LacNAc did not bind siglecs-1, -4, -5, -6, -7, -8, -9, and -10; binding of 6'-O-Su-LacNAc to siglec-8 was stronger than binding of 3'SiaLacNAc. The relative affinity of 3'-O-Su-TF binding to siglecs-1, -4, and -8 was similar to that of 3'SiaTF. 3'-O-Su-Le(c) displayed two-fold weaker binding to siglec-1 and siglec-4 than 3'SiaLe(c). The interaction of soluble siglecs with sulfated oligosaccharides containing sialic acid was also studied. It was shown that siglecs-1, -4, -5, -6, -7, -9, and -10 did not interact with these compounds; binding of 6-O-Su-3'SiaLacNAc and 6-O-Su-3'SiaTF to siglec-8 was weaker than that of the corresponding sulfate-free sialoside probes. Siglec-8 displayed affinity to 6'-O-Su-LacNAc and 6'-O-Su-SiaLe(x), and defucosylation of the latter compound led to an increase in the binding. Sialoside probes containing tyrosine-O-sulfate residue did not display increased affinity to siglecs-1 and -5 compared with glycoconjugates containing only sialoside. Cell-bound siglecs-1, -5, -7, and -9 did not interact with 6-O-Su-3'SiaLacNAc, whereas the sulfate-free probe 3'SiaLacNAc demonstrated binding. In contrast, the presence of sulfate in 6-O-Su-6'SiaLacNAc did not affect binding of the sialoside probe to siglecs. 6'-O-Su-SiaLe(x) displayed affinity to cell-bound siglecs-1 and -5; its isomer 6-O-Su-SiaLe(x) bound more strongly to siglecs-1, -5, and -9 than SiaLe(x).

[Modification of the target cell surface with lipophilic glycoconjugates and interaction of the modified cells with natural killer cells]. / Modifikatsiia poverkhnosti kletok s pomoshch'iu lipofil'nykh glikokon''iugatov i vzaimodeistvie modifitsirovannykh kletok s natural'nymi killerami.

An experimental model system involving the modification of carbohydrate composition of the target cell surface with neoglycolipids was developed for studying the role of surface carbohydrates of target cells in the NK-cell-mediated cytotoxicity. The polymeric glycoconjugates of the Glyc-PAA-PEA and Glyc-PAA(Flu)-PEA types (where Glyc was an oligosaccharide residue, PAA poly(acrylamide) polymer, and PEA the phosphatidylethanolamine residue, and Flu fluorescein residue) capable of incorporation into the cell membrane were synthesized. The optimum structures of neoglycoconjugates and conditions for their incorporation into K562 and Raji cell lines, which differ in their sensitivity to the NK-cell-mediated lysis were selected. The mechanism of association of glycoconjugates with the plasma cell membrane and the kinetics of their elimination from the cell surface were investigated using the fluorescent-labeled Glyc-PAA(Flu)-PEA derivatives. The spatial accessibility of the carbohydrate ligands for the interaction with human NK cells was demonstrated. The target cells modified with the Le(x) trisaccharide were shown to be more sensitive to the cytotoxic effect of human NK cells than the intact cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Uncharged P-selectin blockers.

The blocking potency of P- and L-selectin was studied for certain small molecule mannosides and their polyacrylamide (PAA, 30 kDa) conjugates in comparison to SiaLe(x) and fucoidan. Two experimental systems were used: (1) solid phase static assay based on recombinant selectins, and (2) P-selectin dependent rat peritoneal inflammation. betaMan-SC6H4NO2- p was four times more potent P-selectin inhibitor as compared to SiaLe(x). Docking of this molecule onto the P-selectin carbohydrate-binding site demonstrated that a nitro group enabled an electrostatic interaction with residue Lys 84, while the phenyl ring and the CH2 at C-6 contacted the CH2 groups of the same Lys residue. In vivo, betaMan-SC6H4NO2- p blocked experimental inflammation better than SiaLe(x), but significantly lower than fucoidan. In vitro Man-polyacrylic acid conjugates appeared to be very potent inhibitors comparable to fucoidan, uncharged Man-PAA proved rather active, comparable to SiaLe(x)-PAA both in vitro, and in vivo, whereas mannan did not display any P-selectin blocking effect.

[Neoglycoconjugates based on dendrimeric poly(aminoamides)]. / Neoglikokon''iugaty na osnove dendrimernykh poli(aminoamidov).

Neoglycoconjugates containing 4, 8, 16, 32, and 64 terminal residues of B-disaccharide (BDI) or N-acetylneuraminic acid (Neu5Ac) attached to poly(aminoamide)-type dendrimers (PAMAMs) were synthesized. The ability of BDI conjugates to bind natural xenoantibodies (anti-BDI antibodies) and the ability of Neu5Ac conjugates to inhibit the hemagglutinin-mediated adhesion of influenza virus were studied. The biological activity of PAMAM conjugates turned out to be higher than that of free carbohydrate ligands, but less than that of multivalent glycoconjugates based on other types of synthetic polymeric carriers. A conformational analysis of PAMAM matrices and resulting conjugates was performed to determine the statistical distances between carbohydrate ligands. The computations revealed the tendency of the PAMAM chains toward compaction and formation of dense globules. The process results in a decrease in the distances between the carbohydrate ligands in the conjugates and, hence, could affect the ability of glycoconjugates to efficiently bind the polyvalent carbohydrate-recognizing proteins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.

An enzyme-linked lectin assay for alpha1,3-galactosyltransferase.

UDP-Gal:Galbeta1-4GlcNAc alpha1,3-galactosyltransferase (alpha3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galalpha1-3Galbeta1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of alpha3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galbeta1-4GlcNAc (acceptor) are incubated with alpha3GalT in the presence of "cold" UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of alpha3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of alpha3GalT, no enzymatic conversion of Le(x) into GalalphaLe(x) was observed using this assay. Human alphaGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.

[Incorporation of neoglycolipids into K562 cells. Model for the study of carbohydrate dependent lysis of target cells by natural killer cells]. / Vstraivanie neoglikolipidov v kletki K562. Model' dlia izucheniia uglevodozavisimogo lizisa kletok-mishenei estestvennymi killerami.

A model system was developed to study the role of cell surface oligosaccharides in cytotoxicity mediated by natural killer (NK) cells. Polymeric glycoconjugates Glyc-PAA-PE and Glyc-PAA(Flu)-PE (where Glyc is the 3-aminopropyl Le(x) residue, PAA is the poly(N-2-hydroxyethylacrylamide) matrix, Flu is the residue of a fluorescein derivative, and PE is the phosphatidylethanolamine residue) were synthesized, and their association with the membranes of living K562 cells was studied.

[Synthesis of platelet activation factor analogs]. / Sintez analogov faktora aktivatsii trombotsitov.

Novel structural analogues of the platelet activating factor (PAF), rac-1-octadecyl-2-allyl-glycerol-3-phosphocholine and rac-1-octadecyl-2-allyl-1-thioglycerol-3-phosphocholine , have been synthesized. The phospholipids obtained showed neither PAF-agonistic nor PAF-antagonistic activity at the concentration of 10(-5) to 10(-7) M.