Galina V. Pazynina

6-sulfo sialyl Lewis X is the common receptor determinant recognized by H5, H6, H7 and H9 influenza viruses of terrestrial poultry

BackgroundInfluenza A viruses of domestic birds originate from the natural reservoir in aquatic birds as a result of interspecies transmission and adaptation to new host species. We previously noticed that influenza viruses isolated from distinct orders of aquatic and terrestrial birds may differ in their fine receptor-binding specificity by recognizing the structure of the inner parts of Neu5Acα2-3Gal-terminated sialyloligosaccharide receptors. To further characterize these differences, we studied receptor-binding properties of a large panel of influenza A viruses from wild aquatic birds, poultry, pigs and horses.ResultsUsing a competitive solid-phase binding assay, we determined viral binding to polymeric conjugates of sialyloligosaccharides differing by the type of Neu5Acα-Gal linkage and by the structure of the more distant parts of the oligosaccharide chain. Influenza viruses isolated from terrestrial poultry differed from duck viruses by an enhanced binding to sulfated and/or fucosylated Neu5Acα2-3Gal-containing sialyloligosaccharides. Most of the poultry viruses tested shared a high binding affinity for the 6-sulfo sialyl Lewis X (Su-SLex). Efficient binding of poultry viruses to Su-SLex was often accompanied by their ability to bind to Neu5Acα2-6Gal-terminated (human-type) receptors. Such a dual receptor-binding specificity was demonstrated for the North American and Eurasian H7 viruses, H9N2 Eurasian poultry viruses, and H1, H3 and H9 avian-like virus isolates from pigs.ConclusionInfluenza viruses of terrestrial poultry differ from ancestral duck viruses by enhanced binding to sulfated and/or fucosylated Neu5Acα2-3Gal-terminated receptors and, occasionally, by the ability to bind to Neu5Acα2-6Gal-terminated (human-type) receptors. These findings suggest that the adaptation to receptors in poultry can enhance the potential of an avian virus for avian-to-human transmission and pandemic spread.

Simple stereoselective synthesis of α2-6 sialooligosaccharides

The sialylation approach reported by A. Khorlin et. al 30 years ago has been modified into a highly practical procedure for stereoselective α2-6 sialylation of galactopyranose 4,6-diols.

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-Lec displayed two-fold weaker binding to siglec-1 and siglec-4 than 3′SiaLec. 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-SiaLex, 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-SiaLex displayed affinity to cell-bound siglecs-1 and-5; its isomer 6-O-Su-SiaLex bound more strongly to siglecs-1,-5, and-9 than SiaLex.

Neoglycoconjugates Based on Dendrimer Poly(aminoamides)

Neoglycoconjugates containing 4, 8, 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.

Sialoside-binding macrophage lectins in phagocytosis of apoptotic bodies

Elimination of apoptotic bodies is one of the important functions of macrophages. The aim of this work was to study the role of macrophage lectins in this process. Macrophage lectins were probed with neoglycoconjugates Glyc-PAA-fluo where carbohydrate is linked to fluorescein-labeled polyacrylamide (MW 30 kD). It was shown that neoglycoconjugates containing a Neu5Acα2-3Gal fragment bound to macrophages isolated from blood of healthy donors. Besides, carbohydrate chains containing the same fragment were revealed on apoptotic bodies. Phagocytosis of apoptotic bodies by macrophages was inhibited with sialooligosaccharide ligands of siglec-5 and MAbs to siglec-5. Thus, siglec-5 expressed on macrophages could participate in phagocytosis of apoptotic bodies. In addition, the role of siglecs in engulfment of apoptotic bodies by tumor-associated macrophages was studied. The phagocytic potency of macrophages isolated from blood of breast cancer patients was lower than engulfment ability of macrophages obtained from healthy donors and depended on tumor degree. Staining of macrophages obtained from blood of tumor patients with sialylated Glyc-PAA-fluo probes was more intense than that of macrophages from healthy donors; phagocytosis of apoptotic bodies by tumor associated macrophages was inhibited by carbohydrates that are known to be ligands for siglecs.

Carbohydrate specificity of chicken and human tandem-repeat-type galectins-8 in composition of cells

The network of adhesion/growth-regulatory galectins in chicken (chicken galectin, CG) has only one tandemrepeat-type protein, CG8. Using a cell-based assay and probing galectin reactivity with a panel of fluorescent neoglycoconjugates (glycoprobes), its glycan-binding profile was determined. For internal validation, human galectin-8 (HG8) was tested. In comparison to HG8, CG8 showed a rather similar specificity: both galectins displayed high affinity to blood group ABH antigens as well as to 3′-sialylated and 3′-sulfated lactosamine chains. The most remarkable difference was found to be an ability of HG8 (but not CG8) to bind the disaccharide Galβ1-3GlcNAc (Lec) as well as branched and linear oligolactosamines. The glycan-binding profile was shown to be influenced by glycocalix of the cell, where the galectin is anchored. Particularly, glycosidase treatment of galectin-loaded cells led to the change of the profile. Thus, we suppose the involvement of cis-glycans in the interaction of cell-anchored galectins with external glycoconjugates.

Comparative lectinology: delineating glycan-specificity profiles of the chicken galectins using neoglycoconjugates in a cell assay

A major aspect of carbohydrate-dependent galectin functionality is their cross-linking capacity. Using a cell surface as biorelevant platform for galectin binding and a panel of 40 glycans as sensor part of a fluorescent polyacrylamide neoglycopolymer for profiling galectin reactivity, properties of related proteins can be comparatively analyzed. The group of the chicken galectins (CGs) is an especially suited system toward this end due to its relatively small size, compared with mammalian galectins. The experiments reveal particularly strong reactivity toward N-acetyllactosamine repeats for all tested CGs and shared reactivity of CG-1A and CG-2 to histo-blood group ABH determinants. In cross-species comparison, CG-1Bs properties closely resembled those of human galectin-1, as was the case for the galectin-2 (but not galectin-3) ortholog pair. Although binding-site architectures are rather similar, reactivity patterns can well differ.

Glycoprobes as a tool for the study of lectins expressed on tumor cells

Polyacrylamide glycoconjugates, Glyc-PAA, having various tags or labels are convenient tools for analysis of cellular lectins. Adaptation of such glycoprobes for flow cytometry allows us to reveal lectins expressed on cell surface and analyze their carbohydrate specificity as well as functionality. Localization of lectins is visualized by labeling of cells with fluorescein-tagged glycoprobes, Glyc-PAA-fluo, in combination with fluorescent microscopy techniques. Additionally, biotinylated glycoprobes can be immobilized on magnetic particles making it possible to separate a cell population according to its carbohydrate-binding profile. Here, we exemplify application of glycoprobes in the study of cellular siglecs and galectins, as well as lectin patterning of tumor cells. The specificity of sialic acid binding membrane-anchored lectins, siglecs-1, -5, -7, -8 and -9 was determined using this methodology. To study the carbohydrate-binding profile of soluble galactoside-binding lectins, galectins-1 or -3, these were loaded on (initially galectin free) Raji cells and probed using Glyc-PAA-fluo. Lessons learned from this model system allowed us to study the galectin distribution pattern of tumors: cells obtained from mice carrying mammary adenocarcinoma or lymphoma were probed with Glyc-PAA-fluo using flow cytometry. Disaccharide 6OSuLacdiNAc was shown to be the most potent probe for adenocarcinoma cells, demonstrating that 6OSuLacdiNAc-binding molecules accumulate on cell surface in a patch-wise distribution.

Synthesis of N -Acetyllactosamine-Containing Oligosaccharides, Galectin Ligands

The following spacered oligosaccharides were synthesized: GlcNAcβ1-3Galβ1-4GlcNAcβ-sp, GlcNAcβ1-6Galβ1-4GlcNAcβ-sp, GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, and Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp (sp = O(CH2)2NH2). They represent N-acetyllactosamines substituted with N-acetylglycosamine or N-acetyllalctosamine residue at O3, O6, or at both positions of galactose. Glycosylation was achieved by coupling with N-trichloroethoxycarbonyl-protected glucosamine bromide in the presence of silver triflate.

Interactions of antitumour Sialyl Lewis X liposomes with vascular endothelial cells.

Recently, we showed that tetrasaccharide selectin ligand SiaLeX provided targeted delivery of liposomes loaded in the bilayer with melphalan lipophilic prodrug to tumour endothelium followed by severe injury of tumour vessels in a Lewis lung carcinoma model. Here, we study the impact of SiaLeX ligand on the interactions of liposomes with human umbilical vein endothelial cells (HUVEC) using flow cytometry, spectrofluorimetry and confocal microscopy. Liposomes composed of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol ester of melphalan, 8:1:1, by mol, and varying percentages of lipophilic SiaLeX conjugate were labelled with BODIPY-phosphatidylcholine. The increase in SiaLeX content in liposomes led to a proportional increase in their uptake by cytokine-activated cells as opposed to non-activated HUVEC: for 10% SiaLeX liposomes, binding avidity and overall accumulation increased 14- and 6-fold, respectively. The early stages of intracellular traffic of targeted liposomes in the activated cells were monitored by co-localisation with the trackers of organelles. Endocytosis of SiaLeX liposomes occurred mostly via clathrin-independent pathways, which does not contradict the available literature data on E-selectin localisation in the plasma membrane. Using dual fluorescence labelling, with rhodamine-labelled phospholipid and calcein encapsulated at self-quenching concentrations, we found that SiaLeX liposomes undergo rapid (within minutes) internalisation by activated HUVEC accompanied by the disruption of liposomes; non-activated cells consumed a negligible dose of liposomes during at least 1.5h. Our data evidence the selective effect of SiaLeX formulations on activated endothelial cells and indicate their potential for intracellular delivery of melphalan lipophilic prodrug.