Caution, these are glycan sulfates. The features of their interaction with proteins.

In studies of viruses, lectins and especially human blood anti-glycan antibodies using printed glycan array (PGA), sulfated glycans suspiciously often turn out to be the highest-level binders. The binding to sulfated glycan along with parent neutral is easily explained by the similarity of these two glycans, while the unexpected thing is the many times stronger binding. Analysis of data accumulated over almost two decades allows us to explain the observed effect by the Coulomb interaction of the sulfate residue with a positively charged amino acid that accidently appears near the binding site of the neutral glycan backbone.

Specificity of widely used lectins as probed with oligosaccharide and plant polysaccharide arrays.

Glycan-binding specificity was studied for Jacalin, RCA 120, SBA, PHA-L, PHA-E, WGA, UEA, AAL, LTL, LEL, SNA, DSA, LCA, MAH and Con A, lectins widely used in histochemistry. Oligosaccharide- and polysaccharide-based glycan arrays were applied. Expected specificity was confirmed for only 6 of the 15 lectins and the glycan binding profiles of some lectins were dramatically broader than generally accepted.

Recombinant SARS-CoV-2 S Protein Binds to Glycans of the Lactosamine Family in vitro.

Many viruses, beside binding to their main cell target, interact with other molecules that promote virus adhesion to the cell; often, these additional targets are glycans. The main receptor for SARS-CoV-2 is a peptide motif in the ACE2 protein. We studied interaction of the recombinant SARS-CoV-2 spike (S) protein with an array of glycoconjugates, including various sialylated, sulfated, and other glycans, and found that the S protein binds some (but not all) glycans of the lactosamine family.

Synthesis of blood group A and B (type 2) tetrasaccharides. A strategy with fucosylation at the last stage.

The traditionally used strategy for the synthesis of blood group A and B tetrasaccharides includes 2'-O-fucosylation of lactosamine followed by insertion of an α1-3 linked N-acetylgalactosamine or a galactose moiety. Here, we report the synthesis of 3-aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides via an alternative sequence, i.e.

Glycan-binding profile of DC-like cells.

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-"vector" it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling.

Receptor-binding properties of influenza viruses isolated from gulls.

Ducks, gulls and shorebirds represent the major hosts of influenza A viruses (IAVs) in nature, but distinctions of IAVs in different birds are not well defined. Here we characterized the receptor specificity of gull IAVs with HA subtypes H4, H6, H14, H13 and H16 using synthetic sialylglycopolymers. In contrast to duck IAVs, gull IAVs efficiently bound to fucosylated receptors and often preferred sulfated and non-sulfated receptors with Galβ1-4GlcNAc cores over the counterparts with Galβ1-3GlcNAc cores.

Glycan recognition by human blood mononuclear cells with an emphasis on dendritic cells.

Dendritic cells (DCs) play crucial roles in innate and adaptive immune response, for which reason targeting antigen to these cells is an important strategy for improvement of vaccine development. To this end, we explored recognition of DCs lectins by glycans. For selection of the glycan "vector", a library of 229 fluorescent glycoprobes was employed to assess interaction with the CD14CD16CD83 blood mononuclear cell population containing the DCs known for their importance in antigen presentation to T-lymphocytes.

Glycoprotein CA19.9-specific monoclonal antibodies recognize sialic acid-independent glycotope.

A repertoire of monoclonal antibodies was generated by immunization of mice with cancer-associated glycoprotein CA19.9, and two of them were selected as optimal capture and detecting counterparts for sandwich test system for detection of CA19.9.

Synthetic glyco-O-sulfatome for profiling of human natural antibodies.

Our understanding of biological role of glycans O-sulfation remains at the level of beginners due to microheterogeneity, lability and other difficulties of exact structural assignment. Partially, problem of functional investigations, especially determination of glycoepitope specificity of carbohydrate-binding proteins could be solved with the help of synthetic glycans of certain structure. Here we summing up our synthetic efforts in creation of synthetic O-sulfatome, and bring together all the synthesized in our group sulfated glycans, both existing in nature, yet undiscovered but biochemically licit, and completely unnatural.

Function-spacer-lipid constructs of Lewis and chimeric Lewis/ABH glycans. Synthesis and use in serological studies.

Seven lipophilic constructs containing Lewis (Le, Le, Le) or chimeric Lewis/ABH (ALe, BLe, ALe, BLe) glycans were obtained starting from corresponding oligosaccharides in form of 3-aminopropyl glycosides. ALe and BLe pentasaccharides were synthesized via [3 + 1] blockwise approach. The constructs (neoglycolipids, or FSLs) were inserted in erythrocyte membrane, and obtained "kodecytes" were used to map the immunochemical specificity of historical and contemporary monoclonal and polyclonal blood group system Lewis reagents.

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.

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.

Natural Antibodies Against Sialoglycans.

Natural antibodies, part of the innate immunity system, are produced at strictly regulated levels in normal sera without immunization and thus are part of the innate immune system. The best studied natural antibodies are those directed against blood group antigens A and B and xeno-antigens including glycolylneuraminic acid containing Hanganutziu-Deicher (HD) glycolipid. Abnormal levels of anti-glycan antibodies were found in a number of pathologies.

Comparative study of the glycan specificities of cell-bound human tandem-repeat-type galectin-4, -8 and -9.

Adhesion/growth-regulatory galectins (gals) exert their functionality by the cis/trans-cross-linking of distinct glycans after initial one-point binding. In order to define the specificity of ensuing association events leading to cross-linking, we recently established a cell-based assay using fluorescent glycoconjugates as flow cytometry probes and tested it on two human gals (gal-1 and -3). Here we present a systematic study of tandem-repeat-type gal-4, -8 and -9 loaded on Raji cells resulting in the following key insights: (i) all three gals bound to oligolactosamines; (ii) binding to ligands with Galβ1-3GlcNAc or Galβ1-3GalNAc as basic motifs was commonly better than that to canonical Galβ1-4GlcNAc; (iii) all three gals bound to 3'-O-sulfated and 3'-sialylated disaccharides mentioned above better than that to parental neutral forms and (iv) histo-blood group ABH antigens were the highest affinity ligands in both the cell and the solid-phase assay.

Profiling of serum antibodies with printed glycan array: room for data misinterpretation.

Using an example of Galβ1-3GlcNAc (Le(C)) related glycans, we here demonstrate a risk of data misinterpretation when polyclonal antibodies are probed for their glycan-binding specificities with help of a printed glycan array (PGA). Affinity isolation of antibodies from human serum using Le(C)-Sepharose or 3'-O-SuLe(C)-Sepharose in conditions of excess of the adsorbents generated identical material regardless of the affinity ligand, with the antibodies equally capable of binding to Le(C) and to 3'-O-SuLe(C) disaccharides, as well as to 3'-O-SiaLe(C) trisaccharide. More detailed profiling has shown that the isolated antibodies bind to the inner part of Galβ1-3GlcNAc disaccharide.

Chemical synthesis of 6(GlcNAc)- and 6(Gal)-O-sulfated SiaLe(X) tetrasaccharides in spacer-armed form.

Practical synthesis of tetrasaccharide sulfates, 6((GlcNAc))-O-Su-SiaLe(X)-OCH(2)CH(2)CH(2)NH(2) and 6((Gal))-O-Su-SiaLe(X)-OCH(2)CH(2)CH(2)NH(2) (Su( )SO(3)H), selectin ligands, and leu- kocyte trafficking agents is presented. Both sulfates were synthesized starting from the same precursor, protected SiaLe(x), by the conventional procedures of carbohydrate chemistry. The sulfated SiaLe(x) derivative was modified at the spacer group to give 6((Gal))-O-Su-SiaLe(x)- OCH(2)CH(2)CH(2)NH-COCH(2)CH(2)C[triple bond]CH, convenient for "click chemistry" mode conjugation with an azido carrier, particularly, for the synthesis of an immunogen.

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.

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

Background: Influenza 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 Neu5Ac alpha 2-3Gal-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.

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: a case study on galectins-1 and -3 and the impact of assay setting.

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells.

Evolution of the receptor binding phenotype of influenza A (H5) viruses.

Receptor specificity of influenza A/H5 viruses including human 2003-04 isolates was studied. All but two isolates preserved high affinity to Sia2-3Gal (avian-like) receptors. However, two isolates (February, 2003, Hong Kong) demonstrated decreased affinity to Sia2-3Gal and moderate affinity to a Sia2-6Gal (human-like) receptors.

Receptor-binding properties of swine influenza viruses isolated and propagated in MDCK cells.

To study the receptor specificities of H1 and H3 influenza viruses isolated recently from pigs, we employed the analogues of natural receptors, namely sialyloligosaccharides conjugated with polyacrylamide in biotinylated and label free forms. All Madin-Darby canine kidney (MDCK) cell-propagated viruses with human H3 or classical swine H1 hemagglutinins bound only to Neu5Acalpha2-6Galbeta1-bearing polymers, and not to Neu5Acalpha2-3Galbeta1-bearing polymers. This receptor-binding pattern is typical for human influenza viruses and it differs from the previously described receptor-binding specificity of egg-adapted swine influenza viruses.

Receptor specificity of influenza viruses from birds and mammals: new data on involvement of the inner fragments of the carbohydrate chain.

We studied receptor-binding properties of influenza virus isolates from birds and mammals using polymeric conjugates of sialooligosaccharides terminated with common Neu5Ac alpha2-3Gal beta fragment but differing by the structure of the inner part of carbohydrate chain. Viruses isolated from distinct avian species differed by their recognition of the inner part of oligosaccharide receptor. Duck viruses displayed high affinity for receptors having beta1-3 rather than beta1-4 linkage between Neu5Ac alpha2-3Gal-disaccharide and penultimate N-acetylhexosamine residue.

Specificity of human anti-NOR antibodies, a distinct species of "natural" anti-alpha-galactosyl antibodies.

Natural anti-NOR antibodies are common in human sera and agglutinate human erythrocytes of a rare NOR phenotype. The NOR phenotype-related antigens are unique neutral glycosphingolipids recognized by these antibodies and Griffonia simplicifolia IB4 isolectin (GSL-IB4). The oligosaccharide chains of NOR glycolipids are terminated by Galalpha1-4GalNAcbeta1-3Galalpha units.