Search

It was previously shown that hemagglutinin residues Thr155, Glu158, and Ser228 are crucial for the recognition of Neu5Gc. In this study, we demonstrated that the ability to bind the Neu5Gc-terminated receptor is related to the amino acid 145: viruses of years 1972-1999 with Lys145 bind to the receptor, whereas viruses with Asn145 do not. Sporadic appearance and disappearance of the ability to bind Neu5Gc oligosaccharides and the absence of Neu5Gc in the composition of human glycoconjugates indicate the non-adaptive nature of this ability. It was previously shown that unlike H1N1 viruses, H3N2 viruses of years 1968-1989 did not distinguish between Neu5Acα2-6Galβ1-4Glc (6'SL) and Neu5Acα2-6Galβ1-4GlcNAc (6'SLN). H3N2 viruses isolated after 1993 have acquired the ability to distinguish between 6'SL and 6'SLN, similarly to H1N1 viruses. We found that the affinity for 6'SLN has gradually increased from 1992 to 2003. After 2003, the viruses lost the ability to bind a number of sialosides, including 6'SL, that were good receptors for earlier H3N2 viruses, and retained high affinity for 6'SLN only, which correlated with the acquisition of new glycosylation sites at positions 122, 133, and 144, as well as Glu190Asp and Gly225Asp substitutions, in hemagglutinin. These substitutions are also responsible for the receptor-binding phenotype of human H1N1 viruses. We conclude that the convergent evolution of the receptor specificity of the H1N1 and H3N2 viruses indicates that 6'SLN is the optimal natural human receptor for influenza viruses.

Influenza virus neuraminidase inhibitors (NAIs), currently used as anti-influenza drugs, can lead to the appearance of drug-resistant variants. Resistance to NAIs appears due to mutations in the active site of the neuraminidase (NA) molecule that decrease the NA enzymatic activity and sometimes in the hemagglutinin (HA) that decrease its affinity for cell receptors and, therefore, reduce the requirement for NA activity in releasing mature virions from infected cells. Using a set of sialo-oligosaccharides, we evaluated changes in the receptor-binding specificity of the HA and substrate specificity of the NA of influenza B viruses that had acquired resistance to NAIs. The oligosaccharide specificity of two pairs of field influenza B viruses, namely: i) B/Memphis/20/96 and its NAI-resistant variant, B/Memphis/20-152K/96, containing mutation R152K in the NA and 5 amino acid substitutions in the HA1, and ii) B/Hong Kong/45/2005 and its NAI-resistant variant B/Hong Kong/36/2005, containing a single R371K mutation in the NA, was evaluated. Wild type viruses bound strictly to a "human type" receptor, alpha2-6-sialo-oligosaccharide 6;SLN, but desialylated it is approximately 8 times less efficiently than the alpha2-3 sialosaccharides. Both drug-resistant viruses demonstrated the ability to bind to "avian type" receptors, alpha2-3 sialo-oligosaccharides (such as 3;SLN), whereas their affinity for 6;SLN was noticeably reduced in comparison with corresponding wild type viruses. Thus, the development of the NAI resistance in the studied influenza B viruses was accompanied by a readjustment of HA-NA oligosaccharide specificities.

Peptides are the most promising molecules for nanotechnologies. Stable derivatives suitable for supramolecular design can be synthesized even from the simplest amino acid, glycine. Oligoglycines are able to form an unusual (noncanonical) structure, so-called polyglycine II, where peptide helices 3 1 ( φ = -76.9 ° , ψ = 145.3 ° ) form a network of hydrogen bonds with six neighbors. The arrangement of linear oligoglycine in branched so-called di-, tri-, and tetraantennary peptides gives rise to a new class of molecules, which are dis- tinguished by their simplicity, on the one hand, and by ample opportunities for self-assembly into complex supramolecular structures, on the other hand. This article summarizes the reports of synthesis of symmetrical antennary oligoglycines, studies of their self-assembly in plane atomic smooth layers and vesicles, and condi- tions of the assembly and disassembly of the polyglycine II structure in an aqueous medium. Structural features and peculiarities of assembly of the similar supramolecular structures on the surface of mica are described. The possible control of this process for preparing nanopatterns is demonstrated. Prospects are discussed for using this new class of materials in nanodesign. The concept of creating a new type of antiviral agents, supramolecular compounds whose formation from a precursor is promoted by the virus itself, is proposed.

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. Fucose and sulfate substituents at this residue had negative and low effect, respectively, on saccharide binding to duck viruses. By contrast, gull viruses preferentially bound to receptors bearing fucose at N-acetylglucosamine residue, whereas chicken and mammalian viruses demonstrated increased affinity for oligosaccharides that harbored sulfo group at position 6 of (beta1-4)-linked GlcNAc. These data suggest that although all avian influenza viruses preferentially bind to Neu5Ac alpha2-3Gal-terminated receptors, the fine receptor specificity of the viruses varies depending on the avian species. Further studies are required to determine whether observed host-dependent differences in the receptor specificity of avian viruses can affect their ability to infect humans.

Adsorption of a carbohydrate on solid phase is the necessary stage of the immunosorbent assay (ELISA) and analogous methods of the study of carbohydrate-protein interaction. Usually physical adsorption on polystyrene requires a high concentration of conjugated carbohydrate and, thus, enormous consumption of it. In this study, we explored two approaches allowing more rational use of oligosaccharide (Glyc). The first of them is based on the covalent immobilization of neoglycoconjugates on the NH(2)-modified polystyrene; the second one is based on the elevated adherence of high m.w. neoglycoconjugates to polystyrene. Covalent immobilization of polyacrylamide conjugates, Glyc-PAA, provided a possibility to solve the problem, but the nonspecific binding of antibodies in ELISA proved to be unacceptably high. At the same time, the increase of the Glyc-PAA m.w. from 30 kDa to 2,000 kDa allowed a 10-20 fold decrease of its consumption, when using physical adsorption, whereas the assay background remained at the low level. The amount of 2,000 kDa Glyc-PAA that is sufficient for the coating of a standard 96-well plate corresponds to the nanomole level of oligosaccharide, this providing a possibility to use saccharides that are available in a very limited amount when studying the carbohydrate-protein interaction with solid-phase techniques.

The general principle of anti-adhesion therapy is the inhibition of microorganism adhesion to the host cell with the help of a soluble receptor analog. Despite an evident attractiveness of the concept and its long existence, the therapeutics of the ‘post-antibiotic era’ have not yet appeared. This can be explained by the contradictoriness of requirements for anti-adhesion drugs: to be efficient a drug must be multivalent, i.e. large molecule, but to obtain FDA approval it should be a small molecule. A way to overcome this contradiction is self-assembly of glycopeptides. The carbohydrate part of glycopeptide is responsible for binding with the lectin of microorganisms, whereas a simple peptide part is responsible for an association to the so-called tectomers. Depending on the structure, tectomers are formed either spontaneously or upon promotion of a microorganism. In particular, sialopeptide, which is capable of converting to a tectomer only in the presence of the influenza virus, has been obtained. Thus, the new strategy of anti-adhesion therapy can be formulated as follows: (1) identification of oligosaccharide-receptor for a particular virus (bacteria); (2) optimization of the peptide part; (3) conventional trials. The expected advantages of this strategy are the following: (i) no polymer; (ii) a virion completely covered with a tectomer, i.e. blocking is both complete and irreversible; (iii) rapid and rational lead identification and optimization; (iv) minimum side effects; (v) potential for microorganism resistance to natural receptor is lower than in the case of mimetics. Published in 2004.

Influenza A and B viruses isolated in Vero and Madin Darby canine kidney (MDCK) cells as well as in fertilised hen eggs were tested for the specificity of their neuraminidase (NA) activity. Novel glycoconjugates with variations of terminally bound sialic acid mimicking the three main receptor types for influenza viruses were synthesised. These new substrates together with the lectin from Ricinus communis were used in a solid phase microtitre assay for the detection of NA specificity. Egg or MDCK isolated virus strains tended to exhibit highest NA activity against 3'sialyl-bound sialic acid whereas Vero isolated strains favoured 6'sialyl-(N-acetyllactosamine)-bound sialic acid. Differences were more pronounced for influenza A than for influenza B strains.

To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac alpha2-6Galbeta1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acalpha2-6Galbeta1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses.

Our experience in coating of solid surfaces with glycans, mainly for obtaining routine glycoarrays based on immunological plates, is summarized. Three polystyrene coating techniques are described: direct physical adsorption, covalent binding, and immobilization using the biotin tag. Protocols for studies on anticarbohydrate antibodies are considered, with special emphasis on the application niches of different immobilization techniques as related to the specificity of each method of glycan-binding protein assay, as well as the problems of background binding and quantitative estimation of the results.

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.

Specific immunoadsorption of blood group antibodies by synthetic antigens immobilized on support matrices in the peri-transplantation period provides a promising solution to hyperacute rejection risk following ABO-incompatible transplantation. In this study, we investigated binding interactions between anti-A antibodies and synthetic blood group A trisaccharide conjugated with polyacrylamide of different molecular weights (30 and 1000 kDa). The glycopolymers were equipped with biotin tags and deposited on streptavidin- coated sensor chips. The affinity and kinetics of anti-A antibodies binding to glycoconjugates were studied using surface plasmon resonance (SPR). The high molecular weight conjugate (Atri-PAA 1000 -biotin) enhanced antibody binding capacity by two to three fold compared with the low molecular weight conjugate (Atri-PAA 30 -biotin), whereas varying the carbohydrate content in Atri-PAA 1000 -biotin (20 mol % or 50 mol %) did not affect antibody binding capacity of the glycoconjugate. The obtained results suggest that immunoadsorption devices, especially hollow fiber-based antibody filters which are limited in available surface area for antigen immobilization, may greatly benefit from the new synthetic high molecular weight polyacrylamide glycoconjugates. ' 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 91B: 845-854, 2009

Systematic studying of biological processes driven by multipoint high-cooperative carbohydrate recognition requires application of multivalent carbohydrates as tools. In this regard polyacrylamides with various pendant carbohydrate residues and labels are probably the most well advanced class of carbohydrate multimerics. Here we describe a synthetic approach to polyacrylamide-based glycoconjugates with biotin tag, with special emphasis to development of carbohydrate biosensors and arrays.

Potentially hemocompatible alginate-chitiosan microparticles and microcapsules coated with a semipermeable membrane with incorporated glycoconjugates were synthesized. The membrane acts as a barrier, which keeps the incorporated glycoconjugate from going outside but permits antibodies to penetrate inside and specifically bind to antigens, high-molecular polysaccharide conjugates. The supports obtained are highly competitive in sorption capacity with Sepharose modified by the same oligosaccharides.

Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was observed for free oligoglycines H-(Gly)m-OH (m = 3–5). At the same time, oligoglycylamides H-(Gly)m-NH2 (m = 3–5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an additional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolecular clustering of peptide chains, as expected, was observed. This means that the presence of several oligoglycine chains bound to each other in one center is not a necessary prerequisite for polyglycine II-type association.