40 years of glyco-polyacrylamide in glycobiology.

Polyacrylamide conjugates of glycans have long been widely used in many research areas of glycobiology, mainly for immobilizing glycans in solid-phase assays and as multivalent inhibitors. Pending biotin tag allows immobilizing Glyc-PAA quantitatively on any surface, and acts as a tracer for detection of carbohydrate-binding proteins. However, the scope of already realized capabilities of these probes is immeasurably richer than those listed above. This review is not so much about routine as about less common, but not less significant applications. Also, the data on the glycopolymers themselves, their molecular weight, size and polymer chain flexibility are presented, as well as the methods of synthesis, clusterisation and entropy factor in their interaction with proteins.

Are there specific antibodies against Neu5Gc epitopes in the blood of healthy individuals?

Strong discrepancies in published data on the levels and epitope specificities of antibodies against the xenogenic N-glycolyl forms of sialoglycans (Hanganutziu-Deicher Neu5Gcɑ2-3Galß1-4Glc and related antigens) in healthy donors prompted us to carry out a systematic study in this area using the printed glycan array and other methods. This article summarizes and discusses our published and previously unpublished data, as well as publicly available data from the Consortium for Functional Glycomics. As a result, we conclude that (1) the level of antibodies referred to as anti-Neu5Gc in healthy individuals is low; (2) there are antibodies that seem to interact with Neu5Gc-containing epitopes, but in fact they recognize internal fragments of Neu5Gc-containing glycans (without sialic acids), which served as antigens in the assays used and; (3) a population capable of interacting specifically with Neu5Gc (it does not bind the corresponding NAc analogs) does exist, but it binds the monosaccharide Neu5Gc better than the entire glycans containing it. In other words, in healthy donors, there are populations of antibodies capable of binding the Neu5Gc monosaccharide or the inner core -Galß1-4Glc, but very few true anti-Neu5Gcɑ2-3Galß1-4Glc antibodies, i.e., antibodies capable of specifically recognizing the entire trisaccharide.

Biantennary oligoglycines and glyco-oligoglycines self-associating in aqueous medium.

Oligoglycines designed in a star-like fashion, so-called tri- and tetraantennary molecules, were found to form highly ordered supramers in aqueous medium. The formation of these supramers occurred either spontaneously or due to the assistance of a mica surface. The driving force of the supramer formation is hydrogen bonding, the polypeptide chain conformation is related to the folding of helical polyglycine II (PG II). Tri- and tetraantennary molecules are capable of association if the antenna length reach 7 glycine (Gly) residues. Properties of similar biantennary molecules have not been investigated yet, and we compared their self-aggregating potency with similar tri- and tetraantennary analogs. Here, we synthesized oligoglycines of the general formula R-Gly n -Х-Gly n -R (X = -HN-(СН2) m -NH-, m = 2, 4, 10; n = 1-7) without pendant ligands (R = H) and with two pendant sialoligands (R = sialic acid or sialooligosaccharide). Biantennary oligoglycines formed PG II aggregates, their properties, however, differ from those of the corresponding tri- and tetraantennary oligoglycines. In particular, the tendency to aggregate starts from Gly4 motifs instead of Gly7. The antiviral activity of end-glycosylated peptides was studied, and all capable of assembling glycopeptides demonstrated an antiviral potency which was up to 50 times higher than the activity of peptide-free glycans.

Hydroxamate-directed access to ß-Kdo glycosides.

The reaction repertoire for forming transient aziridinone or azaoxyallyl cations from α-halohydroxamate is conceptually extended to design Kdo-glycosyl donors by installing the hydroxamate moiety at an anomeric centre, which is shown to be highly effective for stereoselective access to ß-Kdo glycosides. The pivotal roles of hydroxamate over amide are revealed in control experiments.

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection.

Multiplex suspension array for human anti-carbohydrate antibody profiling.

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.

Effect of pH-Regulation on the Capture of Lipopolysaccharides from E. coli EH100 by Four-Antennary Oligoglycines in Aqueous Medium.

Bacterial lipopolysaccharides (LPS) are designated as endotoxins, because they cause fever and a wide range of pathologies in humans. It is important to develop effective methodologies to detect trace quantities of LPS in aqueous systems. The present study develops a fine-tuning procedure for the entrapment of trace quantities of LPS from E. coli EH100. The capture agents are self-assemblies (tectomers) formed by synthetic four-antennary oligoglycine (C-(CH2-NH-Gly7)4, T4). Based on previously performed investigations of bulk and adsorption-layer properties of aqueous solutions containing T4 and LPS, the optimal conditions for the entrapment interactions are further fine-tuned by the pH regulation of aqueous systems. A combined investigation protocol is developed, including dynamic light scattering, profile analysis tensiometry, microscopic thin-liquid-film techniques, and transmission electron microscopy. The key results are: (1) two types of complexes between T4 and LPS are generated-amphiphilic species and "sandwich-like" hydrophilic entities; the complexes are smaller at lower pH, and larger at higher pH; (2) an optimum range of pH values is established within which the whole quantity of the LPS is entrapped by the tectomers, namely pH = 5.04-6.30. The obtained data substantiate the notion that T4 may be used for an effective capture and the removal of traces of endotoxins in aqueous systems.

Shift in oligosaccharide specificities of hemagglutinin and neuraminidase of influenza B viruses resistant to neuraminidase inhibitors.

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.

Smart Complex Fluids Based on Two-Antennary Oligoglycines.

Antennary oligoglycines are synthetic products, obtained as a result of preliminary molecular design. Equal-length antennae are built of glycine residues joined through the C end to an oligoamine branching core with an amine group at the N terminus exposed outside. The results of systematic research on the properties of aqueous solutions containing two-antennary oligoglycine with four glycine portions are reported. The central feature is the competition between amphiphilic self-assembly and formation of polyglycine II motifs. A combined procedure is developed to characterize bulk and interfacial structures and coatings. It includes registration of bulk aggregates, examination of interfacial layers at solution/air and solution/solid boundaries, drainage, and stability of liquid films. The obtained results provide new insight into the structure-property relationships in these smart fluids and give essential hints about key factors allied to possible applications in medicine, pharmaceuticals, and environmental protection.

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

BACKGROUND: Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. RESULTS: We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. CONCLUSIONS: Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients.

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2-6 vs. α2-3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers.

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. Swine virus isolates with avian-like H1 and H3 hemagglutinins displayed distinct receptor specificity by binding to both Neu5Acalpha2-6Gal- and Neu5Acalpha2-3Gal-containing receptors. These viruses, as well as egg-adapted swine and turkey viruses with a classical swine HA, differed from the related duck viruses by increased affinity to sulfated sialyloligosaccaride, Su-SiaLe(x). Except for avian-like H3 viruses, none of the studied swine viruses bound to Neu5Gc-containing sialoglycopolymers, suggesting that binding to these sialic acid species abundantly expressed in pigs may not be essential for virus replication in this host.

PEGylation of microbead surfaces reduces unspecific antibody binding in glycan-based suspension array.

Glycan-based suspension array (SGA) is an "in-house" developed multi-target immunoassay, employing commercially available fluorescent microbeads as a solid support for unique chemically synthesized glycopolymers which capture naturally occurring human anti-glycan antibodies. SGA is a sensitive and reliable tool for the high-throughput screening of anti-glycan antibody alterations characteristic for a vast number of human diseases including cancer. However, unspecific background binding, for instance binding of non-target antibodies, is a common obstacle in such immunoassays. In an attempt to reduce unspecific background binding of serum (or plasma) antibodies, we prepared glycosylated microbeads modified with linear poly(ethylene glycols) (PEGs) of different lengths. We compared several kinds of PEG modifications: (a) partial side-chain substitution of glycopolymers by PEGs of different lengths, (b) end-point addition of biotin-linked PEGs to glycopolymer-coupled beads, and (c) linking of heterobifunctional PEGs to the bead surface prior to glycopolymer immobilization. Among the various modifications investigated, the direct modification of the bead surface with linear heterobifunctional PEGs, consisting of 23- and 60PEG-units significantly reduced the background binding. The end-point addition of biotin-linked PEGs, especially in the case of PEG consisting from 50PEG-units, helped to repel non-target binding caused by endogenous biotin. We observed unspecific binding predominantly for antibodies of IgG but of IgM class. The novel design of fluorescent microbeads allows the detection of human anti-glycan antibodies with increased specificity and opens new horizons for practical application of SGA as a diagnostic tool.

Immobilization of polyacrylamide-based glycoconjugates on solid phase in immunosorbent assays.

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.

Biotinylated multivalent glycoconjugates for surface coating.

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.

High molecular weight blood group A trisaccharide-polyacrylamide glycoconjugates as synthetic blood group A antigens for anti-A antibody removal devices.

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.

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. 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.

High molecular weight neoglycoconjugates for solid phase assays.

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.

Multimeric glycotherapeutics: new paradigm.

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.