Antibodies Against Unusual Forms of Sialylated Glycans.

Previous studies have shown that in the blood of healthy donors (1) there are no natural antibodies against sialylated glycoproteins, which contain Neu5Acα (N-acetylneuraminic acid) as the most widespread form of human sialic acid, and (2) there is a moderate level of antibodies capable of binding unnatural oligosaccharides, where Neu5Ac is beta-linked to a typical mammalian glycan core. In the present study, we investigated antibodies against ßNeu5Ac in more detail and verified the presence of Kdn (2-keto-3-deoxy- D-glycero-D-galacto-nonulosonic acid) as a possible cause behind their appearance in humans, taking into account the expected cross-reactivity to Kdn glycans, which are found in bacterial glycoconjugates in both the α- and ß-forms. We observed the binding of peripheral blood immunoglobulins to sialyllactosamines (where "sialyl" is Kdn or neuraminic acid) in only a very limited number of donors, while the binding to monosaccharide Kdn occurred in all samples, regardless of the configuration of the glycosidic bond of the Kdn moiety. In some individuals, the binding level of some of the immunoglobulins was high. This means that bacterial Kdn glycoconjugates are very unlikely to induce antibodies to ßNeu5Ac glycans in humans. To determine the reason for the presence of these antibodies, we focused on noninfectious pathologies, as well as on a normal state in which a significant change in the immune system occurs: namely, pregnancy. As a result, we found that 2/3 of pregnant women have IgM in the blood against Neu5Acß2-3Galß1-4GlcNAcß. Moreover, IgG class antibodies against Neu5Acß2-3Galß1-4GlcNAcß and Neu5Acß2-6Galß1-4GlcNAcß were also detected in eluates from the placenta. Presumably, these antibodies block fetal antigens.

Changes in the Receptor-Binding Properties of H3N2 Viruses during Long-Term Circulation in Humans.

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 Neu5Acα2-6Galß1-4Glc (6'SL) and Neu5Acα2-6Galß1-4GlcNAc (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.

High-resolution atomic force microscopy study of hexaglycylamide epitaxial structures on graphite.

Two types of hexaglycylamide (HGA) epitaxial lamellar structures coexisting on the surface of highly oriented pyrolytic graphite (HOPG) exposed to water solutions were studied by high-resolution atomic force microscopy (AFM). Lamellae are distinguished by growth direction and by morphology. The lamellae of the first type (L1) produced by depositions from more dilute solutions are close-packed with a period of ∼5.2 nm, twice the HGA molecular length, and form highly ordered domains morphologically similar to the lamellar domains of alkanes. The less-ordered lamellae of the second type (L2) appear at intermediate and large HGA concentrations and demonstrate variable lamellar width, morphological diversity, and a tendency to merge. The interlamellar separation in the domains of close-packed L2 lamellae varies with the discrete increment ∼2.5 nm; the most frequently observed value is ∼7.5-8.0 nm corresponding to the triple HGA molecular length. The growth directions of lamellae of each type have sixfold rotational symmetry indicating epitaxy with graphite; however, the rosettes of L1 and L2 lamellae orientations are misaligned by 30°. The molecular modeling of possible HGA epitaxial packing arrangements on graphite and their classification have been conducted, and the energetically preferable structures are selected. On this basis, the structural models of the L1 and L2 lamellae are proposed explaining the experimentally observed peculiarities as follows: (1) the L1 and L2 lamellae are respectively parallel and antiparallel ß-sheets with two HGA molecules in the unit cell oriented normally to the lamellae boundaries, (2) HGA molecules in L1 and L2 lamellae have different orientations with respect to the graphite lattice, respectively along the directions <1120> and <1010>, (3) L1 lamella is the assembly of two hydrogen-bonded parallel ß-sheets oriented head-to-head, (4) L2 lamellae are assemblies of several molecular rows (antiparallel ß-sheets) cross-linked by hydrogen bonds. The AFM observations indicate that the covering of the hydrophobic graphite by the dense, closely packed, well-ordered monolayers of hydrophilic oligopeptide is possible.

[Oligoglycine surface structures: molecular dynamics simulation].

The full-atomic molecular dynamics (MD) simulation of adsorption mode for diantennary oligoglycines [H-Gly4-NH(CH2)5]2 onto graphite and mica surface is described. The resulting structure of adsorption layers is analyzed. The peptide second structure motives have been studied by both STRIDE (structural identification) and DSSP (dictionary of secondary structure of proteins) methods. The obtained results confirm the possibility of polyglycine II (PGII) structure formation in diantennary oligoglycine (DAOG) monolayers deposited onto graphite surface, which was earlier estimated based on atomic-force microscopy measurements.

[Alginate-chitosan microspheres for the specific sorption of antibodies].

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 carriers obtained are highly competitive in sorption capacity with Sepharose modified by the same oligosaccharides.

[Spontaneous and promoted association of linear oligoglycines].

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.

Polymer-bound 6' sialyl-N-acetyllactosamine protects mice infected by influenza virus.

To develop a mouse model for testing receptor attachment inhibitors of human influenza viruses, the human clinical virus isolate in MDCK cells A/NIB/23/89M (H1N1) was adapted to mice by serial passaging through mouse lungs. The adaptation enhanced the viral pathogenicity for mice, but preserved the virus receptor binding phenotype, preferential binding to 2-6-linked sialic acid receptors and low affinity for 2-3-linked receptors. Sequencing of the HA gene of the mouse-adapted virus A/NIB/23/89-MA revealed a loss of the glycosylation sites in positions 94 and 163 of HA1 and substitutions 275Asp-->Gly in HA1 and 145Asn-->Asp in HA2. The four mouse strains tested differed significantly in their sensitivity to A/NIB/23/89-MA with the sensitivity increasing in the order of BALB/cJCitMoise, C57BL/6LacSto, CBA/CaLacSto and A/SnJCitMoise strains. Testing of protective efficacy of the polyacrylamide conjugate bearing Neu5Acalpha2-6Galbeta1-4GlcNAc trisaccharide under conditions of lethal or sublethal virus infection demonstrated a strong protective effect of this preparation. In particular, aerosol treatment of mice with the polymeric attachment inhibitor on 24-110 h after infection completely prevented mortality in sensitive animals and lessened disease symptoms in more resistant mouse strains.

Polymeric inhibitor of influenza virus attachment protects mice from experimental influenza infection.

Synthetic sialic acid-containing macromolecules inhibit influenza virus attachment to target cells and suppress the virus-mediated hemagglutination and neutralize virus infectivity in cell culture. To test the protective effects of attachment inhibitors in vivo, mice were infected with mouse-adapted influenza virus A/Aichi/2/68 (H3N2) and treated with synthetic polyacrylamide-based sialylglycopolymer PAA-YDS bearing moieties of (Neu5Acalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1)2-3,6Manbeta1-4GlcNAcbeta1-4GlcNAc. Single intranasal inoculations with PAA-YDS 30 min before or 10 min after infection increased the survival of mice (P<0.01). Multiple treatments with aerosolized PAA-YDS on days 2-5 post infection also increased survival (P<0.01), alleviated disease symptoms, and decreased lesions in the mouse lungs. These data suggest that synthetic polyvalent inhibitors of virus attachment can be used for prevention and treatment of influenza.

[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.

[Polymerization of 4-nitrophenylacrylate under microwave heating conditions]. / Polimerizatsiia 4-nitrofenilakrilata v usloviiakh mikrovolnovogo nagreva.

The 2,2'-azodiisobutyronitrile-induced radical polymerization in solution of 4-nitrophenyl acrylate was performed under microwave heating at a frequency of 2.45 GHz. This approach allows the control of the molecular mass of poly(4-nitrophenyl acrylate) used in the synthesis of multivalent glycoconjugates. It was found that the polymerization of 4-nitrophenyl acrylate under microwave irradiation results in products with a narrower molecular mass distribution than at conventional heating.