Nicolai V. Bovin

Endogenous lectins as targets for drug delivery

To minimize side effects of drugs it would be ideal to target them exclusively to those cell types which require treatment. As a means to this end prototypical cellular recognition systems pique our interest to devise biomimetic strategies. Since oligosaccharides of glycoconjugates outmatch other information-carrying biomolecules (proteins, nucleic acids) in theoretical storage capacity by far, work on the sugar code can spark off development of effective targeting devices. Conjugation of custom-made glycan epitopes to proteins or biocompatible non-immunogenic polymeric scaffolds produces neoglycoconjugates with purpose-adaptable properties. In the interplay with endogenous receptors such as lectins, suitable oligosaccharides such as histo-blood group trisaccharides as parts of neoglycoconjugates have already proven their practical applications in histopathology. Elucidation of the structure of cell lectins with currently five main families aids to tailor ligand characteristics rationally. They include the types of functional groups and their topological presentation to optimize the bimolecular binding as well as the optimal spatial clustering and spacer characteristics to exploit cooperativity. Indeed, the potent trivalent cluster glycosides designed for the C-type asialoglycoprotein receptors furnish an instructive example how to turn the theoretical guideline on ligand modification into nM-affinity. By placing emphasis on tissue lectins as targets of neoglycoconjugate-mediated drug delivery, the long-term perspective is opened to likewise test members of these families themselves for routing of therapeutic payloads, aiming at cell addressins. This review illustrates the conceivable potential which work on the sugar code with custom-made neoglycoconjugates and tissue lectins can have in store for drug delivery.

Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides

Several types of polymeric glycoconjugates,N-substituted polyacrylamides, have been synthesized by the reaction of activated polymers with ω-aminoalkylglycosides: (i) (carbohydrate-spacer)n-polyacrylamide, ‘pseudopolysaccharides’; (ii) (carbohydrate-spacer)n-phosphatidylethanolaminem-polyacrylamide, neoglycolipids, derivatives of phosphatidylethanolamine; (iii) (carbohydrate-spacer)n-biotinm-polyacrylamide, biotinylated probes; (iv) (carbohydrate-spacer)n-polyacrylamide-(macroporous glass), affinity sorbents based on macroporous glass, covalently coated with polyacrylamide. An almost quantitative yield in the conjugation reaction makes it possible to insert in the conjugate a predetermined quantity of the ligand(s).Pseudopolysaccharides proved to be a suitable form of antigen for activation of polystyrene and poly(vinyl chloride) plates (ELISA) and nitrocellulose membranes (dot blot), being advantageous over traditional neoglycoproteins. Polyvalent glycolipids insert well in biological membranes: their physical properties, particularly solubility, can be changed in a desired direction. Biotinylated derivatives were used as probes for detection and analysis of lectins.

Adaptation of Pandemic H1N1 Influenza Viruses in Mice

ABSTRACT The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouse-adapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to α2,3 together with decreasing binding to α2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals.

Ganglioside GD3 expression on target cells can modulate NK cell cytotoxicity via siglec-7-dependent and -independent mechanisms

Siglec‐7 is a sialic acid binding receptor with inhibitory potential, expressed on human NK cells and monocytes. It has an unusual binding preference for α2,8‐linked disialic acids, such as those displayed by ganglioside GD3. Here we have investigated whether siglec‐7‐GD3 interactions are able to modulate NK cell cytotoxicity. Using synthetic polyacrylamide glycoprobes, siglec‐7 was found to be masked at the NK cell surface but it could be unmasked by sialidase treatment of NK cells. GD3 synthase‐transfected P815 target cells expressed high levels of GD3 and bound strongly to recombinant siglec‐7‐Fc protein. Surprisingly, GD3 synthase‐transfected P815 cells were killed more effectively by untreated cells in a siglec‐7‐independent manner. However, following sialidase treatment of NK cells, a siglec‐7‐dependent inhibition of killing was observed. These findings have important implications for NK cell cytotoxicity against tumor cells like melanoma that express high levels of GD3 ganglioside.

Polyacrylamide-based glycoconjugates as tools in glycobiology

This review describes the synthesis, physicochemical characteristics and application for studying carbohydrate-binding proteins of polyacrylamide (PAA) type neoglycoconjugates. An approach to the synthesis of conjugates based on the interaction of activated polyacrylic acid with ω-aminoalkyl glycosides has been developed. Both the molecules of Glyc-PAA and the conjugates bearing various labels and effectors, as well as sorbents, and glycosurfaces can be designed using this method. Examples of the application of the conjugates as tools for the study of lectins, antibodies, and glycosyltransferases in glycobiology, cytochemistry and histochemistry are described along with the prospects of the further development of the presented approach in glycotechnology and medicine.

In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal(alpha)1-3Gal column.

The major role of anti-alphaGal antibodies in the hyperacute rejection of pig organs by humans and baboons has been clearly demonstrated. Spacered alpha-galactose disaccharide (Gal(alpha1)-3Gal) hapten was produced by chemical synthesis and covalently attached to a flexible, hydrophilic polymer (PAA), which in turn was covalently coupled to macroporous glass beads, forming an immunoadsorbent that is mechanically and chemically stable and can be sterilized. The extracorporeal immunoadsorption (EIA) of anti-alphaGal antibodies using this column has been investigated in vivo in 3 baboons. In Baboon 1 (which had hyperacutely rejected a pig heart transplant 4 months previously, was not splenectomized, and did not receive any pharmacologic immunosuppression) the levels of anti-alphaGal antibody and antipig IgM and IgG, as well as serum cytotoxicity, fell significantly after each of 3 EIAs but were not eliminated. Serum cytotoxicity, antipig immunoglobulin and anti-alphaGal antibody rose steeply within 24 hr of the final EIA, suggesting that the return of cytotoxicity was associated with anti-alphaGa1 antibody. In Baboons 2 and 3 (which were immunologically naive and splenectomized, and received triple drug immunosuppressive therapy) serum cytotoxicity was totally eliminated and anti-alphaGal antibody and antipig IgM and IgG levels were greatly reduced by courses of EIA. In Baboon 2, cytotoxicity and all antibody levels remained negligible for approximately one week after the final (fourth) daily EIA. In Baboon 3, cytotoxicity and antibody levels were maintained low by intermittent EIA (over a period of 13 days) for almost 3 weeks, although antipig IgM began to rebound 4 days after the final EIA. We conclude that, in an immunosuppressed, splenectomized baboon, repeated EIA using a specific alphaGal disaccharide column will reduce antipig and anti-alphaGal antibody levels and serum cytotoxicity significantly for several days. This reduction in cytotoxicity will almost certainly be sufficient to delay the hyperacute rejection of a transplanted pig organ, but further studies are required to investigate whether it will be sufficient to allow accommodation to develop.

Membrane-Associated Heparan Sulfate Proteoglycans Are Involved in the Recognition of Cellular Targets by NKp30 and NKp46

Lysis of virus-infected and tumor cells by NK cells is mediated via natural cytotoxicity receptors (NCRs). We have recently shown that the NKp44 and NKp46 NCRs, but not the NKp30, recognize viral hemagglutinins. In this study we explored the nature of the cellular ligands recognized by the NKp30 and NKp46 NCRs. We demonstrate that target cell surface heparan sulfate proteoglycans (HSPGs) are recognized by NKp30 and NKp46 and that 6-O-sulfation and N-acetylation state of the glucose building unit affect this recognition and lysis by NK cells. Tumor cells expressing cell surface heparanase, CHO cells lacking membranal heparan sulfate and glypican-1-suppressed pancreatic cancer cells manifest reduced recognition by NKp30 and NKp46 and are lysed to a lesser extent by NK cells. Our results are the first clue for the identity of the ligands for NKp30 and NKp46. Whether the ligands are particular HSPGs, unusual heparan sulfate epitopes, or a complex of HSPGs and either other protein or lipid moieties remains to be further explored.

Flow-enhanced adhesion regulated by a selectin interdomain hinge.

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin–ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.

Structural Basis for the Interaction between Human Milk Oligosaccharides and the Bacterial Lectin Pa-Iil of Pseudomonas Aeruginosa.

One of the mechanisms contributing to the protection by breast-feeding of the newborn against enteric diseases is related to the ability of human milk oligosaccharides to prevent the attachment of pathogenic bacteria to the duodenual epithelium. Indeed, a variety of fucosylated oligosaccharides, specific to human milk, form part of the innate immune system. In the present study, we demonstrate the specific blocking of PA-IIL, a fucose-binding lectin of the human pathogen Pseudomonas aeruginosa, by milk oligosaccharides. Two fucosylated epitopes, Lewis a and 3-fucosyl-lactose (Lewis x glucose analogue) bind to the lectin with dissociation constants of 2.2x10(-7) M and 3.6x10(-7) M respectively. Thermodynamic studies indicate that these interactions are dominated by enthalpy. The entropy contribution is slightly favourable when binding to fucose and to the highest-affinity ligand, Lewis a. The high-resolution X-ray structures of two complexes of PA-IIL with milk oligosaccharides allow the precise determination of the conformation of a trisaccharide and a pentasaccharide. The different types of interaction between the oligosaccharides and the protein involve not only hydrogen bonding, but also calcium- and water-bridged contacts, allowing a rationalization of the thermodynamic data. This study provides important structural information about compounds that could be of general application in new therapeutic strategies against bacterial infections.

Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.