Showing papers on "Sialic acid published in 2012"

Multifarious roles of sialic acids in immunity

Abstract: Sialic acids are a diverse family of monosaccharides widely expressed on all cell surfaces of vertebrates and so-called “higher” invertebrates, and on certain bacteria that interact with vertebrates. This overview surveys examples of biological roles of sialic acids in immunity, with emphasis on an evolutionary perspective. Given the breadth of the subject, the treatment of individual topics is brief. Subjects discussed include biophysical effects regulation of factor H; modulation of leukocyte trafficking via selectins; Siglecs in immune cell activation; sialic acids as ligands for microbes; impact of microbial and endogenous sialidases on immune cell responses; pathogen molecular mimicry of host sialic acids; Siglec recognition of sialylated pathogens; bacteriophage recognition of microbial sialic acids; polysialic acid modulation of immune cells; sialic acids as pathogen decoys or biological masks; modulation of immunity by sialic acid O-acetylation; sialic acids as antigens and xeno-autoantigens; antisialoglycan antibodies in reproductive incompatibility; and sialic-acid–based blood groups.

A broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza hemagglutinin

Abstract: The invention features a novel influenza antibody that specifically binds to influenza hemagglutinin and reduces or inhibits hemagglutinin binding to sialic acid. The invention also provides methods, compositions, and kits featuring the novel antibody and its use in preventing or treating influenza infection.

Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation

Abstract: One of the fastest growing fields in the pharmaceutical industry is the market for therapeutic glycoproteins. Today, these molecules play a major role in the treatment of various diseases, and include several protein classes, i.e., clotting factors, hormones, cytokines, antisera, enzymes, enzyme inhibitors, Ig-Fc-Fusion proteins, and monoclonal antibodies. Optimal glycosylation is critical for therapeutic glycoproteins, as glycans can influence their yield, immunogenicity and efficacy, which impact the costs and success of such treatments. While several mammalian cell expression systems currently used can produce therapeutic glycoproteins that are mostly decorated with human-like glycans, they can differ from human glycans by presenting two structures at the terminal and therefore most exposed position. First, natural human N-glycans are lacking the terminal Gal 1-3Gal (alpha-Gal) modification; and second, they do not contain the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). All humans spontaneously express antibodies against both of these glycan structures, risking increased immunogenicity of biotherapeutics carrying such non-human glycan epitopes. However, in striking contrast to the alpha-Gal epitope, exogenous Neu5Gc can be metabolically incorporated into human cells and presented on expressed glycoproteins in several possible epitopes. Recent work has demonstrated that this non-human sialic acid is found in widely varying amounts on biotherapeutic glycoproteins approved for treatment of various medical conditions. Neu5Gc on glycans of these medical agents likely originates from the production process involving the non-human mammalian cell lines and/or the addition of animal-derived tissue culture supplements. Further studies are needed to fully understand the impact of Neu5Gc in biotherapeutic agents. Similar concerns apply to human cells prepared for allo- or auto-transplantation, that have been grown in animal-derived tissue culture supplements.

Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome

Abstract: Fluorinated, cell-permeable analogs of sialic acid and fucose are processed by monosaccharide salvage pathways to generate sialyl- and fucosyltransferase inhibitors intracellularly. These compounds serve as important new tools to dissect the role of glycan modifications within complex biological systems.

A broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza hemagglutinin

Abstract: The invention features a novel influenza antibody that specifically binds to influenza hemagglutinin and reduces or inhibits hemagglutinin binding to sialic acid. The invention also provides methods, compositions, and kits featuring the novel antibody and its use in preventing or treating influenza infection.

Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin

Abstract: The hemagglutinin (HA) of influenza A(H3N2) virus responsible for the 1968 influenza pandemic derived from an avian virus. On introduction into humans, its receptor binding properties had changed from a preference for avian receptors (α2,3-linked sialic acid) to a preference for human receptors (α2,6-linked sialic acid). By 2001, the avidity of human H3 viruses for avian receptors had declined, and since then the affinity for human receptors has also decreased significantly. These changes in receptor binding, which correlate with increased difficulties in virus propagation in vitro and in antigenic analysis, have been assessed by virus hemagglutination of erythrocytes from different species and quantified by measuring virus binding to receptor analogs using surface biolayer interferometry. Crystal structures of HA–receptor analog complexes formed with HAs from viruses isolated in 2004 and 2005 reveal significant differences in the conformation of the 220-loop of HA1, relative to the 1968 structure, resulting in altered interactions between the HA and the receptor analog that explain the changes in receptor affinity. Site-specific mutagenesis shows the HA1 Asp-225→Asn substitution to be the key determinant of the decreased receptor binding in viruses circulating since 2005. Our results indicate that the evolution of human influenza A(H3N2) viruses since 1968 has produced a virus with a low propensity to bind human receptor analogs, and this loss of avidity correlates with the marked reduction in A(H3N2) virus disease impact in the last 10 y.

Spike Protein VP8* of Human Rotavirus Recognizes Histo-Blood Group Antigens in a Type-Specific Manner

Abstract: Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.

Sialic acid metabolism and sialyltransferases: natural functions and applications.

Abstract: Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases.

Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

Abstract: Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for α2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For α2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with α2-6- and α2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for α2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

A Phase II Study of DAS181, a Novel Host Directed Antiviral for the Treatment of Influenza Infection

Abstract: (See the editorial commentary by Ison, on pages 1806–8.) Influenza remains an infection with potential for significant morbidity and mortality. The 1918 influenza pandemic resulted in 40 million to 100 million deaths [1]. Persistent genetic changes in the virus result in highly susceptible populations and a need to develop epitope-specific vaccines yearly [2]. Additionally, immunization can be less effective in older individuals and those with compromised immune status [3, 4]. Antiviral drugs are available to treat influenza but can have reduced efficacy depending on the prevalence of viral resistance and the population being treated. Furthermore, the appearance of avian influenza (H5N1), with mortality possibly reaching 50% of those infected, supports the need for more effective treatment modalities [5]. Because all influenza viruses bind to sialic acids on respiratory epithelial cells, blockage of this interaction has the potential to decrease the magnitude of infection of all influenza variants [6]. DAS181 (Fludas) is a sialidase catalytic domain/amphiregulin glycosaminoglycan binding sequence fusion protein that cleaves both the Neu5Ac α(2,3)- and Neu5Ac α(2,6)-Gal linkages of sialic acid on host cells. DAS181 is administered as an inhalable dry powder to deliver sialidase to the pulmonary epithelium for cleavage of sialic acids, which renders the cells inaccessible to infection by virus [7]. Given the conserved nature of influenza binding to respiratory epithelium, a host-directed approach to the treatment of influenza may be applicable to the treatment of all influenza subtypes. Preclinical in vitro and in vivo studies demonstrated that DAS181 has activity against a number of seasonal influenza strains including those containing the H274Y mutation (conferring resistance to oseltamivir), highly pathogenic avian influenza strains (H5N1), and pandemic 2009 influenza A (H1N1) [8–10]. Three phase 1 studies have examined different formulations of DAS181 administered as single and multiple doses in healthy participants. In these phase 1 clinical trials, DAS181 was found to be well tolerated in all treatment groups, and no serious adverse events were observed.

Influenza virus binds its host cell using multiple dynamic interactions.

Abstract: Influenza virus belongs to a wide range of enveloped viruses The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al (1992) Biochemistry 31:9609–9621], indicating a multivalent binding mode Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus–cell binding quantitatively at the molecular level

Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic Acid receptors.

Abstract: Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both α2-6- and α2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward α2-3 sialosides. D151G NA binds the 3' sialyllactosamine (3'-SLN) and 6'-SLN sialosides with equilibrium dissociation constant (K(D)) values of 30.0 μM and 645 μM, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to α2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.

Analysis and Functional Consequences of Increased Fab-Sialylation of Intravenous Immunoglobulin (IVIG) after Lectin Fractionation

Abstract: It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab’)2 and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

The role of sialic acid as a modulator of the anti-inflammatory activity of IgG.

Abstract: Immunoglobulin G (IgG) molecules can have two completely opposing activities. They can be very potent pro-inflammatory mediators on the one hand, directing the effector functions of the innate immune system towards infected cells, tumor cells or healthy tissues in the case of autoimmune diseases. On the other hand, a mixture of IgG molecules purified from the blood of ten thousands of healthy donors is used as an anti-inflammatory treatment for many autoimmune diseases since several decades. It has become evident only recently that certain residues in the sugar moiety attached to the IgG constant fragment can dramatically alter the pro- and anti-inflammatory activities of IgG. This review will focus on sialic acid residues as a modulator of the anti-inflammatory activity and provide an overview of situations where serum IgG glycosylation and sialylation is altered and which molecular and cellular pathways may be involved in this immunomodulatory pathway.

Tumour suppressor p16INK4a – anoikis‐favouring decrease in N/O‐glycan/cell surface sialylation by down‐regulation of enzymes in sialic acid biosynthesis in tandem in a pancreatic carcinoma model

Abstract: Tumour suppressor p16INK4a is known to exert cell-cycle control via cyclin-dependent kinases. An emerging aspect of its functionality is the orchestrated modulation of N/O-glycosylation and galectin expression to induce anoikis in human Capan-1 pancreatic carcinoma cells. Using chemoselective N/O-glycan enrichment technology (glycoblotting) and product characterization, we first verified a substantial decrease in sialylation. Tests combining genetic (i.e. transfection with α2,6-sialyltransferase-specific cDNA) or metabolic (i.e. medium supplementation with N-acetylmannosamine to track down a bottleneck in sialic acid biosynthesis) engineering with cytofluorometric analysis of lectin binding indicated a role of limited substrate availability, especially for α2,6-sialylation, which switches off reactivity for anoikis-triggering homodimeric galectin-1. Quantitative MS analysis of protein level changes confirmed an enhanced galectin-1 presence along with an influence on glycosyltransferases (β1,4-galactosyltransferase-IV, α2,3-sialyltransferase-I) and detected p16INK4a-dependent down-regulation of two enzymes in the biosynthesis pathway for sialic acid [i.e. the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) and N-acetylneuraminic acid 9-phosphate synthase] (P < 0.001). By contrast, quantitative assessment for the presence of nuclear CMP-N-acetylneuraminic acid synthase (which is responsible for providing the donor for enzymatic sialylation that also acts as feedback inhibitor of the epimerase activity of GNE) revealed a trend for an increase. Partial restoration of sialylation in GNE-transfected cells supports the implied role of sialic acid availability for the glycophenotype. Fittingly, the extent of anoikis was reduced in double-transfected (p16INK4a/GNE) cells. Thus, a second means of modulating cell reactivity to the growth effector galectin-1 is established in addition to the common route of altering α2,6-sialyltransferase expression: regulating enzymes of the pathway for sialic acid biosynthesis.

Sialic Acid on the Neuronal Glycocalyx Prevents Complement C1 Binding and Complement Receptor-3-Mediated Removal by Microglia

Abstract: Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures. Neuronal processes are marked by complement C1 before they are removed in development or during disease processes. Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function. Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia. The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx. Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells. The removal of the desialylated neurites was mediated via the complement receptor-3 (CR3; CD11b/CD18). Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.

IVIg-mediated amelioration of ITP in mice is dependent on sialic acid and SIGNR1.

Abstract: Intravenous immunoglobulin G (IVIg) therapy is widely used to treat autoimmune and inflammatory diseases. Recent evidence suggests that in mice, splenic resident cells might be important for the anti-inflammatory activity of IVIg in a model of serum transfer arthritis. Splenectomized human immunothrombocytopenia (ITP) patients, however, still respond to IVIg therapy. To investigate whether the requirement of the spleen is essential for mouse ITP, we used a passive model of induced ITP and demonstrated that IVIg activity was functional in splenectomized animals. Further analysis showed that the IVIg-mediated amelioration of platelet phagocytosis was fully dependent on terminal sialic acid residues in the IVIg preparation and could be blocked with a specific ICAM3 grabbing nonintegrin-related 1 (SIGNR1) specific antibody. These results suggest that, similar to the human system, a spleen-independent but sialic acid- and SIGNR1-dependent pathway is responsible for IVIg-mediated suppression of autoantibody-dependent platelet depletion in mice.

Biosynthesis of the major brain gangliosides GD1a and GT1b

Abstract: Gangliosides—sialylated glycosphingolipids—are the major glycoconjugates of nerve cells. The same four structures—GM1, GD1a, GD1b and GT1b—comprise the great majority of gangliosides in mammalian brains. They share a common tetrasaccharide core (Galβ1–3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer) with one or two sialic acids on the internal galactose and zero (GM1 and GD1b) or one (GD1a and GT1b) α2–3-linked sialic acid on the terminal galactose. Whereas the genes responsible for the sialylation of the internal galactose are known, those responsible for terminal sialylation have not been established in vivo. We report that St3gal2 and St3gal3 are responsible for nearly all the terminal sialylation of brain gangliosides in the mouse. When brain ganglioside expression was analyzed in adult St3gal1-, St3gal2-, St3gal3- and St3gal4-null mice, only St3gal2-null mice differed significantly from wild type, expressing half the normal amount of GD1a and GT1b. St3gal1/2-double-null mice were no different than St3gal2-single-null mice; however, St3gal2/3-double-null mice were >95% depleted in gangliosides GD1a and GT1b. Total ganglioside expression (lipid-bound sialic acid) in the brains of St3gal2/3-double-null mice was equivalent to that in wild-type mice, whereas total protein sialylation was reduced by half. St3gal2/3-double-null mice were small, weak and short lived. They were half the weight of wild-type mice at weaning and displayed early hindlimb dysreflexia. We conclude that the St3gal2 and St3gal3 gene products (ST3Gal-II and ST3Gal-III sialyltransferases) are largely responsible for ganglioside terminal α2-3 sialylation in the brain, synthesizing the major brain gangliosides GD1a and GT1b.

Receptor-binding Profiles of H7 Subtype Influenza Viruses in Different Host Species

Abstract: Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4 (6-O-HSO3)GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO3)GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry.

Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus.

Abstract: Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4- O -acetylated sialic acid (Neu4,5Ac 2 ). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs.

Recognition of sialylated poly-N-acetyllactosamine chains on N- and O-linked glycans by human and avian influenza A virus hemagglutinins.

Abstract: The initial stages of influenza A virus infection are mediated by the binding of the viral hemagglutinin (HA) to sialylated glycan receptors on host epithelial cells.[1] The specificity of the HA is believed a key determinant of viral host range.[2] While all 16 influenza HA subtypes are found in avian viruses, only three are found in viruses adapted to humans (H1, H2 and H3), each resulting in a major pandemic. HAs from avian and human viruses are characterized by their preference for α2–3 and α2–6 linked sialic acids, respectively. Studies now suggest that other elements of sialoglycan sequence are also important factors of HA specificity that contribute to the species barrier.[3] Recently, human and swine respiratory epithelial cells were shown to express sialylated N-glycans with extended poly-N-acetyllactosamine (poly-LacNAc) chains.[4] Poly-LacNAc chains are Galβ1–4GlcNAcβ1–3 tandem repeats that extend N- and O-linked glycans of glycoproteins and contribute to the biology mediated by glycan binding proteins.[5] Sasisekharan and coworkers have suggested that human HAs bind preferentially to extended α2–6 sialosides and may be critically important for viral adaptation to humans.[4a, 6] Studies on the preference of influenza HAs for extended glycans have employed synthetic sialosides that are linear terminal fragments of natural N- and O-linked glycans, which differ in their core structure and are often branched.[4a, 7] To more fully address the influence of poly-LacNAc chains on HA specificity in the context of natural glycans, we have synthesized a series of sialylated poly-LacNAc structures on intact O- (4–9, 16–21) and N-linked glycan (10–12, 22–24) cores (Figure 1). These sialosides were incorporated into a custom glycan microarray alongside the linear terminal fragments (1–3, 13–15) for analysis of specificities of human and avian influenza HAs. Figure 1 Structures of sialylated poly-LacNAc linear (L) fragments (1–3, 13–15) and the same sequences elaborated on O-linked (O2, O3, O4) (4–9, 16–21) and N-linked (N) glycan cores (10–12, 22–24). Several groups have reported chemical and chemo-enzymatic syntheses of poly-LacNAc structures.[8] For synthesis of extended natural N- and O-linked glycans, our strategy relied on enzymatic elaboration of advanced core structures. The α2–3 a n d α 2–6 sialoside targets comprised O-linked (Cores 2–4) and N-linked cores with up to two and three LacNAc repeats, respectively. Representative syntheses for N- (11 and 23) and Core-2 O-linked glycans (5 and 17) are described in Scheme 1. Key LacNAc extensions were attained by alternating reactions using recombinant Helicobacter pylori β1–3-N-acetylglucosaminyltransferase (β1–3GlcNAcT)[8b] and the bacterial β1–4-galactosyltransferase/UDP-4′-Gal-epimerase fusion protein (GalT-GalE).[9] Reaction of N-glycan 25 with UDP-GlcNAc (4 eq.) using β1–3GlcNAcT followed by treatment with UDP-Glc (4 eq.) and GalT-GalE allowed efficient construction of LacNAc on both antennae affording 27 (Scheme 1a). Divergent sialylation of 27 using rat α2–3-sialyltransferase (rST3Gal-III) or human α2–6-sialyltransferase (hST6Gal-I), with CMP-Neu5Ac gave the desired α2–3 11 and α2–6 23 products, respectively. The synthesis of O-linked cores 3–4 and the tri-LacNAc N-linked glycans were conducted following similar conditions (Schemes S1–S6 in the Supporting information). Scheme 1 a. Enzymatic transformations of 25 to 11 and 23 a) β1–3GlcNAcT, UDP-GlcNAc; b) GalT-GalE, UDP-Glc; c) rST3Gal-III, CMP-Neu5Ac; d) hST6Gal-I, CMP-Neu5Ac. Core-2 O-linked glycans are commonly extended with poly-LacNAc off the β1–6 branch. Initial galactosylation of 28 added Galβ1–4 to GlcNAc giving 29 (Scheme 1b). As both branches of 29 present terminal Gal, two sites were potentially reactive for GlcNAc addition. Regioselective reaction on the β1–6 branch was anticipated as β1–3GlcNAcT demonstrates higher selectivity for Galβ1–4GlcNAc substrates. Thus, under controlled conditions using UDP-GlcNAc (2 eq.), selective elongation of the β1–6 branch was achieved to afford 30.[10] NMR and MS analysis confirmed addition of a single GlcNAc unit. The asialo di-LacNAc structure 31 was prepared by reaction of 30 with UDP-Glc catalyzed by GalT-GalE. Finally, selective sialylation of 31 was performed with either rST3Gal-III or hST6Gal-I and CMP-Neu5Ac (2 eq). Both sialyltransferases show preference for Galβ1–4GlcNAc substrates and gave 5 and 17, respectively. The mono-sialylated products were confirmed by NMR and MS analysis. The 24 glycans in the sialoside library (Figure 1) contain either the terminal Neu5Acα2–3Gal (1–12) or Neu5Acα2–6Gal (13–24) sequence. A glycan microarray was constructed from this library to study the binding properties of influenza A virus HA.[7a, 11] The aglycone of each sialoside was equipped with a free amine for direct printing on N-hydroxy succinimde-activated slides (Figure S1 in the Supporting information). Recombinant HAs from selected avian and human influenza A viruses were then screened to assess the effects on HA binding of both length and presentation of sialylated poly-LacNAc. As expected, the avian HAs preferentially recognized α2–3-linked sialosides (Figure 2 and Figure S2 in the Supporting information). However, while H4 (A/duck/Czech/1/56) bound strongly to nearly all α2–3 structures, other avian HAs showed more selective binding patterns. For instance, H3 (A/duck/Ukr/1/63), a progenitor of the 1968 Hong Kong pandemic,[12] only bound the linear glycans (2, 3), and the O- (8) and N-linked (10) glycans. Remarkably, all avian HAs, including H5 (A/Vietnam/1203/04), a highly pathogenic human isolate of the bird flu,[13] showed strong preference for short N-linked structures, binding strongly to 10, and reduced or no binding to the longer glycans (11, 12). Figure 2 Glycan microarray binding analyses as measured by fluorescence intensity for avian and human influenza A recombinant hemagglutinins. All HAs were evaluated at 15 µg/ml except for A/SC and A/Beijing, which were evaluated at 150 µg/ml. See ... Although human HAs demonstrated classic preference for α2–6-sialosides, they exhibited varied fine specificity for the extended N- and O-linked glycans (Figure 2 and Figure S2 in the Supporting Information). As previously reported, the human HAs bound best to the linear sialosides with di- and tri-LacNAc extensions (13–15).[4] Significantly, however, the same sequences were not uniformly recognized when presented on N- and O-linked glycan cores. For instance, while the H1 (A/SC/1/18) and the H2 (A/Japan/305/57) HAs bound strongly to the linear sialoside with the di-LacNAc extension (14), they bound poorly to the same sequence presented on Core 3 (19) and Core 4 (21) glycans. Surprisingly, these same two HAs exhibited strong binding to N-glycans with the di-LacNAc sequence (23), but dramatically reduced binding to the same sequence with the tri-LacNAc repeat (24). In summary, we have synthesized a panel of novel glycans containing sialylated poly-LacNAc on intact N- and O-linked glycan cores as candidates of the natural glycan receptors of influenza viruses. While all avian and human virus HAs retained their basic α2–3 and α2–6 linkage specificity, respectively, the N- and O-linked glycan cores differentially impacted the ability of individual HAs to recognize the sialic acid as a receptor. The lack of a consistent recognition pattern for human HAs suggests that the fine specificity of the virus for receptor(s) may drift under antigenic selective pressure, while retaining the ability to bind to a subset of α2–6-sialosides sufficient to mediate infection and transmission. It should also be noted that the branched N-linked and Core 4 O-linked glycans produced with our synthetic strategy are symmetric di-sialylated glycans. However, glycans extended on a single branch also occur in nature.[4a, 4b] Thus, it will also be of interest to investigate the role of asymmetric glycans on influenza receptor biology.

Influenza A virus entry into cells lacking sialylated N-glycans

Abstract: Influenza A virus (IAV) enters host cells after attachment of its hemagglutinin (HA) to surface-exposed sialic acid. Sialylated N-linked glycans have been reported to be essential for IAV entry [Chu VC, Whittaker GR (2004) Proc Natl Acad Sci USA 102:18153–18158], thereby implicating the requirement for proteinaceous receptors in IAV entry. Here we show, using different N-acetylglucosaminyl transferase 1 (GnT1)-deficient cells, that N-linked sialosides can mediate, but are not required for, entry of IAV. Entry into GnT1-deficient cells was fully dependent on sialic acid. Although macropinocytic entry appeared to be affected by the absence of sialylated N-glycans, dynamin-dependent entry was not affected at all. However, binding of HA to GnT1-deficient cells and subsequent entry of IAV were reduced by the presence of serum, which could be reversed by back-transfection of a GnT1-encoding plasmid. The inhibitory effect of serum was significantly increased by inhibition of the viral receptor-destroying enzyme neuraminidase (NA). Our results indicate that decoy receptors on soluble serum factors compete with cell surface receptors for binding to HA in the absence of sialylated N-glycans at the cell surface. This competition is particularly disturbed by the additional presence of NA inhibitors, resulting in strongly reduced IAV entry. Our results indicate that the balance between HA and NA is important not only for virion release, but also for entry into cells.

Entry of Influenza A Virus with a α2,6-Linked Sialic Acid Binding Preference Requires Host Fibronectin

Abstract: The receptor binding specificity of influenza A virus is one of the major determinants of viral tropism and host specificity. In general, avian viral hemagglutinin prefers to bind to α2,3-linked sialic acid, whereas the human viral hemagglutinin prefers to bind to α2,6-linked sialic acid. Here, we demonstrate that host fibronectin protein plays an important role in the life cycle of some influenza A viruses. Treating cells with anti-fibronectin antibodies or fibronectin-specific small interfering RNA can inhibit the virus replication of human H1N1 influenza A viruses. Strikingly, these inhibitory effects cannot be observed in cells infected with H5N1 viruses. By using reverse genetics techniques, we observed that the receptor binding specificity, but not the origin of the hemagglutinin subtype, is responsible for this differential inhibitory effect. Changing the binding preference of hemagglutinin from α2,6-linked sialic acid to α2,3-linked sialic acid can make the virus resistant to the anti-fibronectin antibody treatment and vice versa. Our further characterizations indicate that anti-fibronectin antibody acts on the early phase of viral replication cycle, but it has no effect on the initial binding of influenza A virus to cell surface. Our subsequent investigations further show that anti-fibronectin antibody can block the postattachment entry of influenza virus. Overall, these results indicate that the sialic acid binding preference of influenza viral hemagglutinin can modulate the preferences of viral entry pathways, suggesting that there are subtle differences between the virus entries of human and avian influenza viruses.

Terminal sialic acids are an important determinant of pulmonary endothelial barrier integrity

Abstract: The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Preliminary studies have demonstrated structural differences in the glycocalyx of pulmonary artery endothelial cells compared with pulmonary microvascular endothelial cells. Herein we begin to probe in more detail structural and functional attributes of endothelial cell-surface carbohydrates. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. We observed that, although pulmonary microvascular endothelial cells express similar amounts of total sialic acids as pulmonary artery endothelial cells, the nature of the sialic acid linkages differs between the two cell types such that pulmonary artery endothelial cells express both α(2,3)- and α(2,6)-linked sialic acids on the surface (i.e., surficially), whereas microvascular endothelial cells principally express α(2,3)-linked sialic acids. To determine whether sialic acids play a role in endothelial barrier function, cells were treated with neuraminidases to hydrolyze sialic acid moieties. Disruption of cell-cell and cell-matrix adhesions was observed following neuraminidase treatment, suggesting that terminal sialic acids promote endothelial barrier integrity. When we measured transendothelial resistance, differential responses of pulmonary artery and microvascular endothelial cells to neuraminidase from Clostridium perfringens suggest that the molecular architecture of the sialic acid glycomes differs between these two cell types. Collectively our observations reveal critical structural and functional differences of terminally linked sialic acids on the pulmonary endothelium.