Showing papers on "Sialic acid published in 2010"
TL;DR: Resistance to one of the two main antiviral drugs is differentially acquired by the two distinct subsets of neuraminidase as a consequence of structural differences in the enzyme active site between the two phylogenetic groups.
538 citations
TL;DR: A large library of sialoside standards and derivatives in amounts sufficient for structure-activity relationship studies are provided and sialoglycan microarrays provide an efficient platform for quick identification of preferred ligands for sialic acid-binding proteins.
Abstract: Sialic acids are a subset of nonulosonic acids, which are nine-carbon α-keto aldonic acids. Natural existing sialic acid-containing structures are presented in different sialic acid forms, various sialyl linkages, and on diverse underlying glycans. They play important roles in biological, pathological, and immunological processes. Sialobiology has been a challenging and yet attractive research area. Recent advances in chemical and chemoenzymatic synthesis, as well as large-scale E. coli cell-based production, have provided a large library of sialoside standards and derivatives in amounts sufficient for structure−activity relationship studies. Sialoglycan microarrays provide an efficient platform for quick identification of preferred ligands for sialic acid-binding proteins. Future research on sialic acid will continue to be at the interface of chemistry and biology. Research efforts not only will lead to a better understanding of the biological and pathological importance of sialic acids and their diversi...
455 citations
TL;DR: It is shown that the Neu5Gc content of cultured human and nonhuman cell lines and their secreted glycoproteins can be reduced by adding a human sialic acid to the culture medium, which may be relevant to improving the half-life, efficacy and immunogenicity of glycoprotein therapeutics.
Abstract: Recombinant glycoproteins produced in animal cell lines often bear the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc). Ghaderi et al. show that two monoclonal antibodies in clinical use differ with respect to addition of Neu5Gc and propose that drug developers should consider the consequences of the Neu5Gc modification.
353 citations
TL;DR: An efficient synthesis of sialic-acid-terminated glycerol dendron to chemically functionalize 2 nm and 14 nm gold nanoparticles (AuNPs) is described, which allows a new type of molecular-imaging activity-correlation and is of particular relevance for further application in alternative antiviral therapy.
Abstract: An efficient synthesis of sialic-acid-terminated glycerol dendron to chemically functionalize 2 nm and 14 nm gold nanoparticles (AuNPs) is described. These nanoparticles are highly stable and show high activity towards the inhibition of influenza virus infection. As the binding of the viral fusion protein hemagglutinin to the host cell surface is mediated by sialic acid receptors, a multivalent interaction with sialic-acid-functionalized AuNPs is expected to competitively inhibit viral infection. Electron microscopy techniques and biochemical analysis show a high binding affinity of the 14 nm AuNPs to hemagglutinin on the virus surface and, less efficiently, to isolated hemagglutinin. The functionalized AuNPs are nontoxic to the cells under the conditions studied. This approach allows a new type of molecular-imaging activity-correlation and is of particular relevance for further application in alternative antiviral therapy.
254 citations
21 Jul 2010
TL;DR: It is suggested that the oligosaccharides are formed from lactose by the concerted action of glycosyltransferases, which are responsible for elongation and branching of the Galβ1-4GlcNAc group in the sugar chains of glycoconjugates on the surface of epithelial cells.
Abstract: Comparative study of the oligosaccharide profiles of individual human milk revealed the presence of three different patterns. Four oligosaccharides containing the Fucalpha1-2Gal group were missing in the milk of non-secretor, and three oligosaccharides containing the Fucalpha1-4GlcNAc group were missing in the milk of Lewis negative individuals. Disappearance of some major oligosaccharides in these samples led to the finding of five novel minor oligosaccharides, which were hidden under the missing oligosaccharides. Following these studies, structures of many novel milk oligosaccharides were elucidated. At least 13 core oligosaccharides were found in these oligosaccharides. By adding alpha-fucosyl residues and sialic acid residues to these core oligosaccharides, more than one hundred oligosaccharides were formed. All these oligosaccharides contain lactose at their reducing termini. This evidence, together with the deletion phenomena found in the milk oligosaccharides of non-secretor and Lewis negative individuals, suggested that the oligosaccharides are formed from lactose by the concerted action of glycosyltransferases, which are responsible for elongation and branching of the Galbeta1-4GlcNAc group in the sugar chains of glycoconjugates on the surface of epithelial cells. Therefore, oligosaccharides in human milk could include many structures, starting from the Galbeta1-4GlcNAc group in the sugar chains of various glycoconjugates. Many lines of evidence recently indicated that virulent enteric bacteria and viruses start their infection by binding to particular sugar chains of glycoconjugates on the target cell surfaces. Therefore, milk oligosaccharides could be useful for developing drugs, which inhibit the infection of bacteria and viruses.
242 citations
TL;DR: The extensive presence of SA α2,3-Gal and SAα2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection.
Abstract: A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid α2,3-galactose (SAα2,3-Gal) linked receptors, whereas human strains bind to sialic acid α2,6-galactose (SAα2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAα2,3-GalG(1-3)GalNAc and SAα2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II) and Sambucus nigra agglutinin (SNA) respectively. Both SAα2,3-Gal and SAα2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon). Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAα2,3-Gal and SAα2,6-Gal receptors from duodenum to colon in the pig. The extensive presence of SAα2,3-Gal and SAα2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors in the pig intestines highlights a need for clarification on the presence of SA receptors in the human intestinal tract.
185 citations
TL;DR: The approach provided a new rationale for the label-free, noninvasive, and real-time determination of SA, and can also be applied to the analysis of tumor malignancy and the degree of metastasis.
Abstract: Sialic acid (SA) is an anionic monosaccharide that frequently occurs at the termini of glycan chains and provides many opportunities for the assessment of both normal and pathological cell processes. It is generally present in tumorassociated carbohydrate antigens, including those clinically approved as tumor markers. Accordingly, the overexpression of SA on cell membranes has been implicated in the malignant and metastatic phenotypes of various types of cancer. Therefore, SA is an important molecular target for diagnostic and therapeutic approaches. The installation of SA-specific ligands enables reagents to target highly sialylated or tumor cells. Alternatively, monitoring of the cellsurface expression of SA should provide rational indexes of dynamic changes in pathological conditions and other SAassociated biological events. We previously developed a method for the potentiometric detection of SA by exploiting the reversible and specific nature of the binding between phenylboronic acid (PBA) and SA. A gold electrode modified with PBA and with a carefully optimized dissociation constant (or pKa value) was able to quantify SA present in the free state as well as cell-surface SA under physiological aqueous conditions. The observed ability of the electrode to differentiate altered levels of SA expression on the surface of rabbit erythrocytes is relevant to the diagnosis of insulin-dependent diabetes mellitus (IDDM). The approach provided a new rationale for the label-free, noninvasive (enzyme-free and operative on living cells), and real-time determination of SA. Herein we show that the technique can also be applied to the analysis of tumor malignancy and the degree of metastasis. PBA derivatives are able to form reversible cyclic boronates with 1,2-diols, 1,3-diols, and polyols: hallmark structures of the majority of glycans. Because of this property, PBA has quite a history as a synthetic ligand for these molecules. It is usually observed that these complexes have a stabilizing effect only if PBA is disassociated (at pH values above the pKa value), [6] whereas those formed between nondissociated PBA and sugars are unstable with high susceptibility to hydrolysis. However, as an exception, a complex formed between nondissociated PBA and SA is remarkably stable owing to its special binding modalities, some aspects of which have been clarified previously. As a result, a PBA with an appropriate pKa value can provide a molecular basis for selective recognition of SA among other saccharides under physiological conditions (see the Supporting Information). A procedure for the surface modification of a gold electrode with PBA was described previously. Briefly, a self-assembled monolayer (SAM) of 10-carboxy-1-decanethiol was first formed on a gold electrode. Next, a reaction between the terminal carboxyl groups and 3-aminophenylboronic acid resulted in the introduction of meta-amidesubstituted PBA on the SAM surface. Both quartz crystal microbalance (QCM) and ellipsometric measurements confirmed stoichiometric monolayer formation at each step of the reaction (see the Supporting Information). The surface PBA moiety had an apparent pKa value of about 9.5, as judged from pH-dependent changes in its threshold voltage (VT; see the Supporting Information). We could therefore safely conclude that it was not dissociated at the physiological pH value (7.4) and would be SA-specific under such conditions. The electrode was then linked to a field-effect-transistor (FET) gate for the real-time monitoring of charge-density changes on the electrode. In this configuration, a carboxyl anion of SA can be detected as a positive-direction shift of the VT value of the FET. Owing to the nature of the field effect, FET-based charge detection is possible only within a distance corresponding to the electrical double layer or the Debye length, which is no greater than a few nanometers even under conditions of minimized ionic strength. This requirement should be compatible with the purpose of detecting cellsurface SA moieties, which generally dominate the termini of the glycan chains, as described earlier. Besides, the tumoror metastasis-associated overexpression of SA is usually found in the form of polysialylation. Such a sequential arrangement of target SA units (as an SA homopolymer) on the glycanchain termini may help to enable the precise reflection of altered levels of SA expression. Moreover, the fact that the technique is limited to short detection distances could beneficially restrict charge detection to molecules that are truly (covalently) bound to the electrode surface within the vicinity of the Debye length (i.e., PBA-bound SA) and exclude other charges bound through nonspecific or noncovalent interactions. To demonstrate the ability of the electrode to assess malignancy or metastasis of a tissue specimen, metastatic murine melanoma cells expressing luciferase (B16-F10-Luc[*] Dr. A. Matsumoto, Dr. H. Cabral, N. Sato, Dr. K. Kataoka, Dr. Y. Miyahara Centre for NanoBio Integration, The University of Tokyo Hongo 7-3-1, Bunkyo-ku, Tokyo (Japan) Fax: (+ 81)29-860-4506 E-mail: miyahara.yuji@nims.go.jp
174 citations
TL;DR: Too little is known on the exact mechanism and on predisposing factors for reassortment to assess the true role of the pig in the emergence of novel influenza viruses, so this system offers a valuable alternative to study virus and/or host properties required for adaptation or reassortment of influenza viruses.
Abstract: Background
Throughout the history of human influenza pandemics, pigs have been considered the most likely "mixing vessel" for reassortment between human and avian influenza viruses (AIVs). However, the replication efficiencies of influenza viruses from various hosts, as well as the expression of sialic acid (Sia) receptor variants in the entire porcine respiratory tract have never been studied in detail. Therefore, we established porcine nasal, tracheal, bronchial and lung explants, which cover the entire porcine respiratory tract with maximal similarity to the in vivo situation. Subsequently, we assessed virus yields of three porcine, two human and six AIVs in these explants. Since our results on virus replication were in disagreement with the previously reported presence of putative avian virus receptors in the trachea, we additionally studied the distribution of sialic acid receptors by means of lectin histochemistry. Human (Siaα2-6Gal) and avian virus receptors (Siaα2-3Gal) were identified with Sambucus Nigra and Maackia amurensis lectins respectively.
157 citations
TL;DR: The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages, and the knowledge and tools generated in this study hold the key to the development of a new generation ofPRRSV vaccines.
Abstract: The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP(3), GP(4) and GP(5) envelope glycoproteins, only the M/GP(5) glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP(5). These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.
149 citations
TL;DR: A glycomic method is reported that selectively amidatesalpha2,6-linked sialic acids, while those that are alpha2,3-linked undergo spontaneous lactonization, which aids in increasing the metastatic potential of tumor cells.
Abstract: A number of alterations to the normal glycomic profile have been previously described for a number of diseases and disorders, thus underscoring the medical importance of studying the glycans associated with proteins present in biological samples An important alteration in cancer progression is an increased level of α2,6-sialylation, which aids in increasing the metastatic potential of tumor cells Here we report a glycomic method that selectively amidates α2,6-linked sialic acids, while those that are α2,3-linked undergo spontaneous lactonization Following subsequent permethylation, MALDI-TOF MS analysis revealed that many sialylated glycans present on glycoproteins found in blood serum featured increased levels of α2,6-sialylation in breast cancer samples On the basis of the altered ratios of α2,3-linked to α2,6-linked sialic acids, many of these glycans became diagnostically relevant when they did not act as such indicators when based on traditional glycomic profiling alone
140 citations
TL;DR: A new sialylation reagent 1 is reported that employs an N-acetyl-5-N,4-O-carbonyl protection with dibutyl phosphate as the leaving group and the new donor was applied to develop efficient strategies to use to create natural sialosides.
Abstract: N-Acetyl neuraminic acid (Neu5Ac) is most frequently found at the terminal end of glycoconjugates on the cell surface. This terminally exposed position allows Neu5Ac-containing conjugates to be exploited as receptors for viruses and bacteria, in addition to governing a wide variety of biological processes, such as tumor metastasis, cell differentiation, and cell–cell interactions. In naturally occurring sialosides, Neu5Ac is found linked to galactosides through the aACHTUNGTRENNUNG(2!3) or aACHTUNGTRENNUNG(2!6) linkage in Nand O-linked glycoproteins, and also to N-acetylgalactosamine through the aACHTUNGTRENNUNG(2!6) linkage in O-linked glycoproteins. In addition, polysialosides formed by the aACHTUNGTRENNUNG(2!8) or a ACHTUNGTRENNUNG(2!9) linkages are constituents of glycoproteins and glycolipids. The biological significance of sialoside receptors has driven research for their efficient synthesis. This has included significant efforts directed toward the development of sialic acid donors for efficient a-sialylation, including the use of anomeric leaving groups, such as halides, phosphites, sulfides, xanthates, and phenyltrifluoroacetimidates, the introduction of an auxiliary group at C-1 and C-3, the modification of the Nacetyl functional group at C-5, or the optimized combinations of the leaving group with positional modification. However, high yielding a-selective sialylation is still problematic owing to the presence of the C-1 electron-withdrawing carboxyl group at the tertiary anomeric center and the lack of a participating group at C-3 to direct the stereochemical outcome of glycosylation. The development of new strategies for the convergent synthesis of saccharides is a major focus in carbohydrate chemistry. Approaches such as armed–disarmed; onepot; reactivity-based, programmable one-pot; solidphase; orthogonal; and pre-activation methods have been developed to improve efficiency, with the ultimate goal of developing an automated method for oligosaccharide synthesis. The basic concept of these strategies involves the selective activation of one donor in the presence of an acceptor with the same or different leaving groups, so the newly formed product can be directly elongated without further aglycon leaving group adjustment. However, these approaches are not applicable to all glycosides. For example, sialic acid thioglycosides have limitations in our programmable reactivity-based, one-pot strategy due to their poor and narrow range of anomeric reactivity values (RRVs). To resolve the problem, sialylated disaccharides have been used as building blocks. However, application of this strategy is limited by the lack of an efficient and a-selective sialic acid donor that possesses a leaving group orthogonal to the thioglycoside. Herein, we report a new sialylation reagent 1 that employs an N-acetyl-5-N,4-O-carbonyl protection with dibutyl phosphate as the leaving group. Furthermore, the new donor was applied to develop efficient strategies to use to create natural sialosides. Many researchers have focused on the modification of the N-acetyl functional group of sialic acid for highly efficient a-sialylation. However, there is no general donor for different acceptors. The choice of C-5 modification to be used is highly dependent on protecting groups and the nature of glycosyl acceptors. There is an excellent review on C-5 modification by De Meo. Crich and co-workers have shown that the N-acetyl-5-N,4-O-carbonyl thiosialoside can [a] C.-H. Hsu, Dr. K.-C. Chu, Y.-S. Lin, Dr. J.-L. Han, Y.-S. Peng, Dr. C.-T. Ren, Prof. C.-Y. Wu, Prof. C.-H. Wong The Genomics Research Center, Academia Sinica (Taiwan) 128 Academia Road, Section 2, Nankang, Taipei 115 (Taiwan) Fax: (+886)2-2785-3852 E-mail : cyiwu@gate.sinica.edu.tw chwong@gate.sinica.edu.tw [b] C.-H. Hsu, Prof. C.-H. Wong Department of Chemistry, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) [c] C.-H. Hsu, Prof. C.-Y. Wu, Prof. C.-H. Wong Chemical Biology and Molecular Biophysics Taiwan International Graduate Program, Academia Sinica 128 Academia Road, Section 2, Nankang, Taipei 115 (Taiwan) [d] C.-H. Hsu Institute of Bioinformatics and Structural Biology National Tsing-Hua University, Hsin-Chu (Taiwan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200903035.
TL;DR: The mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 is determined by mobility shift and enzyme inhibition assays and shows that microbe-associated NAs can directly activate LTGF -β and that TGF- β plays a pivotal role protecting the host from influenza pathogenesis.
Abstract: Transforming growth factor-beta (TGF-β), a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β). We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA) protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3) except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus-infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis.
TL;DR: Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.
Abstract: The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-A resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.
TL;DR: It is shown that trace exogenous Neu5Gc can be incorporated into cell surface lipooligosaccharides of nontypeable Haemophilus influenzae (NTHi), a human-specific commensal/pathogen, and postnatally appearing anti-Neu 5Gc antibodies appear coincident with antibodies against NTHi.
Abstract: The nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc) is metabolically incorporated into human tissues from certain mammalian-derived foods, and this occurs in the face of an anti-Neu5Gc “xeno-autoantibody” response. Given evidence that this process contributes to chronic inflammation in some diseases, it is important to understand when and how these antibodies are generated in humans. We show here that human anti-Neu5Gc antibodies appear during infancy and correlate with weaning and exposure to dietary Neu5Gc. However, dietary Neu5Gc alone cannot elicit anti-Neu5Gc antibodies in mice with a humanlike Neu5Gc deficiency. Other postnatally appearing anti-carbohydrate antibodies are likely induced by bacteria expressing these epitopes; however, no microbe is known to synthesize Neu5Gc. Here, we show that trace exogenous Neu5Gc can be incorporated into cell surface lipooligosaccharides (LOS) of nontypeable Haemophilus influenzae (NTHi), a human-specific commensal/pathogen. Indeed, infant anti-Neu5Gc antibodies appear coincident with antibodies against NTHi. Furthermore, NTHi that express Neu5Gc-containing LOS induce anti-Neu5Gc antibodies in Neu5Gc-deficient mice, without added adjuvant. Finally, Neu5Gc from baby food is taken up and expressed by NTHi. As the flora residing in the nasopharynx of infants can be in contact with ingested food, we propose a novel model for how NTHi and dietary Neu5Gc cooperate to generate anti-Neu5Gc antibodies in humans.
TL;DR: It is concluded that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.
Abstract: Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca 2+ -dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.
TL;DR: These findings support the importance of the glycocalyx, notably the terminal sialic acids, in the regulation of apoptotic cell clearance, and suggest changes in surface glycosylation can either directly mediate cellular engulfment or enhance phagocytosis by cooperation with further engulfment signals.
Abstract: The silent clearance of apoptotic cells is essential for cellular homeostasis in multicellular organisms, and several mediators of apoptotic cell recognition have been identified. However, the distinct mechanisms involved are not fully deciphered yet. We analyzed alterations of the glycocalyx on the surfaces of apoptotic cells and its impact for engulfment. After apoptosis induction of lymphocytes, a decrease of α2,6-terminal sialic acids and sialic acids in α2,3-linkage with galactose was observed. Similar changes were to be found on the surface of apoptotic membrane blebs released during early stages of apoptosis, whereas later released blebs showed no impaired, but rather an increased, exposure of sialic acids. We detected an exposure of fucose residues on the surface of apoptotic-cell-derived membrane blebs. Cleavage by neuraminidase of sialic acids, as well as lectin binding to sialic acids on the surfaces, enhanced the engulfment of apoptotic cells and blebs. Interestingly, even viable lymphoblasts were engulfed in an autologous cell system after neuraminidase treatment. Similarly, the engulfment of resting apoptotic lymphocytes was augmented after neuraminidase treatment. However, the engulfment of resting viable lymphocytes was not significantly enhanced after neuraminidase treatment. Our findings support the importance of the glycocalyx, notably the terminal sialic acids, in the regulation of apoptotic cell clearance. Thus, depending on cell type and activation status, changes in surface glycosylation can either directly mediate cellular engulfment or enhance phagocytosis by cooperation with further engulfment signals.
TL;DR: These studies provide a correlation between binding affinity to glycan receptors with terminal alpha 2,6-linked sialic acid and the efficiency of respiratory droplet transmission for pandemic H2N2 influenza viruses.
Abstract: Influenza viruses of the H2N2 subtype have not circulated among humans in over 40 years. The occasional isolation of avian H2 strains from swine and avian species coupled with waning population immunity to H2 hemagglutinin (HA) warrants investigation of this subtype due to its pandemic potential. In this study we examined the transmissibility of representative human H2N2 viruses, A/Albany/6/58 (Alb/58) and A/El Salvador/2/57 (ElSalv/57), isolated during the 1957/58 pandemic, in the ferret model. The receptor binding properties of these H2N2 viruses was analyzed using dose-dependent direct glycan array-binding assays. Alb/58 virus, which contains the 226L/228S amino acid combination in the HA and displayed dual binding to both alpha 2,6 and alpha 2,3 glycan receptors, transmitted efficiently to naive ferrets by respiratory droplets. Inefficient transmission was observed with ElSalv/57 virus, which contains the 226Q/228G amino acid combination and preferentially binds alpha 2,3 over alpha 2,6 glycan receptors. However, a unique transmission event with the ElSalv/57 virus occurred which produced a 226L/228G H2N2 natural variant virus that displayed an increase in binding specificity to alpha 2,6 glycan receptors and enhanced respiratory droplet transmissibility. Our studies provide a correlation between binding affinity to glycan receptors with terminal alpha 2,6-linked sialic acid and the efficiency of respiratory droplet transmission for pandemic H2N2 influenza viruses.
TL;DR: The proposed strategy demonstrated that mannose exhibited high expression levels in both normal and cancer cells, while sialic acid was more abundant in cancer cells as compared to normal ones, indicating that sIALic acid could serve as a potential biomarker for early cancer detection.
Abstract: In this article, we report a novel lectin-based biosensor for electrochemical assay of cancer-associated glycosylation by comparative study of mannose and sialic acid expression on normal and cancer cells derived from human lung, liver, and prostate. Using a sandwich format, high sensitivity and selectivity were achieved by combining the lectin-based biosensor with the {lectin−Au−Th} bioconjugates featuring lectin and thionine (Th) labels linked to gold nanoparticles (AuNPs) for signal amplification. The proposed strategy demonstrated that mannose exhibited high expression levels in both normal and cancer cells, while sialic acid was more abundant in cancer cells as compared to normal ones. The results were in good agreement with those from fluorescent microscopy studies. The differences in the two glycan expression indicated that sialic acid could serve as a potential biomarker for early cancer detection. The lectin-based biosensor was also successfully used to quantify cancer cells and evaluate the aver...
TL;DR: It is suggested that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development.
Abstract: Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4(+) and TLR4(-) mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.
TL;DR: Novel ficolin ligands such as sulfated N-acetyllactosamine and gangliosides are revealed and precise insights are provided into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr271 in this respect.
TL;DR: A new paradigm is suggested for understanding the role of the structure of sialylated glycan receptors in influenza virus pathogenesis and is critical for efficient human adaptation and human-human transmission of influenza A viruses.
Abstract: Influenza A viruses, members of the Orthomyxoviridae family, are responsible for annual seasonal influenza epidemics and occasional global pandemics. The binding of viral coat glycoprotein hemagglutinin (HA) to sialylated glycan receptors on host epithelial cells is the critical initial step in the infection and transmission of these viruses. Scientists believe that a switch in the binding specificity of HA from Neu5Acα2-3Gal linked (α2-3) to Neu5Acα2-6Gal linked (α2-6) glycans is essential for the crossover of the viruses from avian to human hosts. However, studies have shown that the classification of glycan binding preference of HA based on sialic acid linkage alone is insufficient to establish a correlation between receptor specificity of HA and the efficient transmission of influenza A viruses. A recent study reported extensive diversity in the structure and composition of α2-6 glycans (which goes beyond the sialic acid linkage) in human upper respiratory epithelia and identified different glycan structural topologies. Biochemical examination of the multivalent HA binding to these diverse sialylated glycan structures also demonstrated that high affinity binding of HA to α2-6 glycans with a characteristic umbrella-like structural topology is critical for efficient human adaptation and human-human transmission of influenza A viruses. This review summarizes studies which suggest a new paradigm for understanding the role of the structure of sialylated glycan receptors in influenza virus pathogenesis.
TL;DR: N-stearoyl peptides inhibited infections by the A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains of influenza virus, indicating that these peptides mimic the sialic acid structure.
Abstract: Influenza is an infectious disease caused by the influenza virus, and each year many people suffer from this disease. Hemagglutinin (HA) in the membrane of type A influenza viruses recognizes sialylglycoconjugate receptors on the host cell surface at an initial step in the infection process; consequently, HA inhibitors are considered potential candidates for antiviral drugs. We identified peptides that bind to receptor-binding sites through a multiple serial selection from phage-displayed random peptide libraries. Using the HA of the H1 and H3 strains as target proteins, we obtained peptides that bind to both HAs. The binding affinities of peptides for these HAs were improved by secondary and tertiary selections from the corresponding sublibraries. A docking simulation suggested that, similar to sialic acid, the peptides are recognized by the receptor-binding site in HA, which indicates that these peptides mimic the sialic acid structure. N-stearoyl peptides inhibited infections by the A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains of influenza virus. Such HA-inhibitors are promising candidates for novel antiviral drugs.
TL;DR: It is reported here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum, and that merozoites interact directly with CR1 on the ERYthrocyte surface and with sCR1-coated microspheres.
Abstract: Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1-coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine.
TL;DR: It is shown, through a multidisciplinary approach, that novel sialic acid-based derivatives can exploit this structural difference and selectively inhibit the activity of group-1 sialidases, and this is the first direct proof that compounds may be developed to selectively target the pandemic A/H1N1, avian A/ H5N1 and other group- 1 sIALidase-containing viruses, based on an open 150-loop conformation of the enzyme.
Abstract: Influenza virus sialidase has an essential role in the virus' life cycle. Two distinct groups of influenza A virus sialidases have been established, that differ in the flexibility of the '150-loop', providing a more open active site in the apo form of the group-1 compared to group-2 enzymes. In this study we show, through a multidisciplinary approach, that novel sialic acid-based derivatives can exploit this structural difference and selectively inhibit the activity of group-1 sialidases. We also demonstrate that group-1 sialidases from drug-resistant mutant influenza viruses are sensitive to these designed compounds. Moreover, we have determined, by protein X-ray crystallography, that these inhibitors lock open the group-1 sialidase flexible 150-loop, in agreement with our molecular modelling prediction. This is the first direct proof that compounds may be developed to selectively target the pandemic A/H1N1, avian A/H5N1 and other group-1 sialidase-containing viruses, based on an open 150-loop conformation of the enzyme.
TL;DR: It is concluded that sialidase‐mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS‐induced cytokine production, thereby contributing to development of adaptive immune responses.
Abstract: Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1-4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6, IL-12p40, and TNF-α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1⁻/⁻ and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production, thereby contributing to development of adaptive immune responses.
TL;DR: A family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii are identified, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion.
TL;DR: A simple and rapid method to modify the sialic acids of sialylated glycans in the presence of methylamine and (7-azabenzotriazol-1-yloxy) trispyrrolidinophosphonium hexafluorophosphate to enable the successful characterization of N-glycans of sera from different species by MALDI-MS.
Abstract: Neutralization of carboxylic acid is an important means to avoid sialic acid dissociation when sialylated glycans are analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In this paper, we describe a simple and rapid method to modify the sialic acids of sialylated glycans in the presence of methylamine and (7-azabenzotriazol-1-yloxy) trispyrrolidinophosphonium hexafluorophosphate (PyAOP). After methylamidation, sialylated glycans can be analyzed by MALDI-MS without loss of the sialic acid moiety. The electrospray ionization mass spectrometry (ESI-MS) and MALDI-MS analysis of both 3'- and 6'-sialyllactose derivatives indicated that the quantitative conversion of sialic acids was achieved, regardless of their linkage types. This derivatization strategy was further validated with the N-glycans released from three standard glycoproteins (fetuin, human acid glycoprotein, and bovine acid glycoprotein) containing different types of complex glycans. Most importantly, this derivatization method enabled the successful characterization of N-glycans of sera from different species (human, mouse, and rat) by MALDI-MS. Because of the mild reaction conditions, the modification in sialic acid residues can be retained. This improvement makes it possible to detect sialylated glycans containing O-acetylated sialic acid moieties using MALDI-MS in positive-ion mode.
TL;DR: This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor, and because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in G BS disease.
Abstract: In Campylobacter jejuni-induced Guillain-Barre syndrome (GBS), molecular mimicry between C. jejuni lipooligosaccharide (LOS) and host gangliosides leads to the production of cross-reactive antibodies directed against the peripheral nerves of the host. Currently, the presence of surface exposed sialylated LOS in C. jejuni is the single known bacterial pathogenesis factor associated with the development of GBS. Using a unique, well-characterized strain collection, we demonstrate that GBS-associated C. jejuni strains bind preferentially to sialoadhesin (Sn, Siglec-1, or CD169), a sialic acid receptor found on a subset of macrophages. In addition, using a whole-cell enzyme-linked immunosorbent assay (ELISA), C. jejuni strains with sialylated LOS bound exclusively to soluble Sn. Mass spectrometry revealed that binding was sialic acid-linkage specific with a preference for α(2,3)-linked sialic acid attached to the terminal galactose of the LOS chain as seen in the gangliosides GD1a, GM1b, and GM3. This molecular interaction was also related to functional consequences as a GBS-associated C. jejuni strain that bound Sn in a whole-cell ELISA adhered to surface-expressed Sn of Sn-transfected CHO cells but was unable to adhere to wild-type CHO cells. Moreover, a sialic acid-negative mutant of the same C. jejuni strain was unable to bind Sn-transfected CHO cells. This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor (P = 0.014). Moreover, because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in GBS disease.
TL;DR: Data indicate that NeuAcα2–6-terminated polylactosamine and sialylated N-glycans are important determinants for influenza viruses to infect SRECs, which has implications for transmission of viruses between humans and pigs and for pigs as possible adaptation hosts of novel human influenza viruses.
TL;DR: Retroviral complementation of the patients' fibroblasts with the wild-type COG6-cDNA led to normalization of the COG complex-depending retrograde protein transport after Brefeldin A treatment, demonstrated by immunofluorescence analysis.
Abstract: Deficiency of subunit 6 of the conserved oligomeric Golgi (COG6) complex causes a new combined N- and O-glycosylation deficiency of the congenital disorders of glycosylation, designated as CDG-IIL (COG6-CDG). The index patient presented with a severe neurologic disease characterized by vitamin K deficiency, vomiting, intractable focal seizures, intracranial bleedings and fatal outcome in early infancy. Analysis of oligosaccharides from serum transferrin by HPLC and mass spectrometry revealed the loss of galactose and sialic acid residues, whereas import and transfer of these sugar residues into Golgi-enriched vesicles or onto proteins, respectively, were normal to slightly reduced. Western blot examinations combined with gel filtration chromatography studies in patient-derived skin fibroblasts showed a severely reduced expression of the mentioned subunit and the occurrence of COG complex fragments at the expense of the integral COG complex. Sequencing of COG6-cDNA and COG6 gene resulted in a homozygous mutation (c.G1646T), leading to amino acid exchange p.G549V in the COG6 protein. Retroviral complementation of the patients' fibroblasts with the wild-type COG6-cDNA led to normalization of the COG complex-depending retrograde protein transport after Brefeldin A treatment, demonstrated by immunofluorescence analysis.