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1
Tiralongo, Joe, Therese Wohlschlager, Evelin Tiralongo, and Milton J. Kiefel. "Inhibition of Aspergillus fumigatus conidia binding to extracellular matrix proteins by sialic acids: a pH effect?" Microbiology 155, no. 9 (September 1, 2009): 3100–3109. http://dx.doi.org/10.1099/mic.0.026997-0.
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Infection by Aspergillus fumigatus, which causes the life-threatening disease invasive aspergillosis, begins with the inhalation of conidia that adhere to and germinate in the lung. Previous studies have shown that A. fumigatus conidia express high levels of the negatively charged 9-carbon sugar sialic acid, and that sialic acid appears to mediate the binding of A. fumigatus conidia to basal lamina proteins. However, despite the ability of sialic acid to inhibit adherence of A. fumigatus conidia, the exact mechanism by which this binding occurs remains unresolved. Utilizing various free sialic acids and other carbohydrates, sialic acid derivatives, sialoglycoconjugates, glycoproteins, α-keto acid related compounds and amino acids we have found that the binding of A. fumigatus conidia to type IV collagen and fibrinogen was inhibited by (i) glycoproteins (in a sialic acid-independent manner), and (ii) free sialic acids, glucuronic acid and α-keto acid related compounds. However, inhibition by the latter was found to be the result of a shift in pH from neutral (pH 7.4) to acidic (less than pH 4.6) induced by the relatively high concentrations of free sialic acids, glucuronic acid and α-keto acid related compounds used in the binding assays. This suggests that previous reports describing inhibition of A. fumigatus conidia binding by free sialic acid may actually be due to a pH shift similar to that shown here. As previously reported, we found that A. fumigatus conidia express only N-acetylneuraminic acid, the most common sialic acid found in nature. However, A. fumigatus appears to do so by an alternative mechanism to that seen in other organisms. We report here that A. fumigatus (i) does not incorporate sialic acid obtained from the environment, (ii) does not synthesize and incorporate sialic acid from exogenous N-acetylmannosamine, and (iii) lacks homologues of known sialic acid biosynthesizing enzymes.
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Gangi Setty, Thanuja, Christine Cho, Sowmya Govindappa, Michael A. Apicella, and S. Ramaswamy. "Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site." Acta Crystallographica Section D Biological Crystallography 70, no. 7 (June 24, 2014): 1801–11. http://dx.doi.org/10.1107/s139900471400830x.
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Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteriaFusobacterium nucleatum,Pasteurella multocidaandVibrio choleraeand their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of theHaemophilus influenzaeprotein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.
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Li, Qiongyu, Yixuan Xie, Gege Xu, and Carlito B. Lebrilla. "Identification of potential sialic acid binding proteins on cell membranes by proximity chemical labeling." Chemical Science 10, no. 24 (2019): 6199–209. http://dx.doi.org/10.1039/c9sc01360a.
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Hayakawa, Toshiyuki, Takashi Angata, Elliott H. Margulies, Tarjei Mikkelsen, Eric D. Green, and Ajit Varki. "Gene Conversion of Sialic Acid Binding Domains in CD33-Related Siglecs by Adjacent Pseudogenes: A Novel Mechanism To Change Sialic Acid Binding Specificity." Blood 104, no. 11 (November 16, 2004): 1471. http://dx.doi.org/10.1182/blood.v104.11.1471.1471.
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Abstract Siglecs (sialic acid-binding immunoglobulin superfamily lectins) are a family of cell surface receptors involved in regulating the immune response. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules found primarily on cells of the innate immune system. All are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that includes two tyrosine-based signaling motifs. Available data suggest an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. Nine of the 13 known primate Siglec genes along with 14 Siglec pseudogenes comprise the CD33-related Siglec gene cluster on human chromosome 19. Gene conversion is a mechanism for copying part of a genomic sequence into another, contributing to genetic diversity. Pseudogenes are known to play role in generating functional diversity of related genes (e.g., antibody diversity via gene conversion in chickens). We recently analyzed genomic sequences of the CD33-related Siglec gene cluster in three primates (human, chimpanzee and baboon) and found evidence for rapid evolution in this gene family (Angata et al., PNAS, in press). Additional evolutionary studies using distance-based phylogenetic trees shows evidence for three partial gene conversions between Siglec genes and adjacent Siglec pseudogenes. All three involve the coding regions for extracellular domains that mediate sialic acid recognition, and two involve a pseudogene converting a known Siglec gene. Functional analyses using recombinant proteins show marked differences in sialic acid-binding properties between the converted Siglec and its non-converted ortholog. These findings suggest that gene conversion with pseudogenes has contributed to the rapid functional evolution of the Siglecs, and provides a novel mechanism for changing sialic acid binding specificity. We hypothesize that this mechanism allows for rapid evolutionary adjustments in the recognition of endogenous sialic acids as “self”, a potential factor in controlling the innate immune response.
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Zhao, Chuankuo, and Juan Pu. "Influence of Host Sialic Acid Receptors Structure on the Host Specificity of Influenza Viruses." Viruses 14, no. 10 (September 28, 2022): 2141. http://dx.doi.org/10.3390/v14102141.
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Influenza viruses need to use sialic acid receptors to invade host cells, and the α-2,3 and α-2,6 sialic acids glycosidic bonds linking the terminal sialic acids are generally considered to be the most important factors influencing the cross-species transmission of the influenza viruses. The development of methods to detect the binding of influenza virus HA proteins to sialic acid receptors, as well as the development of glycobiological techniques, has led to a richer understanding of the structure of the sialylated glycan in influenza virus hosts. It was found that, in addition to the sialic acid glycosidic bond, sialic acid variants, length of the sialylated glycan, Gal-GlcNAc-linked glycosidic bond within the sialylated glycan, and sulfation/fucosylation of the GlcNAc within the sialylated glycan all affect the binding properties of influenza viruses to the sialic acid receptors, thus indirectly affecting the host specificity of influenza viruses. This paper will review the sialic acid variants, internal structural differences of sialylated glycan molecules that affect the host specificity of influenza viruses, and distribution characteristics of sialic acid receptors in influenza virus hosts, in order to provide a more reliable theoretical basis for the in-depth investigation of cross-species transmission of influenza viruses and the development of new antiviral drugs.
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Schwegmann-Weßels, Christel, Gert Zimmer, Hubert Laude, Luis Enjuanes, and Georg Herrler. "Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins." Journal of Virology 76, no. 12 (June 15, 2002): 6037–43. http://dx.doi.org/10.1128/jvi.76.12.6037-6043.2002.
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ABSTRACT The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.
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Zeng, Fu-Yue, and Hans-Joachim Gabius. "Sialic Acid-Binding Proteins: Characterization, Biological Function and Application." Zeitschrift für Naturforschung C 47, no. 9-10 (October 1, 1992): 641–53. http://dx.doi.org/10.1515/znc-1992-9-1001.
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The last decade has witnessed steadily growing support for the notion that the carbohydrate portion of glycoconjugates is not merely an inert structural addition to the protein or lipid backbone. Considerable attention has been given to the chemical composition of glycosidic residues in such heteropolymers. Sialic acids are frequently occurring components of oligosaccharide side chains in glycoconjugates of most higher animals and a few microorganisms. They appear to play an important role as ligands in glycobiological interactions. Mediation of a proposed protein-carbohydrate recognition will necessarily involve a binding protein with the respective specificity. Such proteins thus are able to serve as receptors for certain types of carbohydrate moieties like sialic acids in vivo. Various members of this class of proteins have already proven their value as analytical tools in studying expression and localization of defined sialoglycoconjugates. These proteins attract much attention due to both their functions in situ and their potential as laboratory tools in glycoconjugate research in areas like biochemistry or histology. We present a survey of the purification, characterization and application of this class of proteins to illustrate the status of knowledge and the current directions of research in this field.
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Gaytán, Meztlli O., Anirudh K. Singh, Shireen A. Woodiga, Surina A. Patel, Seon-Sook An, Arturo Vera-Ponce de León, Sean McGrath, et al. "A novel sialic acid-binding adhesin present in multiple species contributes to the pathogenesis of Infective endocarditis." PLOS Pathogens 17, no. 1 (January 19, 2021): e1009222. http://dx.doi.org/10.1371/journal.ppat.1009222.
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Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP- isolates also bind the cryptic receptor β-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.
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Schwegmann-Wessels, Christel, Gert Zimmer, Bernd Schröder, Gerhard Breves, and Georg Herrler. "Binding of Transmissible Gastroenteritis Coronavirus to Brush Border Membrane Sialoglycoproteins." Journal of Virology 77, no. 21 (November 1, 2003): 11846–48. http://dx.doi.org/10.1128/jvi.77.21.11846-11848.2003.
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ABSTRACT Transmissible gastroenteritis coronavirus (TGEV) is a porcine pathogen causing enteric infections that are lethal for suckling piglets. The enterotropism of TGEV is connected with the sialic acid binding activity of the viral surface protein S. Here we show that, among porcine intestinal brush border membrane proteins, TGEV recognizes a mucin-type glycoprotein designated MGP in a sialic acid-dependent fashion. Virus binding assays with cryosections of the small intestine from a suckling piglet revealed the binding of TGEV to mucin-producing goblet cells. A nonenteropathogenic mutant virus that lacked a sialic acid binding activity was unable to bind to MGP and to attach to goblet cells. Our results suggest a role of MGP in the enteropathogenicity of TGEV.
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Marshall, P., A. Hasegawa, E. A. Davidson, V. Nussenzweig, and M. Whitlow. "Interaction between complement proteins C5b-7 and erythrocyte membrane sialic acid." Journal of Experimental Medicine 184, no. 4 (October 1, 1996): 1225–32. http://dx.doi.org/10.1084/jem.184.4.1225.
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The initial phase of membrane attack by complement is the interaction between C5b6, C7, and the cell membrane that leads to the insertion of C5b-7. Here we investigate the role of sialic acid residues in the assembly of C5b-7 intermediates on erythrocyte cell membranes. We find that C5b6 binds to glycophorin, whereas C5 or C6 does not bind, and desialylation of the glycophorin abolishes C5b6 binding. Complement lysis is inhibited by either masking glycophorin sialic acid with F(ab) fragments of an mAb, or by removal of the sialylated region of glycophorin by mild trypsinization. Gangliosides inhibit C5b-7 deposition when added to the aqueous phase. Asialogangliosides and synthetic gangliosides lacking the carboxylic acid residue have no inhibitory activity. We conclude that C5b6 binds to sialylated molecules on the erythrocyte surface. We propose a new model of membrane attack in which C5b6 initially binds to membranes via ionic forces. C7 then binds to C5b6, disrupting the ionic interaction and leading to the exposure of hydrophobic domains. Sialic acid is known to inhibit complement activation. Thus, these findings reveal a paradoxical role for sialic acid in complement attack; the presence of sialic acid inhibits the generation of C5b6, but once the membrane attack pathway is initiated, sialic acid enhances complement lysis.
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Baker, H. M., M. Chung, I. Basu, E. N. Baker, and J. D. Fraser. "Two staphylococcal sialic acid binding proteins from the superantigen superfamily." Acta Crystallographica Section A Foundations of Crystallography 64, a1 (August 23, 2008): C357. http://dx.doi.org/10.1107/s0108767308088594.
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Bensing, Barbara A., José A. López, and Paul M. Sullam. "The Streptococcus gordonii Surface Proteins GspB and Hsa Mediate Binding to Sialylated Carbohydrate Epitopes on the Platelet Membrane Glycoprotein Ibα." Infection and Immunity 72, no. 11 (November 2004): 6528–37. http://dx.doi.org/10.1128/iai.72.11.6528-6537.2004.
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ABSTRACT Platelet binding by Streptococcus gordonii strain M99 is dependent on expression of the cell wall-anchored glycoprotein GspB. This large cell surface protein is exported from the M99 cytoplasm via a dedicated transport system that includes SecA2 and SecY2. GspB is highly similar to Hsa, a protein expressed by S. gordonii Challis that has been characterized as a sialic acid binding hemagglutinin. In this study, we compared the contribution of GspB and Hsa to the adherence of S. gordonii to selected glycoproteins. Our results indicate that GspB can mediate binding to a variety of sialylated glycoproteins. GspB facilitates binding to carbohydrates bearing sialic acid in either α(2-3) or α(2-6) linkages, with a slight preference for α(2-3) linkages. Furthermore, GspB readily mediates binding to sialic acid residues on immobilized glycocalicin, the extracellular portion of the platelet membrane glycoprotein (GP) Ibα (the ligand binding subunit of the platelet von Willebrand factor receptor complex GPIb-IX-V). Although Hsa is required for the binding of S. gordonii Challis to sialic acid, most of the Hsa expressed by Challis is retained in the cytoplasm. The deficiency in export is due, at least in part, to a nonsense mutation in secA2. Hsa export can be enhanced by complementation with secA2 from M99, which also results in significantly greater binding to sialylated glycoproteins, including glycocalicin. The combined results indicate that GspB and Hsa contribute similar binding capabilities to M99 and Challis, respectively, but there may be subtle differences in the preferred epitopes to which these adhesins bind.
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Warner, Thomas G., and Laura A. Lee. "An azidoaryl thioglycoside of sialic acid. A potential photoaffinity probe of sialidases and sialic acid-binding proteins." Carbohydrate Research 176, no. 2 (May 1988): 211–18. http://dx.doi.org/10.1016/0008-6215(88)80132-0.
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Cohen, Miriam, Nancy Hurtado-Ziola, and Ajit Varki. "ABO blood group glycans modulate sialic acid recognition on erythrocytes." Blood 114, no. 17 (October 22, 2009): 3668–76. http://dx.doi.org/10.1182/blood-2009-06-227041.
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Abstract ABH(O) blood group polymorphisms are based on well-known intraspecies variations in structures of neutral blood cell surface glycans in humans and other primates. Whereas natural antibodies against these glycans can act as barriers to blood transfusion and transplantation, the normal functions of this long-standing evolutionary polymorphism remain largely unknown. Although microbial interactions have been suggested as a selective force, direct binding of lethal pathogens to ABH antigens has not been reported. We show in this study that ABH antigens found on human erythrocytes modulate the specific interactions of 3 sialic acid-recognizing proteins (human Siglec-2, 1918SC influenza hemagglutinin, and Sambucus nigra agglutinin) with sialylated glycans on the same cell surface. Using specific glycosidases that convert A and B glycans to the underlying H(O) structure, we show ABH antigens stabilize sialylated glycan clusters on erythrocyte membranes uniquely for each blood type, generating differential interactions of the 3 sialic acid-binding proteins with erythrocytes from each blood type. We further show that by stabilizing such structures ABH antigens can also modulate sialic acid-mediated interaction of pathogens such as Plasmodium falciparum malarial parasite. Thus, ABH antigens can noncovalently alter the presentation of other cell surface glycans to cognate-binding proteins, without themselves being a direct ligand.
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Brinkman-Van der Linden, Els C. M., Takashi Angata, Shirley A. Reynolds, Leland D. Powell, Stephen M. Hedrick, and Ajit Varki. "CD33/Siglec-3 Binding Specificity, Expression Pattern, and Consequences of Gene Deletion in Mice." Molecular and Cellular Biology 23, no. 12 (June 15, 2003): 4199–206. http://dx.doi.org/10.1128/mcb.23.12.4199-4206.2003.
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ABSTRACT Mouse CD33/Siglec-3 (mCD33) is the apparent ortholog of human CD33/Siglec-3 (hCD33), a member of the Siglec (sialic acid-binding Ig superfamily lectin) family of sialic acid-recognizing cell-surface lectins. We examined the binding specificity and expression pattern of mCD33 and explored its functions by generating mice deficient in this molecule. Like hCD33, mCD33 is expressed on myeloid precursors in the bone marrow, albeit mostly in the more mature stages of the granulocytic lineage. Moreover, unlike hCD33, mCD33 in peripheral blood is primarily expressed on granulocytes. Also, unlike hCD33, mCD33 did not bind to α2-3- or α2-6-linked sialic acids on lactosamine units. Instead, it showed distinctive sialic acid-dependent binding only to the short O-linked glycans of certain mucins and weak binding to the sialyl-Tn epitope. Binding was enhanced by removal of 9-O-acetyl groups and attenuated by truncation of the glycerol-like side chain of sialic acids. Mice deficient in CD33 were viable and fertile in a controlled-access specific-pathogen-free vivarium, showed no major morphological or histological abnormalities, had no changes in bone marrow or peripheral leukocyte subpopulations, and had very minor differences in biochemical and erythrocyte parameters. Cellular responses to intraperitoneally injected proinflammatory stimulants, as well as subsequent interleukin-6 secretion, were also apparently unaffected. These results indicate substantial species differences in CD33 expression patterns and ligand recognition and suggest functional degeneracy between mCD33 and the other CD33-related Siglec proteins expressed on cells of the myeloid lineage.
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Ryan, Patricia A., Vijaykumar Pancholi, and Vincent A. Fischetti. "Group A Streptococci Bind to Mucin and Human Pharyngeal Cells through Sialic Acid-Containing Receptors." Infection and Immunity 69, no. 12 (December 1, 2001): 7402–12. http://dx.doi.org/10.1128/iai.69.12.7402-7412.2001.
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ABSTRACT The first step in the colonization of group A streptococci (Streptococcus pyogenes) is adherence to pharyngeal epithelial cells. Prior to adherence to their target tissue, the first barrier that the streptococci encounter is the mucous layer of the respiratory tract. The present study was undertaken to characterize the interaction between mucin, the major glycoprotein component of mucus, and streptococci. We report here that S. pyogenes is able to bind to bovine submaxillary mucin in solid-phase microtiter plate assays. Western blots probed with 125I-labeled mucin and a panel of monoclonal antibodies revealed that the streptococcal M protein is one of two cell wall-associated proteins responsible for this binding. The binding was further localized to the N-terminal portion of the M molecule. Further analysis revealed that the M protein binds to the sialic acid moieties on mucin, and this interaction seems to be based on M-protein conformation rather than specific amino acid sequences. We found that sialic acid also plays a critical role in the adherence of an M6 streptococcal strain to the Detroit 562 human pharyngeal cell line and have identified α2-6-linked sialic acid as an important sialylated linkage for M-protein recognition. Western blot analysis of extracted pharyngeal cell membrane proteins identified three potential sialic acid-containing receptors for the M protein. The results are the first to show that sialic acid not only is involved in the binding of the streptococci to mucin but also plays an important role in adherence of group A streptococci to the pharyngeal cell surface.
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Connaris, Helen, Toru Takimoto, Rupert Russell, Susan Crennell, Ibrahim Moustafa, Allen Portner, and Garry Taylor. "Probing the Sialic Acid Binding Site of the Hemagglutinin-Neuraminidase of Newcastle Disease Virus: Identification of Key Amino Acids Involved in Cell Binding, Catalysis, and Fusion." Journal of Virology 76, no. 4 (February 15, 2002): 1816–24. http://dx.doi.org/10.1128/jvi.76.4.1816-1824.2002.
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ABSTRACT We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.
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Chen, Yin, David J. Friedman, Mayank Saraswat, Michael J. Shapiro, Drew Wilfahrt, Akhilesh Pandey, and Virginia Smith Shapiro. "Sialylation of CD44 by ST8sia6 is required for recognition by the inhibitory receptor Siglec-7." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 176.13. http://dx.doi.org/10.4049/jimmunol.208.supp.176.13.
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Abstract Glycans decorate almost all proteins and lipids on the cell surface, and they play a critical role in cell-cell interactions. Terminal sialic acid addition to glycans helps the immune system distinguish between “self” and “non-self.” The enzyme ST8sia6 adds alpha-2,8 disialic acids to cell surface receptors, and this sialic acid addition promotes binding to the inhibitory receptor Siglec-E. We have previously shown ST8sia6 overexpression in tumors accelerates tumor growth in a Siglec-E dependent manner. Proteomic analysis in murine tumor lines identified CD44 as a target for ST8sia6. CD44 expression on tumors is associated with metastasis and chemoresistance. Since humans and mice have different sialic acid structures, we examined whether ST8sia6 makes CD44 a ligand to the ortholog of Siglec-E in human cells, Siglec-7. When HEK293T cells overexpress ST8sia6 and CD44 together, Siglec-7 binding was significantly increased compared to ST8sia6 or CD44 overexpression alone. Thus, ST8sia6 has a conserved role from mouse to human in decorating CD44 with sialic acid. Our study proposes that ST8sia6 sialylation of CD44 could suppress immune responses and enables cancer progression in human. Supported by grant: 2T32AI007425-23 to DJF and R01 CA243545-01S1 to V.S.S.
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Thuy-Boun, Peter S., and Dennis W. Wolan. "A glycal-based photoaffinity probe that enriches sialic acid binding proteins." Bioorganic & Medicinal Chemistry Letters 29, no. 18 (September 2019): 2609–12. http://dx.doi.org/10.1016/j.bmcl.2019.07.054.
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Hurtado-Ziola, Nancy, Justin L. Sonnenburg, and Ajit Varki. "Differential Expression and Function of the CD33-Related Siglecs between Humans and Great Apes." Blood 104, no. 11 (November 16, 2004): 1466. http://dx.doi.org/10.1182/blood.v104.11.1466.1466.
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Abstract The Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are a recently discovered family of mammalian glycan-binding proteins that have been shown to recognize the terminal sialic acids of glycoproteins and glycolipids. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules, which are thought to be primarily expressed on cells of the innate immune system. All CD33rSiglecs are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that usually has two tyrosine-based signaling motifs, one of which conforms to a canonical negative regulatory ITIM motif. Although the true function of the CD33rSiglecs has yet to be discovered, available data are most consistent with an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. CD33rSiglecs also interact with sialic acids on the same cell surface, typically resulting in “masking” of their sialic acid-binding sites. Our recent studies have shown that humans and non-human primates have a similar clustered localization of CD33rSiglec genes, and that true orthologs can generally be identified within each cluster (Angata et al., PNAS, in press). However, humans no longer express CMP-sialic acid hydroxylase (CMAH) the enzyme required to generate one of the potential CD33rSiglec sialic acid ligands called N-glycolylneuraminic acid (Neu5Gc), from its precursor N-acetylneuraminic acid (Neu5Ac). This genetic change occurred after our last common ancestor with the great apes, and dramatically altered the “Sialome” (the sialic acid makeup of a specific species) of humans when compared to that of the great apes. While great ape blood cells express about equal amounts of Neu5Ac and Neu5Gc, human blood cells express almost exclusively Neu5Ac. We also recently discovered that preferential recognition of Neu5Gc is the ancestral condition of most or all of the great ape (chimpanzee and gorilla) CD33rSiglecs (Sonnenburg JL, Altheide TK, Varki A. Glycobiology.14:339–46, 2004). We therefore reasoned that the sudden and major change in the sialome of our hominid ancestors could have had a significant impact on the evolution, binding specificities and expression patterns of CD33rSiglecs. Indeed, we have found that all human CD33rSiglecs can recognize both Neu5Ac and Neu5Gc. This presumably represents an evolutionarily-selected “relaxation” in binding specificity that was necessary to “remask” the Siglecs that had lost their Neu5Gc ligands. Also, there are differences in CD33rSiglec expression on monocytes and neutrophils between humans and great apes (chimp, bonobo, gorilla and orangutan). Furthermore, while great ape cells often show multiple populations with different signal intensities, humans express a single bright peak for each Siglec in flow cytometry. Surprisingly, while humans showed almost no CD33rSiglec expression on lymphocytes, the great apes show a moderate to high expression of some Siglecs on these cells. Total leukocyte expression of some CD33rSiglecs also shows differences between humans and great apes. Overall, CD33rSiglecs appear to be rapidly evolving in primates, with an apparent further acceleration of changes in humans. Additional studies are needed to define the mechanistic details, as well as the implications for human health and disease.
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Gonzalez-Gil, Anabel, and Ronald L. Schnaar. "Siglec Ligands." Cells 10, no. 5 (May 20, 2021): 1260. http://dx.doi.org/10.3390/cells10051260.
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A dense and diverse array of glycans on glycoproteins and glycolipids decorate all cell surfaces. In vertebrates, many of these carry sialic acid, in a variety of linkages and glycan contexts, as their outermost sugar moiety. Among their functions, glycans engage complementary glycan binding proteins (lectins) to regulate cell physiology. Among the glycan binding proteins are the Siglecs, sialic acid binding immunoglobulin-like lectins. In humans, there are 14 Siglecs, most of which are expressed on overlapping subsets of immune system cells. Each Siglec engages distinct, endogenous sialylated glycans that initiate signaling programs and regulate cellular responses. Here, we explore the emerging science of Siglec ligands, including endogenous sialoglycoproteins and glycolipids and synthetic sialomimetics. Knowledge in this field promises to reveal new molecular pathways controlling cell physiology and new opportunities for therapeutic intervention.
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Chappell, James D., Joy L. Duong, Benjamin W. Wright, and Terence S. Dermody. "Identification of Carbohydrate-Binding Domains in the Attachment Proteins of Type 1 and Type 3 Reoviruses." Journal of Virology 74, no. 18 (September 15, 2000): 8472–79. http://dx.doi.org/10.1128/jvi.74.18.8472-8479.2000.
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ABSTRACT The reovirus attachment protein, ς1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The ς1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of ς1 that binds cell surface carbohydrate. Chimeric and truncated ς1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-ς1 antibodies, and oligomerization indicates that the chimeric and truncated ς1 proteins are properly folded. To assess carbohydrate binding, recombinant ς1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated ς1 proteins, the sialic acid-binding domain of type 3 ς1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted β-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of ς1 protein purified from virions. In contrast, the homologous region of T1L ς1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 ς1 tail. Furthermore, our findings indicate that T1L and T3D ς1 proteins contain different arrangements of receptor-binding domains.
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VONLAUFEN, N., A. NAGULESWARAN, C. GIANINAZZI, and A. HEMPHILL. "Characterization of the fetuin-binding fraction ofNeospora caninumtachyzoites and its potential involvement in host-parasite interactions." Parasitology 134, no. 6 (February 12, 2007): 805–17. http://dx.doi.org/10.1017/s0031182006002186.
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SUMMARYTerminal sialic acid residues on surface-associated glycoconjugates mediate host cell interactions of many pathogens. Addition of sialic acid-rich fetuin enhanced, and the presence of the sialidiase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reduced, the physical interaction ofNeospora caninumtachyzoites and bradyzoites with Vero cell monolayers. Thus,Neosporaextracts were subjected to fetuin-agarose affinity chromatography in order to isolate components potentially interacting with sialic acid residues. SDS-PAGE and silver staining of the fetuin binding fraction revealed the presence of a single protein band of ∼65 kDa, subsequently named NcFBP (Neospora caninumfetuin-binding protein), which was localized at the apical tip of the tachyzoites and was continuously released into the surrounding medium in a temperature-independent manner. NcFBP readily interacted with Vero cells and bound to chondroitin sulfate A and C, and anti-NcFBP antibodies interfered in tachyzoite adhesion to host cell monolayers. In additon, analysis of the fetuin binding fraction by gelatin substrate zymography was performed, and demonstrated the presence of two bands of 96 and 140 kDa exhibiting metalloprotease-activity. The metalloprotease activity readily degraded glycosylated proteins such as fetuin and bovine immunoglobulin G heavy chain, whereas non-glycosylated proteins such as bovine serum albumin and immunoglobulin G light chain were not affected. These findings suggest that the fetuin-binding fraction ofNeospora caninumtachyzoites contains components that could be potentially involved in host-parasite interactions.
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Urbanek, Kelly, Danica M. Sutherland, Robert C. Orchard, Craig B. Wilen, Jonathan J. Knowlton, Pavithra Aravamudhan, Gwen M. Taylor, Herbert W. Virgin, and Terence S. Dermody. "Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection." Journal of Virology 95, no. 2 (October 21, 2020): e01571-20. http://dx.doi.org/10.1128/jvi.01571-20.
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ABSTRACTEngagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA−, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA−, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA− in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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Crocker, Paul R., and Jiquan Zhang. "New I-type lectins of the CD 33-related siglec subgroup identified through genomics." Biochemical Society Symposia 69 (October 1, 2002): 83–94. http://dx.doi.org/10.1042/bss0690083.
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Siglecs are sialic-acid-binding proteins of the Ig superfamily that are involved in cell–cell interactions and signalling. In recent years, several novel siglecs that are highly related to CD33/Siglec-2 have been identified through genomics and functional screens. In addition to their distinct sialic-acid-binding properties, most of these novel siglecs bear tyrosine-based signalling motifs that are typically found in inhibitory receptors of the immune system. The restricted expression patterns of CD33-related siglecs in the haemopoietic and immune systems suggests that they are involved in regulating leucocyte activation during inflammatory and immune responses.
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Zárate, Selene, Pedro Romero, Rafaela Espinosa, Carlos F. Arias, and Susana López. "VP7 Mediates the Interaction of Rotaviruses with Integrin αvβ3 through a Novel Integrin-Binding Site." Journal of Virology 78, no. 20 (October 15, 2004): 10839–47. http://dx.doi.org/10.1128/jvi.78.20.10839-10847.2004.
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ABSTRACT Rotavirus entry is a complex multistep process that depends on the trypsin cleavage of the virus spike protein VP4 into polypeptides VP5 and VP8 and on the interaction of these polypeptides and of VP7, the second viral surface protein, with several cell surface molecules, including integrin αvβ3. We characterized the effect of the trypsin cleavage of VP4 on the binding to MA104 cells of the sialic acid-dependent virus strain RRV and its sialic acid-independent variant, nar3. We found that, although the trypsin treatment did not affect the attachment of these viruses to the cell surface, their binding was qualitatively different. In contrast to the trypsin-treated viruses, which initially bound to the cell surface through VP4, the non-trypsin-treated variant nar3 bound to the cell through VP7. Amino acid sequence comparison of the surface proteins of rotavirus and hantavirus, both of which interact with integrin αvβ3 in an RGD-independent manner, identified a region shared by rotavirus VP7 and hantavirus G1G2 protein in which six of nine amino acids are identical. This region, which is highly conserved among the VP7 proteins of different rotavirus strains, mediates the binding of rotaviruses to integrin αvβ3 and probably represents a novel binding motif for this integrin.
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Greenberger, L. M., and K. H. Pfenninger. "Membrane glycoproteins of the nerve growth cone: diversity and growth regulation of oligosaccharides." Journal of Cell Biology 103, no. 4 (October 1, 1986): 1369–82. http://dx.doi.org/10.1083/jcb.103.4.1369.
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A subcellular fraction prepared from fetal rat brain and enriched in growth cone membranes is analyzed for its lectin-binding proteins. Growth-associated glycoproteins are identified by comparing the growth cone glycoproteins with those of synaptosomes. Protein was resolved in one- or two-dimensional gels, electroblotted, and blots probed with radioiodinated concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinins I and II. In one-dimensional gels, each lectin recognizes approximately 20 polypeptides (with substantial overlap) most of which migrate diffusely and have relatively high molecular masses (range 30-200 kD). The seven major Coomassie-staining proteins of the membrane fraction (34-52 kD) are not the major lectin-binding proteins. In two-dimensional gels, the lectin-binding proteins are either streaked across the pH gradient or exist as multiple spots, indicating broad charge heterogeneity. Seven wheat germ agglutinin- and Ricinus communis agglutinin II-binding glycoproteins are present in greater abundance in growth cone fractions compared with synaptosomes. Most notably, an acidic, sialic acid-rich protein (27-30 kD, pI 4.0; termed gp27-30) is most abundant at postnatal day 4, but absent from adult brain. The protein's very acidic isoelectric point is due, at least in part, to its high sialic acid content. Growth regulation of specific protein-linked oligosaccharides suggests that they play a special role in growth cone function. In addition, the great diversity of growth cone glycoproteins from whole brain suggests glycoprotein heterogeneity among growth cones from different neuron types.
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Rustmeier, Strebl, and Stehle. "The Symmetry of Viral Sialic Acid Binding Sites–Implications for Antiviral Strategies." Viruses 11, no. 10 (October 14, 2019): 947. http://dx.doi.org/10.3390/v11100947.
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Virus infections are initiated by the attachment of the viral particle to protein or carbohydrate receptors on the host cell. Sialic acid-bearing glycan structures are prominently displayed at the cell surface, and, consequently, these structures can function as receptors for a large number of diverse viruses. Structural biology research has helped to establish the molecular bases for many virus–sialic acid interactions. Due to the icosahedral 532 point group symmetry that underlies many viral capsids, the receptor binding sites are frequently arranged in a highly symmetric fashion and linked by five-fold, three-fold, or two-fold rotation axes. For the inhibition of viral attachment, one emerging strategy is based on developing multivalent sialic acid-based inhibitors that can simultaneously engage several of these binding sites, thus binding viral capsids with high avidity. In this review, we will evaluate the structures of non-enveloped virus capsid proteins bound to sialylated glycan receptors and discuss the potential of these structures for the development of potent antiviral attachment inhibitors.
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Mitchell, Jennifer, and Paul M. Sullam. "Streptococcus mitis Phage-Encoded Adhesins Mediate Attachment to α2-8-Linked Sialic Acid Residues on Platelet Membrane Gangliosides." Infection and Immunity 77, no. 8 (June 8, 2009): 3485–90. http://dx.doi.org/10.1128/iai.01573-08.
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ABSTRACT The direct binding of bacteria to human platelets contributes to the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 is mediated in part by two bacteriophage-encoded proteins, PblA and PblB. However, the platelet membrane receptor for these adhesins has been unknown. In this study, we demonstrate that these proteins mediate attachment of bacterial cells to sialylated gangliosides on the platelet cell surface. Desialylation of human platelet monolayers reduced adherence of SF100, whereas treatment of the platelets with N- or O-glycanases did not affect platelet binding. Treatment of platelets with sialidases having different linkage specificities showed that removal of α2-8-linked sialic acids resulted in a marked reduction in bacterial binding. Preincubation of SF100 with ganglioside GD3, a glycolipid containing α2-8-linked sialic acids that is present on platelet membranes, blocked subsequent binding of this strain to these cells. In contrast, GD3 had no effect on the residual binding of platelets by strain PS344, an isogenic ΔpblA ΔpblB mutant. Preincubating platelets with specific monoclonal antibodies to ganglioside GD3 also inhibited binding of SF100 to platelets, but again, they had no effect on binding by PS344. When the direct binding of S. mitis strains SF100 and PS344 to immobilized gangliosides was tested, binding of PS344 to GD3 was reduced by 70% compared to the parent strain. These results indicated that platelet binding by SF100 is mediated by the interaction of PblA and PblB with α2-8-linked sialic acids on ganglioside GD3.
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Kiser, Zachary Monroe, Greta L. Becker, Julia Nguyen, Anel Lizcano, John D. Belcher, Ajit P. Varki, and Gregory M. Vercellotti. "Decreased Erythrocyte Binding Capability for Neutrophil Siglec-9 Is a Source of Oxidative Stress in Sickle Cell Disease." Blood 132, Supplement 1 (November 29, 2018): 3650. http://dx.doi.org/10.1182/blood-2018-99-113579.
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Abstract Introduction Oxidative stress and inflammation promote hemolysis and vaso-occlusion in sickle cell disease (SCD). Erythrocytes can play a pro-oxidative and anti-oxidative role in disease-associated inflammation through iron-driven free radical production and endogenous anti-oxidants. In SCD, HbS accelerated auto-oxidation, iron de-compartmentalization, and inflammatory cell-derived oxidants drive this oxidative stress. CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids in "Self-Associated Molecular Patterns" (SAMPs) and typically inhibit innate immune cell functions via cytosolic signaling. Recent studies have shown that Siglec-9 on human neutrophils in circulating blood interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactivity, including reactive oxygen species (ROS) production. Modification of erythrocyte membrane sialic acids interferes with their ability to inhibit neutrophil activation and oxidative burst. As erythrocytes age and undergo cellular damage there is a loss of membrane bound sialoglycoproteins. Several studies have indicated that this reduction in the sialome is accelerated on the sickle erythrocyte. We hypothesize that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and that decreased binding of neutrophil Siglec-9 to sickle erythrocyte sialic acid enhances neutrophil activation and oxidative burst. Methods & Results Binding of recombinant Siglec-9-Fc protein to AA (n=9) and SS erythrocytes (n=7) was measured using flow cytometry. SS erythrocytes displayed significantly less Siglec-9-Fc binding 45% ± 11.9 (mean ± SEM) compared to AA erythrocytes 82% ± 5.4 (p=.03). Treatment of AA erythrocytes with neuraminidase to remove sialic acid decreased binding to 4% ± 7.9. Neutrophil ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Neutrophils were purified from AA donors (n=5) and SS (n=11) or AA erythrocytes (n=5) were added to the neutrophils at a ratio of 50 erythrocytes to 1 neutrophil. Oxidative burst was stimulated using phorbol 12-myristate 13-acetate (PMA). AA erythrocytes decreased PMA-stimulated neutrophil ROS production by 84% ± 6.7. In contrast, SS erythrocytes decreased PMA-stimulated neutrophil ROS production by 53% ± 6.8 (p=0.03,). Recent studies have shown that neutrophil extracellular trap (NET) formation is pathogenic in SCD. We added AA and SS erythrocytes (50:1) to neutrophils stimulated with PMA and NET formation was assessed using Syto Orange, Syto Green and confocal microscopy. PMA-stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation. In contrast, AA erythrocytes treated with neuraminidase to remove sialic acid had increased NET formation. PMA-stimulated neutrophils incubated with SS erythrocytes showed increased NET formation. Conclusions A constant disease state of oxidative stress and inflammation underlies SCD pathophysiology. These data demonstrate that SS erythrocytes with decreased membrane sialic acid are deficient in binding to neutrophil Siglec-9. The decreased binding of SS erythrocytes to neutrophil Siglec-9 diminishes the ability of SS erythrocytes to properly modulate neutrophil activation, which may contribute to the oxidative stress and increased state of basal inflammation inherent to SCD. The overall reduction in number of RBCs in SS may also be a factor? Figure. Figure. Disclosures Belcher: CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding.
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Cavaldesi, Michaela, Maddalena Caruso, Olga Sthandier, Paolo Amati, and Marie Isabelle Garcia. "Conformational Changes of Murine Polyomavirus Capsid Proteins Induced by Sialic Acid Binding." Journal of Biological Chemistry 279, no. 40 (October 2004): 41573–79. http://dx.doi.org/10.1074/jbc.m405995200.
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Liu, Yang, Pengwei Huang, Ming Tan, Yiliu Liu, Jacek Biesiada, Jarek Meller, Alejandro A. Castello, Baoming Jiang, and Xi Jiang. "Rotavirus VP8*: Phylogeny, Host Range, and Interaction with Histo-Blood Group Antigens." Journal of Virology 86, no. 18 (July 3, 2012): 9899–910. http://dx.doi.org/10.1128/jvi.00979-12.
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The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.
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Iwabuchi, K., I. Nagaoka, A. Someya, and T. Yamashita. "Type IV collagen-binding proteins of neutrophils: possible involvement of L-selectin in the neutrophil binding to type IV collagen." Blood 87, no. 1 (January 1, 1996): 365–72. http://dx.doi.org/10.1182/blood.v87.1.365.365.
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Abstract To isolate type IV collagen-binding proteins, 125I-labeled human- neutrophil extracts were chromatographed on a type IV collagen- Sepharose column. The affinity chromatography-separated fraction contained the four radioactive proteins with apparent molecular masses of 28, 49, 67, and 95 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis indicated that the 95-kD proteins contained both L-selectin and nonspecific cross-reacting antigen 90 (NCA90), and that the 67-kD protein was the 67-kD elastin/laminin-binding protein (67BP). The data obtained with the type IV collagen-affinity chromatography and the immunoaffinity chromatographies using anti-L-selectin and anti-NCA90 monoclonal antibodies (MoAbs) have shown that L-selectin is closely associated with 67BP and the 49-kD protein, and that NCA90 is associated with 67BP, the 28-kD and 49-kD proteins. Among these binding proteins, sialic acid residues were contained in 67BP, L-selectin, and NCA90, but not in the 28-kD and 49-kD proteins. Sialidase treatment completely abolished both the binding affinity of the type IV collagen-binding proteins to type IV collagen and the neutrophil adherence to type IV collagen-coated plastic. Thus, the sialic acid residues of 67BP, L- selectin, and NCA90 seem to be important for the binding of neutrophils to type IV collagen. Furthermore, L-selectin IgG chimeric protein directly bound to type IV collagen-Sepharose column, and anti-L- selectin MoAb DREG56 inhibited the neutrophil adherence to type IV collagen-coated plastic by 51%. These observations suggest that L- selectin likely plays a role in the neutrophil binding to type IV collagen, although neutrophils have several kinds of adhesion molecules for type IV collagen such as L-selectin, NCA90, 67BP, and the 28-kD and 49-kD proteins.
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Iwabuchi, K., I. Nagaoka, A. Someya, and T. Yamashita. "Type IV collagen-binding proteins of neutrophils: possible involvement of L-selectin in the neutrophil binding to type IV collagen." Blood 87, no. 1 (January 1, 1996): 365–72. http://dx.doi.org/10.1182/blood.v87.1.365.bloodjournal871365.
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To isolate type IV collagen-binding proteins, 125I-labeled human- neutrophil extracts were chromatographed on a type IV collagen- Sepharose column. The affinity chromatography-separated fraction contained the four radioactive proteins with apparent molecular masses of 28, 49, 67, and 95 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis indicated that the 95-kD proteins contained both L-selectin and nonspecific cross-reacting antigen 90 (NCA90), and that the 67-kD protein was the 67-kD elastin/laminin-binding protein (67BP). The data obtained with the type IV collagen-affinity chromatography and the immunoaffinity chromatographies using anti-L-selectin and anti-NCA90 monoclonal antibodies (MoAbs) have shown that L-selectin is closely associated with 67BP and the 49-kD protein, and that NCA90 is associated with 67BP, the 28-kD and 49-kD proteins. Among these binding proteins, sialic acid residues were contained in 67BP, L-selectin, and NCA90, but not in the 28-kD and 49-kD proteins. Sialidase treatment completely abolished both the binding affinity of the type IV collagen-binding proteins to type IV collagen and the neutrophil adherence to type IV collagen-coated plastic. Thus, the sialic acid residues of 67BP, L- selectin, and NCA90 seem to be important for the binding of neutrophils to type IV collagen. Furthermore, L-selectin IgG chimeric protein directly bound to type IV collagen-Sepharose column, and anti-L- selectin MoAb DREG56 inhibited the neutrophil adherence to type IV collagen-coated plastic by 51%. These observations suggest that L- selectin likely plays a role in the neutrophil binding to type IV collagen, although neutrophils have several kinds of adhesion molecules for type IV collagen such as L-selectin, NCA90, 67BP, and the 28-kD and 49-kD proteins.
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Kosik, Ivan, and Jonathan W. Yewdell. "Influenza Hemagglutinin and Neuraminidase: Yin–Yang Proteins Coevolving to Thwart Immunity." Viruses 11, no. 4 (April 16, 2019): 346. http://dx.doi.org/10.3390/v11040346.
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Influenza A virions possess two surface glycoproteins—the hemagglutinin (HA) and neuraminidase (NA)—which exert opposite functions. HA attaches virions to cells by binding to terminal sialic acid residues on glycoproteins/glycolipids to initiate the infectious cycle, while NA cleaves terminal sialic acids, releasing virions to complete the infectious cycle. Antibodies specific for HA or NA can protect experimental animals from IAV pathogenesis and drive antigenic variation in their target epitopes that impairs vaccine effectiveness in humans. Here, we review progress in understanding HA/NA co-evolution as each acquires epistatic mutations to restore viral fitness to mutants selected in the other protein by host innate or adaptive immune pressure. We also discuss recent exciting findings that antibodies to HA can function in vivo by blocking NA enzyme activity to prevent nascent virion release and enhance Fc receptor-based activation of innate immune cells.
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Jackson, Shawn, Ana de las Heras Sanchez, Hafiz Ahmed, Arun Ammayappan, Vikram Vakharia, and Gerardo Vasta. "Galectin binding to and expression modulation by viruses in zebrafish (89.51)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 89.51. http://dx.doi.org/10.4049/jimmunol.184.supp.89.51.
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Abstract The galectin family of β-galactoside-binding proteins is evolutionarily conserved. Galectins are known to interact with and affect the infectivity and/or pathogenicity of several viruses. We hypothesize that galectin interactions with viruses influence anti-viral immune responses. We investigate the interactions of zebrafish galectins with the glycosylated (G) protein of the rhabdovirus IHNV. We (a) characterized the carbohydrates present on IHNV proteins, and (b) identified direct interactions between zebrafish galectins (Drgal) and IHNV. We assessed the effects of immune challenge on (c) galectin and (d) cytokine expression in zebrafish. Several IHNV proteins exhibit complex glycosylation patterns. One or multiple IHNV proteins exhibit terminally linked mannose, sialic acid terminally linked to galactose, galactose-β(1-3)-GalNAc, sialic acid terminally linked to galactose or GalNAc, and galactose-β(1-4)-GlcNAc. The proto type Drgal1-L2, the chimera type Drgal3-L1 and the tandem-repeat type Drgal9-L2 all appear to bind to one or more IHNV proteins. All tested Drgal appear to bind to the IHNV G protein. ZFL cells endogenously express mRNA encoding Drgal1-L2, -L3, and -L4, Drgal3-L1 and -L2, and Drgal9-L1 mRNA. Drgal mRNA expression in ZFL cells was not induced by treatment with polyI:C. ZFL cells were found to endogenously express mRNA encoding Mx, TNFα, and IFNαβ. Treatment of these cells with polyI:C induced the expression of mRNA encoding IL-10 and IFNγ2.
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Powell, L. D., S. W. Whiteheart, and G. W. Hart. "Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction." Journal of Immunology 139, no. 1 (July 1, 1987): 262–70. http://dx.doi.org/10.4049/jimmunol.139.1.262.
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Abstract The Ia+ B cell lymphoma, AKTB-1b, fails to stimulate thymic lymphocytes in a one-way mixed lymphocyte reaction unless pretreated with sialidase or inhibitors of N-linked oligosaccharide processing. A comparison of different sialidases and sialyltransferases suggests that the removal of only a subset of total surface sialic acid, rather than net desialylation of the cell surface, is required. Three sialidases were compared, including Vibrio cholerae (VC) and Clostridium perfringens (CP), which will cleave alpha 2-3, alpha 2-6, and alpha 2-8, sialic acid linkages, and Newcastle Disease virus (NDV), which will remove only alpha 2-3 and alpha 2-8 linked sialic acid. When treated with equivalent units of sialidase, CP-, VC-, and NDV-treated cells were 24-fold, sixfold, and threefold better stimulators than untreated cells. In contrast, VC released 1.3-fold and 2.5-fold more sialic acid per cell than did CP or NDV, respectively. Furthermore, VC was superior in reducing the levels of binding of the sialic acid-specific lectin, Limulus polyphemus agglutinin, in exposing Gal beta 1-3GalNAc and Gal beta 1-4GlcNAc residues, and in desialylating gangliosides. Two-dimensional gel analysis indicated that VC and CP were both equal and superior to NDV in the desialylation of iodinatable cell-surface proteins, including H-2Kk, I-A beta k, and a highly sialylated 65,000 dalton protein of unknown identity. Maximal resialylation of CP-treated cells with exogenously added CMP-NANA and either the alpha 2-3(Gal beta 1-3GalNAc) or alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase did not reduce the stimulatory capacity of these cells. However, resialylation of VC-treated cells with just CMP-NANA alone resulted in 49% reversal of their stimulatory capacity, and no additional reversal could be achieved with either of the sialyltransferases. Although the alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase was capable of adding back approximately 10% of the sialic acid removed, the endogenous activity added back approximately 0.1% of the total sialic acid removed. SDS-PAGE gels of the sialylated cells indicated that the exogenously added sialyltransferase labeled many different proteins, whereas the endogenous activity labeled far fewer proteins, predominantly in 46,000 and 25,000 m.w. range. Both the desialylation and resialylation data suggest that the sialidase-dependent stimulation is due to the desialylation of specific membrane structures. Together with previous studies, these data suggest that the sialic acids involved are probably alpha 2-6 linked to N-linked glycosyl moieties.
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Padler-Karavani, Vered, Nancy Hurtado-Ziola, Andrea Verhagen, Justin Sonnenburg, Xi Chen, Ajit Varki, and Takashi Angata. "Rapid evolution of the binding specificities and expression patterns of CD33-related Siglecs in primates (181.4)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 181.4. http://dx.doi.org/10.4049/jimmunol.188.supp.181.4.
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Abstract Siglecs (Sialic acid-binding Immunoglobulin-like Lectins) are vertebrate sialic acid (Sia) binding proteins mostly expressed in hematopoietic and immune cells. They are type-I proteins with an extracellular N-terminal Sia-binding Ig-like V-set domain, one or more C2-set Ig-like domains, a transmembrane domain and a C-terminal cytoplasmic tail. CD33-related Siglecs (CD33rSiglecs) are a genetically clustered subgroup. Prior comparative genomic analysis indicated accelerated evolution of CD33rSiglecs selectively occurring in the Sia-binding V-set domain, suggesting that this domain is under the greatest selection pressure. To gain further insight into this rapid evolution of CD33rSiglecs, we prepared recombinant form of the N-terminal domains of CD33rSiglecs from human, chimpanzee, and baboon, and compared their sialoglycan binding-specificities. Using ELISA on a panel of gangliosides or analysis on a sialoglycan microarray we show that binding patterns are widely divergent between the three species. Some show preference to Neu5Gc over Neu5Ac, especially in chimpanzees and baboons, indicating an evolutionary adjustment to the loss of Neu5Gc expression in humans. Expression patterns of CD33rSiglecs on circulating blood cells of humans and related great apes also show evidence for rapid evolution, and overall expression is greater in apes compared to human. The selection mechanisms driving this rapid evolution will be discussed.
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Hidaka, H., and N. H. Fidge. "Affinity purification of the hepatic high-density lipoprotein receptor identifies two acidic glycoproteins and enables further characterization of their binding properties." Biochemical Journal 284, no. 1 (May 15, 1992): 161–67. http://dx.doi.org/10.1042/bj2840161.
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Several high-density lipoprotein (HDL)-binding proteins, candidates for the putative HDL receptor, have recently been identified, including two membrane proteins: HB1 of 120 kDa and HB2 of 100 kDa, present in rat and human liver plasma membranes respectively. Further insights into their function however, have been hampered by poor recoveries of these hydrophobic peptides, and the present work was undertaken to improve yields and enable a more detailed investigation of their properties. A significant improvement has been achieved using two affinity chromatographic procedures, one exploiting the glycoprotein nature of the proteins and the other exploiting their ligand properties, which in combination resulted in considerable enrichment of HB1 and HB2. Thus DEAE-Sephacel fractionation (0.05-0.2 M-NaCl) of CHAPS-solubilized plasma membranes yielded active HDL-binding proteins which bound to concanavalin A-Sepharose or wheat-germ-lectin-Sepharose columns and retained their binding activity after eluting with methyl-alpha-D-mannoside or N-acetylglucosamine respectively. These glycoproteins were further purified by affinity chromatography using apo-HDL-Sepharose columns. Final purification required preparative SDS/PAGE. Investigation of the carbohydrate moieties of the proteins using glycosidases and two-dimensional gel electrophoresis revealed pI values ranging from 4.6 to 4.9 and from 4.5 to 4.7 for HB1 and HB2 respectively, which after treatment with neuraminidase shifted towards basic pH (5.4-5.7 and 5.3-5.5 respectively). The molecular masses were decreased to 115 kDa and 95 kDa respectively, demonstrating that sialic acid residues contributed significantly to the negative charge of the glycosylated peptides. Treatment with the enzyme peptide N-glycosidase F (N-glycanase) resulted in a decrease in molecular mass of HB1 and HB2 to 105 kDa and 80 kDa respectively, but endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment was not effective. Neither neuraminidase nor N-glycanase treatment destroyed activity, suggesting that sialic acids or N-linked oligosaccharides are not important determinants of HDL binding. Digestion of plasma membranes with trypsin or Pronase resulted in a loss of activity of both HB1 and HB2 that was not influenced by prior treatment with neuraminidase, suggesting that sialic acid residues play no protective role against proteolytic cleavage of HDL receptor proteins.
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Gangi Setty, Thanuja, and S. Ramaswamy. "Structural and Functional Characterization of Periplasmic Sialic Acid Binding Proteins from Pathogenic Bacteria." Biophysical Journal 116, no. 3 (February 2019): 151a. http://dx.doi.org/10.1016/j.bpj.2018.11.841.
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Wu, W., P. H. Harley, J. A. Punt, S. O. Sharrow, and K. P. Kearse. "Identification of CD8 as a peanut agglutinin (PNA) receptor molecule on immature thymocytes." Journal of Experimental Medicine 184, no. 2 (August 1, 1996): 759–64. http://dx.doi.org/10.1084/jem.184.2.759.
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Differentiation of most T lymphocytes occurs within the thymus and is characterized by variable expression of CD4/CD8 coreceptor molecules, increased surface density of T cell antigen receptor (TCR) alpha beta proteins, and decreased expression of glycan chains recognized by the galactose-specific lectin peanut agglutinin (PNA). Although appreciated for several decades that PNA agglutination is useful for the physical separation of immature and mature thymocyte sub-populations, the identity of specific PNA-binding glycoproteins expressed on immature thymocytes remains to be determined. In the current report, we studied the expression of PNA-specific glycans on immature and mature T cells and used lectin affinity chromatography and immunoprecipitation techniques to characterize PNA-binding glycoproteins on thymocytes. Our data demonstrate that PNA-specific glycans are localized on a relatively small subset of thymocyte surface proteins, several of which were specifically identified, including CD43, CD45, and suprisingly, CD8 molecules. CD8 alpha and CD8 alpha' proteins bound to PNA in the absence of CD8 beta expression showing that O-glycans on CD8 beta glycoproteins are not necessary for PNA binding and that glycosylation of CD8 alpha and CD8 alpha' proteins proceeds effectively in the absence of CD8 beta. Finally, we demonstrate that PNA binding of CD8 is developmentally regulated by sialic acid addition as CD8 proteins from mature T cells bound to PNA only after sialidase treatment. These studies identify CD8 as a PNA receptor molecule on immature thymocytes and show that PNA binding of CD8 on immature and mature T cells is developmentally regulated by sialic acid modification.
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Strotmeier, Jasmin, Kwangkook Lee, Anne K. Völker, Stefan Mahrhold, Yinong Zong, Johannes Zeiser, Jie Zhou, et al. "Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner." Biochemical Journal 431, no. 2 (September 28, 2010): 207–16. http://dx.doi.org/10.1042/bj20101042.
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The extraordinarily high toxicity of botulinum neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven BoNT (botulinum neurotoxin) serotypes A–G inhibit acetylcholine release leading to flaccid paralysis. Uptake of BoNT/A, B, E, F and G requires a dual interaction with gangliosides and the synaptic vesicle proteins synaptotagmin or SV2 (synaptic vesicle glycoprotein 2), whereas little is known about the cell entry mechanisms of the serotypes C and D, which display the lowest amino acid sequence identity compared with the other five serotypes. In the present study we demonstrate that the neurotoxicity of BoNT/D depends on the presence of gangliosides by employing phrenic nerve hemidiaphragm preparations derived from mice expressing the gangliosides GM3, GM2, GM1 and GD1a, or only GM3 [a description of our use of ganglioside nomenclature is given in Svennerholm (1994) Prog. Brain Res. 101, XI–XIV]. High-resolution crystal structures of the 50 kDa cell-binding domain of BoNT/D alone and in complex with sialic acid, as well as biological analyses of single-site BoNT/D mutants identified two carbohydrate-binding sites. One site is located at a position previously identified in BoNT/A, B, E, F and G, but is lacking the conserved SXWY motif. The other site, co-ordinating one molecule of sialic acid, resembles the second ganglioside-binding pocket (the sialic-acid-binding site) of TeNT (tetanus neurotoxin).
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Cassidy, L. F., D. S. Lyles, and J. S. Abramson. "Depression of polymorphonuclear leukocyte functions by purified influenza virus hemagglutinin and sialic acid-binding lectins." Journal of Immunology 142, no. 12 (June 15, 1989): 4401–6. http://dx.doi.org/10.4049/jimmunol.142.12.4401.
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Abstract Infection of polymorphonuclear leukocytes (PMNL) with influenza virus causes depression of PMNL metabolic and bactericidal activities. The studies reported here were undertaken to determine whether the hemagglutinin (HA) glycoprotein of influenza virus mediates this depression. PMNL were incubated with purified HA and the oxidative responses to exogenous stimuli were measured. The results indicate that HA, in either liposomes or protein aggregates referred to as rosettes, depressed PMNL oxidative responses. Depression was observed within 2 min of initial interaction of HA with PMNL and lasted more than 2 h. The membrane fusion activity of HA requires proteolytic cleavage of the HA, whereas the receptor binding activity does not. There was no difference in the ability of virions with cleaved or uncleaved HA to depress PMNL responses suggesting that the fusion event is not required for PMNL dysfunction. Inasmuch as the HA glycoprotein binds to sialic acid-containing receptors on the surface of the PMNL, we tested whether other sialic acid-specific binding proteins can mediate the reduction of PMNL responses. Sialic acid-specific lectins from Limulus polyphemus or Limax flavus were incubated with PMNL before measuring their responses to secondary stimulus. Depression was observed upon incubation with the lectins similar to that seen upon incubation with the HA or influenza virus. These results suggest that attachment of influenza virus to sialic acid-containing receptors is responsible at least in part, for suppressing PMNL oxidative responses.
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López-Bueno, Alberto, Mari-Paz Rubio, Nathan Bryant, Robert McKenna, Mavis Agbandje-McKenna, and José M. Almendral. "Host-Selected Amino Acid Changes at the Sialic Acid Binding Pocket of the Parvovirus Capsid Modulate Cell Binding Affinity and Determine Virulence." Journal of Virology 80, no. 3 (February 1, 2006): 1563–73. http://dx.doi.org/10.1128/jvi.80.3.1563-1573.2006.
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ABSTRACT The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 Å away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.
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Chi, Kaijun, Huilin Xu, Hanjie Li, Ganglong Yang, Xiaoman Zhou, and Xiao-Dong Gao. "Expression of a Siglec-Fc Protein and Its Characterization." Biology 12, no. 4 (April 10, 2023): 574. http://dx.doi.org/10.3390/biology12040574.
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The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines.
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Mohammed, Soran, and Natalie Ferry. "Characterization of Sialic Acid Affinity of the Binding Domain of Mistletoe Lectin Isoform One." International Journal of Molecular Sciences 22, no. 15 (July 31, 2021): 8284. http://dx.doi.org/10.3390/ijms22158284.
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Sialic acid (Sia) is considered as one of the most important biomolecules of life since its derivatives and terminal orientations on cell membranes and macromolecules play a major role in many biological and pathological processes. To date, there is only a limited number of active molecules that can selectively bind to Sia and this limitation has made the study of this glycan challenging. The lectin superfamily is a well-known family of glycan binding proteins, which encompasses many strong glycan binding peptides with diverse glycan affinities. Mistletoe lectin (ML) is considered one of the most active members of lectin family which was initially classified in early studies as a galactose binding lectin; more recent studies have suggested that the peptide can also actively bind to Sia. However, the details with respect to Sia binding of ML and the domain responsible for this binding are left unanswered because no comprehensive studies have been instigated. In this study, we sought to identify the binding domain responsible for the sialic acid affinity of mistletoe lectin isoform I (MLI) in comparison to the binding activity of elderberry lectin isoform I (SNA), which has long been identified as a potent Sia binding lectin. In order to execute this, we performed computational carbohydrate-protein docking for MLB and SNA with Neu5Ac and β-Galactose. We further analyzed the coding sequence of both lectins and identified their glycan binding domains, which were later cloned upstream and downstream to green fluorescent protein (GFP) and expressed in Escherichia coli (E. coli). Finally, the glycan affinity of the expressed fusion proteins was assessed by using different biochemical and cell-based assays and the Sia binding domains were identified.
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Urano-Tashiro, Yumiko, Ayako Yajima, Eizo Takashima, Yukihiro Takahashi, and Kiyoshi Konishi. "Binding of the Streptococcus gordonii DL1 Surface Protein Hsa to the Host Cell Membrane Glycoproteins CD11b, CD43, and CD50." Infection and Immunity 76, no. 10 (August 4, 2008): 4686–91. http://dx.doi.org/10.1128/iai.00238-08.
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ABSTRACTInfective endocarditis is frequently attributed to oral streptococci. The mechanisms of pathogenesis, however, are not well understood, although interaction between streptococci and phagocytes are thought to be very important. A highly expressed surface component ofStreptococcus gordonii, Hsa, which has sialic acid-binding activity, contributes to infective endocarditis in vivo. In the present study, we found thatS. gordoniiDL1 binds to HL-60 cells differentiated into monocytes, granulocytes, and macrophages. Using a glutathioneS-transferase (GST) fusion to the NR2 domain, which is the sialic acid-binding region of Hsa, we confirmed that the Hsa NR2 domain also binds to differentiated HL-60 cells. To identify which sialoglycoproteins on the surface of differentiated HL-60 cells are receptors for Hsa, intrinsic membrane proteins were assessed by bacterial overlay and far-Western blotting.S. gordoniiDL1 adhered to 100- to 150-kDa proteins, a reaction that was abolished by neuraminidase treatment. These sialoglycoproteins were identified as CD11b, CD43, and CD50 by GST pull-down assay and immunoprecipitation with each specific monoclonal antibody. These data suggest thatS. gordoniiDL1 Hsa specifically binds to three glycoproteins as receptors and that this interaction may be the initial bacterial binding step in infective endocarditis by oral streptococci.
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Büll, Christian, Rebecca Nason, Lingbo Sun, Julie Van Coillie, Daniel Madriz Sørensen, Sam J. Moons, Zhang Yang, et al. "Probing the binding specificities of human Siglecs by cell-based glycan arrays." Proceedings of the National Academy of Sciences 118, no. 17 (April 23, 2021): e2026102118. http://dx.doi.org/10.1073/pnas.2026102118.
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Siglecs are a family of sialic acid–binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2–3(6-O-sulfo)Galβ1–4GlcNAc (6′-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer’s disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid–binding proteins.
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Miller-Podraza, H., T. Larsson, J. Nilsson, S. Teneberg, M. Matrosovich, and L. Johansson. "Epitope dissection of receptor-active gangliosides with affinity for Helicobacter pylori and influenza virus." Acta Biochimica Polonica 45, no. 2 (June 30, 1998): 439–49. http://dx.doi.org/10.18388/abp.1998_4238.
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Receptor-active gangliosides with affinity for Helicobacter pylori and influenza virus were chemically modified and analyzed by negative ion fast atom bombardment mass spectrometry (FAB MS) or electron ionization mass spectrometry (EI MS) after permethylation. Derivatizations included mild periodate oxidation of the sialic acid glycerol tail or conversion of the carboxyl group to primary alcohol or amides. The modified gangliosides were then tested for binding affinity using thin-layer plates overlaid with labeled microbes or microbe-derived proteins. Mild periodate oxidation, which shortens sialic acid tail without destruction of sugar cores, abolished or drastically reduced binding of H. pylori and avian influenza virus to sialyl-3-paragloboside (S-3-PG). The same effect was observed in the case of binding of the human influenza virus to receptor-active gangliosides of human leukocytes. Conversion of S-3-PG or leukocyte gangliosides to primary alcohols or amides also abolished the binding. However, mild periodate oxidation had no effect on binding of NAP (neutrophil-activating protein of H. pylori) to the active ganglioside.
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Perdicchio, Maurizio, Juan M. Ilarregui, Marleen I. Verstege, Lenneke A. M. Cornelissen, Sjoerd T. T. Schetters, Steef Engels, Martino Ambrosini, et al. "Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells." Proceedings of the National Academy of Sciences 113, no. 12 (March 3, 2016): 3329–34. http://dx.doi.org/10.1073/pnas.1507706113.
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Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4+ and CD8+ T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen–loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E–mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro–established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance.