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Paul R Crocker - One of the best experts on this subject based on the ideXlab platform.
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Role of macrophage Sialoadhesin in host defense against the sialylated pathogen group B Streptococcus
Journal of Molecular Medicine, 2014Co-Authors: Yung-chi Chang, Paul R Crocker, Joshua Olson, Aaron Louie, Ajit Varki, Victor NizetAbstract:Several bacterial pathogens decorate their surfaces with sialic acid (Sia) residues within cell wall components or capsular exopolysaccharides. Sialic acid expression can promote bacterial virulence by blocking complement activation or by engagement of inhibitory sialic acid-binding immunoglobulin-like lectins (Siglecs) on host leukocytes. Expressed at high levels on splenic and lymph node macrophages, Sialoadhesin (Sn) is a unique Siglec with an elongated structure that lacks intracellular signaling motifs. Sialoadhesin allows macrophage to engage certain sialylated pathogens and stimulate inflammatory responses, but the in vivo significance of Sialoadhesin in infection has not been shown. We demonstrate that macrophages phagocytose the sialylated pathogen group B Streptococcus (GBS) and increase bactericidal activity via Sialoadhesin-sialic-acid-mediated recognition. Sialoadhesin expression on marginal zone metallophillic macrophages in the spleen trapped circulating GBS and restricted the spread of the GBS to distant organs, reducing mortality. Specific IgM antibody responses to GBS challenge were also impaired in Sialoadhesin-deficient mice. Thus, Sialoadhesin represents a key bridge to orchestrate innate and adaptive immune defenses against invasive sialylated bacterial pathogens. Key message Sialoadhesin is critical for macrophages to phagocytose and clear GBS. Increased GBS organ dissemination in the Sialoadhesin-deficient mice. Reduced anti-GBS IgM production in the Sialoadhesin-deficient mice.
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Peripheral prion disease pathogenesis is unaltered in the absence of Sialoadhesin (Siglec-1/CD169)
Immunology, 2014Co-Authors: Barry Bradford, Paul R Crocker, Neil A. MabbottAbstract:Summary Prions are a unique group of pathogens, which are considered to comprise solely of an abnormally folded isoform of the cellular prion protein. The accumulation and replication of prions within secondary lymphoid organs is important for their efficient spread from the periphery to the brain where they ultimately cause neurodegeneration and death. Mononuclear phagocytes (MNP) play key roles in prion disease pathogenesis. Some MNP appear to facilitate the propagation of prions to and within lymphoid tissues, whereas others may aid their clearance by phagocytosis and by destroying them. Our recent data show that an intact splenic marginal zone is important for the efficient delivery of prions into the B-cell follicles where they subsequently replicate upon follicular dendritic cells before infecting the nervous system. Sialoadhesin is an MNP-restricted cell adhesion molecule that binds sialylated glycoproteins. Sialoadhesin is constitutively expressed upon splenic marginal zone metallophilic and lymph node sub-capsular sinus macrophage populations, where it may function to bind sialylated glycoproteins, pathogens and exosomes in the blood and lymph via recognition of terminal sialic acid residues. As the prion glycoprotein is highly sialylated, we tested the hypothesis that Sialoadhesin may influence prion disease pathogenesis. We show that after peripheral exposure, prion pathogenesis was unaltered in Sialoadhesindeficient mice; revealing that lymphoid sequestration of prions is not mediated via Sialoadhesin. Hence, although an intact marginal zone is important for the efficient uptake and delivery of prions into the B-cell follicles of the spleen, this is not influenced by Sialoadhesin expression by the MNP within it.
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ORIGINAL ARTICLE Role of macrophage Sialoadhesin in host defense against the sialylated pathogen group B Streptococcus
2014Co-Authors: Yung-chi Chang, Paul R Crocker, Joshua Olson, Aaron Louie, Ajit Varki, Victor NizetAbstract:Several bacterial pathogens decorate their surfaces with sialic acid (Sia) residues within cell wall components or capsular exopolysaccharides. Sialic acid expression can promote bacterial virulence by blocking complement activation or by engagement of inhibitory sialic acid-binding immunoglobulin-like lectins (Siglecs) on host leukocytes. Expressed at high levels on splenic and lymph node macrophages, Sialoadhesin (Sn) is a unique Siglec with an elongated structure that lacks intracellular signal-ing motifs. Sialoadhesin allows macrophage to engage certain sialylated pathogens and stimulate inflammatory responses, but the in vivo significance of Sialoadhesin in infection has not been shown. We demonstrate that macrophages phagocytose the sialylated pathogen group B Streptococcus (GBS) and increase bactericidal activity via Sialoadhesin-sialic-acid-mediated recog-nition. Sialoadhesin expression on marginal zone metallophillic macrophages in the spleen trapped circulating GBS and restrict-ed the spread of the GBS to distant organs, reducing mortality. Specific IgM antibody responses to GBS challenge were also impaired in Sialoadhesin-deficient mice. Thus, Sialoadhesin rep-resents a key bridge to orchestrate innate and adaptive immune defenses against invasive sialylated bacterial pathogens. Key message & Sialoadhesin is critical for macrophages to phagocytose and clear GBS. & Increased GBS organ dissemination in the Sialoadhesin-deficient mice. & Reduced anti-GBS IgM production in the Sialoadhesin-deficient mice
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Printed in the United Kingdom Developmental Regulation of Sialoadhesin (Sheep Erythrocyte Receptor), a Macrophage-Cell Interaction Molecule Expressed in Lymphohemopoietic Tissues
2013Co-Authors: Lynn Morris, Paul R Crocker, Maxine Hill, Siamon GordonAbstract:Stromal macrophages in lymphohemopoietic tissues express novel macrophagerestricted plasma membrane receptors involved in nonphagocytic interactions with other hemopoietic cells. One such receptor with lectinlike specificity for sialylated glycoconjugates on sheep erythrocytes and murine hemopoietic cells has been characterized immunochemically and termed Sialoadhesin. We have examined Sialoadhesin expression during mouse development to learn more about its regulation and function. Immunocytochemical, rosetting, and Western blot studies show that Sialoadhesin is first detected on fetal liver macrophages on day 18 of development, 7 days after numerous F4/80 macrophages are found within erythroblastic islands. In spleen and bone marrow, Sialoadhesin appears between day 18 and birth, in parallel with myeloid development. Strongly labeled macrophages in the marginal zone of spleen, characteristic of adult lymphoid tissues, appeared gradually between 1-4 weeks after birth, as the white pulp became enlarged. Isolation of fetal liver macrophages at day 14 confirmed that Sialoadhesin was not involved in the binding of erythroblasts, which is mediated by a distinct cationdependen
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antigen delivery to macrophages using liposomal nanoparticles targeting Sialoadhesin cd169
PLOS ONE, 2012Co-Authors: Weihsu C Chen, Paul R Crocker, Norihito Kawasaki, Corwin M Nycholat, Shoufa Han, Julie Pilotte, James C PaulsonAbstract:Sialoadhesin (Sn, Siglec-1, CD169) is a member of the sialic acid binding Ig-like lectin (siglec) family expressed on macrophages. Its macrophage specific expression makes it an attractive target for delivering antigens to tissue macrophages via Sn-mediated endocytosis. Here we describe a novel approach for delivering antigens to macrophages using liposomal nanoparticles displaying high affinity glycan ligands of Sn. The Sn-targeted liposomes selectively bind to and are internalized by Sn-expressing cells, and accumulate intracellularly over time. Our results show that ligand decorated liposomes are specific for Sn, since they are taken up by bone marrow derived macrophages that are derived from wild type but not Sn−/− mice. Importantly, the Sn-targeted liposomes dramatically enhance the delivery of antigens to macrophages for presentation to and proliferation of antigen-specific T cells. Together, these data provide insights into the potential of cell-specific targeting and delivery of antigens to intracellular organelles of macrophages using Sn-ligand decorated liposomal nanoparticles.
Hans Nauwynck - One of the best experts on this subject based on the ideXlab platform.
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monoclonal antibody binding to the macrophage specific receptor Sialoadhesin alters the phagocytic properties of human and mouse macrophages
Cellular Immunology, 2017Co-Authors: Marjorie De Schryver, Hans Nauwynck, Davie Cappoen, Dirk Elewaut, Louis Maes, Guy Caljon, Paul Cos, Peter DelputteAbstract:Sialoadhesin (Sn) is a surface receptor expressed on macrophages in steady state conditions, but during inflammation, Sn can be upregulated both on macrophages and on circulating monocytes. It was shown for different species that Sn becomes internalized after binding with monoclonal antibodies. These features suggest that Sn is a potential target for immunotherapies. In this study, human and mouse macrophages were treated with anti-Sn monoclonal antibodies or F(ab′)2 fragments and the effect of their binding to Sn on phagocytosis was analyzed. Binding of antibodies to Sn resulted in delayed and reduced phagocytosis of fluorescent beads. No effect was observed on Fc-mediated phagocytosis or phagocytosis of bacteria by human macrophages. In contrast, an enhanced phagocytosis of bacteria by mouse macrophages was detected. These results showed that stimulation of Sn could have different effects on macrophage phagocytosis, depending both on the type of phagocytosis and cellular background.
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development and characterization of new species cross reactive anti Sialoadhesin monoclonal antibodies
Antibodies, 2016Co-Authors: Marjorie De Schryver, Hans Nauwynck, Hanne Van Gorp, Inge Hoebeke, Louis Maes, Paul Cos, Bauke De Maeyer, Karen Ooms, Isabel Pintelon, Peter DelputteAbstract:Sialoadhesin (Sn) is a surface receptor expressed on a subset of macrophages in steady state conditions. During inflammation and diseases, Sn is highly upregulated on macrophages and blood monocytes. Therefore, therapies using monoclonal antibodies (mAbs) to target Sn-positive (Sn+) cells are a potential strategy for targeted treatment. It has been shown that Sn internalizes after binding with a mAb, though it is not clear whether this is species-specific. In this study, new Sn-specific mAbs were developed and analyzed for cross-reactivity between species. In addition, the newly developed mAbs were compared to mAbs used in previous research for their epitope recognition and other Sn-specific characteristics. Both species-specific and cross-reactive antibodies could be identified. Furthermore, sialic acid-binding of red blood cells (RBC) could be inhibited with mAbs recognizing different epitopes and all mAb showed internalization of Sn. The newly developed mAbs can be used as novel tools for Sn research and further analysis of Sn internalization in different species.
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porcine Sialoadhesin a newly identified xenogeneic innate immune receptor
American Journal of Transplantation, 2012Co-Authors: Linda G Brock, Peter Delputte, Hans Nauwynck, Joshua P Waldman, Michael A ReesAbstract:Extracorporeal porcine liver perfusion is being developed as a bridge to liver allotransplantation for patients with fulminant hepatic failure. This strategy is limited by porcine Kupffer cell destruction of human erythrocytes, mediated by lectin binding of a sialic acid motif in the absence of antibody and complement. Sialoadhesin, a macrophage restricted lectin that binds sialic acid, was originally described as a sheep erythrocyte binding receptor. Given similarities between Sialoadhesin and the unidentified macrophage lectin in our model, we hypothesized porcine Sialoadhesin contributed to recognition of human erythrocytes. Two additional types of macrophages were identified to bind human erythrocytes—spleen and alveolar. Expression of Sialoadhesin was confirmed by immunofluorescence in porcine tissues and by flow cytometry on primary macrophages. A stable transgenic cell line expressing porcine Sialoadhesin (pSn CHO) bound human erythrocytes, while a Sialoadhesin mutant cell line did not. Porcine macrophage and pSn CHO recognition of human erythrocytes was inhibited approximately 90% by an antiporcine Sialoadhesin monoclonal antibody and by human erythrocyte glycoproteins. Furthermore, this binding was substantially reduced by sialidase treatment of erythrocytes. These data support the hypothesis that porcine Sialoadhesin is a xenogeneic receptor that mediates porcine macrophage binding of human erythrocytes in a sialic acid-dependent manner.
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porcine Sialoadhesin cd169 siglec 1 is an endocytic receptor that allows targeted delivery of toxins and antigens to macrophages
PLOS ONE, 2011Co-Authors: Peter Delputte, Hanne Van Gorp, Herman W. Favoreel, Inge Hoebeke, Iris Delrue, Bruno Verhasselt, Eric Cox, Hannah L Dewerchin, Frank Verdonck, Hans NauwynckAbstract:Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since Sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via Sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, Sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that Sialoadhesin is an endocytic receptor. In this report, we show that porcine Sialoadhesin-specific antibodies and F(ab')2 fragments trigger Sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine Sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine Sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the Sialoadhesin endocytosis mechanism, two Sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine Sialoadhesin-specific immunotoxins efficiently kill Sialoadhesin-expressing macrophages. Furthermore, porcine Sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand Sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to Sialoadhesin-expressing macrophages.
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susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of Sialoadhesin and cd163
BMC Biotechnology, 2010Co-Authors: Iris Delrue, Peter Delputte, Hanne Van Gorp, Jan Van Doorsselaere, Hans NauwynckAbstract:Background Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. In vivo, the virus infects a subpopulation of tissue macrophages. In vitro, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145. The latter is currently used for vaccine production. However, since virus entry in Marc-145 cells is different compared to entry in primary macrophages, specific epitopes associated with virus entry could potentially alter upon growth on Marc-145 cells. To avoid this, we constructed CHO and PK15 cell lines recombinantly expressing the PRRSV receptors involved in virus entry into macrophages, Sialoadhesin (Sn) and CD163 (CHOSn-CD163 and PK15Sn-CD163) and evaluated their potential for production of PRRSV.
Peter Delputte - One of the best experts on this subject based on the ideXlab platform.
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monoclonal antibody binding to the macrophage specific receptor Sialoadhesin alters the phagocytic properties of human and mouse macrophages
Cellular Immunology, 2017Co-Authors: Marjorie De Schryver, Hans Nauwynck, Davie Cappoen, Dirk Elewaut, Louis Maes, Guy Caljon, Paul Cos, Peter DelputteAbstract:Sialoadhesin (Sn) is a surface receptor expressed on macrophages in steady state conditions, but during inflammation, Sn can be upregulated both on macrophages and on circulating monocytes. It was shown for different species that Sn becomes internalized after binding with monoclonal antibodies. These features suggest that Sn is a potential target for immunotherapies. In this study, human and mouse macrophages were treated with anti-Sn monoclonal antibodies or F(ab′)2 fragments and the effect of their binding to Sn on phagocytosis was analyzed. Binding of antibodies to Sn resulted in delayed and reduced phagocytosis of fluorescent beads. No effect was observed on Fc-mediated phagocytosis or phagocytosis of bacteria by human macrophages. In contrast, an enhanced phagocytosis of bacteria by mouse macrophages was detected. These results showed that stimulation of Sn could have different effects on macrophage phagocytosis, depending both on the type of phagocytosis and cellular background.
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development and characterization of new species cross reactive anti Sialoadhesin monoclonal antibodies
Antibodies, 2016Co-Authors: Marjorie De Schryver, Hans Nauwynck, Hanne Van Gorp, Inge Hoebeke, Louis Maes, Paul Cos, Bauke De Maeyer, Karen Ooms, Isabel Pintelon, Peter DelputteAbstract:Sialoadhesin (Sn) is a surface receptor expressed on a subset of macrophages in steady state conditions. During inflammation and diseases, Sn is highly upregulated on macrophages and blood monocytes. Therefore, therapies using monoclonal antibodies (mAbs) to target Sn-positive (Sn+) cells are a potential strategy for targeted treatment. It has been shown that Sn internalizes after binding with a mAb, though it is not clear whether this is species-specific. In this study, new Sn-specific mAbs were developed and analyzed for cross-reactivity between species. In addition, the newly developed mAbs were compared to mAbs used in previous research for their epitope recognition and other Sn-specific characteristics. Both species-specific and cross-reactive antibodies could be identified. Furthermore, sialic acid-binding of red blood cells (RBC) could be inhibited with mAbs recognizing different epitopes and all mAb showed internalization of Sn. The newly developed mAbs can be used as novel tools for Sn research and further analysis of Sn internalization in different species.
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porcine Sialoadhesin a newly identified xenogeneic innate immune receptor
American Journal of Transplantation, 2012Co-Authors: Linda G Brock, Peter Delputte, Hans Nauwynck, Joshua P Waldman, Michael A ReesAbstract:Extracorporeal porcine liver perfusion is being developed as a bridge to liver allotransplantation for patients with fulminant hepatic failure. This strategy is limited by porcine Kupffer cell destruction of human erythrocytes, mediated by lectin binding of a sialic acid motif in the absence of antibody and complement. Sialoadhesin, a macrophage restricted lectin that binds sialic acid, was originally described as a sheep erythrocyte binding receptor. Given similarities between Sialoadhesin and the unidentified macrophage lectin in our model, we hypothesized porcine Sialoadhesin contributed to recognition of human erythrocytes. Two additional types of macrophages were identified to bind human erythrocytes—spleen and alveolar. Expression of Sialoadhesin was confirmed by immunofluorescence in porcine tissues and by flow cytometry on primary macrophages. A stable transgenic cell line expressing porcine Sialoadhesin (pSn CHO) bound human erythrocytes, while a Sialoadhesin mutant cell line did not. Porcine macrophage and pSn CHO recognition of human erythrocytes was inhibited approximately 90% by an antiporcine Sialoadhesin monoclonal antibody and by human erythrocyte glycoproteins. Furthermore, this binding was substantially reduced by sialidase treatment of erythrocytes. These data support the hypothesis that porcine Sialoadhesin is a xenogeneic receptor that mediates porcine macrophage binding of human erythrocytes in a sialic acid-dependent manner.
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porcine Sialoadhesin cd169 siglec 1 is an endocytic receptor that allows targeted delivery of toxins and antigens to macrophages
PLOS ONE, 2011Co-Authors: Peter Delputte, Hanne Van Gorp, Herman W. Favoreel, Inge Hoebeke, Iris Delrue, Bruno Verhasselt, Eric Cox, Hannah L Dewerchin, Frank Verdonck, Hans NauwynckAbstract:Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since Sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via Sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, Sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that Sialoadhesin is an endocytic receptor. In this report, we show that porcine Sialoadhesin-specific antibodies and F(ab')2 fragments trigger Sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine Sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine Sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the Sialoadhesin endocytosis mechanism, two Sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine Sialoadhesin-specific immunotoxins efficiently kill Sialoadhesin-expressing macrophages. Furthermore, porcine Sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand Sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to Sialoadhesin-expressing macrophages.
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susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of Sialoadhesin and cd163
BMC Biotechnology, 2010Co-Authors: Iris Delrue, Peter Delputte, Hanne Van Gorp, Jan Van Doorsselaere, Hans NauwynckAbstract:Background Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. In vivo, the virus infects a subpopulation of tissue macrophages. In vitro, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145. The latter is currently used for vaccine production. However, since virus entry in Marc-145 cells is different compared to entry in primary macrophages, specific epitopes associated with virus entry could potentially alter upon growth on Marc-145 cells. To avoid this, we constructed CHO and PK15 cell lines recombinantly expressing the PRRSV receptors involved in virus entry into macrophages, Sialoadhesin (Sn) and CD163 (CHOSn-CD163 and PK15Sn-CD163) and evaluated their potential for production of PRRSV.
Sorge Kelm - One of the best experts on this subject based on the ideXlab platform.
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saturation transfer difference nmr and computational modeling of a Sialoadhesin sialyl lactose complex
Carbohydrate Research, 2004Co-Authors: Anirban Bhunia, Sorge Kelm, V Jayalakshmi, Andrew J Benie, Oliver Schuster, Rama N Krishna, Thomas PetersAbstract:Abstract The siglecs are a family of I-type lectins binding to sialic acids on the cell surface. Sialoadhesin (siglec-1) is expressed at much higher levels in inflammatory macrophages and specifically binds to α-2,3-sialylated N-acetyl lactosamine residues of glycan chains. The terminal disaccharide α- d -Neu5Ac-(2 → 3)-β- d -Gal is thought to be the main epitope recognized by Sialoadhesin. To understand the basis of this biological recognition reaction we combined NMR experiments with a molecular modeling study. We employed saturation transfer difference (STD) NMR experiments to characterize the binding epitope of α-2,3-sialylated lactose, α- d -Neu5Ac-(2 → 3)-β- d -Gal-(1 → 4)- d -Glc 1 to Sialoadhesin at atomic resolution. The experimental results were compared to a computational docking model and to X-ray data of a complex of sialyl lactose and Sialoadhesin. The data reveal that Sialoadhesin mainly recognizes the N-acetyl neuraminic acid and a small part of the galactose moiety of 1 . The crystal structure of a complex of Sialoadhesin with sialyl lactose 1 was used as a basis for a modeling study using the FlexiDock algorithm. The model generated was very similar to the original crystal structure. Therefore, the X-ray data were used to predict theoretical STD values utilizing the CORCEMA-STD protocol. The good agreement between experimental and theoretical STD values indicates that a combined modeling/STD NMR approach yields a reliable structural model for the complex of Sialoadhesin with α- d -Neu5Ac-(2 → 3)-β- d -Gal-(1 → 4)- d -Glc 1 in aqueous solution.
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Structure-Guided Design of Sialic Acid-Based Siglec Inhibitors and Crystallographic Analysis in Complex with Sialoadhesin
Structure (London England : 1993), 2003Co-Authors: Nathan R. Zaccai, Sorge Kelm, Paul R Crocker, Reinhard Brossmer, Katsumi Maenaka, Taeko Maenaka, E. Yvonne JonesAbstract:Abstract The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec Sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against Sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC 50 values (relative to methyl-α-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-α-9- N -benzoyl-amino-9-deoxy-Neu5Ac (BENZ), Me-α-9- N -(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-α-9- N -(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.
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molecular analysis of sialoside binding to Sialoadhesin by nmr and site directed mutagenesis
Biochemical Journal, 1999Co-Authors: Sorge Kelm, Paul R Crocker, Mary Vinson, Kurt DrickamerAbstract:The molecular interactions between Sialoadhesin and sialylated ligands have been investigated by using proton NMR. Addition of ligands to the 12 kDa N-terminal immunoglobulin-like domain of Sialoadhesin result in resonance shifts in the protein spectrum that have been used to determine the affinities of Sialoadhesin for several sialosides. The results indicate that alpha2, 3-sialyl-lactose and alpha2,6-sialyl-lactose bind respectively 2- and 1.5-fold more strongly than does alpha-methyl-N-acetylneuraminic acid (alpha-Me-NeuAc). The resonances corresponding to the methyl protons within the N-acetyl moiety of sialic acid undergo upfield shifting and broadening during titrations, reflecting an interaction of this group with Trp2 in Sialoadhesin as observed in co-crystals of the terminal domain with bound ligand. This resonance shift was used to measure the affinities of mutant and wild-type forms of Sialoadhesin in which the first three domains are fused to the Fc region of human IgG1. Substitution of Arg97 by alanine completely abrogated measurable interaction with alpha-Me-NeuAc, whereas a conservative substitution with lysine resulted in a 10-fold decrease in affinity. These results provide the first direct measurement of the affinity of Sialoadhesin for sialosides and confirm the critical importance of the conserved arginine in interactions between sialosides and members of the siglec family of sialic acid-binding, immunoglobulin-like lectins.
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Functional groups of sialic acids involved in binding to siglecs (Sialoadhesins) deduced from interactions with synthetic analogues
European journal of biochemistry, 1998Co-Authors: Sorge Kelm, Rainer Isecke, Reinhard Brossmer, Hans-jürgen Gross, Karen Strenge, Roland SchauerAbstract:The siglecs, formerly called Sialoadhesins, are a family of I-type lectins binding to sialic acids on the cell surface. Five members of this family have been identified: Sialoadhesin, myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), CD22 and CD33. We have investigated the relevance of substituents at position C-9 and in the N-acetyl group of N-acetylneuraminic acid, using a series of synthetic sialic-acid analogues either on resialylated human erythrocytes or as free A-glycosides in hapten inhibition. All five siglecs require the hydroxy group at C-9 for binding, suggesting hydrogen bonding of this substituent with the binding site. Remarkable differences were found among the proteins in their specificity for modifications of the N-acetyl group. Whereas Sialoadhesin, MAG and SMP do not tolerate a hydroxy group as in N-glycolylneuraminic acid, they bind to halogenated acetyl residues. In the case of MAG, N-fluoroacetylneuraminic acid is bound about 17-fold better than N-acetylneuraminic acid. In contrast, human and murine CD22 both show good affinity for N-glycolylneuraminic acid, but only human CD22 bound the halogenated compounds. In conclusion, our data indicate that interactions of the hydroxy group at position 9 and the N-acyl substituent contribute significantly to the binding strength.
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sialic acid recognition by Sialoadhesin and related lectins
Trends in Glycoscience and Glycotechnology, 1997Co-Authors: M Vinson, Sorge Kelm, A P May, E Y Jones, Stuart Mucklow, Paul R CrockerAbstract:シアル酸はヒトの細胞表面に豊富にあり、負の電荷を持つことにより、好ましくない細胞間相互作用を妨げる働きを助けている。免疫グロブリンスーパーファミリーであるシアロアドヘシンサブセットの最近の発見により (これには、シアロアドヘシン、CD22、ミエリン関連糖タンパク質 (MAG) やCD33が含まれる)、シアル酸が、変化に富んだ生理システムの中で、細胞間相互作用を促進させる手段でもある可能性が出てきた。これら膜タンパク質のそれぞれは、認識されるシアル酸の種類と一つ内側の糖への結合の両方に対して、明確な特異性を表す。これらは、保存されたジスルフィド結合によって保持されるNH2末端側の二つのIgドメインにおいて、高い度合いで似通った配列を持っている。ドメインの1と2 (NH2, 末端より数えて) は遺伝子複製を通じて、ファミリーの中の現メンバーを生み出した先祖遺伝子を表しているように思われる。端を切り取った組換えタンパク質作成と、部位特異的変異誘発によって、シアロアドヘシンとCD22のNH2末端にあるVセットドメインのCFC′C″面が、シアル酸の結合部位を含んでいることが示された。最近になって、リガンドとして用いた3′-シアリルラクトースと複合体を作っているシアルアドヘシンのVセットドメインの構造がX線結晶学によって確かなものになった。シアロアドヘシンとそれ以外のシアル酸結合タンパク質によるシアル酸の認識における類似性及び差異について述べる。
Hans J. Nauwynck - One of the best experts on this subject based on the ideXlab platform.
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Preferential use of Siglec-1 or Siglec-10 by type 1 and type 2 PRRSV strains to infect PK15S1–CD163 and PK15S10–CD163 cells
Veterinary Research, 2018Co-Authors: Jiexiong Xie, Bo Yang, Ivan Trus, Isaura Christiaens, Bert Devriendt, Tingting Cui, Ruifang Wei, Hans J. NauwynckAbstract:AbstractCellular entry mediators define whether the cell is permissive to PRRSV infection. Porcine Sialoadhesin (pSn, Siglec-1) and CD163 are main entry mediators facilitating infection of porcine macrophages by PRRSV. Recently, Siglec-10 was demonstrated to be an alternative receptor for PRRSV. To examine if virulence and pathogenicity of PRRSV strains could be correlated with the use of different Siglecs, a PK15 cell line recombinantly expressing Siglec-1 and CD163 (PK15S1–CD163) and a PK15 cell line recombinantly expressing Siglec-10 and CD163 (PK15S10–CD163) were used to compare the virus replication of 7 genotype 1 subtype 1 strains (G1s1), 2 genotype 1 subtype 3 (G1s3) strains and 5 genotype 2 (G2) strains. Some strains (08VA (G1s1), 13V117 (G1s1), 17V035 (G1s1), VR2332 (G2)) were poor virus producers (
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Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages
2016Co-Authors: Peter Delputte*. L. ¤b, Hanne Van Gorp, Herman W. Favoreel, Inge Hoebeke, Iris Delrue, Bruno Verhasselt, Eric Cox, Hans J. NauwynckAbstract:Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since Sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via Sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, Sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that Sialoadhesin is an endocytic receptor. In this report, we show that porcine Sialoadhesin-specific antibodies and F(ab’)2 fragments trigger Sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine Sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine Sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the Sialoadhesin endocytosis mechanism, two Sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine Sialoadhesin-specific immunotoxins efficiently kill Sialoadhesin-expressing macrophages. Furthermore, porcine Sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand Sialoadhesin functionality an
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Different clinical, virological, serological and tissue tropism outcomes of two new and one old Belgian type 1 subtype 1 porcine reproductive and respiratory virus (PRRSV) isolates
Veterinary Research, 2015Co-Authors: Ilias S Frydas, Ivan Trus, Lise K Kvisgaard, Caroline Bonckaert, Vishwanatha Rap Reddy, Lars E Larsen, Hans J. NauwynckAbstract:In this study, the pathogenic behavior of PRRSV 13V091 and 13V117, isolated in 2013 from two different Belgian farms with enzootic respiratory problems shortly after weaning in the nursery, were compared with the Belgian strain 07V063 isolated in 2007. Full-length genome sequencing was performed to identify their origin. Twelve weeks-old pigs were inoculated intranasally (IN) with 13V091, 13V117 or 07V063 (9 pigs/group). At 10 days post inoculation (dpi), 4 animals from each group were euthanized and tissues were collected for pathology, virological and serological analysis. 13V091 infection resulted in the highest respiratory disease scores and longest period of fever. Gross lung lesions were more pronounced for 13V091 (13%), than for 13V117 (7%) and 07V063 (11%). The nasal shedding and viremia was also most extensive with 13V091. The 13V091 group showed the highest virus replication in conchae, tonsils and retropharyngeal lymph nodes. 13V117 infection resulted in the lowest virus replication in lymphoid tissues. 13V091 showed higher numbers of Sialoadhesin^− infected cells/mm^2 in conchae, tonsils and spleen than 13V117 and 07V063. Neutralizing antibody response with 07V063 was stronger than with 13V091 and 13V117. It can be concluded that (i) 13V091 is a highly pathogenic type 1 subtype 1 PRRSV strain that replicates better than 07V063 and 13V117 and has a strong tropism for Sialoadhesin^− cells and (ii) despite the close genetic relationship between 13V117 and 07V063, 13V117 has an increased nasal replication and shedding, but a decreased replication in lymphoid tissues compared to 07V063.
