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Roy Bicknell - One of the best experts on this subject based on the ideXlab platform.
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proteomics based screening of the endothelial heparan sulfate interactome reveals that c type Lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Author(s): Sandoval, Daniel R; Gomez Toledo, Alejandro; Painter, Chelsea D; Tota, Ember M; Sheikh, M Osman; West, Alan MV; Frank, Martin M; Wells, Lance; Xu, Ding; Bicknell, Roy; Corbett, Kevin D; Esko, Jeffrey D | Abstract: Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-Type Lectin 14a (CLEC14A), a member of the C-Type Lectin family that modulates angiogenesis. We found that the C-Type Lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
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proteomics based screening of the endothelial heparan sulfate interactome reveals that c type Lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-Type Lectin 14a (CLEC14A), a member of the C-Type Lectin family that modulates angiogenesis. We found that the C-Type Lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
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proteomics based screening of the endothelial heparan sulfate interactome reveals that c type Lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-Type Lectin 14a (CLEC14A), a member of the C-Type Lectin family that modulates angiogenesis. We found that the C-Type Lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
Gordon D. Brown - One of the best experts on this subject based on the ideXlab platform.
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signalling c type Lectin receptors microbial recognition and immunity
Cellular Microbiology, 2014Co-Authors: Claire J Hoving, Gordon D. Brown, Gillian J. WilsonAbstract:Signalling C-Type Lectin receptors (CLRs) are crucial in shaping the immune response to fungal pathogens, but comparably little is known about the role of these receptors in bacterial, viral and parasitic infections. CLRs have many diverse functions depending on the signalling motifs in their cytoplasmic domains, and can induce endocytic, phagocytic, antimicrobial, pro-inflammatory or anti-inflammatory responses which are either protective or not during an infection. Understanding the role of CLRs in shaping anti-microbial immunity offers great potential for the future development of therapeutics for disease intervention. In this review we will focus on the recognition of bacterial, viral and parasitic pathogens by CLRs, and how these receptors influence the outcome of infection. We will also provide a brief update on the role of CLRs in antifungal immunity.
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The Dectin-2 family of C-Type Lectin-like receptors: an update
International immunology, 2013Co-Authors: Bernhard Kerscher, Janet A. Willment, Gordon D. BrownAbstract:Myeloid and non-myeloid cells express members of the C-Type Lectin-like receptor (CTLR) family, which mediate crucial cellular functions during immunity and homeostasis. Of relevance here is the dendritic cell-associated C-Type Lectin-2 (Dectin-2) family of CTLRs, which includes blood dendritic cell antigen 2 (BDCA-2), dendritic cell immunoactivating receptor (DCAR), dendritic cell immunoreceptor (DCIR), Dectin-2, C-Type Lectin superfamily 8 (CLECSF8) and macrophage-inducible C-Type Lectin (Mincle). These CTLRs possess a single extracellular conserved C-Type Lectin-like domain and are capable of mediating intracellular signalling either directly, through integral signalling domains, or indirectly, by associating with signalling adaptor molecules. These receptors recognize a diverse range of endogenous and exogenous ligands, and can function as pattern recognition receptors for several classes of pathogens including fungi, bacteria and parasites, driving both innate and adaptive immunity. In this review, we summarize our knowledge of each of these receptors, highlighting the exciting discoveries that have been made in recent years.
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c type Lectin like receptors of the dectin 1 cluster ligands and signaling pathways
International Reviews of Immunology, 2013Co-Authors: Anthony Plato, Janet A. Willment, Gordon D. BrownAbstract:Innate immunity is constructed around genetically encoded receptors that survey the intracellular and extracellular environments for signs of invading microorganisms. These receptors recognise the invader and through complex intracellular networks of molecular signaling, they destroy the threat whilst instructing effective adaptive immune responses. Many of these receptors, like the Toll-like receptors in particular, are well-known for their ability to mediate downstream responses upon recognition of exogenous or endogenous ligands; however, the emerging family known as the C-Type Lectin-like receptors contains many members that have a huge impact on immune and homeostatic regulation. Of particular interest here are the C-Type Lectin-like receptors that make up the Dectin-1 cluster and their intracellular signaling motifs that mediate their functions. In this review, we aim to draw together current knowledge of ligands, motifs and signaling pathways, present downstream of Dectin-1 cluster receptors, and discuss how these dictate their role within biological systems.
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c type Lectin receptors orchestrate antifungal immunity
Nature Immunology, 2012Co-Authors: Sarah E Hardison, Gordon D. BrownAbstract:Immunity to pathogens critically requires pattern-recognition receptors (PRRs) to trigger intracellular signaling cascades that initiate and direct innate and adaptive immune responses. For fungal infections, these responses are primarily mediated by members of the C-Type Lectin receptor family. In this Review, we highlight recent advances in the understanding of the roles and mechanisms of these multifunctional receptors, explore how these PRRs orchestrate antifungal immunity and briefly discuss progress in the use of these receptors as targets for antifungal and other vaccines.
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c type Lectin receptors in antifungal immunity
Trends in Microbiology, 2008Co-Authors: Janet A. Willment, Gordon D. BrownAbstract:Fungal infections represent a significant health burden, especially in immunocompromised individuals, yet many of the underlying immunological mechanisms involved in the recognition and control of these pathogens are unclear. The identification of the Toll-like receptors (TLRs) has shed new insights on innate microbial recognition and the initiation of immune responses; however, recent evidence indicates that the 'non-TLR' receptors also have a significant role in these processes, particularly in antifungal immunity. Of interest are members of the C-Type Lectin-receptor family, including the mannose receptor, dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), Dectin-1, Dectin-2 and the colLectins. Here, we review the roles of each of these receptors, describing how they contribute to fungal recognition, uptake and killing and also participate in the induction and/or modulation of the host immune response.
Daniel R Sandoval - One of the best experts on this subject based on the ideXlab platform.
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proteomics based screening of the endothelial heparan sulfate interactome reveals that c type Lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Author(s): Sandoval, Daniel R; Gomez Toledo, Alejandro; Painter, Chelsea D; Tota, Ember M; Sheikh, M Osman; West, Alan MV; Frank, Martin M; Wells, Lance; Xu, Ding; Bicknell, Roy; Corbett, Kevin D; Esko, Jeffrey D | Abstract: Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-Type Lectin 14a (CLEC14A), a member of the C-Type Lectin family that modulates angiogenesis. We found that the C-Type Lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
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proteomics based screening of the endothelial heparan sulfate interactome reveals that c type Lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-Type Lectin 14a (CLEC14A), a member of the C-Type Lectin family that modulates angiogenesis. We found that the C-Type Lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
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proteomics based screening of the endothelial heparan sulfate interactome reveals that c type Lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-Type Lectin 14a (CLEC14A), a member of the C-Type Lectin family that modulates angiogenesis. We found that the C-Type Lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
Huan Zhang - One of the best experts on this subject based on the ideXlab platform.
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a scallop c type Lectin from argopecten irradians aictl5 with activities of lipopolysaccharide binding and gram negative bacteria agglutination
Fish & Shellfish Immunology, 2012Co-Authors: Xiaoyan Song, Jianmin Zhao, Lingling Wang, Limei Qiu, Huan Zhang, Zhi Zhou, Mengqiang Wang, Linsheng Song, Chunlin WangAbstract:C-Type Lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-Type Lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-Type Lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-Type Lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent Lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of Lectin.
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aictl 6 a novel c type Lectin from bay scallop argopecten irradians with a long c type Lectin like domain
Fish & Shellfish Immunology, 2011Co-Authors: Huan Zhang, Jialong Yang, Xiaoyan Song, Lingling Wang, Limei Qiu, Pengfei Kong, Lin Liu, Ying Zhang, Lihua Qiu, Linsheng SongAbstract:C-Type Lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel C-Type Lectin gene from scallop Argopecten irradians (designated as AiCTL-6) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of AiCTL-6 was 1080 bp. The open reading frame encoded a polypeptide of 307 amino acids, including a signal sequence and a C-Type Lectin-like domain (CTLD) of 150 amino acid residues longer than any usual CTLD. It contained six conserved cysteine residues involved in the formation of three internal disulfide bridges and an EPD (Glu(269)-Pro(270)-Asp(271)) motif at the Ca(2+)-binding site 2. The deduced amino acid sequence of AiCTL-6 showed high similarity to members of C-Type Lectin superfamily. By fluorescent quantitative real-time PCR, AiCTL-6 mRNA was found mainly in hepatopancreas and gill, and marginally expressed in other tissues. After the scallops were challenged by Listonella anguillarum for 6 h, the mRNA expression of AiCTL-6 was up-regulated significantly to 7.2-fold compared to the blank group. While at 9 h post Micrococcus luteus challenge, its expression level was 60.1 times higher than that of the blank group. The functional activity of AiCTL-6 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiCTL-6 could agglutinate Gram-negative bacteria E. coli TOP10F', Gram-positive bacteria M. luteus and Staphylococcus aureus. These results collectively suggested that AiCTL-6, as a novel member of C-Type Lectin family, contributed to the host defense mechanisms against invading microorganism in A. irradians. (C) 2009 Elsevier Ltd. All rights reserved.
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an ancient c type Lectin in chlamys farreri cflec 2 that mediate pathogen recognition and cellular adhesion
Developmental and Comparative Immunology, 2010Co-Authors: Jialong Yang, Xiumei Wei, Lingling Wang, Limei Qiu, Huan Zhang, Zhi Zhou, Lin Liu, Leilei Wang, Linsheng SongAbstract:C-Type Lectins are a superfamily of Ca(2+) dependent carbohydrate-recognition proteins which play significant diverse roles in nonself-recognition and clearance of invaders. In the present study, a C-Type Lectin (CfLec-2) from Zhikong scallop Chlamys farreri was selected to investigate its functions in innate immunity. The mRNA expression of CfLec-2 in hemocytes was significantly up-regulated (P<0.01) after scallops were stimulated by LPS, PGN or β-glucan, and reached the highest expression level at 12h post-stimulation, which was 72.5-, 23.6- or 43.8-fold compared with blank group, respectively. The recombinant CfLec-2 (designated as rCfLec-2) could bind LPS, PGN, mannan and zymosan in vitro, but it could not bind β-glucan. Immunofluorescence assay with polyclonal antibody specific for CfLec-2 revealed that CfLec-2 was mainly located in the mantle, kidney and gonad. Furthermore, rCfLec-2 could bind to the surface of scallop hemocytes, and then initiated cellular adhesion and recruited hemocytes to enhance their encapsulation in vitro, and this process could be specifically blocked by anti-rCfLec-2 serum. These results collectively suggested that CfLec-2 from the primitive deuterostome C. farreri could perform two distinct immune functions, pathogen recognition and cellular adhesion synchronously, while these functions were performed by colLectins and seLectins in vertebrates, respectively. The synchronous functions of pathogen recognition and cellular adhesion performed by CfLec-2 tempted us to suspect that CfLec-2 was an ancient form of C-Type Lectin, and apparently the differentiation of these two functions mediated by C-Type Lectins occurred after mollusk in phylogeny.
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cflec 4 a multidomain c type Lectin involved in immune defense of zhikong scallop chlamys farreri
Developmental and Comparative Immunology, 2009Co-Authors: Huan Zhang, Xiaoyan Song, Jianmin Zhao, Lingling Wang, Linsheng Song, Hao Wang, Limei QiuAbstract:C-Type Lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel multidomain C-Type Lectin gene from scallop Chlamys farreri (designated as Cflec-4) was cloned by RACE approach based on EST analysis. The full-length cDNA of Cflec-4 was of 2086 bp. The open reading frame was of 1830bp and encoded a polypeptide of 609 amino acids, including a signal sequence and four dissimilar carbohydrate-recognition domains (CRDs). The deduced amino acid sequence of Cflec-4 shared high similarities to other C-Type Lectin family members. The phylogenetic analysis revealed the divergence between the three N-terminal CRDs and the C-terminal one, suggesting that the four CRDs in Cflec-4 originated by repeated duplication of different primordial CRD. The potential tertiary structure of each CRD in Cflec-4 was typical double-loop structure with Ca2+-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. The tissue distribution of Cflec-4 mRNA was examined by fluorescent quantitative real-time PCR. In the healthy scallops, the Cflec-4 transcripts could be only detected in gonad and hepatopancreas, whereas in the Listonella anguillarum challenged scallops, it could be also detected in hemocytes. These results collectively suggested that Cflec-4 was involved in the immune defense of scallop against pathogen infection and provided new insight into the evolution of C-Type Lectin superfamily.
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a novel c type Lectin cflec 3 from chlamys farreri with three carbohydrate recognition domains
Fish & Shellfish Immunology, 2009Co-Authors: Huan Zhang, Xiaoyan Song, Jianmin Zhao, Lingling Wang, Limei Qiu, Hao Wang, Ming Cong, Linsheng SongAbstract:C-Type Lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, the gene of a C-Type Lectin with multiple carbohydrate-recognition domains (CRDs) from scallop Chlamys farreri (designated as Cflec-3) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of Cflec-3 was of 2256 bp. The open reading frame encoded a polypeptide of 516 amino acids, including a signal sequence and three CRDs. The deduced amino acid sequence of Cflec-3 showed high similarity to members of C-Type Lectin superfamily. By fluorescent quantitative real-time PCR, the Cflec-3 mRNA was mainly detected in hepatopancreas, adductor, mantle, and marginally in gill, gonad and hemocytes of healthy scallops. After scallops were challenged by Listonella anguillarum, the mRNA level of Cflec-3 in hemocytes was up-regulated and was significantly higher than that of blank at 8 h and 12 h post-challenge. The function of Cflec-3 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombined Cflec-3 (rCflec-3) agglutinated Gram-negative bacteria Pseudomonas stutzeri. The agglutinating activity was calcium-dependent and could be inhibited by D-mannose. These results collectively suggested that Cflec-3 was involved in the immune response against microbe infection and contributed to nonself-recognition and clearance of bacterial pathogens in scallop. (C) 2009 Elsevier Ltd. All rights reserved.
Christoph Rademacher - One of the best experts on this subject based on the ideXlab platform.
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high affinity sugar ligands of c type Lectin receptor langerin
Biochimica et Biophysica Acta, 2018Co-Authors: Tetsuya Hirayama, Masahiro Nagata, Hendra S Ismanto, Bernd Lepenies, Jonas Aretz, Yasuhiko Kizuka, Reiko Fujinawa, Yoshiki Yamaguchi, Christoph RademacherAbstract:Abstract Background Langerin, a C-Type Lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application. Methods We performed binding analysis between purified long and short forms of langerin and a series of KS disaccharide components. We also chemically synthesized oligomeric derivatives of L4 to develop a new high-affinity ligand of langerin. Results We show that the binding critically requires the 6-sulfation of Gal and that the long form of langerin displays higher affinity than the short form. The synthesized trimeric (also designated as triangle or Tri) and polymeric (pendant) L4 derivatives displayed over 1000-fold higher affinity toward langerin than monomeric L4. The pendant L4, but not the L4 monomer, was found to effectively transduce langerin signaling in a model cell system. Conclusions L4 is a specific ligand for langerin. Oligomerization of L4 unit increased the affinity toward langerin. General significance These results suggest that oligomeric L4 derivatives will be useful for clarifying the langerin functions and for the development of new glycan-based anti-inflammatory drugs.
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intradomain allosteric network modulates calcium affinity of the c type Lectin receptor langerin
Journal of the American Chemical Society, 2016Co-Authors: Jonas Hanske, Stevan Aleksic, Martin Ballaschk, Marcel Jurk, Elena Shanina, Monika Beerbaum, Peter Schmieder, Bettina G Keller, Christoph RademacherAbstract:Antigen uptake and processing by innate immune cells is crucial to initiate the immune response. Therein, the endocytic C-Type Lectin receptors serve as pattern recognition receptors, detecting pathogens by their glycan structures. Herein, we studied the carbohydrate recognition domain of Langerin, a C-Type Lectin receptor involved in the host defense against viruses such as HIV and influenza as well as bacteria and fungi. Using a combination of nuclear magnetic resonance and molecular dynamics simulations, we unraveled the molecular determinants underlying cargo capture and release encoded in the receptor architecture. Our findings revealed receptor dynamics over several time scales associated with binding and release of the essential cofactor Ca2+ controlled by the coupled motions of two loops. Applying mutual information theory and site-directed mutagenesis, we identified an allosteric intradomain network that modulates the Ca2+ affinity depending on the pH, thereby promoting fast ligand release.
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computational and experimental prediction of human c type Lectin receptor druggability
Frontiers in Immunology, 2014Co-Authors: Jonas Aretz, Eike-christian Wamhoff, Jonas Hanske, Dario Heymann, Christoph RademacherAbstract:Mammalian C-Type Lectin receptors (CTLRS) are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies, and lymphocyte homing. Despite a great interest in modulating CTLR recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 CTLRs using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable.To further explore these findings, we employed a fluorine-based nuclear magnetic resonance screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rateD 13.5%). A surface plasmon resonance-based follow-up assay confirmed 18 of these fragments (47%) and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7 and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular, with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of CTLRs can be developed.
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computational and experimental prediction of human c type Lectin receptor druggability
Frontiers in Immunology, 2014Co-Authors: Jonas Aretz, Eike-christian Wamhoff, Jonas Hanske, Dario Heymann, Christoph RademacherAbstract:Mammalian C-Type Lectin receptors (CTLRS) are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies, and lymphocyte homing. Despite a great interest in modulating CTLR recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 CTLRs using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable.To further explore these findings, we employed a fluorine-based nuclear magnetic resonance screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rateD 13.5%). A surface plasmon resonance-based follow-up assay confirmed 18 of these fragments (47%) and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7 and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular, with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of CTLRs can be developed.
