Dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin(DC-SIGN;CD209)has an important role in mediating adherence of Mycobacteria species,including M.tuberculosis and M.bovis BCG to human dend...Dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin(DC-SIGN;CD209)has an important role in mediating adherence of Mycobacteria species,including M.tuberculosis and M.bovis BCG to human dendritic cells and macrophages,in which these bacteria can survive intracellularly.DC-SIGN is a C-type lectin,and interactions with mycobacterial cells are believed to occur via mannosylated structures on the mycobacterial surface.Recent studies suggest more varied modes of binding to multiple mycobacterial ligands.Here we identify,by affinity chromatography and mass-spectrometry,four novel ligands of M.bovis BCG that bind to DC-SIGN.The novel ligands are chaperone protein DnaK,60 kDa chaperonin-1(Cpn60.1),glyceraldehyde-3 phosphate dehydrogenase(GAPDH)and lipoprotein lprG.Other published work strongly suggests that these are on the cell surface.Of these ligands,lprG appears to bind DC-SIGN via typical proteinglycan interactions,but DnaK and Cpn60.1 binding do not show evidence of carbohydrate-dependent interactions.LprG was also identified as a ligand for DC-SIGNR(L-SIGN;CD299)and the M.tuberculosis orthologue of lprG has been found previously to interact with human toll-like receptor 2.Collectively,these findings offer new targets for combating mycobacterial adhesion and within-host survival,and reinforce the role of DCSIGN as an important host ligand in mycobacterial infection.展开更多
Surfactant proteins A(SP-A)and D(SP-D),both members of the collectin family,play a well established role in apoptotic cell recognition and clearance.Recent in vitro data show that SP-A and SP-D interact with apoptotic...Surfactant proteins A(SP-A)and D(SP-D),both members of the collectin family,play a well established role in apoptotic cell recognition and clearance.Recent in vitro data show that SP-A and SP-D interact with apoptotic neutrophils in a distinct manner.SP-A and SP-D bind in a Ca^(2+)-dependent manner to viable and early apoptotic neutrophils whereas the much greater interaction with late apoptotic neutrophils is Ca^(2+)-independent.Cell surface molecules on the apoptotic target cells responsible for these interactions had not been identified and this study was done to find candidate target molecules.Myeloperoxidase(MPO),a specific intracellular defense molecule of neutrophils that becomes exposed on the outside of the cell upon apoptosis,was identified by affinity purification,mass-spectrometry and western blotting as a novel binding molecule for SP-A and SP-D.To confirm its role in recognition,it was shown that purified immobilised MPO binds SP-A and SP-D,and that MPO is surface-exposed on late apoptotic neutrophils.SP-A and SP-D inhibit binding of an anti-MPO monoclonal Ab to late apoptotic cells.Fluorescence microscopy confirmed that anti-MPO mAb and SP-A/SP-D colocalise on late apoptotic neutrophils.Desmoplakin was identified as a further potential ligand for SP-A,and neutrophil defensin as a target for both proteins.展开更多
Proteins of the complement system are known to interact with many charged substances.We recently characterized binding of C1q and factor H to immobilized and liposomal anionic phospholipids.Factor H inhibited C1q bind...Proteins of the complement system are known to interact with many charged substances.We recently characterized binding of C1q and factor H to immobilized and liposomal anionic phospholipids.Factor H inhibited C1q binding to anionic phospholipids,suggesting a role for factor H in regulating activation of the complement classical pathway by anionic phospholipids.To extend this finding,we examined interactions of C1q and factor H with lipid A,a well-characterized activator of the classical pathway.We report that C1q and factor H both bind to immobilized lipid A,lipid A liposomes and intact Escherichia coli TG1.Factor H competes with C1q for binding to these targets.Furthermore,increasing the factor H:C1q molar ratio in serum diminished C4b fixation,indicating that factor H diminishes classical pathway activation.The recombinant forms of the Cterminal,globular heads of C1q A,B and C chains bound to lipid A and E.coli in a manner qualitatively similar to native C1q,confirming that C1q interacts with these targets via its globular head region.These observations reinforce our proposal that factor H has an additional complement regulatory role of down-regulating classical pathway activation in response to certain targets.This is distinct from its role as an alternative pathway downregulator.We suggest that under physiological conditions,factor H may serve as a downregulator of bacterially-driven inflammatory responses,thereby finetuning and balancing the inflammatory response in infections with Gram-negative bacteria.展开更多
Mannan-binding lectin(MBL)is a soluble innate immune protein that binds to glycosylated targets.MBL acts as an opsonin and activates complement,contributing to the destruction and clearance of infecting microorganisms...Mannan-binding lectin(MBL)is a soluble innate immune protein that binds to glycosylated targets.MBL acts as an opsonin and activates complement,contributing to the destruction and clearance of infecting microorganisms.Hepatitis C virus(HCV)encodes two envelope glycoproteins E1 and E2,expressed as non-covalent E1/E2 heterodimers in the viral envelope.E1 and E2 are potential ligands for MBL.Here we describe an analysis of the interaction between HCV and MBL using recombinant soluble E2 ectodomain fragment,the full-length E1/E2 heterodimer,expressed in vitro,and assess the effect of this interaction on virus entry.A binding assay using antibody capture of full length E1/E2 heterodimers was used to demonstrate calcium dependent,saturating binding of MBL to HCV glycoproteins.Competition with various saccharides further confirmed that the interaction was via the lectin domain of MBL.MBL binds to E1/E2 representing a broad range of virus genotypes.MBL was shown to neutralize the entry into Huh-7 cells of HCV pseudoparticles(HCVpp)bearing E1/E2 from a wide range of genotypes.HCVpp were neutralized to varying degrees.MBL was also shown to neutralize an authentic cell culture infectious virus,strain JFH-1(HCVcc).Furthermore,binding of MBL to E1/E2 was able to activate the complement system via MBL-associated serine protease 2.In conclusion,MBL interacts directly with HCV glycoproteins,which are present on the surface of the virion,resulting in neutralization of HCV particles.展开更多
文摘Dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin(DC-SIGN;CD209)has an important role in mediating adherence of Mycobacteria species,including M.tuberculosis and M.bovis BCG to human dendritic cells and macrophages,in which these bacteria can survive intracellularly.DC-SIGN is a C-type lectin,and interactions with mycobacterial cells are believed to occur via mannosylated structures on the mycobacterial surface.Recent studies suggest more varied modes of binding to multiple mycobacterial ligands.Here we identify,by affinity chromatography and mass-spectrometry,four novel ligands of M.bovis BCG that bind to DC-SIGN.The novel ligands are chaperone protein DnaK,60 kDa chaperonin-1(Cpn60.1),glyceraldehyde-3 phosphate dehydrogenase(GAPDH)and lipoprotein lprG.Other published work strongly suggests that these are on the cell surface.Of these ligands,lprG appears to bind DC-SIGN via typical proteinglycan interactions,but DnaK and Cpn60.1 binding do not show evidence of carbohydrate-dependent interactions.LprG was also identified as a ligand for DC-SIGNR(L-SIGN;CD299)and the M.tuberculosis orthologue of lprG has been found previously to interact with human toll-like receptor 2.Collectively,these findings offer new targets for combating mycobacterial adhesion and within-host survival,and reinforce the role of DCSIGN as an important host ligand in mycobacterial infection.
基金This work was financially supported by the Medical Research Council,UK.
文摘Surfactant proteins A(SP-A)and D(SP-D),both members of the collectin family,play a well established role in apoptotic cell recognition and clearance.Recent in vitro data show that SP-A and SP-D interact with apoptotic neutrophils in a distinct manner.SP-A and SP-D bind in a Ca^(2+)-dependent manner to viable and early apoptotic neutrophils whereas the much greater interaction with late apoptotic neutrophils is Ca^(2+)-independent.Cell surface molecules on the apoptotic target cells responsible for these interactions had not been identified and this study was done to find candidate target molecules.Myeloperoxidase(MPO),a specific intracellular defense molecule of neutrophils that becomes exposed on the outside of the cell upon apoptosis,was identified by affinity purification,mass-spectrometry and western blotting as a novel binding molecule for SP-A and SP-D.To confirm its role in recognition,it was shown that purified immobilised MPO binds SP-A and SP-D,and that MPO is surface-exposed on late apoptotic neutrophils.SP-A and SP-D inhibit binding of an anti-MPO monoclonal Ab to late apoptotic cells.Fluorescence microscopy confirmed that anti-MPO mAb and SP-A/SP-D colocalise on late apoptotic neutrophils.Desmoplakin was identified as a further potential ligand for SP-A,and neutrophil defensin as a target for both proteins.
文摘Proteins of the complement system are known to interact with many charged substances.We recently characterized binding of C1q and factor H to immobilized and liposomal anionic phospholipids.Factor H inhibited C1q binding to anionic phospholipids,suggesting a role for factor H in regulating activation of the complement classical pathway by anionic phospholipids.To extend this finding,we examined interactions of C1q and factor H with lipid A,a well-characterized activator of the classical pathway.We report that C1q and factor H both bind to immobilized lipid A,lipid A liposomes and intact Escherichia coli TG1.Factor H competes with C1q for binding to these targets.Furthermore,increasing the factor H:C1q molar ratio in serum diminished C4b fixation,indicating that factor H diminishes classical pathway activation.The recombinant forms of the Cterminal,globular heads of C1q A,B and C chains bound to lipid A and E.coli in a manner qualitatively similar to native C1q,confirming that C1q interacts with these targets via its globular head region.These observations reinforce our proposal that factor H has an additional complement regulatory role of down-regulating classical pathway activation in response to certain targets.This is distinct from its role as an alternative pathway downregulator.We suggest that under physiological conditions,factor H may serve as a downregulator of bacterially-driven inflammatory responses,thereby finetuning and balancing the inflammatory response in infections with Gram-negative bacteria.
文摘Mannan-binding lectin(MBL)is a soluble innate immune protein that binds to glycosylated targets.MBL acts as an opsonin and activates complement,contributing to the destruction and clearance of infecting microorganisms.Hepatitis C virus(HCV)encodes two envelope glycoproteins E1 and E2,expressed as non-covalent E1/E2 heterodimers in the viral envelope.E1 and E2 are potential ligands for MBL.Here we describe an analysis of the interaction between HCV and MBL using recombinant soluble E2 ectodomain fragment,the full-length E1/E2 heterodimer,expressed in vitro,and assess the effect of this interaction on virus entry.A binding assay using antibody capture of full length E1/E2 heterodimers was used to demonstrate calcium dependent,saturating binding of MBL to HCV glycoproteins.Competition with various saccharides further confirmed that the interaction was via the lectin domain of MBL.MBL binds to E1/E2 representing a broad range of virus genotypes.MBL was shown to neutralize the entry into Huh-7 cells of HCV pseudoparticles(HCVpp)bearing E1/E2 from a wide range of genotypes.HCVpp were neutralized to varying degrees.MBL was also shown to neutralize an authentic cell culture infectious virus,strain JFH-1(HCVcc).Furthermore,binding of MBL to E1/E2 was able to activate the complement system via MBL-associated serine protease 2.In conclusion,MBL interacts directly with HCV glycoproteins,which are present on the surface of the virion,resulting in neutralization of HCV particles.
