A classical function of Clq is to bind immune complexes and initiate complement activation producing membrane lytic complexes, opsonins and anaphylatoxins. This classical pathway of complement activation is also elici...A classical function of Clq is to bind immune complexes and initiate complement activation producing membrane lytic complexes, opsonins and anaphylatoxins. This classical pathway of complement activation is also elicited when Clq binds some other ligands. Besides complement activation, Clq also regulates cell differentiation, adhesion, migration, activation and survival. Clq deficiency is associated with autoimmunity as well as increased susceptibility to infections. In this article, we discuss the basic properties of Clq, its expression, and classical and regulatory functions. Cellular ＆ Molecular Immunology.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
Complement proteins in blood recognize charged particles.The anionic phospholipid(aPL)cardiolipin binds both complement proteins C1q and factor H.C1q is an activator of the complement classical pathway,while factor H ...Complement proteins in blood recognize charged particles.The anionic phospholipid(aPL)cardiolipin binds both complement proteins C1q and factor H.C1q is an activator of the complement classical pathway,while factor H is an inhibitor of the alternative pathway.To examine opposing effects of C1q and factor H on complement activation by aPL,we surveyed C1q and factor H binding,and complement activation by aPL,either coated on microtitre plates or in liposomes.Both C1q and factor H bound to all aPL tested,and competed directly with each other for binding.All the aPL activated the complement classical pathway,but negligibly the alternative pathway,consistent with accepted roles of C1q and factor H.However,in this system,factor H,by competing directly with C1q for binding to aPL,acts as a direct regulator of the complement classical pathway.This regulatory mechanism is distinct from its action on the alternative pathway.Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range.Thus factor H,which is regarded as a down-regulator only of the alternative pathway,has a distinct role in downregulating activation of the classical complement pathway by aPL.A factor H homologue,β2-glycoprotein-1,also strongly inhibits C1q binding to cardiolipin.Recombinant globular domains of C1q A,B and C chains bound aPL similarly to native C1q,confirming that C1q binds aPL via its globular heads.展开更多
The role of surfactant protein A(SP-A)in the recognition and clearance of apoptotic cells is well established,but to date,it is still not clear which surface molecules of apoptotic cells are involved in the process.He...The role of surfactant protein A(SP-A)in the recognition and clearance of apoptotic cells is well established,but to date,it is still not clear which surface molecules of apoptotic cells are involved in the process.Here we present evidence that phosphatidylserine(PS)is a relevant binding molecule for human SP-A.The binding is Ca^(2+)-dependent and is not inhibited by mannose,suggesting that the sugar-binding site of the carbohydrate recognition domain(CRD)of SP-A is not involved.Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells,and this was consistent for Jurkat cells and neutrophils.Supporting these data,confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells.However,we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface,as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.展开更多
The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis.In addition,SR-AI modulates macrophage activation through cell signaling.However,inves...The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis.In addition,SR-AI modulates macrophage activation through cell signaling.However,investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors.Therefore,we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling.On 293T cells,SR-AI could respond to E.coli DH5α,leading to NF-κB activation and IL-8 production.However,this requires E.coli DH5αto be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion.Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5αand blocks DH5αstimulation of SR-AI signaling.Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b.By mutagenesis,The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α.These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.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A classical function of Clq is to bind immune complexes and initiate complement activation producing membrane lytic complexes, opsonins and anaphylatoxins. This classical pathway of complement activation is also elicited when Clq binds some other ligands. Besides complement activation, Clq also regulates cell differentiation, adhesion, migration, activation and survival. Clq deficiency is associated with autoimmunity as well as increased susceptibility to infections. In this article, we discuss the basic properties of Clq, its expression, and classical and regulatory functions. Cellular ＆ Molecular Immunology.
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Complement proteins in blood recognize charged particles.The anionic phospholipid(aPL)cardiolipin binds both complement proteins C1q and factor H.C1q is an activator of the complement classical pathway,while factor H is an inhibitor of the alternative pathway.To examine opposing effects of C1q and factor H on complement activation by aPL,we surveyed C1q and factor H binding,and complement activation by aPL,either coated on microtitre plates or in liposomes.Both C1q and factor H bound to all aPL tested,and competed directly with each other for binding.All the aPL activated the complement classical pathway,but negligibly the alternative pathway,consistent with accepted roles of C1q and factor H.However,in this system,factor H,by competing directly with C1q for binding to aPL,acts as a direct regulator of the complement classical pathway.This regulatory mechanism is distinct from its action on the alternative pathway.Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range.Thus factor H,which is regarded as a down-regulator only of the alternative pathway,has a distinct role in downregulating activation of the classical complement pathway by aPL.A factor H homologue,β2-glycoprotein-1,also strongly inhibits C1q binding to cardiolipin.Recombinant globular domains of C1q A,B and C chains bound aPL similarly to native C1q,confirming that C1q binds aPL via its globular heads.
基金This work was financially supported by the Medical Research Council,UK.
文摘The role of surfactant protein A(SP-A)in the recognition and clearance of apoptotic cells is well established,but to date,it is still not clear which surface molecules of apoptotic cells are involved in the process.Here we present evidence that phosphatidylserine(PS)is a relevant binding molecule for human SP-A.The binding is Ca^(2+)-dependent and is not inhibited by mannose,suggesting that the sugar-binding site of the carbohydrate recognition domain(CRD)of SP-A is not involved.Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells,and this was consistent for Jurkat cells and neutrophils.Supporting these data,confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells.However,we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface,as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.
基金This project is support by the Singapore Biomedical Research Council grant R-182-000-089-305National Medical Research Council grants R-364-000-019-213.JWKG received a scholarship from the National University of Singapore.
文摘The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis.In addition,SR-AI modulates macrophage activation through cell signaling.However,investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors.Therefore,we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling.On 293T cells,SR-AI could respond to E.coli DH5α,leading to NF-κB activation and IL-8 production.However,this requires E.coli DH5αto be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion.Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5αand blocks DH5αstimulation of SR-AI signaling.Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b.By mutagenesis,The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α.These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.
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.
