Fixation of the first component of complement by immune complexes: effect of reduction and fragmentation of antibody.
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ABSTRACT:
SUBMITTER: Press EM
PROVIDER: S-EPMC1165615 | biostudies-other | 1975 Jul
REPOSITORIES: biostudies-other
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Project description:The activation of subcomponents C1r and C1s in the first component of complement, C1, when bound to antibody-antigen complexes was investigated. Activation was followed both by the splitting of the peptide chains of subcomponents C1r and C1s and by the development of proteolytic activity. For the maximum rate of activation to occur, all components must be present in approximate molar proportions of antibody: C1q:C1r:C1s of 13:1:5:5. For activation of subcomponent C1s, subcomponents C1r or C1r, but not C1r inactivated with iPr2P-F (di-isopropyl phosphorofluorideate), are effective. For activation of subcomponent C1r, subcomponents C1s, C1s or C1s inactivated with iPr2P-F are effective. Subcomponent C1s is activated by C1r, and C1r is activated autocatalytically, probably through the formation of an intermediary C1r. in which the peptide chain is unsplit but a conformational change caused by interaction with the other components has led to the formation of a catalytic site able to split subcomponent C1r to C1r.
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Project description:The interaction of C1 inhibitor with complement component C1 bound to immune complexes was examined by using 125I-labelled C1 subcomponents. The inhibitor binds rapidly to subcomponent C1s, and more slowly to subcomponent C1r. Formation of the C1r-C1 inhibitor complex causes rapid dissociation of subcomponents C1r and C1s from the antibody-antigen-component C1 aggregate. The rate and extent of this release are proportional to C1 Inhibitor concentration and are also dependent on ionic strength. Results obtained with purified C1 Inhibitor, plasma or serum as source of C1 Inhibitor are all closely comparable. Only slight dissociation of subcomponent C1q is observed under the same range of conditions. The implications of the release phenomenon are discussed in relation to the structure of component C1 and the possibility of differential turnover of C1 subcomponents.
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Project description:Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.
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Project description:Age-related macular degeneration (AMD) is one of the main causes of visual impairment in adults. Visual deterioration is more prominent in neovascular AMD with choroidal neovascularization (CNV). Clinical and postmortem studies suggested that complement system activation might induce CNV. In this study, we demonstrated that an anti-mouse complement component 5 (C5) antibody targeting MG4 domain of ? chain effectively inhibited CNV which was induced by laser photocoagulation in mice. The targeted epitope of this anti-C5 antibody was different from that of currently utilized anti-C5 antibody (eculizumab) in the MG7 domain in which a single nucleotide polymorphism (R885H/C) results in poor response to eculizumab. Even with targeting MG4 domain, this anti-C5 antibody reduced production of C5a, monocyte chemoattractant protein-1 and vascular endothelial growth factor to prevent infiltration of F4/80-positive cells into CNV lesions and formation of CNV. Furthermore, anti-C5 antibody targeting MG4 domain induced no definite toxicity in normal retina. These results demonstrated that anti-C5 antibody targeting MG4 domain inhibited CNV in neovascular AMD.
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Project description:1. Haemolytic activity corresponding to that of the first component of complement (C1) was synthesized and secreted by all nine human fibroblast cell lines examined. No activity was found in the culture media of a variety of other human cell lines. 2. The component-C1 haemolytic activity secreted by the fibroblast lines behaved in an identical manner, in most respects, with that of the component-C1 haemolytic activity of human serum. The component-C1 haemolytic activity secreted by fibroblasts, however, was less susceptible to inhibition by rabbit fragment F(ab')(2) anti-(human subcomponent C1q) than was the component-C1 haemolytic activity of human serum. 3. Biosynthesis of fibroblast component-C1 haemolytic activity was inhibited by the presence of cycloheximide and regained on its removal. 4. Incorporation of radioactivity into proteins secreted by the fibroblasts and release of component-C1 haemolytic activity by the fibroblasts both increased in a linear manner until several days after the cultures had reached a state of confluent growth. 5. Radioactivity was incorporated into subcomponents C1q, C1r and C1s, as judged by the formation of specific immunoprecipitates and by absorption with immune aggregates. 6. The immunoprecipitates formed by using antisera against subcomponents C1r and C1s were run on polyacrylamide gels in sodium dodecyl sulphate, and this provided convincing physiochemical evidence for the biosynthesis of these subcomponents de novo. 7. The results obtained with immunoprecipitates formed by using anti-(subcomponent C1q) suggest that subcomponent C1q may be synthesized and secreted by fibroblast cell lines in vitro, in a form with a higher molecular weight than that of subcomponent C1q which is isolated by conventional techniques of protein fractionation from fresh serum.
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Project description:The heptoseless mutant of Escherichia coli, E. coli D31 m4, binds C1q and C1 at 0 degrees C and at low ionic strength (I0.07). Under these conditions, the maximum C1q binding averages 3.0 X 10(5) molecules per bacterium, with a Ka of 1.4 X 10(8) M-1. Binding involves the collagen-like region of C1q, as shown by the capacity of C1q pepsin-digest fragments to bind to E. coli D31 m4, and to compete with native C1q. Proenzyme and activated forms of C1 subcomponents C1r and C1s and their Ca2+-dependent association (C1r-C1s)2 do not bind to E. coli D31 m4. In contrast, the C1 complex binds very effectively, with an average fixation of 3.5 X 10(5) molecules per bacterium, and a Ka of 0.25 X 10(8) M-1, both comparable with the values obtained for C1q binding. C1 bound to E. coli D31 m4 undergoes rapid activation at 0 degrees C. The activation process is not affected by C1-inhibitor, and only slightly inhibited by p-nitrophenyl p'-guanidinobenzoate. No turnover of the (C1r-C1s)2 subunit is observed. Once activated, C1 is only partially dissociated by C1-inhibitor. Our observations are in favour of a strong association between C1 and the outer membrane of E. coli D31 m4, involving mainly the collagen-like moiety of C1.
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Project description:The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development.
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