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Timothy A Springer - One of the best experts on this subject based on the ideXlab platform.
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complement receptor 3 forms a compact high affinity complex with IC3b
Journal of Immunology, 2021Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, Mehmet Sen, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment IC3b and, to a lesser extent, C3dg. Although the interaction between the IC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-IC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between IC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for IC3b compared with 515 nM for the IC3b thioester domain, whereas experiments monitoring binding of IC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-IC3b interaction. Small angle x-ray scattering analysis revealed that IC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, IC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the IC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the IC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by IC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.
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complement receptor 3 forms a compact high affinity complex with IC3b
bioRxiv, 2020Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin M2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells. The receptor recognizes cells opsonized with the complement fragment IC3b and to a lesser extend C3dg. While the interaction between the IC3b thioester domain and the ligand binding CR3 M I-domain is now structurally well characterized, additional CR3-IC3b interactions lack structural insight. Using an integrated structural biology approach, we analyze the interaction between IC3b and the headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments found an affinity of 30 nM of CR3 for IC3b compared to 515 nM for the IC3b thioester domain. The IC3b1 intermediate formed during factor I degradation is shown to be a CR3 headpiece ligand in addition to IC3b and C3dg. Small angle x-ray scattering analysis reveals that in solution the IC3b-CR3 complex is more compact than either of the individual proteins and prior models of the complex derived by electron microscopy. Overall, the data suggest that the IC3b-CR3 complex is structurally ordered and governed by high affinity. The identification of significant CR3-IC3b interactions outside the IC3b TE domain appears as a promising target for future therapeutics interfering specifically with the formation of the CR3-IC3b complex.
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complement receptor 3 forms a compact high affinity complex with IC3b
bioRxiv, 2020Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, Mehmet Sen, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment IC3b and to a lesser extent C3dg. While the interaction between the IC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-IC3b interactions required for phagocytosis of complement opsonized objects remain obscure. Here we analyzed the interaction between IC3b and the 150 kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of CR3 for IC3b compared to 515 nM for the IC3b thioester domain. Small angle x-ray scattering analysis revealed that IC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, IC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the IC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the IC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by IC3b than by C3dg and pave the way for development of specific therapeutics for treatment of inflammatory and neurodegenerative diseases that do not interfere with recognition of non-complement CR3 ligands.
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distinct recognition of complement IC3b by integrins αxβ2 and αmβ2
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Jianchuan Wang, Jiahuai Wang, Timothy A SpringerAbstract:Recognition by the leukocyte integrins αXβ2 and αMβ2 of complement IC3b-opsonized targets is essential for effector functions including phagocytosis. The integrin-binding sites on IC3b remain incompletely characterized. Here, we describe negative-stain electron microscopy and biochemical studies of αXβ2 and αMβ2 in complex with IC3b. Despite high homology, the two integrins bind IC3b at multiple distinct sites. αXβ2 uses the αX αI domain to bind IC3b on its C3c moiety at one of two sites: a major site at the interface between macroglobulin (MG) 3 and MG4 domains, and a less frequently used site near the C345C domain. In contrast, αMβ2 uses its αI domain to bind IC3b at the thioester domain and simultaneously interacts through a region near the αM β-propeller and β2 βI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXβ2 and the binding site of αMβ2 on IC3b. Distinctive binding sites on IC3b by integrins αXβ2 and αMβ2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors.
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binding between the integrin αxβ2 cd11c cd18 and heparin
Journal of Biological Chemistry, 2007Co-Authors: Thomas Vorupjensen, Robert J Linhardt, Lianli Chi, Louise C Gjelstrup, Uffe Birk Jensen, Craig A Jewett, Can Xie, Motomu Shimaoka, Timothy A SpringerAbstract:Abstract The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor αXβ2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin αXβ2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the αX I domain to the complement fragment IC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of IC3b, with a 1.3 μm affinity for the αX I domain. In studies of direct binding by the αX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the αXβ2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation.
Gordon D Ross - One of the best experts on this subject based on the ideXlab platform.
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-Glucan Functions as an Adjuvant for Monoclonal Antibody Immunotherapy by Recruiting Tumoricidal Granulocytes as Killer Cells
2015Co-Authors: Gordon D RossAbstract:The tumor-killing mechanisms available to monoclonal antibodies (mAbs; e.g., antagonism of growth factor receptors, antibody-dependent cell-mediated cytotoxicity) limit efficacy. Previous studies suggested that i.v. -glucan might function as an adjuvant for antitumor mAbs. -Glucan had been shown to function via the IC3b-receptor complement receptor 3 (CR3; CD11b/CD18) thereby enhancing leukocyte killing of tumor cells coated with IC3b via naturally occurring antitumor antibod-ies. Therapy with -glucans was limited by levels of natural antibodies and by tumor escape through elimination of antigen-positive cells. Ac-cordingly, it was hypothesized that -glucan responses could be improved by combined administration with antitumor mAbs. Five tumor models were explored in BALB/c or C57Bl/6 mice using tumors that expressed either high levels of naturally occurring antigens (e.g., GD2 ganglioside) or recombinant human MUC1. In comparison with antitumor mAb or -glucan alone, combined treatment with mAb plus -glucan produced significantly greater tumor regression in all models that included mam-mary, s.c., and hepatic tumors. Tumor-free survival only occurred in models that incorporated stable expression of the target antigen. -Glucan enhancement of the mAb tumoricidal response did not occur in mice deficient in either leukocyte CR3 (CD11b/) or serum C3, confirming the requirement for CR3 on leukocytes and IC3b on tumors. Granulocytes appeared to be primarily responsible for tumoricidal activity, because -glucan therapeutic responses did not occur in granulocyte-depleted mice. These data suggest that the therapeutic efficacy of mAbs known to activate complement (e.g., Herceptin, Rituxan, and Erbitux) could be significantly enhanced if they were combined with -glucan
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cr3 cd11b cd18 a phagocyte and nk cell membrane receptor with multiple ligand specificities and functions
Clinical and Experimental Immunology, 2008Co-Authors: Gordon D Ross, V VětvickaAbstract:The C3 receptor CR3 is expressed on phagocytic cells, minor subsets of B and T cells, and natural killer (NK) cells. It has important functions both as an adhesion molecule and a membrane receptor mediating recognition of diverse ligands such as intercellular adhesion molecule-1 (ICAM-1) and fixed IC3b. The receptor is capable of undergoing an activation event that regulates both its specificity for various ligands and its ability to mediate phagocytosis or extracellular cytotoxicity. Certain bacteria express carbohydrates or lipopolysaccharides (LPS) that can bind to and activate CR3, allowing the receptor to assume its activated state. Soluble beta-glucan derived from the yeast Saccharomyces cerevisiae is a particularly potent stimulator of CR3, and produces an activated state of the receptor that permits neutrophil phagocytosis of IC3b-coated erythrocytes or NK, cell cytotoxicity of IC3b-coated tumour cells, that are normally resistant to NK cells.
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mechanism by which orally administered 1 3 glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models 1
Journal of Immunology, 2004Co-Authors: Feng Hong, Jun Yan, Jarek T Baran, Daniel J Allendorf, Richard Hansen, Gary R Ostroff, Pei Xiang Xing, Naikong V Cheung, Gordon D RossAbstract:Antitumor mAb bind to tumors and activate complement, coating tumors with IC3b. Intravenously administered yeast beta-1,3;1,6-glucan functions as an adjuvant for antitumor mAb by priming the inactivated C3b (IC3b) receptors (CR3; CD11b/CD18) of circulating granulocytes, enabling CR3 to trigger cytotoxicity of IC3b-coated tumors. Recent data indicated that barley beta-1,3;1,4-glucan given orally similarly potentiated the activity of antitumor mAb, leading to enhanced tumor regression and survival. This investigation showed that orally administered yeast beta-1,3;1,6-glucan functioned similarly to barley beta-1,3;1,4-glucan with antitumor mAb. With both oral beta-1,3-glucans, a requirement for IC3b on tumors and CR3 on granulocytes was confirmed by demonstrating therapeutic failures in mice deficient in C3 or CR3. Barley and yeast beta-1,3-glucan were labeled with fluorescein to track their oral uptake and processing in vivo. Orally administered beta-1,3-glucans were taken up by macrophages that transported them to spleen, lymph nodes, and bone marrow. Within the bone marrow, the macrophages degraded the large beta-1,3-glucans into smaller soluble beta-1,3-glucan fragments that were taken up by the CR3 of marginated granulocytes. These granulocytes with CR3-bound beta-1,3-glucan-fluorescein were shown to kill IC3b-opsonized tumor cells following their recruitment to a site of complement activation resembling a tumor coated with mAb.
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β glucan functions as an adjuvant for monoclonal antibody immunotherapy by recruiting tumoricidal granulocytes as killer cells
Cancer Research, 2003Co-Authors: Feng Hong, Jun Yan, Jarek T Baran, Daniel J Allendorf, Richard Hansen, Gary R Ostroff, Gordon D RossAbstract:The tumor-killing mechanisms available to monoclonal antibodies (mAbs; e.g., antagonism of growth factor receptors, antibody-dependent cell-mediated cytotoxicity) limit efficacy. Previous studies suggested that i.v. -glucan might function as an adjuvant for antitumor mAbs. Glucan had been shown to function via the IC3b-receptor complement receptor 3 (CR3; CD11b/CD18) thereby enhancing leukocyte killing of tumor cells coated with IC3b via naturally occurring antitumor antibodies. Therapy with -glucans was limited by levels of natural antibodies and by tumor escape through elimination of antigen-positive cells. Accordingly, it was hypothesized that -glucan responses could be improved by combined administration with antitumor mAbs. Five tumor models were explored in BALB/c or C57Bl/6 mice using tumors that expressed either high levels of naturally occurring antigens (e.g., GD2 ganglioside) or recombinant human MUC1. In comparison with antitumor mAb or glucan alone, combined treatment with mAb plus -glucan produced significantly greater tumor regression in all models that included mammary, s.c., and hepatic tumors. Tumor-free survival only occurred in models that incorporated stable expression of the target antigen. -Glucan enhancement of the mAb tumoricidal response did not occur in mice deficient in either leukocyte CR3 (CD11b / ) or serum C3, confirming the requirement for CR3 on leukocytes and IC3b on tumors. Granulocytes appeared to be primarily responsible for tumoricidal activity, because -glucan therapeutic responses did not occur in granulocyte-depleted mice. These data suggest that the therapeutic efficacy of mAbs known to activate complement (e.g., Herceptin, Rituxan, and Erbitux) could be significantly enhanced if they were combined with -glucan.
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beta glucan a specific biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 cd11b cd18
Journal of Immunology, 1999Co-Authors: Jun Yan, Yu Xia, Vaclav Vetvicka, Angela Coxon, Michael C Carroll, Tanya N Mayadas, Gordon D RossAbstract:beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., IC3b) resulting in the priming of this IC3b receptor for cytotoxicity of IC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct IC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack IC3b, but can be restored by tumor-specific Abs that deposit IC3b onto the tumors.
Thomas Vorupjensen - One of the best experts on this subject based on the ideXlab platform.
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complement receptor 3 forms a compact high affinity complex with IC3b
Journal of Immunology, 2021Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, Mehmet Sen, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment IC3b and, to a lesser extent, C3dg. Although the interaction between the IC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-IC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between IC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for IC3b compared with 515 nM for the IC3b thioester domain, whereas experiments monitoring binding of IC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-IC3b interaction. Small angle x-ray scattering analysis revealed that IC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, IC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the IC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the IC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by IC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.
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complement receptor 3 forms a compact high affinity complex with IC3b
bioRxiv, 2020Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin M2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells. The receptor recognizes cells opsonized with the complement fragment IC3b and to a lesser extend C3dg. While the interaction between the IC3b thioester domain and the ligand binding CR3 M I-domain is now structurally well characterized, additional CR3-IC3b interactions lack structural insight. Using an integrated structural biology approach, we analyze the interaction between IC3b and the headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments found an affinity of 30 nM of CR3 for IC3b compared to 515 nM for the IC3b thioester domain. The IC3b1 intermediate formed during factor I degradation is shown to be a CR3 headpiece ligand in addition to IC3b and C3dg. Small angle x-ray scattering analysis reveals that in solution the IC3b-CR3 complex is more compact than either of the individual proteins and prior models of the complex derived by electron microscopy. Overall, the data suggest that the IC3b-CR3 complex is structurally ordered and governed by high affinity. The identification of significant CR3-IC3b interactions outside the IC3b TE domain appears as a promising target for future therapeutics interfering specifically with the formation of the CR3-IC3b complex.
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complement receptor 3 forms a compact high affinity complex with IC3b
bioRxiv, 2020Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, Mehmet Sen, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment IC3b and to a lesser extent C3dg. While the interaction between the IC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-IC3b interactions required for phagocytosis of complement opsonized objects remain obscure. Here we analyzed the interaction between IC3b and the 150 kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of CR3 for IC3b compared to 515 nM for the IC3b thioester domain. Small angle x-ray scattering analysis revealed that IC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, IC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the IC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the IC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by IC3b than by C3dg and pave the way for development of specific therapeutics for treatment of inflammatory and neurodegenerative diseases that do not interfere with recognition of non-complement CR3 ligands.
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structural insight on the recognition of surface bound opsonins by the integrin i domain of complement receptor 3
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Goran Bajic, Thomas Vorupjensen, Laure Yatime, G R AndersenAbstract:Complement receptors (CRs), expressed notably on myeloid and lymphoid cells, play an essential function in the elimination of complement-opsonized pathogens and apoptotic/necrotic cells. In addition, these receptors are crucial for the cross-talk between the innate and adaptive branches of the immune system. CR3 (also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on all macrophages and recognizes IC3b on complement-opsonized objects, enabling their phagocytosis. We demonstrate that the C3d moiety of IC3b harbors the binding site for the CR3 αI domain, and our structure of the C3d:αI domain complex rationalizes the CR3 selectivity for IC3b. Based on extensive structural analysis, we suggest that the choice between a ligand glutamate or aspartate for coordination of a receptor metal ion-dependent adhesion site-bound metal ion is governed by the secondary structure of the ligand. Comparison of our structure to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d:CR2 complex suggests a molecular mechanism for the hand-over of CR3-bound immune complexes from macrophages to CR2-presenting cells in lymph nodes.
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binding between the integrin αxβ2 cd11c cd18 and heparin
Journal of Biological Chemistry, 2007Co-Authors: Thomas Vorupjensen, Robert J Linhardt, Lianli Chi, Louise C Gjelstrup, Uffe Birk Jensen, Craig A Jewett, Can Xie, Motomu Shimaoka, Timothy A SpringerAbstract:Abstract The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor αXβ2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin αXβ2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the αX I domain to the complement fragment IC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of IC3b, with a 1.3 μm affinity for the αX I domain. In studies of direct binding by the αX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the αXβ2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation.
G R Andersen - One of the best experts on this subject based on the ideXlab platform.
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complement receptor 3 forms a compact high affinity complex with IC3b
Journal of Immunology, 2021Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, Mehmet Sen, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment IC3b and, to a lesser extent, C3dg. Although the interaction between the IC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-IC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between IC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for IC3b compared with 515 nM for the IC3b thioester domain, whereas experiments monitoring binding of IC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-IC3b interaction. Small angle x-ray scattering analysis revealed that IC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, IC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the IC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the IC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by IC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.
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complement receptor 3 forms a compact high affinity complex with IC3b
bioRxiv, 2020Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin M2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells. The receptor recognizes cells opsonized with the complement fragment IC3b and to a lesser extend C3dg. While the interaction between the IC3b thioester domain and the ligand binding CR3 M I-domain is now structurally well characterized, additional CR3-IC3b interactions lack structural insight. Using an integrated structural biology approach, we analyze the interaction between IC3b and the headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments found an affinity of 30 nM of CR3 for IC3b compared to 515 nM for the IC3b thioester domain. The IC3b1 intermediate formed during factor I degradation is shown to be a CR3 headpiece ligand in addition to IC3b and C3dg. Small angle x-ray scattering analysis reveals that in solution the IC3b-CR3 complex is more compact than either of the individual proteins and prior models of the complex derived by electron microscopy. Overall, the data suggest that the IC3b-CR3 complex is structurally ordered and governed by high affinity. The identification of significant CR3-IC3b interactions outside the IC3b TE domain appears as a promising target for future therapeutics interfering specifically with the formation of the CR3-IC3b complex.
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complement receptor 3 forms a compact high affinity complex with IC3b
bioRxiv, 2020Co-Authors: Rasmus K Jensen, Goran Bajic, Thomas Vorupjensen, Timothy A Springer, Mehmet Sen, G R AndersenAbstract:Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment IC3b and to a lesser extent C3dg. While the interaction between the IC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-IC3b interactions required for phagocytosis of complement opsonized objects remain obscure. Here we analyzed the interaction between IC3b and the 150 kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of CR3 for IC3b compared to 515 nM for the IC3b thioester domain. Small angle x-ray scattering analysis revealed that IC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, IC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the IC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the IC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by IC3b than by C3dg and pave the way for development of specific therapeutics for treatment of inflammatory and neurodegenerative diseases that do not interfere with recognition of non-complement CR3 ligands.
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structural insight on the recognition of surface bound opsonins by the integrin i domain of complement receptor 3
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Goran Bajic, Thomas Vorupjensen, Laure Yatime, G R AndersenAbstract:Complement receptors (CRs), expressed notably on myeloid and lymphoid cells, play an essential function in the elimination of complement-opsonized pathogens and apoptotic/necrotic cells. In addition, these receptors are crucial for the cross-talk between the innate and adaptive branches of the immune system. CR3 (also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on all macrophages and recognizes IC3b on complement-opsonized objects, enabling their phagocytosis. We demonstrate that the C3d moiety of IC3b harbors the binding site for the CR3 αI domain, and our structure of the C3d:αI domain complex rationalizes the CR3 selectivity for IC3b. Based on extensive structural analysis, we suggest that the choice between a ligand glutamate or aspartate for coordination of a receptor metal ion-dependent adhesion site-bound metal ion is governed by the secondary structure of the ligand. Comparison of our structure to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d:CR2 complex suggests a molecular mechanism for the hand-over of CR3-bound immune complexes from macrophages to CR2-presenting cells in lymph nodes.
John P Atkinson - One of the best experts on this subject based on the ideXlab platform.
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functional analysis of rare genetic variants in complement factor i cfi using a serum based assay in advanced age related macular degeneration
Translational Vision Science & Technology, 2020Co-Authors: Anuja Java, John P Atkinson, Peter Baciu, Rafael Widjajahakim, Yun Ju Sung, Jae Yang, David J Kavanagh, Johanna M SeddonAbstract:Purpose Factor I (FI) is a serine protease regulator of the complement system. Genetic variants in CFI are associated with advanced age-related macular degeneration (AAMD). However, the clinical and functional impact of these variants is unknown. This study assessed the functional significance of rare CFI variants using a serum-based assay. Methods Carriers of rare variants with (n = 78) and without AAMD (n = 28), and noncarriers with (n = 49) and without AMD (n = 44) were evaluated. Function of FI was determined by measuring the proteolytic cleavage of C3b to IC3b, using the cofactor protein, Factor H. Results CFI variants were categorized into three groups based on antigenic and functional assessments. Type 1 variants (n = 18) in 35 patients with AAMD demonstrated low serum FI levels and a corresponding decrease in FI function. Type 2 variants (n = 6) in 7 individuals demonstrated normal serum FI antigenic levels but reduced degradation of C3b to IC3b. Type 3 variants (n = 15) in 64 individuals demonstrated normal antigenic levels and degradation of C3b to IC3b. However, IC3b generation was low when measured per unit of FI. Thus most rare CFI variants demonstrate either low antigenic levels (type 1) or normal levels but reduced function (types 2 or 3). Conclusions Results provide for the first time a comprehensive functional assessment in serum of CFI rare genetic variants and further establish FI's key role in the pathogenesis of AAMD. Translational Relevance Stratifying patients in the clinic with a rare CFI variant will facilitate screening and targeting patients most likely to benefit from complement therapies.
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54 blood concentrations of complement split product IC3b and serum c3 associate with systemic lupus erythematosus disease activity
Lupus science & medicine, 2019Co-Authors: Vibeke Strand, Qiang J Fu, Martin Schmidt, John P AtkinsonAbstract:Background A major unmet need in SLE is the identification of a biomarker that consistently tracks with disease activity. One current approach is measuring complement activation by evaluating consumption of serum C3 and C4. However, since they are acute phase reactants, interpretation of these levels is challenging as serum levels may not decrease until late in a disease flare. IC3b is a proteolytically derived molecule of C3b and increases with complement activation. IC3b/C3 ratio measures complement consumption relative to production, which may provide for a more accurate assessment of complement activation. We hypothesize that blood IC3b and IC3b/C3 levels will provide a more specific and reliable marker of complement activation and disease activity in SLE. Methods 159 consecutive subjects with American College of Rheumatology or Systemic Lupus International Collaborating Clinics classified SLE were enrolled into CASTLE (Complement Activation Signatures in Systemic Lupus Erythematosus), a prospective observational study. Patients with 1–7 study visits were included in this longitudinal analysis. 48 healthy volunteers were enrolled to establish the normal reference IC3b/C3 ratio. Serum C3 and C4 were measured by nephelometry and blood IC3b levels by a lateral flow assay. SLE disease activity was monitored utilizing the Systemic Lupus Erythematosus Disease Activity Index 2K Responder Index-50 instrument. Results IC3b/C3 ratio, double-stranded (ds)DNA antibodies (Abs), and supraphysiologic prednisone dose (>7.5 mg/day) each independently correlated with SLE disease activity, employing multilevel multiple logistic regression analysis. Only the IC3b/C3 ratio was significantly associated with clinically meaningful improvements in disease activity among subjects receiving supraphysiologic doses of prednisone. IC3b/C3 outperformed C3 and C4 levels discriminating both active versus inactive SLE disease and major flares versus no disease activity. iC3/C3, dsDNA Abs, ESR, and supraphysiologic prednisone dose were independently associated with lupus nephritis, while none were associated with SLE rash. The association of IC3b/C3 with nephritis was independent of other observed clinical manifestations. Finally, we observed a stronger association of the IC3b/C3 ratio with SLE disease activity in African-Americans compared to Whites. Conclusions Blood IC3b/C3 correlates with SLE disease activity and clinically meaningful changes. Furthermore, it discriminates between active versus inactive SLE, and major flares compared to those patients without active disease. Differences in the strength of association was observed between races and manifestations. Funding Source(s): Kypha, Inc. and National Institutes of Health (NIH)/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) under Award Number R21AR069833.
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association of blood concentrations of complement split product IC3b and serum c3 with systemic lupus erythematosus disease activity
Arthritis & Rheumatism, 2019Co-Authors: Alfred H J Kim, Vibeke Strand, Martin Schmidt, Nancy L Mathis, Deepali Sen, Robin R Bruchas, Nick Staten, Paul Olson, Chad Stiening, John P AtkinsonAbstract:Objective To examine correlations between blood levels of complement split product IC3b and serum component C3 with clinically meaningful changes in disease activity in patients with systemic lupus erythematosus (SLE). Methods A total of 159 consecutive patients with SLE, diagnosed according to the American College of Rheumatology or Systemic Lupus International Collaborating Clinics classification criteria, were enrolled in CASTLE (Complement Activation Signatures in Systemic Lupus Erythematosus), a prospective observational study. Patients with 1-7 study visits were included in this longitudinal analysis. In addition, 48 healthy volunteers were enrolled to establish a normal reference value for the ratio of blood IC3b to serum C3 concentrations. Serum C3 and C4 levels were measured by nephelometry, and blood IC3b levels were measured by a lateral flow assay. SLE disease activity was monitored with the Responder Index 50 instrument of the SLE Disease Activity Index 2000. Results Relative changes in the IC3b:C3 ratio, levels of anti-double-stranded DNA (anti-dsDNA) antibodies, and use of a supraphysiologic dose of prednisone (>7.5 mg/day) each independently correlated with SLE disease activity, as determined in multilevel multiple logistic regression analyses. Only the IC3b:C3 ratio was significantly associated with clinically meaningful improvements in disease activity among patients with SLE who were receiving a supraphysiologic dose of prednisone. The IC3b:C3 ratio outperformed C3 and C4 levels with regard to discriminating active SLE from inactive SLE, and major flares from no disease activity. The iC3:C3 ratio, anti-dsDNA antibody levels, erythrocyte sedimentation rate, and use of a supraphysiologic prednisone dose were each independently associated with the presence of lupus nephritis, whereas none of these measures was associated with SLE rash. The association of the IC3b:C3 ratio with lupus nephritis was independent of other observed clinical manifestations. Conclusion The ratio of blood IC3b to serum C3 concentrations correlates with the extent of SLE disease activity and with clinically meaningful changes in disease activity in patients with SLE. Furthermore, the IC3b:C3 ratio may discriminate between active and inactive SLE, and between major flares and no active disease.
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dissection of cr1 factor h membrane cofactor protein and factor b binding and functional sites in the third complement component
Journal of Immunology, 1996Co-Authors: John D Lambris, John P Atkinson, Zhege Lao, T J Oglesby, C E Hack, J D BechererAbstract:Previous studies have suggested that the residues 727-768 of human (Hu) C3 contain the binding sites for CR1, factor H, and factor B. Here, we have (1) characterized further some of the C3 structural requirements for its binding to CR1, H, and B, (2) investigated the functions associated with these C3-ligand interactions, and (3) studied the relationship of MCP-binding sites in C3 with those for CR1, H, and B. Hu C3 molecules in which residues 727-768 were deleted (designated C3delta727-768) or substituted with the corresponding segment of cobra venom factor, Xenopus, or trout C3 (chimeric C3s) were expressed in the baculovirus system and analyzed for their reactivity with C3-binding proteins. In contrast to wild-type iC3 which, in the presence of CR1, is cleaved by factor I to IC3b-a and C3c-a and C3dg, all chimeric C3s were cleaved only to IC3b-a. In addition, the cleavage of deleted (C3delta727-768) iC3 to IC3b-a by factor I in the presence of CR1 was significantly reduced, whereas it remained unaltered in the presence of MCP. Cleavage of iC3 to IC3b-a by factor I and H was similar in all expressed C3s except C3delta727-768, whose cleavage was significantly reduced. All of the expressed molecules except C3delta727-768 were capable of forming the fluid-phase alternative pathway C3 convertase, and all reacted with properdin. These results suggest that during cleavage of iC3 by factor I and CR1, or H, CR1 and H bind to at least two sites on C3 and that the MCP binding site(s) on C3b are different from those for CR1. They also indicate that some or all of the C3 residues that are directly involved in, or contribute to, the structure of one of the CR1 and H binding sites are located within residues 727-768. These studies also demonstrate that, although this segment of C3 may be involved in C3-factor B interaction, other residues in addition to 736EE (previously implicated in B binding) must also contribute significantly to this interaction.