ABSTRACT
Complement receptor 1 (CR1) is a membrane glycoprotein with a highly duplicated domain structure able to bind multiple ligands such as C3b and C4b, the activated fragments of complement components C3 and C4, respectively. We have previously used our knowledge of this domain structure to identify CSL040, a soluble extracellular fragment of CR1 containing the long homologous repeat (LHR) domains A, B, and C. CSL040 retains the ability to bind both C3b and C4b but is also a more potent complement inhibitor than other recombinant CR1-based therapeutics. To generate soluble CR1 variants with increased inhibitory potential across all three complement pathways, or variants with activity skewed to specific pathways, we exploited the domain structure of CR1 further by generating LHR domain duplications. We identified LHR-ABCC, a soluble CR1 variant containing a duplicated C3b-binding C-terminal LHR-C domain that exhibited significantly enhanced alternative pathway inhibitory activity in vitro compared to CSL040. Another variant, LHR-BBCC, containing duplications of both LHR-B and LHR-C with four C3b binding sites, was shown to have reduced classical/lectin pathway inhibitory activity compared to CSL040, but comparable alternative pathway activity. Interestingly, multiplication of the C4b-binding LHR-A domain resulted in only minor increases in classical/lectin pathway inhibitory activity. The CR1 duplication variants characterized in these in vitro potency assays, as well as in affinity in solution C3b and C4b binding assays, not only provides an opportunity to identify new therapeutic molecules but also additional mechanistic insights to the multiple interactions between CR1 and C3b/C4b.
Subject(s)
Complement C3b , Protein Domains , Humans , Complement C3b/metabolism , Complement C3b/chemistry , Complement C3b/genetics , Receptors, Complement 3b/metabolism , Receptors, Complement 3b/genetics , Receptors, Complement 3b/chemistry , Complement C4b/metabolism , Complement C4b/genetics , Complement C4b/chemistry , Protein BindingABSTRACT
The complement system is a potent mediator of ischemia-reperfusion injury (IRI), which detrimentally affects the function and survival of transplanted kidneys. Human complement receptor 1 (HuCR1) is an integral membrane protein that inhibits complement activation by blocking the convertases that activate C3 and C5. We have previously reported that CSL040, a truncated form of recombinant soluble HuCR1 (sHuCR1), has enhanced complement inhibitory activity and improved pharmacokinetic properties compared to the parent molecule. Here, we compared the capacity of CSL040 and full-length sHuCR1 to suppress complement-mediated organ damage in a mouse model of warm renal IRI. Mice were treated with two doses of CSL040 or sHuCR1, given 1 h prior to 22 min unilateral renal ischemia and again 3 h later. 24 h after reperfusion, mice treated with CSL040 were protected against warm renal IRI in a dose-dependent manner, with the highest dose of 60 mg/kg significantly reducing renal dysfunction, tubular injury, complement activation, endothelial damage, and leukocyte infiltration. In contrast, treatment with sHuCR1 at a molar equivalent dose to 60 mg/kg CSL040 did not confer significant protection. Our results identify CSL040 as a promising therapeutic candidate to attenuate renal IRI and demonstrate its superior efficacy over full-length sHuCR1 in vivo.
Subject(s)
Kidney/injuries , Receptors, Complement 3b/administration & dosage , Reperfusion Injury/prevention & control , Animals , Complement Activation/drug effects , Disease Models, Animal , Humans , Kidney/drug effects , Kidney/immunology , Kidney Transplantation/adverse effects , Male , Mice , Mice, Inbred C57BL , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Receptors, Complement 3b/chemistry , Reperfusion Injury/etiology , Reperfusion Injury/immunology , SolubilityABSTRACT
Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders.
Subject(s)
Complement Activation , Receptors, Complement 3b/immunology , Animals , Cell Line , Complement C3b/immunology , Complement C4b/immunology , Female , Glomerulonephritis/immunology , Glomerulonephritis/therapy , Humans , Mice , Mice, Inbred C57BL , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/therapeutic use , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic useABSTRACT
BACKGROUND: All antigens described in the KN blood group system are located in the long homologous repeat D (LHR-D) of complement receptor 1 (CR1). While there have been reports that some sera react only with the long homologous repeat C (LHR-C), the antigens in LHR-C are unknown. STUDY DESIGN AND METHODS: Recombinant LHR-C and LHR-D were used to identify antibodies directed against LHR-C of CR1, into which a point mutation was introduced to characterize the underlying blood group antigens. In addition, database studies to define haplotypes of CR1 were performed. RESULTS: Several antisera were identified that were specific against CR1 p.1208His and against CR1 p.1208Arg, located in LHR-C. Fifteen KN haplotypes were found in the Ensembl genome browser. It was shown that due to a linkage disequilibrium anti-CR1 p.1208His may be mistaken for anti-KCAM. CONCLUSION: A novel antithetical KN blood group antigen pair was found at position p.1208 of CR1, for which the names DACY and YCAD are proposed. Antibodies against these two novel antigens seem to contribute to more than a quarter of all KN sera in Europe.
Subject(s)
Blood Group Antigens , Point Mutation , Polymorphism, Genetic , Receptors, Complement 3b , Amino Acid Substitution , Antibodies/chemistry , Antibodies/immunology , Blood Group Antigens/chemistry , Blood Group Antigens/genetics , Blood Group Antigens/immunology , Europe , Humans , Protein Domains , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/genetics , Receptors, Complement 3b/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
Dysregulation of the complement system is involved in the pathogenesis of several diseases, and its inhibition has been shown to be a feasible therapeutic option. Therefore, there is an interest in the development of complement modulators to treat complement activation-related inflammatory pathologies. Mannose-binding lectin (MBL)/ficolin/collectin-associated protein-1 (MAP-1) is a regulatory molecule of the lectin pathway (LP), whereas complement receptor 1 (CD35) and decay-accelerating factor (CD55) are membrane-anchored regulators with effects on the central effector molecule C3. In this study, we developed 2 novel soluble chimeric inhibitors by fusing MAP-1 to the 3 first domains of CD35 (CD351-3) or the 4 domains of CD55 (CD551-4) to modulate the complement cascade at 2 different stages. The constructs showed biologic properties similar to those of the parent molecules. In functional complement activation assays, the constructs were very efficient in inhibiting LP activation at the level of C3 and in the formation of terminal complement complex. This activity was enhanced when coincubated with recombinant LP recognition molecules MBL and ficolin-3. Recombinant MAP-1 fusion proteins, combined with recombinant LP recognition molecules to target sites of inflammation, represent a novel and effective therapeutic approach involving the initiation and the central and terminal effector functions of the complement cascade.-Pérez-Alós, L., Bayarri-Olmos, R., Skjoedt, M.-O., Garred, P. Combining MAP-1:CD35 or MAP-1:CD55 fusion proteins with pattern-recognition molecules as novel targeted modulators of the complement cascade.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , CD55 Antigens , Complement Activation/drug effects , Complement C3 , Receptors, Complement 3b , Receptors, Pattern Recognition , Recombinant Fusion Proteins , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/pharmacology , Animals , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/pharmacology , CD55 Antigens/chemistry , CD55 Antigens/genetics , CD55 Antigens/pharmacology , CHO Cells , Complement C3/chemistry , Complement C3/metabolism , Cricetulus , Humans , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/genetics , Receptors, Pattern Recognition/chemistry , Receptors, Pattern Recognition/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacologyABSTRACT
Regulation of complement activation in the host cells is mediated primarily by the regulators of complement activation (RCA) family proteins that are formed by tandemly repeating complement control protein (CCP) domains. Functional annotation of these proteins, however, is challenging as contiguous CCP domains are found in proteins with varied functions. Here, by employing an in silico approach, we identify five motifs which are conserved spatially in a specific order in the regulatory CCP domains of known RCA proteins. We report that the presence of these motifs in a specific pattern is sufficient to annotate regulatory domains in RCA proteins. We show that incorporation of the lost motif in the fourth long-homologous repeat (LHR-D) in complement receptor 1 regains its regulatory activity. Additionally, the motif pattern also helped annotate human polydom as a complement regulator. Thus, we propose that the motifs identified here are the determinants of functionality in RCA proteins.
Subject(s)
Cell Adhesion Molecules/metabolism , Complement Activation , Complement System Proteins/metabolism , Receptors, Complement 3b/metabolism , Amino Acid Motifs , Animals , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cnidaria/chemistry , Cnidaria/metabolism , Complement System Proteins/chemistry , Complement System Proteins/genetics , Conserved Sequence , Humans , Phylogeny , Protein Conformation , Protein Domains , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/genetics , Structure-Activity Relationship , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
INTRODUCTION: Genome-wide association studies consistently show that single nucleotide polymorphisms (SNPs) in the complement receptor 1 (CR1) gene modestly but significantly alter Alzheimer's disease (AD) risk. Follow-up research has assumed that CR1 is expressed in the human brain despite a paucity of evidence for its function there. Alternatively, erythrocytes contain >80% of the body's CR1, where, in primates, it is known to bind circulating pathogens. METHODS: Multidisciplinary methods were employed. RESULTS: Conventional Western blots and quantitative polymerase chain reaction failed to detect CR1 in the human brain. Brain immunohistochemistry revealed only vascular CR1. By contrast, erythrocyte CR1 immunoreactivity was readily observed and was significantly deficient in AD, as was CR1-mediated erythrocyte capture of circulating amyloid ß peptide. CR1 SNPs associated with decreased erythrocyte CR1 increased AD risk, whereas a CR1 SNP associated with increased erythrocyte CR1 decreased AD risk. DISCUSSION: SNP effects on erythrocyte CR1 likely underlie the association of CR1 polymorphisms with AD risk.
Subject(s)
Amyloid beta-Peptides/metabolism , Polymorphism, Single Nucleotide , Receptors, Complement 3b/genetics , Receptors, Complement 3b/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Apolipoproteins E/genetics , Erythrocytes/metabolism , Female , Genetic Predisposition to Disease , Humans , Male , Microglia/metabolism , Neocortex/metabolism , Prospective Studies , Protein Isoforms , Receptors, Complement 3b/chemistryABSTRACT
Complement receptor type 1 (CR1) is a multi modular membrane receptor composed of 30 homologous complement control protein modules (CCP) organized in four different functional regions called long homologous repeats (LHR A, B, C, and D). CR1 is a receptor for complement-opsonins C3b and C4b and specifically interacts through pairs of CCP modules located in LHR A, B, and C. Defense collagens such as mannose-binding lectin (MBL), ficolin-2, and C1q also act as opsonins and are involved in immune clearance through binding to the LHR-D region of CR1. Our previous results using deletion variants of CR1 mapped the interaction site for MBL and ficolin-2 on CCP24-25. The present work aimed at deciphering the interaction of C1q with CR1 using new CR1 variants concentrated around CCP24-25. CR1 bimodular fragment CCP24-25 and CR1 CCP22-30 deleted from CCP24-25 produced in eukaryotic cells enabled to highlight that the interaction site for both MBL and C1q is located on the same pair of modules CCP24-25. C1q binding to CR1 shares with MBL a main common interaction site on the collagen stalks but also subsidiary sites most probably located on C1q globular heads, contrarily to MBL.
Subject(s)
Complement C1q/chemistry , Mannose-Binding Lectin/chemistry , Peptides/chemistry , Receptors, Complement 3b/chemistry , Complement C1q/genetics , Complement C1q/immunology , Humans , Lectins/chemistry , Lectins/genetics , Lectins/immunology , Mannose-Binding Lectin/genetics , Mannose-Binding Lectin/immunology , Peptides/genetics , Peptides/immunology , Protein Binding , Protein Domains , Protein Structure, Secondary , Receptors, Complement 3b/genetics , Receptors, Complement 3b/immunology , FicolinsABSTRACT
BACKGROUND: Delayed graft function (DGF) is traditionally defined as the requirement for dialysis during the first week after transplantation. DGF is a common complication of renal transplantation, and it negatively affects short- and long-term graft outcomes. Ischaemia reperfusion injury (IRI) is a prime contributor to the development of DGF. It is well established that complement system activation plays a pivotal role in the pathogenesis of IRI. Mirococept is a highly effective complement inhibitor that can be administered ex vivo to the donor kidney just before transplantation. Preclinical and clinical evidence suggests that Mirococept inhibits inflammatory responses that follow IRI. The EMPIRIKAL trial (REC 12/LO/1334) aims to evaluate the efficacy of Mirococept in reducing the incidence of DGF in cadaveric renal transplantation. METHODS/DESIGN: EMPIRIKAL is a multicentre double-blind randomised case-control trial designed to test the superiority of Mirococept in the prevention of DGF in cadaveric renal allografts, as compared to standard cold perfusion fluid (Soltran®). Patients will be randomised to Mirococept or placebo (Pbo) and will be enrolled in cohorts of N = 80 with a maximum number of 7 cohorts. The first cohort will be randomised to 10 mg of Mirococept or Pbo. After the completion of each cohort, an interim analysis will be carried out in order to evaluate the dose allocation for the next cohort (possible doses: 5-25 mg). Immunosuppression therapy, antibiotic and antiviral prophylaxis will be administered as per local centre protocols. The enrolment will take approximately 24 months, and patients will be followed for 12 months. The primary endpoint is DGF, defined as the requirement for dialysis during the first week after transplantation. Secondary endpoints include duration of DGF, functional DGF, renal function at 12 months, acute rejection episodes at 6 and 12 months, primary non-function and time of hospital stay on first admission and in the first year following transplant. Safety evaluation will include the monitoring of laboratory data and the recording of all adverse events. DISCUSSION: The EMPIRIKAL trial is the first study to evaluate the efficacy of an ex vivo administered complement inhibitor (Mirococept) in preventing DGF in cadaveric human renal transplantation. Mirococept has a unique 'cytotopic' property that permits its retention in the organ microvasculature. TRIAL REGISTRATION: ISRCTN registry, ISRCTN49958194 . Registered on 3 August 2012.
Subject(s)
Complement Inactivating Agents/administration & dosage , Delayed Graft Function/prevention & control , Kidney Transplantation/adverse effects , Peptide Fragments/administration & dosage , Receptors, Complement 3b/chemistry , Reperfusion Injury/prevention & control , Allografts , Clinical Protocols , Complement Inactivating Agents/adverse effects , Delayed Graft Function/diagnosis , Delayed Graft Function/immunology , Double-Blind Method , Drug Administration Schedule , Humans , Peptide Fragments/adverse effects , Peptide Fragments/chemistry , Reperfusion Injury/diagnosis , Reperfusion Injury/immunology , Research Design , Time Factors , Treatment Outcome , United KingdomABSTRACT
Alzheimer's is a neurodegenerative disease affecting large populations worldwide characterized mainly by progressive loss of memory along with various other symptoms. The foremost cause of the disease is still unclear, however various mechanisms have been proposed to cause the disease that include amyloid hypothesis, tau hypothesis, and cholinergic hypothesis in addition to genetic factors. Various genes have been known to be involved which are APOE, PSEN1, PSEN2, and APP among others. In the present study, we have used computational methods to examine the pathogenic effects of non-synonymous single nucleotide polymorphisms (SNPs) associated with ABCA7, CR1, MS4A6A, CD2AP, PSEN1, PSEN2, and APP genes. The SNPs were obtained from dbSNP database followed by identification of deleterious SNPs and prediction of their functional impact. Prediction of disease-associated mutations was performed and the impact of the mutations on the stability of the protein was carried out. To study the structural significance of the computationally prioritized mutations on the proteins, molecular dynamics simulation studies were carried out. On analysis, the SNPs with IDs rs76282929 ABCA7; CR1 rs55962594; MS4A6A rs601172; CD2AP rs61747098; PSEN1 rs63750231, rs63750265, rs63750526, rs63750577, rs63750687, rs63750815, rs63750900, rs63751037, rs63751163, rs63751399; PSEN2 rs63749851; and APP rs63749964, rs63750066, rs63750734, and rs63751039 were predicted to be deleterious and disease-associated having significant structural impact on the proteins. The current study proposes a precise computational methodology for the identification of disease-associated SNPs. J. Cell. Biochem. 118: 1471-1479, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
Alzheimer Disease/genetics , Computational Biology/methods , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/genetics , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Dynamics Simulation , Presenilin-1/chemistry , Presenilin-1/genetics , Presenilin-2/chemistry , Presenilin-2/genetics , Protein Stability , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/geneticsABSTRACT
Regulators of complement activation (RCA) inhibit complement-induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i-iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b-binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease-related mutations and immune evasion.
Subject(s)
Complement C3b/chemistry , Complement C3b/metabolism , Binding Sites , CD55 Antigens/chemistry , CD55 Antigens/metabolism , Complement Activation , Humans , Membrane Cofactor Protein/chemistry , Membrane Cofactor Protein/metabolism , Protein Domains , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolismABSTRACT
The regulators of complement activation gene cluster encodes a group of proteins that have evolved to control the amplification of complement at the critical step of C3 activation. Complement receptor 1 (CR1) is the most versatile of these inhibitors with both receptor and regulatory functions. While expressed on most peripheral blood cells, the only epithelial site of expression in the kidney is by the podocyte. Its expression by this cell population has aroused considerable speculation as to its biologic function in view of many complement-mediated renal diseases. The goal of this investigation was to assess the role of CR1 on epithelial cells. To this end, we utilized a Chinese hamster ovary cell model system. Among our findings, CR1 reduced C3b deposition by â¼ 80% during classical pathway activation; however, it was an even more potent regulator (>95% reduction in C3b deposition) of the alternative pathway. This inhibition was primarily mediated by decay accelerating activity. The deposited C4b and C3b were progressively cleaved with a t½ of â¼ 30 min to C4d and C3d, respectively, by CR1-dependent cofactor activity. CR1 functioned intrinsically (i.e, worked only on the cell on which it was expressed). Moreover, CR1 efficiently and stably bound but didn't internalize C4b/C3b opsonized immune complexes. Our studies underscore the potential importance of CR1 on an epithelial cell population as both an intrinsic complement regulator and an immune adherence receptor. These results provide a framework for understanding how loss of CR1 expression on podocytes may contribute to complement-mediated damage in the kidney.
Subject(s)
Complement System Proteins/immunology , Epithelial Cells/metabolism , Kidney/metabolism , Kidney/pathology , Models, Immunological , Receptors, Complement 3b/metabolism , Animals , CHO Cells , Complement C4b , Complement Pathway, Alternative/immunology , Cricetinae , Cricetulus , Erythrocytes/metabolism , Humans , Kinetics , Opsonin Proteins/metabolism , Receptors, Complement 3b/chemistry , TransfectionABSTRACT
The long complement receptor type 1 (CR1) isoform, CR1*2 (S), has been identified as being associated with Alzheimer's disease (AD) risk. We aimed to analyze the phenotypic structural and expression aspects (length and density) of CR1 in erythrocytes of 135 Caucasian subjects (100 AD and 35 controls). CR1 length polymorphism was assessed at protein and gene levels using Western blot and high-resolution melting, respectively. CR1 sites on erythrocytes were enumerated by flow cytometry. CR1 gene analysis, spotting the rs6656401 and rs3818361 polymorphisms, was performed by pyrosequencing. The CR1 density was significantly lower in AD patients expressing the CR1*2 isoform compared with the controls (p = 0.001), demonstrating lower expression of CR1 in CR1*2 carriers. Our data suggested the existence of silent CR1 alleles. Finally, rs6656401 and rs3818361 were strongly associated with CR1 length polymorphism (p < 0.0001). These observations indicate that AD susceptibility is associated with the long CR1 isoform (CR1*2), albeit at a lower density, suggesting that AD results from insufficient clearance of plaque deposits rather than increased inflammation.
Subject(s)
Alzheimer Disease/genetics , Genetic Association Studies , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/genetics , Alleles , Erythrocytes/metabolism , Gene Expression , Genetic Predisposition to Disease/genetics , Heterozygote , Humans , Phenotype , Polymorphism, Genetic , Prospective Studies , Protein Isoforms , Receptors, Complement 3b/blood , RiskSubject(s)
DNA, Complementary/genetics , Receptors, Complement 3b/genetics , Sus scrofa/genetics , Sus scrofa/immunology , 5' Untranslated Regions , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Computational Biology , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Phylogeny , Promoter Regions, Genetic , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Complement 3b/chemistry , Sequence Homology, Amino AcidABSTRACT
To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1-3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1-3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an â¼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.
Subject(s)
Membrane Proteins/metabolism , Merozoites/metabolism , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Receptors, Complement 3b/metabolism , Binding Sites , Complement C3b/chemistry , Complement C3b/genetics , Complement C3b/metabolism , Complement C4b/chemistry , Complement C4b/genetics , Complement C4b/metabolism , Erythrocytes/chemistry , Erythrocytes/metabolism , Erythrocytes/parasitology , HEK293 Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Merozoites/chemistry , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular , Plasmodium falciparum/chemistry , Plasmodium falciparum/genetics , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/genetics , Scattering, Small Angle , Surface Plasmon Resonance , X-Ray DiffractionABSTRACT
Activation of C3, deposition of C3b on the target surface, and subsequent amplification by formation of a C3-cleaving enzyme (C3-convertase; C3bBb) triggers the effector functions of complement that result in inflammation and cell lysis. Concurrently, surface-bound C3b is proteolyzed to iC3b by factor I and appropriate cofactors. iC3b then interacts with the complement receptors (CR) of the Ig superfamily, CR2 (CD21), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) on leukocytes, down-modulating inflammation, enhancing B cell-mediated immunity, and targeting pathogens for clearance by phagocytosis. Using EM and small-angle X-ray scattering, we now present a medium-resolution structure of iC3b (24 Å). iC3b displays a unique conformation with structural features distinct from any other C3 fragment. The macroglobulin ring in iC3b is similar to that in C3b, whereas the TED (thioester-containing domain) domain and the remnants of the CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain have moved to locations more similar to where they were in native C3. A consequence of this large conformational change is the disruption of the factor B binding site, which renders iC3b unable to assemble a C3-convertase. This structural model also justifies the decreased interaction between iC3b and complement regulators and the recognition of iC3b by the CR of the Ig superfamily, CR2, CR3, and CR4. These data further illustrate the extraordinary conformational versatility of C3 to accommodate a great diversity of functional activities.
Subject(s)
Complement C3b/chemistry , Complement C3b/ultrastructure , Microscopy, Electron , Complement C3b/isolation & purification , Humans , Models, Molecular , Protein Structure, Tertiary , Receptors, Complement 3b/chemistry , Scattering, Small Angle , Solutions , X-Ray DiffractionSubject(s)
Integrin alphaXbeta2/blood , Macrophage-1 Antigen/blood , Receptors, Complement 3b/blood , Receptors, Complement 3d/blood , Biomarkers/blood , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry/methods , Humans , Integrin alphaXbeta2/chemistry , Integrin alphaXbeta2/physiology , Leukocyte-Adhesion Deficiency Syndrome/diagnosis , Lupus Erythematosus, Systemic/diagnosis , Macrophage-1 Antigen/chemistry , Macrophage-1 Antigen/physiology , Phagocytosis , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/physiology , Receptors, Complement 3d/chemistry , Receptors, Complement 3d/physiology , Reference Values , Specimen HandlingABSTRACT
CR1 (Complement Receptor type 1, C3bR, CD35) is a polymorphic glycoprotein expressed on erythrocytes, leukocytes and glomerular podocytes. It consists of extracellular, transmembrane and cytoplasmic domains. Soluble form of CR1 (sCR1), lacking the transmembrane and cytoplasmic domains, is present in serum. CR1 belongs to the Regulator of Complement Activation (RCA) family, which is characterized by the appearance of small consensus repeats (SCR). Gene for CR1 is localized on chromosome 1q32. Polymorphism of erythrocyte CR1 is connected with the difference in length of molecule (molecular weight), level of the expression of CR1 (number of receptors) on red blood cells and the Knops blood group antigens. CR1 is a receptor for C3b and C4b and plays an important role in the removal of immune complexes coated with C3b and C4b. It also regulates the complement cascade activation by preventing formation of classical and alternative pathway convertases and by acting as a cofactor for factor I mediated cleavage of C3b to iC3b, C3c i C3dg. CR1 takes part in pathogenesis and development of various autoimmune and infection diseases.
Subject(s)
Receptors, Complement 3b/chemistry , Receptors, Complement 3b/physiology , Animals , Autoimmune Diseases/metabolism , Blood Group Antigens/metabolism , Erythrocytes/immunology , Erythrocytes/metabolism , Humans , Infections/metabolism , Polymorphism, Genetic , Receptors, Complement 3b/geneticsABSTRACT
The complement system is comprised of soluble and cell surface associated proteins that recognize exogenous, altered, or potentially harmful endogenous ligands. In recent years, the complement system--particularly component C3 and its receptors--have been demonstrated to be a key link between innate and adaptive immunity. Complement receptor type 1 (CR1), the receptor for C3b/C4b complement peptides, has emerged as a molecule of immense interest in gaining insight to the susceptibility, pathophysiology, diagnosis, prognosis and therapy of such diseases. In this review, we wish to briefly bring forth the structure, genetic polymorphisms, expression and biological functions of CR1.
Subject(s)
Gene Expression Regulation/immunology , Inflammation Mediators/physiology , Polymorphism, Genetic/genetics , Receptors, Complement 3b/chemistry , Receptors, Complement 3b/genetics , Animals , Autoimmune Diseases/diagnosis , Autoimmune Diseases/genetics , Autoimmune Diseases/physiopathology , Autoimmune Diseases/therapy , Complement Factor I/metabolism , Genetic Predisposition to Disease , Humans , Inflammation Mediators/metabolism , Polymorphism, Genetic/physiology , Receptors, Complement 3b/biosynthesis , Receptors, Complement 3b/physiologyABSTRACT
CRIg is a recently discovered complement C3 receptor expressed on a subpopulation of tissue-resident macrophages. The extracellular IgV domain of CRIg (CRIg-ECD) holds considerable promise as a potential therapeutic because it selectively inhibits the alternative pathway of complement by binding to C3b and inhibiting proteolytic activation of C3 and C5. However, CRIg binds weakly to the convertase subunit C3b (K(D) = 1.1 microm), and thus a relatively high concentration of protein is required to reach nearly complete complement inhibition. To improve therapeutic efficacy while minimizing risk of immunogenicity, we devised a phage display strategy to evolve a high affinity CRIg-ECD variant with a minimal number of mutations. Using the crystal structure of CRIg in complex with C3b as a guide for library design, we isolated a CRIg-ECD double mutant (Q64R/M86Y, CRIg-v27) that showed increased binding affinity and improved complement inhibitory activity relative to CRIg-ECD. In a mouse model of arthritis, treatment with a Fc fusion of CRIg-v27 resulted in a significant reduction in clinical scores compared with treatment with an Fc fusion of CRIg-ECD. This study clearly illustrates how phage display technology and structural information can be combined to generate proteins with nearly natural sequences that act as potent complement inhibitors with greatly improved therapeutic efficacy.