Complement Control Protein Repeats Research Articles

The interaction between factor H and Neisseria meningitidis

Neisseria meningitidis is a leading cause of meningitis and sepsis. The bacterium possesses a number of mechanisms that enable it to evade the immune system and cause disease. Complement is an integral part of the immune system and is essential for protection against the meningococcus. Factor H (fH) is a negative regulator of the alternative pathway of complement and is a 155kDa protein composed of 20 domains known as complement control protein (CCP) repeats. This regulator is recruited by endothelial cells to reduce complement activation, and by some microbial pathogens, including N.

Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates.

Complement is an essential component of the innate and acquired immune system1, and consists of a series of proteolytic cascades that are initiated by the presence of micro-organisms. In health, activation of complement is precisely controlled through membrane-bound and soluble plasma-regulatory proteins including factor H (fH)2, a 155 kDa protein composed of twenty domains (termed complement control protein repeats, or CCPs). Many pathogens have evolved the ability to avoid immune- killing by recruiting host complement regulators3 and several pathogens have adapted to avoid complement-mediated killing by sequestering fH to their surface4. Here we present the first structure of a complement regulator in complex with its pathogen surface-protein ligand. This reveals how the important human pathogen Neisseria meningitidis subverts immune responses by mimicking the host, using protein instead of charged-carbohydrate chemistry to recruit the host complement regulator, factor H. The structure also indicates the molecular basis of the host-specificity of the interaction between factor H and the meningococcus, and informs attempts to develop novel therapeutics and vaccines.

The Role of Decay-accelerating Factor as a Receptor for Helicobacter pylori and a Mediator of Gastric Inflammation

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

Advanced Generation Adenoviral Virotherapy Agents Embody Enhanced Potency Based upon CAR-Independent Tropism

In this current issue of Clinical Cancer Research , Reddy et al. ([1][1]) constructed an adenoviral virotherapy agent that uses the human E2F-1 gene promoter to regulate the adenoviral E1A gene. The E2F transcription factor plays an important role in cell proliferation and is repressed by the

Synergy between Two Active Sites of Human Complement Receptor Type 1 (CD35) in Complement Regulation: Implications for the Structure of the Classical Pathway C3 Convertase and Generation of More Potent Inhibitors

The extracellular domain of the complement receptor type 1 (CR1; CD35) consists entirely of 30 complement control protein repeats (CCPs). CR1 has two distinct functional sites, site 1 (CCPs 1-3) and two copies of site 2 (CCPs 8-10 and CCPs 15-17). In this report we further define the structural requirements for decay-accelerating activity (DAA) for the classical pathway (CP) C3 and C5 convertases and, using these results, generate more potent decay accelerators. Previously, we demonstrated that both sites 1 and 2, tandemly arranged, are required for efficient DAA for C5 convertases. We show that site 1 dissociates the CP C5 convertase, whereas the role of site 2 is to bind the C3b subunit. The intervening CCPs between two functional sites are required for optimal DAA, suggesting that a spatial orientation of the two sites is important. DAA for the CP C3 convertase is increased synergistically if two copies of site 1, particularly those carrying DAA-increasing mutations, are contained within one protein. DAA in such constructs may exceed that of long homologous repeat A (CCPs 1-7) by up to 58-fold. To explain this synergy, we propose a dimeric structure for the CP C3 convertase on cell surfaces. We also extended our previous studies of the amino acid requirements for DAA of site 1 and found that the CCP 1/CCP 2 junction is critical and that Phe82 may contact the C3 convertases. These observations increase our understanding of the mechanism of DAA. In addition, a more potent decay-accelerating form of CR1 was generated.

Human thyroperoxidase folds in one complex B-cell immunodominant region

Human thyroperoxidase (TPO) ectodomain is successively made of myeloperoxidase-, complement control protein repeat-, and epidermal growth factor-like gene modules. However, the TPO immunodominant region targeted by autoantibodies from patients with an autoimmune thyroid disease has not been mapped on the molecule. Here, we used two purified recombinant TPO peptides produced in eukaryotic cells, which correspond to the major first and the further two gene modules of TPO. We compared by ELISA their respective immunoreactivity with that of the recombinant soluble TPO containing all the three gene modules. We used well-characterized murine and human TPO monoclonal antibodies and human autoantibodies affinity-purified from a large pool of patients' sera. We found that the TPO immunodominant region was susceptible to denaturation and required the integrity of the molecule to be correctly expressed. We concluded that TPO B-cell autoepitopes are made by amino acids from the three gene modules, which fold in one highly conformational immunodominant region.

Bulk Production and Functional Analyses of Mouse CD55's Native and Deglycosylated Active Domains

We report the use of methylotrophic yeast Pichia pastoris as a host to efficiently express complement control protein repeats (CCPs) 1–4 of mouse decay accelerating factor (DAF, CD55) as a soluble protein. With this system, the mouse DAF CCP1-4-active-domain-containing module linked to a 6× His tag at its C terminus was secreted into the culture supernatant at 15 mg/L after 24 h of induction with methanol. A mouse DAF CCP1–4 mutant protein in which its two potential N-glycosylation sites were deleted by changing Asn187 and Asn262 to Gln was also produced. Using Ni2+-immobilized agarose affinity chromatography, the recombinant mouse DAF modules with their 6× His tags could be one-step isolated to SDS-PAGE purity. Polyclonal antibody against native mouse DAF CCP1–4 was raised by immunizing NZW rabbits with the purified product. Measurements of the bioactivities of the wild-type and mutant mouse DAF proteins in C3b uptake assays showed no differences in regulatory activities in either the classical or the alternative pathways. With the use of the mutant DAF protein, small rod-shaped crystals were produced and preliminary data obtained. The production of large quantities of functional recombinant mouse DAF CCP1–4 modules and their antibody offers the opportunity to study DAF structure and DAF function in vivo.

Characterization of the active sites in decay-accelerating factor.

Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2-3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2-3 of DAF were oriented according to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.

Structure–function relationships of complement receptor type 1

Human complement receptor type 1 (CR1) is a large, multifunctional glycoprotein which is a member of the regulators of complement activation family. Like other members of this family, it is composed mainly of tandemly arranged modules, each about 60-70 amino acids long, known as complement control protein repeats (CCPs). Each domain folds independently and contains a hydrophobic core wrapped in beta sheets. These domains mediate interactions with C3/C4-derived fragments. CR1 is the most versatile inhibitor of both classical and alternative pathway C3 and C5 convertases due to its decay-accelerating activity and co-factor activity for C3b/C4b cleavage. Moreover, CR1 plays a major role in immune complex clearance due to its high affinity for C3b and C4b. CR1 is an excellent model to study structure-function relationships because its functions are mediated by two distinct but highly homologous sites, each composed of three CCPs. CR1 derivatives carrying just one active site were used to define critical sequences/amino acids. This was achieved by testing functional profiles of the proteins carrying a mutated active site produced by substituting peptides/amino acids with their counterparts from the other site. These mutated proteins, of which we analyzed over 100, permitted the fine mapping of the functional sites. CR1 on primate erythrocytes varies in size. In most cases it is smaller and has fewer active sites than does human CR1. This variation was used to determine that increased copy number (3,000 to 20,000 versus 300 for human CR1) compensates for a smaller size. Moreover, studies of primate CR1 led to the finding that subtle differences in the critical areas, as compared to human sites, produce active sites with a broader functional repertoire. These alterations ensure that short CR1 forms possess similar biologic activities to the large CR1 forms. There is much interest in producing therapeutic agents to inhibit unwanted complement activation. Based on these structure-function analyses, smaller and more potent complement inhibitors derived from CR1 can be produced.

Attachment of Neisseria gonorrhoeae to the cellular pilus receptor CD46: identification of domains important for bacterial adherence.

Pili of Neisseria gonorrhoeae mediate binding of the bacteria to human host cells. Membrane cofactor protein (MCP or CD46), a human cell-surface protein involved in regulation of complement activation, acts as a cellular pilus receptor. In this work, we examined which domains of CD46 mediate bacterial adherence. The CD46 expression was quantified and characterized in human epithelial cell lines. N. gonorrhoeae showed the highest adherence to ME180 cells, which have BC1 as the dominant phenotype. The BC isoforms of CD46 were expressed in all cell lines tested. The adherence was not enhanced by high expression of other isoforms, showing that the BC domain of CD46 is important in adherence of N. gonorrhoeae to human cells. To characterize the pilus-binding site within the CD46 molecule, a set of CD46-BC1 deletion constructs were transfected into COS-7 cells. Piliated N. gonorrhoeae attached well to CD46-BC1-expressing COS-7 cells. We show that the complement control protein repeat 3 (CCP-3) and the serine-threonine-proline (STP)-rich domain of CD46 are important for efficient adherence to host cells. Further, partial deletion of the cytoplasmic tail of CD46 results in low bacterial binding, indicating that the cytoplasmic tail takes part in the process of establishing a stable interaction between N. gonorrhoeae and host cells.

Structure/function studies of human decay‐accelerating factor

The decay-accelerating factor (DAF) contains four complement control protein repeats (CCPs) with a single N-linked glycan positioned between CCPs 1 and 2. In previous studies we found that the classical pathway regulatory activity of DAF resides in CCPs 2 and 3 while its alternative pathway regulatory activity resides in CCPs 2, 3 and 4. Molecular modelling of the protein predicted that a positively charged surface area on CCPs 2 and 3 (including KKK125-127) and nearby exposed hydrophobic residues (L147F148) on CCP3 may function as ligand-binding sites. To assess the roles of the N-linked glycan and the above two sets of amino acids in the function of DAF, we mutated N61 to Q, KKK125-127 to TTT and L147F148 to SS. Following expression of the mutated cDNAs in Chinese hamster ovary cells, the glycosylphosphatidylinositol (GPI)-anchored mutant proteins were affinity purified and their functions were assessed. In initial assays, the proteins were incorporated into sheep and rabbit erythrocytes and the effects of the mutations on regulation of classical and alternative C3 convertase activity were quantified by measuring C3b deposition. Since DAF also functions on C5 convertases, comparative haemolytic assays of cells bearing each mutant protein were performed. Finally, to establish if spatial orientation between DAF and the convertases on the cell surface played any role in the observed effects, fluid-phase C3a generation assays were performed. All three assays gave equivalent results and showed that the N-linked glycan of DAF is not involved in its regulatory function; that L147F148 in a hydrophobic area of CCP3 is essential in both classical and alternative pathway C3 convertase regulation; and that KKK125-127 in the positively charged pocket between CCPs 2 and 3 is necessary for the regulatory activity of DAF on the alternative pathway C3 convertase but plays a lesser role in its activity on the classical pathway enzyme.

Conservation in decay accelerating factor (DAF) structure among primates

The decay accelerating factor (DAF, CD55) protects self cells from activation of autologous complement on their surfaces. It functions to disable the C3 convertases, the central amplification enzymes of the cascade. Its active site(s) are contained within four ∼60 amino acid long units, termed complement control protein repeats (CCPs), which are suspended above the cell surface on a 68 amino acid long serine/threonine (S/T)-rich cushion that derives from three exons. We previously proposed a molecular model of human DAF’s four CCPs in which certain amino acids were postulated to be recognition sites for the interaction between DAF and the C3 convertases. In the current study, we characterized DAF in five non-human primates: the great apes, gorilla and common chimpanzee, and the Old World monkeys: hamadryas baboon, Rhesus macaque, and patas monkey. Amino acid homology to human DAF was approximately 98% for the two great apes and 83% for the three Old World monkeys. The above cited putative ligand interactive residues were found to be fully conserved in all of the non-human primates, although there were amino acid changes outside of these areas. In the chimpanzee, alternative splicing of the S/T region was found potentially to be the source of multiple protein isoforms in erythrocytes, whereas in the patas monkey, similar alternative splicing was observed but only one protein band was seen. Interestingly, a Rhesus macaque was found to exhibit a phenomenon paralleling the human Cromer Dr(a-) blood group, in which a 44-base pair deletion in CCP3 leads to a frameshift and early STOP codon.

Decay Accelerating Activity of Complement Receptor Type 1 (CD35): TWO ACTIVE SITES ARE REQUIRED FOR DISSOCIATING C5 CONVERTASES

The goal of this study was to identify the site(s) in CR1 that mediate the dissociation of the C3 and C5 convertases. To that end, truncated derivatives of CR1 whose extracellular part is composed of 30 tandem repeating modules, termed complement control protein repeats (CCPs), were generated. Site 1 (CCPs 1-3) alone mediated the decay acceleration of the classical and alternative pathway C3 convertases. Site 2 (CCPs 8-10 or the nearly identical CCPs 15-17) had one-fifth the activity of site 1. In contrast, for the C5 convertase, site 1 had only 0.5% of the decay accelerating activity, while site 2 had no detectable activity. Efficient C5 decay accelerating activity was detected in recombinants that carried both site 1 and site 2. The activity was reduced if the intervening repeats between site 1 and site 2 were deleted. The results indicate that, for the C5 convertases, decay accelerating activity is mediated primarily by site 1. A properly spaced site 2 has an important auxiliary role, which may involve its C3b binding capacity. Moreover, using homologous substitution mutagenesis, residues important in site 1 for dissociating activity were identified. Based on these results, we generated proteins one-fourth the size of CR1 but with enhanced decay accelerating activity for the C3 convertases.

Viral complement regulatory proteins

The inactivation of complement provides cells and tissues critical protection from complement-mediated attack and decreases the associated recruitment of other inflammatory mediators. In an attempt to evade the host immune response, viruses have evolved two mechanisms to acquire complement regulatory proteins. They can directly seize the host cell complement regulators onto their outer envelope and/or they can produce their own proteins which are either secreted into the neighboring intercellular space or expressed as membrane-bound proteins on the infected host cell. The following review will concentrate on the viral homologues of the mammalian complement regulatory proteins, specifically those containing complement control protein (CCP) repeats.

Membrane Cofactor Protein: Importance ofN- andO-Glycosylation for Complement Regulatory Function

AbstractMembrane cofactor protein (MCP; CD46) is a type 1 membrane glycoprotein that inhibits complement activation on host cells. It also is a measles virus (MV) receptor, an adherence factor for group A Streptococcus pyogenes, and a cellular pilus receptor for pathogenic Neisseria. The amino terminus of MCP consists of four complement control protein (CCP) repeats, three of which (CCP-1, -2, and -4) possess N-glycans. Immediately following the CCP modules is an alternatively spliced region for extensive O-glycosylation (termed the STP domain). Previous studies established that the N-glycan of CCP-2 is essential for MV binding and infection and that the splicing variants of the STP domain not only affect MV binding and fusion, but also differentially protect against complement-mediated cytolysis. In this report, we dissect the role of these carbohydrates on complement regulatory function. We constructed, expressed, and characterized proteins deleting these carbohydrates. For MCP-mediated protection against cytolysis, the N-glycans of CCP-2 and -4 were necessary, the STP segment influenced but was not essential, and the N-glycan of CCP-1 was not required. In addition, the rate and magnitude of cell surface cleavage of C4b to C4c and C4d by MCP and factor I correlated with cytoprotection. These studies expand the structure-function understanding of the active sites of MCP and elucidate an important role for carbohydrates in its function, a finding consistent with their conservation in the MCP of other species.

Structure-Function Analysis of the Active Sites of Complement Receptor Type 1

Two functionally distinct but homologous sites in complement receptor type 1 (CR1) (CD35) were further characterized by homologous substitution mutagenesis of two CR1 derivatives, each containing one site. In both sites, reducing negative and/or increasing positive charge augmented interaction with iC3/C3b and C4b, supporting a role of ionic forces in the binding reaction. In one case, substitution of Asp at the end of complement control protein repeat (CCP) 2 with an Asn transformed the protein, with negligible cofactor activity and iC3 binding, into a mutant with activities similar to native CR1. Consequently, this protein, one-fourth the size of CR1, is a therapeutic candidate for a complement inhibitor. Another important observation is that the residues between two CCPs contribute to activity, probably because they influence positioning of one CCP relative to the next. The initial characterization of the third CCP of an active site led to identification of three peptides necessary for binding. In line with earlier findings for the first two CCPs, interactions with iC3/C3b are similar but not identical to those with C4b, implying overlapping but distinct binding domains. Moreover, changes in cofactor activity usually, but not always, parallel alterations in binding, indicating that these two activities are separable. We also mapped epitopes for a blocking and a function enhancing monoclonal antibody. Their effects can be explained by epitope location. The first antibody binds near functionally important residues. The second may shield inhibitory (negatively charged) residues. These results represent a comprehensive analysis of the active sites of CR1, which is built of modules found in more than 50 mammalian proteins.

Primary sequence of baboon CR1 demonstrates concerted evolution within the CR1 gene

Complement receptor type one (CR1) in primates has several remarkable structural features including a size polymorphism ( M r 190000, 220000, 250000 and 280000) in man, multiple size variants ( M r 55000–220000) among non-human primates, and a partial amino-terminal duplication (CR1-like gene) that appears to encode the short (55000–70000) forms expressed on primate erythrocytes. In general, these short CR1 forms, some of which are GPI anchored, are expressed on erythrocytes and the 220000 molecular weight CR1 form is expressed on PBMC, except in man, where only the 220000 molecular weight form has been detected. In addition, the M r 220000 human CR1 sequence carries several long internal repeats of up to 99% homology. It has been suggested that the highest homology is maintained by gene conversion and/or unequal crossover. To address further the evolutionary and biologic implications of these multiple forms, a 6 kb cDNA encoding baboon CR1 220 was identified by RTPCR using human CR1 primers. Its sequence contains the expected 30 complement control protein repeats (CCP) and demonstrates an overall homology to human CR1 of 95.4% at the nucleotide level and 93.2% at the amino acid level. As in human CR1, the first 28 CCP maintain the characteristic “seven CCP-long homologous repeats (LHR)” organization. Analysis of baboon CR1 220 indicates that horizontal or concerted evolution has maintained a high degree (>98%) of identity between corresponding CCP within the LHRs from CCP 4 to CCP 19, while this homology region extends from CCP 3 to CCP 18 in man. In contrast, substitutions occurring in other CCP are not propagated to the corresponding sites of other LHR. Sequence differences in CCP 1, 2 and 3 are likely to be related to the acquisition of enhanced C3b binding capability by this amino-terminal region of the protein. Thus, the sequence data strongly support the hypotheses that gene conversion and or unequal crossover events have driven the evolution of the protein in regions of high homology while selective forces, probably ligand binding requirements, have maintained the regions of divergence.

Complete nucleotide and deduced amino acid sequence of human beta 2-glycoprotein I.

The nucleotide and complete amino acid sequence for the human beta 2-glycoprotein I (beta 2I) was derived by sequencing the cDNA clone pB2I-1. In addition to the 326 amino acid residues of the mature protein this clone codes for a putative leader peptide and contains sequence representing 5' and 3' untranslated regions. When this amino acid sequence was compared with the previously published primary sequence, three major amino acid substitutions were found, two involving cysteine residues. These substitutions lead to a new alignment of the complement control protein (CCP) repeats present in beta 2I and a prediction of the complete disulphide bond organization. Northern-blot analysis indicates that hepatocytes are a major site of biosynthesis for this protein. A transcription signal of about 1.5 kb was detected by using RNA from HepG2 cells.