Short Consensus Repeats Research Articles

Localization of the third heparin-binding site in the human complement regulator factor H 1

Complement factor H (fH) plays a pivotal role in regulating the alternative pathway, allowing complement activation to proceed on foreign surfaces, whilst protecting surrounding host cell surfaces from complement-mediated damage. Host cell recognition is mediated by polyanions such as sialic acid and glycosaminoglycans (GAGs), which promote a high affinity interaction between fH and C3b deposited on host cell surfaces. Factor H is composed of 20 short consensus repeats (SCRs); two heparin-binding sites have been identified within SCR 7 and SCR 20 and a third site is thought to exist within or near SCR 13. Using an extensive series of recombinant fH fragments and heparin affinity chromatography, we have localized the third heparin-binding domain to SCR 9. A recombinant fH fragment containing both SCR 7 and SCR 9 exhibited higher affinity for heparin than SCR 7 alone, suggesting that the individual heparin-binding sites interact simultaneously with heparin to create a higher avidity interaction. Recombinant fragments containing SCR 9 bound to endothelial cells, indicating that this domain is capable of interacting with polyanions within a physiologically relevant environment. In addition, the three heparin-binding sites exhibited differences in their specificity for certain GAGs, suggesting that the individual binding domains may possess separate GAG recognition functions.

Demonstration of Factor H-Like Protein 1 Binding toTreponema denticola, a Pathogen Associated with Periodontal Disease in Humans

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.

Binding of Complement Factor H to Endothelial Cells Is Mediated by the Carboxy-Terminal Glycosaminoglycan Binding Site

Factor H (FH), the major fluid phase regulator of the alternative complement pathway, mediates protection of plasma-exposed host structures. It has recently been shown that short consensus repeats 19 to 20 of FH are mutational hot spots associated with atypical hemolytic uremic syndrome (aHUS), a disease with endothelial cell damage. Domain 20 of FH contains binding sites for heparin, C3b, and the cleavage product C3d. To study the role of these binding sites in target recognition, we performed site-directed mutagenesis in domain 20 and assayed the resulting recombinant proteins. The mutant FH15-20A (substitutions R1203E, R1206E, and R1210S) bound neither heparin nor endothelial cells. Similarly, an aHUS-derived mutant FH protein (E1172Stop, lacking domain 20) failed to bind endothelial cells and showed impaired binding to heparin. Binding of FH to endothelial cells was inhibited by heparin and a specific monoclonal antibody that inhibited heparin but not C3d binding, demonstrating that the heparin site on domains 19 to 20 mediates interaction of FH to endothelial cells. Binding of FH15-20 to heparin was inhibited by several cell surface- and basement membrane-associated glycosaminoglycans, suggesting that binding site specificity is not restricted to heparin. Thus, defective heparin/glycosaminoglycan-binding site on domains 19 to 20 of FH most probably mediates complement-induced endothelial cell damage in aHUS.

Regulator of Complement Activation (RCA) Locus in Chicken: Identification of Chicken RCA Gene Cluster and Functional RCA Proteins

A 150-kb DNA fragment, which contains the gene of the chicken complement regulatory protein CREM (formerly named Cremp), was isolated from a microchromosome by screening bacterial artificial chromosome library. Within 100 kb of the cloned region, three complete genes encoding short consensus repeats (SCRs, motifs with tandemly arranged 60 aa) were identified by exon-trap method and 3'- or 5'-RACE. A chicken orthologue of the human gene 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2, which exists in close proximity to the regulator of complement activation genes in humans and mice, was located near this chicken SCR gene cluster. Moreover, additional genes encoding SCR proteins appeared to be present in this region. Three distinct transcripts were detected in RNA samples from a variety of chicken organs and cell lines. Two novel genes named complement regulatory secretory protein of chicken (CRES) and complement regulatory GPI-anchored protein of chicken (CREG) besides CREM were identified by cloning corresponding cDNA. Based on the predicted primary structures and properties of the expressed molecules, CRES is a secretory protein, whereas CREG is a GPI-anchored membrane protein. CREG and CREM were protected host cells from chicken complement-mediated cytolysis. Likewise, a membrane-bound form of CRES, which was artificially generated, also protected host cells from chicken complement. Taken together, the chicken possesses an regulator of complement activation locus similar to those of the mammals, and the gene products function as complement regulators.

Evolutionary history of orthopoxvirus proteins similar to human complement regulators

Orthopoxviruses include many important pathogens such as variola major virus, camelpox, buffalopox, monkeypox, cowpox, and variola minor viruses. This group of viruses also includes vaccinia virus, which is extensively used in human vaccine development. Genomes of orthopoxviruses encode proteins with sequences similar to human regulators of complement activation (RCA) that contain tandem short consensus repeats (SCRs). We employed phylogenetic tree analysis to evaluate the structural relationships among SCRs of orthopoxvirus RCA-like proteins and those of human complement regulators. The human complement RCA proteins analyzed were factor H (FH), C4 binding protein alpha chain, membrane cofactor protein (MCP), decay accelerating factor (DAF), and complement receptors type 1 (CR1) and 2 (CR2). Sequences of key poxvirus regulators of complement activation, vaccinia virus complement control protein (VCP), smallpox inhibitor of complement enzymes (SPICE), and cowpox inflammation modulatory protein (IMP) were similar to SCRs 1 through 5 of C4 binding protein, alpha chain, and they were also clustered with other homologous repeats of MCP, DAF, CR1, CR2, and FH. Phylogenetic clustering of RCA sequences suggested that poxvirus complement regulators VCP, SPICE, and IMP arose from a single ancestral sequence that shared similarity with all human regulators of complement activation. Any changes in poxvirus complement regulators leading to the enhancement of their ability to regulate complement activation likely resulted from new mutations in the viral lineages.

The Distal Short Consensus Repeats 1 and 2 of the Membrane Cofactor Protein CD46 and Their Distance from the Cell Membrane Determine Productive Entry of Species B Adenovirus Serotype 35

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.

Human Factor H-Related Protein 5 Has Cofactor Activity, Inhibits C3 Convertase Activity, Binds Heparin and C-Reactive Protein, and Associates with Lipoprotein

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5-7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5-7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.

Method for Selecting and Enriching Cells Expressing Low Affinity Ligands for Cell Surface Receptors

The affinity of protein-protein interaction in biological systems ranges from extremely strong, such as that between avidin and biotin (Ka = approximately 10 15 M-1) to the very weak, such as E-selectin and E-selectin ligand-1 (Ka = approximately 7.4 × 104 M-1S-1) (1). It is known that the interactions mediated by the surface receptors of immune cells are often of low-affinity and transient in nature (2). Despite weak affinities, cell surface interactions among immune cells have been shown to play an important role in many functions such as surveillance, immunosynapse formation, and activation (2). It is therefore desirable to devise a way to select and enrich cells expressing low-affinity ligands for specific immune cell surface receptors. Here we use the well-studied interaction between CD97 and CD55 as a model system to describe a simple method for this purpose. Both CD97 and CD55 are cell surface proteins expressed predominantly on leukocytes (3–5). The binding of CD97 to CD55 has been demonstrated to be both of low affinity (86 μM) and transient (off-rate; 0.6/s) (6). We and others have previously shown that the interaction is mediated exclusively by the epidermal growth factor (EGF)-like domains of CD97 and the short consensus repeats (SCR) of CD55 in a Ca2+-dependent fashion (6,7). Furthermore, a CD97-like molecule, EMR2, has been shown not to bind CD55 even though it contains highly homologous EGF-like domains (97.5% identity), therefore providing an ideal negative control (8). Our method is based on (i) the efficient isolation and coupling of Fc-fusion proteins to protein AGconjugated paramagnetic beads (Protein A/G Dynabeads®; Dynal A.S., Oslo, Norway) (Figure 1A) and (ii) the simple capture and separation of ligand-positive cells from the Fcfusion protein-coated paramagnetic beads (Figure 1B). The coupling of the Fc-fusion proteins to the protein AG-conjugated paramagnetic beads generates a multivalent protein probe, thereby increasing binding efficiency (Figure 1, A and B). As the major CD55-binding CD97 isoform contains three EGF-domains, we fused the three EGF-domains immediately upstream of the mouse Fc region (Figure 1A). CD97(EGF-1,2,5)-mFc and EMR2(EGF-1,2,5)-mFc fusion proteins were produced as described previously in transiently transfected HEK293T cells (Figure 1C). Protein AG-conjugated paramagnetic beads were washed three times in binding buffer [0.5% bovine serum albumin (BSA) in Hank’s Balanced Salt solution (HBSS)], reconstituted in the binding buffer to the original concentration (10 mg/mL), and incubated at 4°C for 30 min with or without 2 μg mFc-fusion protein/10 μL beads/reaction. Following the removal of unbound proteins by magnetic separation, the fusion protein-bead complexes were then resuspended in 20 μL binding buffer/reaction. K562 cells (CD55+) were washed, adjusted to a cell density of 3 × 106 cells/mL, and incubated at 4°C with the protein-bead

Epitope-Mapping Studies Define Two Major Neutralization Sites on the Vaccinia Virus Extracellular Enveloped Virus Glycoprotein B5R

Vaccinia extracellular enveloped virus (EEV) is critical for cell-to-cell and long-range virus spread both in vitro and in vivo. The B5R gene encodes an EEV-specific type I membrane protein that is essential for efficient EEV formation. The majority of the B5R ectodomain consists of four domains with homology to short consensus repeat domains followed by a stalk. Previous studies have shown that polyclonal antibodies raised against the B5R ectodomain inhibit EEV infection. In this study, our goal was to elucidate the antigenic structure of B5R and relate this to its function. To do this, we produced multimilligram quantities of vaccinia virus B5R as a soluble protein [B5R(275t)] using a baculovirus expression system. We then selected and characterized a panel of 26 monoclonal antibodies (MAbs) that recognize B5R(275t). Five of these MAbs neutralized EEV and inhibited comet formation. Two other MAbs were able only to neutralize EEV, while five others were able only to inhibit comet formation. This suggests that the EEV neutralization and comet inhibition assays measure different viral functions and that at least two different antigenic sites on B5R are important for these activities. We further characterized the MAbs and the antigenic structure of B5R(275t) by peptide mapping and by reciprocal MAb blocking studies using biosensor analysis. The epitopes recognized by neutralizing MAbs were localized to SCR1-SCR2 and/or the stalk of B5R(275t). Furthermore, the peptide and blocking data support the concept that SCR1 and the stalk may be in juxtaposition and may be part of the same functional domain.

Hemolytic Uremic Syndrome

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy defined by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute renal failure. The most frequent form is associated with infections by Shiga-like toxin-producing bacteria (STEC-HUS). Rarer cases are triggered by neuraminidase-producing Streptococcus pneumoniae (pneumococcal-HUS). The designation of aHUS is used to refer to those cases in which an infection by Shiga-like toxin-producing bacteria or S. pneumoniae can be excluded. Studies performed in the last two decades have documented that hyperactivation of the complement system is the pathogenetic effector mechanism leading to the endothelial damage and the microvascular thrombosis in aHUS. Recent data suggested the involvement of the complement system in the pathogenesis of STEC-HUS and pneumococcal-HUS as well. Clinical signs and symptoms may overlap among the different forms of HUS; however, pneumococcal-HUS and aHUS have a worse prognosis compared with STEC-HUS. Early diagnosis and identification of underlying pathogenetic mechanism allows instating specific support measures and therapies. In clinical trials in patients with aHUS, complement inhibition by eculizumab administration leads to a rapid and sustained normalization of hematological parameters with improvement in long-term renal function. This review summarizes current concepts about the epidemiological findings, the pathological and clinical aspects of STEC-HUS, pneumococcal-HUS, and aHUS, and their diagnosis and management.

Electrostatic Modeling Predicts the Activities of Orthopoxvirus Complement Control Proteins

Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the alpha' chain of C3b between residues 954-955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.

Domain V of β2-Glycoprotein I Binds Factor XI/XIa and Is Cleaved at Lys317-Thr318

The fifth domain (DV) of beta2-glycoprotein I (beta2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). Beta2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA. Following proteolytic cleavage of DV by plasmin, beta2GPI retains binding to FXI but not to phospholipids. Native beta2GPI, but not cleaved beta2GPI, inhibits activation of FXI by thrombin and factor XIIa, attenuating a positive feedback mechanism for additional thrombin generation. In this report, we have defined the FXI/FXIa binding site on beta2GPI using site-directed mutagenesis. We show that the positively charged residues Lys284, Lys286, and Lys287 in DV are essential for the interaction of beta2GPI with FXI/FXIa. We also demonstrate that FXIa proteolytically cleaves beta2GPI at Lys317-Thr318 in DV. Thus, FXIa cleavage of beta2GPI in vivo during thrombus formation may accelerate FXI activation by decreasing the inhibitory effect of beta2GPI.

Analysis of SERF in Thai blood donors

Abstract The Cromer blood group system consists of nine high-prevalence and three low-prevalence antigens carried on decay-accelerating factor (DAF). We recently described one of these Cromer high-prevalence antigens, SERF, the absence of which was found in a Thai woman. The lack of SERF antigen in this proband was associated with a substitution of nucleotide 647C>T in exon 5 of DAF, which is predicted to be a change of proline to leucine at amino acid position 182 in short consensus repeat (SCR) 3 of DAF. This study reports on PCR-RFLP analysis of the SERF allele with BstNI restriction endonuclease on more than one thousand Thai blood donor samples. One new donor homozygous (647T) and 21 donors heterozygous (647C/T) for the SERF allele were found. Among this cohort of random Thai blood donors, the SERF allele frequency was 1.1 percent. Thus, like other alleles in the Cromer blood group system, SERF is found in a certain ethnic group. Immunohematology 2005, 21:66–69.

Mutational Analysis of the Complement Receptor Type 2 (CR2/CD21)–C3d Interaction Reveals a Putative Charged SCR1 Binding Site for C3d

We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.

Genomic analysis reveals a duplication of eight rather than seven short consensus repeats in primate CR1 and CR1L: evidence for an additional set shared between CR1 and CR2

We report the discovery of previously unrecognised short consensus repeats (SCRs) within human and chimpanzee CR1 and CR1L. Analysis of available genomic, protein and expression databases suggests that these are actually genomic remnants of SCRs previously reported in other complement control proteins (CCPs). Comparison with the nucleotide motifs of the 11 defined subfamilies of SCRs justifies the designation g-like because of the close similarity to the g subfamily found in CR2 and MCP. To date, we have identified five such SCRs in human and chimpanzee CR1, one in human and chimpanzee CR1L, but none in either rat or mouse Crry in keeping with the number of internal duplications of the long homologous repeat (LHR) found in CR1 and CR1L. In fact, at the genomic level, the ancestral LHR must have contained eight SCRs rather than seven as previously thought. Since g-like SCRs are found immediately downstream of d SCRs, we suggest that there must have been a functional dg set which has been retained by CR2 and MCP but which is degenerate in CR1 or CR1L. Interestingly, dg is also present in the CR2 component of mouse CR1. The degeneration of the g SCR must have occurred prior to the formation of primate CR1L and prior to the duplication events which resulted in primate CR1. In this context, the apparent conservation of g-like SCRs may be surprising and may suggest the existence of mechanisms unrelated to protein coding. These results provide examples of the many processes which have contributed to the evolution of the extensive repertoire of CCPs.

Interdomain Contact Regions and Angles Between Adjacent Short Consensus Repeat Domains

The short consensus repeat domain (SCR, complement control protein module, sushi-domain) is a structural unit found in multiple adjacent copies in more than 40 human proteins. Each bead-like domain is composed of approximately 60 residues and the adjacent domains are connected in a head-to-tail fashion with linkers that consist of two to 12 amino acid residues. Based on experimentally determined structures the neighbouring SCR domains interact with each other at the so-called hinge or interdomain contact region. The functions mediated by the SCR domains have been studied using mutagenesis but the possible effects of the mutations on the hinge regions and interdomain angles have not been analysed. In this study, the linker and three loops in conserved locations were found to be responsible for the interdomain contact regions of all the solved experimental structures. The interdomain contact regions were identified in sequences of 140 human SCR domain pairs, and distinct hydrophobic and charge features were found in different subsets of SCR proteins and functional domains. To compare the possible associations of the interdomain contact region characteristics to the interdomain orientations all the experimentally solved SCR structures were subjected to a uniform calculation of tilt, twist, and skew angles that define the interdomain orientation. The twist and skew angles were found to have a linear correlation and the spatial location of one loop of the N-terminal domain (N#1) was found to have an effect on the skew angle. Thus, we describe location of the interdomain contact regions in primary structures of SCR domains and report that the orientation of adjacent SCR domains is not random and depends partially on the interdomain contact regions. On the basis of these results, mutations within the interdomain contact regions and subsequent loss-of-function effects caused by changes in the interdomain orientation can be avoided in mutagenesis studies.

Constitutive expression and alternative splicing of the exons encoding SCRs in Sp152, the sea urchin homologue of complement factor B. Implications on the evolution of the Bf/C2 gene family.

The purple sea urchin, Strongylocentrotus purpuratus, possesses a non-adaptive immune system including elements homologous to C3 and factor B (Bf) of the vertebrate complement system. SpBf is composed of motifs typical of the Bf/C2 protein family. Expression of Sp152 (encodes SpBf) was identified in the phagocyte type of coelomocyte in addition to gut, pharynx and esophagus, which may have been due to the presence of these coelomocytes in and on all tissues of the animal. Sp152 expression in coelomocytes was constitutive and non-inducible based on comparisons between pre- and post-injection with lipopolysaccharide or sterile seawater. The pattern of five short consensus repeats (SCRs) in SpBf has been considered ancestral compared to other deuterostome Bf/C2 proteins that contain either three or four SCRs. Three alternatively spliced messages were identified for Sp152 and designated Sp152Delta1, Sp152Delta4, and Sp152Delta1+Delta4, based on which of the five SCRs were deleted. Sp152Delta4 had an in-frame deletion of SCR4, which would encode a putative SpBfDelta4 protein with four SCRs rather than five. On the other hand, both Sp152Delta1 and Sp152Delta1+Delta4 had a frame-shift that introduced a stop codon six amino acids downstream of the splice site for SCR1, and would encode putative proteins composed only of the leader. Comparisons between the full-length SpBf and its several splice variants with other Bf/C2 proteins suggested that the early evolution of this gene family may have involved a combination of gene duplications and deletions of exons encoding SCRs.

Complement components C5 and C7: recombinant factor I modules of C7 bind to the C345C domain of C5.

Studies reported over 30 years ago revealed that latent, nonactivated C5 binds specifically and reversibly to C6 and C7. These reversible reactions are distinct from the essentially nonreversible associations with activated C5b that occur during assembly of the membrane attack complex, but they likely involve some, perhaps many, of the same molecular contacts. We recently reported that these reversible reactions are mediated by the C345C (NTR) domain at the C terminus of the C5 alpha-chain. Earlier work by others localized the complementary binding sites to a tryptic fragment of C6 composed entirely of two adjacent factor I modules (FIMs), and to a larger fragment of C7 composed of its homologous FIMs as well as two adjoining short consensus repeat modules. In this work, we expressed the tandem FIMs from C7 in bacteria. The mobility on SDS-polyacrylamide gels, lack of free sulfhydryl groups, and atypical circular dichroism spectrum of the recombinant product rC7-FIMs were all consistent with a native structure. Using surface plasmon resonance, we found that rC7-FIMs binds specifically to both C5 and the rC5-C345C domain with K(D) approximately 50 nM, and competes with C7 for binding to C5, as expected for an active domain. These results indicate that, like C6, the FIMs alone in C7 mediate reversible binding to C5. Based on available evidence, we suggest a model for an irreversible membrane attack complex assembly in which the C7 FIMs, but not those in C6, are bound to the C345C domain of C5 within the fully assembled complex.

A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice

Studies in gene-targeted mice have demonstrated that factor B of the alternative complement pathway plays an important role in several disease models, but an exogenous inhibitor of factor B has not previously been available. We have developed an inhibitory monoclonal antibody directed against a critical epitope on mouse factor B and have tested it in a model of antiphospholipid (aPL) antibody (Ab)-induced fetal loss. Gene-targeted factor B-deficient mice (fB −/−) were injected with a fusion protein comprised of the second and third short consensus repeat (SCR) domains of mouse factor B linked to a mouse IgG 1 Fc domain. Hybridomas were made from splenocytes of the immunized mouse. One mAb, designated 1379, produced an IgG 1 antibody that inhibited alternative pathway activation in vitro and in vivo by preventing formation of the C3bBb complex. Strikingly, this mAb inhibited alternative pathway activation in serum from mice, rats, humans, monkeys, pigs and horses. Fab fragments made from this mAb also inhibited alternative pathway activation. Epitope mapping demonstrated that this antibody binds to factor B within the third SCR domain. When mAb 1379 was administered to mice that also received human IgG containing antiphospholipid antibodies, it provided significant protection from antiphospholipid antibody-induced complement activation and fetal loss. Thus, this mAb to factor B has broad species reactivity and effectively inhibits alternative pathway activation. The mAb protects mice in an in vivo model of antiphospholipid antibody syndrome, demonstrating the therapeutic potential for the inhibition of factor B in this disease.

Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli.

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.