Complement activation in factor D-deficient mice.

To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balance in vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activation in vitro by CVF demonstrated that in factor D-deficient serum the alpha chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg(2+), whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.

Human C-reactive protein: expression, structure, and function.

C-reactive protein (CRP) is an acute-phase protein featuring a homopentameric structure and Ca-binding specificity for phosphocholine (PCh). Expression of CRP is regulated mainly at the transcriptional level with interleukin-6 being the principal inducer of the gene during the acute phase. The crystal structure of CRP has been determined and the topology and chemical composition of its ligand-binding site determined. The wide distribution of PCh in polysaccharides of pathogens and in cellular membranes allows CRP to recognize a range of pathogenic targets as well as membranes of damaged and necrotic host cells. CRP bound to a multivalent ligand can efficiently initiate the assembly of a C3 convertase through the classical pathway and thus decorate the surface of the ligand with opsonic complement fragments. However, the protein does not favor the formation of a C5 convertase and therefore, CRP-initiated complement activation does not mediate acute inflammatory reactions and membrane damage. CRP also interacts with Fc receptors on phagocytic cells and acts as an opsonin. Other CRP-initiated signals through interactions with neutrophil Fc receptors have an overall anti-inflammatory effect. Thus, the main biological function of CRP appears to be host defense against bacterial pathogens and clearance of apoptotic and necrotic cells. Protection from lethal bacterial infection, from complement-induced alveolitis, and from endotoxemia has been confirmed in vivo using transgenic mice. Additional functions, including participation in atherogenesis and pathogenesis of myocardial injury after myocardial infarction have been reported. However, the weight of the evidence is that CRP like other acute-phase proteins is a component first line of innate host defense.

Structural biology of the alternative pathway convertase.

Complement convertases are bimolecular complexes expressing protease activity only against C3 and C5. Their action is necessary for production of the biological activities of the complement system. Formation of these complexes proceeds through sequential protein-protein interactions and proteolytic cleavages of high specificity. Recent structural, mutational and functional data on factors D and B have significantly enhanced our understanding of the assembly, action, and regulation of the alternative pathway convertase. These processes were shown to depend critically on conformational changes, only some of which are reversible. The need for such changes is dictated by the zymogen-like configurations of the active centers of these unique serine proteases. The structural determinants of some of these changes have been defined from structural and mutational analyses of the two enzymes. Transition of factor D from the zymogen-like to the catalytically active conformation is completely reversible, while the active conformation of the catalytic center of the Bb fragment of factor B is irreversibly attenuated to a great extent on dissociation of the convertase complex. Both mechanisms contribute to the regulation of the proteolytic activity of these enzymes. Additional studies are necessary for a complete description of the elegant mechanisms mediating these processes.

Topology and structure of the C1q-binding site on C-reactive protein.

The host defense functions of human C-reactive protein (CRP) depend to a great extent on its ability to activate the classical complement pathway. The aim of this study was to define the topology and structure of the CRP site that binds C1q, the recognition protein of the classical pathway. We have previously reported that residue Asp(112) of CRP plays a major role in the formation of the C1q-binding site, while the neighboring Lys(114) hinders C1q binding. The three-dimensional structure of CRP shows the presence of a deep, extended cleft in each protomer on the face of the pentamer opposite that containing the phosphocholine-binding sites. Asp(112) is part of this marked cleft that is deep at its origin but becomes wider and shallower close to the inner edge of the protomer and the central pore of the pentamer. The shallow end of the pocket is bounded by the 112-114 loop, residues 86-92 (the inner loop), the C terminus of the protomer, and the C terminus of the pentraxin alpha-helix 169-176, particularly Tyr(175). Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp(112) and Tyr(175) are important contact residues for C1q binding, that Glu(88) influences the conformational change in C1q necessary for complement activation, and that Asn(158) and His(38) probably contribute to the correct geometry of the binding site. Thus, it appears that the pocket at the open end of the cleft is the C1q-binding site of CRP.

Human C-reactive protein is protective against fatal Salmonella enterica serovar typhimurium infection in transgenic mice.

C-reactive protein (CRP) is an acute-phase protein with a well-known association with infection and other inflammatory conditions. We have shown that expression of human CRP by CRP transgenic (CRPtg) mice is protective against lethal infection by Streptococcus pneumoniae, an effect likely mediated by CRP's ability to bind to this gram-positive pathogen. In the present study we tested whether CRPtg mice are resistant to infection with Salmonella enterica serovar Typhimurium, a gram-negative pathogen that causes the murine equivalent of typhoid fever. CRPtg mice experimentally infected with a virulent Typhimurium strain lived longer and had significantly lower mortality than their non-tg littermates. The greater resistance of CRPtg mice could be attributed to significantly increased early (0 to 4 h) blood clearance of salmonellae and significantly decreased numbers of bacteria in the liver and spleen on day 7 postinfection. In addition, 14 days after infection with an avirulent Salmonella strain, the serum titer of anti-Salmonella immunoglobulin G antibodies was higher in CRPtg than non-tg mice. This study provides unequivocal evidence that CRP plays an important role in vivo in host defense against salmonellae during the early stages of infection. In addition, as the beneficial effect of CRP includes enhancement of the host's humoral immune response, CRP may also contribute indirectly to host defense during later stages of infection.

Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component.

The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.

Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding.

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.

High prevalence of complement component C6 deficiency among African-Americans in the south-eastern USA.

Complement component C6 is a part of the membrane attack complex that forms a pore-like structure in cell membranes following complement activation. Deficiency of terminal complement components including C6 predisposes individuals to infection with Neisseriae. Using polymerase chain reaction/single-strand conformation polymorphism analysis followed by DNA sequencing, we screened genomic DNA from 200 randomly chosen blacks and an equal number from whites for three loss-of-function C6 mutations. Ten blacks and two whites were found to be heterozygous for one of the mutations. Two of the mutations, 1195delC and 1936delG, were found exclusively in black individuals. A third previously undescribed mutation, 878delA, was found at equal frequency among the two groups. The difference between the two groups was significant (P = 0.027), indicating that C6 deficiency due to these three mutations is more common among blacks than whites in the local area, principally Jefferson County, Alabama. In addition, three previously undescribed point mutations, two of which result in amino acid substitutions, were identified within exon 6. A review of the county health department records over the past 6 years revealed a higher incidence of meningococcal meningitis in blacks due to serogroups Y and W-135 which paralleled the difference in the estimated prevalence of C6 deficiency. Among black residents of the county (n = 235 598) there were 15 cases of meningitis due to these two serogroups, compared with two cases in the white population (n = 422 604) (P = 0.002). We conclude that C6 deficiency is more common among blacks than whites in the south-eastern United States, with a frequency approaching 1 in 1600 black individuals.

New structural motifs on the chymotrypsin fold and their potential roles in complement factor B.

Factor B and C2 are two central enzymes for complement activation. They are multidomain serine proteases and require cofactor binding for full expression of proteolytic activities. We present a 2.1 A crystal structure of the serine protease domain of factor B. It shows a number of structural motifs novel to the chymotrypsin fold, which by sequence homology are probably present in C2 as well. These motifs distribute characteristically on the protein surface. Six loops surround the active site, four of which shape substrate-binding pockets. Three loops next to the oxyanion hole, which typically mediate zymogen activation, are much shorter or absent. Three insertions including the linker to the preceding domain bulge from the side opposite to the active site. The catalytic triad and non-specific substrate-binding site display active conformations, but the oxyanion hole displays a zymogen-like conformation. The bottom of the S1 pocket has a negative charge at residue 226 instead of the typical 189 position. These unique structural features may play different roles in domain-domain interaction, cofactor binding and substrate binding.

The Arthus reaction in rodents: species-specific requirement of complement.

We induced reverse passive Arthus (RPA) reactions in the skin of rodents and found that the contribution of complement to immune complex-mediated inflammation is species specific. Complement was found to be necessary in rats and guinea pigs but not in C57BL/6J mice. In rats, within 4 h after initiation of an RPA reaction, serum alternative pathway hemolytic titers decreased significantly below basal levels, whereas classical pathway titers were unchanged. Thus the dermal reaction proceeds coincident with systemic activation of complement. The serine protease inhibitor BCX 1470, which blocks the esterolytic and hemolytic activities of the complement enzymes Cls and factor D in vitro, also blocked development of RPA-induced edema in the rat. These data support the proposal that complement-mediated processes are of major importance in the Arthus reaction in rats and guinea pigs, and suggest that BCX 1470 will be useful as an anti-inflammatory agent in diseases where complement activation is known to be detrimental.

Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.

Genetic deficiencies of the complement protein C3 occur naturally in humans and animal models and have been induced in mice by targeted deletion of the C3 gene. The study of these deficiencies has provided evidence for C3 functions in immune responses. C3 deficient mice were generated by replacing the 5'-flanking region of the C3 gene with the neomycin-resistance (neo) gene. Serum from these mice had no detectable C3 protein or complement activity. Challenge with Streptococcus pneumoniae revealed approximately 2000-fold increase in bacteremia as compared to littermate controls. C3 mRNA was absent in the liver, but it was detected in the lung, kidney, fat tissue, heart and spleen. Metabolic labeling of the lung tissue and peritoneal macrophages showed synthesis of pro-C3, but no post-synthetic intracellular processing of the protein and no secretion of mature C3. cDNA analysis at the cap site indicated that extrahepatic transcription of the targeted gene was initiated in the neo cassette, close to the C3/neo junction and predicted a primary translation product lacking the leader peptide. The data indicate that these mice provide a good animal model for the study of complete C3 deficiencies and a potential probe for tissue-specific C3 gene regulatory elements.

C-reactive protein: structural biology and host defense function.

Human C-reactive protein is a Ca2+-binding acute phase-protein with binding specificity for phosphocholine. Recent crystallographic and mutagenesis studies have provided a solid understanding of the structural biology of the protein, while experiments using transgenic mice have confirmed its host-defense function. The protein consists of five identical protomers in cyclic symmetry. On one face of each protomer there is a binding site for phosphocholine consisting of two Ca2+ ions that ligate the phosphate group and a hydrophobic pocket that accommodates the methyl groups of phosphocholine. On the opposite face is a deep cleft formed by parts of the N and C termini and bordered by an alpha-helix. Mutational studies indicate that the C1q-binding site of the molecule is located at the open end of this cleft with Asp112 and Tyr175 representing contact residues. Using human C-reactive protein transgenic mice, we investigated the host defense functions of the protein. Transgenic mice infected with Streptococcus pneumoniae had increased lifespan and lowered mortality compared to wild-type mice. This was attributable to an up to 400-fold reduction in bacteremia mediated mainly by the interaction of C-reactive protein with complement. A complement-independent host protective effect was also demonstrated.

Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.

The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D.

Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity.

Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.

A novel type II complement C2 deficiency allele in an African-American family.

A 9-yr-old African-American male presenting with severe recurrent pyogenic infections was found to have C2 deficiency (C2D). Analysis of his genomic DNA demonstrated that he carried one type I C2D allele associated with the HLA-A25, B18, DR15 haplotype. Screening all 18 exons of the C2 gene by exon-specific PCR/single-strand conformation polymorphism indicated abnormal bands in exons 3, 7, and 6, the latter apparently caused by the 28-bp deletion of the typical type I C2D allele. Nucleotide (nt) sequencing of the PCR-amplified exons 3 and 7 revealed a heterozygous G to A transition at nt 392, causing a C111Y mutation, and a heterozygous G to C transversion at nt 954, causing a E298D mutation and a polymorphic MaeII site. Cys111 is the invariable third half-cystine of the second complement control protein module of C2. Pulse-chase biosynthetic labeling experiments indicated that the C111Y mutant C2 was retained by transfected COS cells and secreted only in minimal amounts. Therefore, this mutation causes a type II C2D. In contrast, the E298D mutation affected neither the secretion of C2 from transfected cells nor its specific hemolytic activity. Analysis of genomic DNA from members of the patient's family indicated that 1) the proband as well as one of his sisters inherited the type I C2D allele from their father and the novel type II C2D allele from their mother; 2) the polymorphic MaeII site caused by the G954C transversion is associated with the type I C2D allele; and 3) the novel C111Y mutation is associated in this family with the haplotype HLA-A28, B58, DR12.

Testosterone and IL-6 requirements for human C-reactive protein gene expression in transgenic mice.

In vitro, IL-6 is the main inducer of the human C-reactive protein (CRP) gene, and IL-1 and steroids can enhance this effect. However, in mice, IL-6 is necessary but not sufficient for induction of the human CRP transgene, and testosterone is required for its constitutive expression by males. To examine the relative contributions of testosterone and IL-6 in the regulation of CRP gene expression, we produced CRP-transgenic (CRPtg), IL-6-deficient (IL-6-/-) mice. Male CRPtg/IL-6-/- mice expressed CRP constitutively, but CRP levels were not increased after injection of LPS. However, acute-phase CRP levels were attained after injection of IL-6. In contrast, female CRPtg/IL-6-/- mice did not express CRP constitutively or after administration of LPS, IL-6, IL-1, or IL-6 plus IL-1. Like males, testosterone-treated CRPtg/IL-6-/- females expressed CRP constitutively, and their transgene responded to injection of IL-6. The endogenous acute-phase protein serum amyloid P (SAP) was expressed constitutively equally by male and female IL-6-/- mice, responded minimally to LPS, and did not respond to either IL-6 or IL-1 alone. Acute-phase levels of SAP were induced in IL-6-/- mice by injection of IL-6 together with IL-1 or LPS. We conclude that in vivo, both constitutive and IL-6-dependent acute-phase expression of the CRP transgene require testosterone. In contrast, testosterone is not required for expression of the SAP gene, which requires IL-1 plus IL-6 for acute-phase induction.

Susceptibility locus for IgA deficiency and common variable immunodeficiency in the HLA-DR3, -B8, -A1 haplotypes.

BACKGROUND: A common genetic basis for IgA deficiency (IgAD) and common variable immunodeficiency (CVID) is suggested by their occurrence in members of the same family and the similarity of the underlying B cell differentiation defects. An association between IgAD/CVID and HLA alleles DR3, B8, and A1 has also been documented. In a search for the gene(s) in the major histocompatibility complex (MHC) that predispose to IgAD/CVID, we analyzed the extended MHC haplotypes present in a large family with 8 affected members. MATERIALS AND METHODS: We examined the CVID proband, 72 immediate relatives, and 21 spouses, and determined their serum immunoglobulin concentrations. The MHC haplotype analysis of individual family members employed 21 allelic DNA and protein markers, including seven newly available microsatellite markers. RESULTS: Forty-one (56%) of the 73 relatives by common descent were heterozygous and nine (12%) were homozygous for a fragment or the entire extended MHC haplotype designated haplotype 1 that included HLA- DR3, -C4A-0, -B8, and -A1. The remarkable prevalence of haplotype 1 was due in part to marital introduction into the family of 11 different copies of the haplotype, eight sharing 20 identical genotype markers between HLA-DR3 and HLA-B8, and three that contained fragments of haplotype 1. CONCLUSION: Crossover events within the MHC indicated a susceptibility locus for IgAD/CVID between the class III markers D821/D823 and HLA-B8, a region populated by 21 genes that include tumor necrosis factor alpha and lymphotoxins alpha and beta. Inheritance of at least this fragment of haplotype 1 appears to be necessary for the development of IgAD/CVID in this family.