John E. Volanakis

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.

Three dimensional structure of human C-reactive protein.

The structure of the classical acute phase reactant human C-reactive protein provides evidence that phosphocholine binding is mediated through calcium and a hydrophobic pocket centred on Phe 66. The residue Glu 81 is suitably positioned to interact with the choline group. A cleft on the pentameric face opposite to that containing the calcium site may have an important functional role. The structure provides insights into the molecular mechanisms by which this highly conserved plasma protein, for which no polymorphism or deficiency state is known, may exert its biological role.

COMPLEMENT ACTIVATION BY C-REACTIVE PROTEIN COMPLEXES*

The serum complement system represents a major element of host defense mechanisms against invading pathogens. As such, it functions in concert with other elements of host defense mechanisms both of an immune or a nonimmune nature to preserve and/or restore normal function and health. The initial observations leading to the discovery of complement were made in the late 1800~’-~ when it was found that killing of bacteria by specific antibodies required an additional, nonspecific, thermolabile factor present in all normal sera. This discovery led to the concept that this nonspecific factor assisted the antibodies to carry out their bacteriolytic function. Hence, the term complement which literally means “that which completes or brings to perfection.” As a result of active research in the area during the last 25 years, the original concept of immune bacteriolysis has been completely revised. It is now well established that complement proteins are actually responsible for the formation of lethal cell lesions, whereas the role of antibody is to identify the invading cells and to initiate complement activation. Further, it is also well established that complement can kill bacteria and other pathogens such as protozoa, fungi, and viruses as well as cells of higher organisms even in the absence of antibody. More importantly, it is now well accepted that the contributions of complement to host defense are much broader than killing of cells. The system mediates a number of other functions essential to host defense such as release of histamine from mast cells, chemotactic attraction of phagocytic cells, and opsonization of cells and particles. Complement is a highly organized and tightly regulated system which like other mediator systems in the blood, such as the coagulation system, the fibrinolytic system, and the kinin-generating system, is activated in. a sequential, “cascade-like’’ fashion. Fourteen distinct proteins participate in the activation sequence and at least six other proteins are responsible for the regulation and control of the system (recently reviewed in 4-7). This apparent complexity is contrasted by the functibnal simplicity of the complement system, which becomes evident when its participation in host defense is considered. With few possible exceptions, all the complement-derived biological activities that are relevant to host-defense are derived from two proteins, C3 and C5. These two complement components are structurally very similar. They both have a molecular weight of approximately 185,000.8~’0 Each has two disulfide-linked polypeptide chains termed (Y (110,000) and /3 (75,000). Their biosynthetic precursors are singlepolypeptide chain molecules.yl*lz Finally, available limited amino acid sequence data indicate extensive sequence homologies between C3 and C5.13 The structural similarities between the two proteins make it likely that they represent gene

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 Asp112 of CRP plays a major role in the formation of the C1q-binding site, while the neighboring Lys114 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. Asp112 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 α-helix 169–176, particularly Tyr175. Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp112 and Tyr175 are important contact residues for C1q binding, that Glu88 influences the conformational change in C1q necessary for complement activation, and that Asn158 and His38 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.

Major histocompatibility complex class III genes and susceptibility to immunoglobulin A deficiency and common variable immunodeficiency.

We have proposed that significant subsets of individuals with IgA deficiency (IgA-D) and common variable immunodeficiency (CVID) may represent polar ends of a clinical spectrum reflecting a single underlying genetic defect. This proposal was supported by our finding that individuals with these immunodeficiencies have in common a high incidence of C4A gene deletions and C2 rare gene alleles. Here we present our analysis of the MHC haplotypes of 12 IgA-D and 19 CVID individuals from 21 families and of 79 of their immediate relatives. MHC haplotypes were defined by analyzing polymorphic markers for 11 genes or their products between the HLA-DQB1 and the HLA-A genes. Five of the families investigated contained more than one immunodeficient individual and all of these included both IgA-D and CVID members. Analysis of the data indicated that a small number of MHC haplotypes were shared by the majority of immunodeficient individuals. At least one of two of these haplotypes was present in 24 of the 31 (77%) immunodeficient individuals. No differences in the distribution of these haplotypes were observed between IgA-D and CVID individuals. Detailed analysis of these haplotypes suggests that a susceptibility gene or genes for both immunodeficiencies are located within the class III region of the MHC, possibly between the C4B and C2 genes.

C-reactive protein: purification by affinity chromatography and physicochemical characterization

Abstract A new method for obtaining C-reactive protein from ascites fluid in highly purified form and high yields has been described. It is based on the Ca2+-dependent affinity of this protein for phosphorylcholine. Purified C-reactive protein was shown to be homogeneous by electrophoretic, immunochemical and ultracentrifugal criteria. By sedimentation equilibrium a molecular weight of 118,000 was calculated in neutral buffer. In 5 M guanidine hydrochloride the apparent molecular weight was 24,300.

Human C-Reactive Protein Is Protective against Fatal Salmonella enterica Serovar Typhimurium Infection in Transgenic Mice

ABSTRACT 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 byStreptococcus pneumoniae, an effect likely mediated by CRPs ability to bind to this gram-positive pathogen. In the present study we tested whether CRPtg mice are resistant to infection withSalmonella 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 hosts humoral immune response, CRP may also contribute indirectly to host defense during later stages of infection.

Renal filtration and catabolism of complement protein D

Complement protein D, a serine protease participating in the formation of the C3 convertase of the alternative complement pathway, has the lowest molecular weight (23,750) and serum concentration of all complement proteins. In normal serum, D is the rate-limiting protease of the alternative pathway of complement activation. We report that the serum concentrations of D in 20 patients with chronic renal failure (mean +/- S.D., 0.42 +/- 0.28 mg per deciliter) and in 16 patients on long-term dialysis (1.53 +/- 0.39 mg per deciliter) were significantly higher (P less than 0.001) than in 22 healthy adults (0.18 +/- 0.04 mg per deciliter). In chronic renal failure the serum concentration of D correlated with that of creatinine (r = 0.75, P less than 0.001). The serum concentrations of D found in patients with renal failure reached and in some cases exceeded those at which the protease is no longer rate-limiting. Thus, enhanced activity of the alternative pathway of complement should be expected in patients with advanced renal failure. Urinary D was undetectable (less than 0.2 micrograms per deciliter) in 17 normal adults and either undetectable or below the concentration expected from the degree of proteinuria in 10 patients with nephrotic syndrome. However, in a patient with Fanconis syndrome the urinary concentration of D (1.3 mg per deciliter) was an order of magnitude higher than the serum concentration, representing 0.5 per cent of the total protein. The urinary D in this patient had normal hemolytic activity, antigenicity, and size. These results indicate that D is filtered through the glomerular membrane and is probably catabolized in the proximal renal tubules.

C-reactive protein

Over the past few years substantial insight was gained into the biol ogy and biochemistry of human C-reactive protein (CRP). X-ray crystallography in conjunction with mutational analyses, generated the three-dimensional structure of the protein and indicated the topology and structure of ligand-binding sites. Using human CRP transgenic mice infected withStreptococcus pneumoniae, we obtained data that clearly established CRP as an important host defense molecule. Studies using the same mice revealed a previ ously unknown testosterone-dependence of constitutive expression of human CRP. In this article we provide a brief overview of these recent findings.

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.