Is the membrane attack complex of complement an enzyme?

Summary Recent studies on the functional activities of the membrane attack complex of complement, C5b-9, are reviewed. A new speculative hypothesis has been advanced to account for the ability of complement to mediate lysis of various targets. This hypothesis has three major elements: 1) that the membrane attack complex is an enzyme; 2) that the substrate for this putative enzyme is a membrane constituent; 3) that the substrate specificity of the putative enzyme is dependent on the species source of individual complement components within the C5b-9 complex.Abbreviations E = sheep red cells - A = rabbit IgM anti-Forssman antibody - Hu or hu = human - GP or gp = guinea pig     

How do we get a perfect complement of digits?

A crucial issue in limb development is how a correct set of precisely shaped digits forms in the digital plate. This process relies on patterning across the anterior-posterior axis of the limb bud, which is under the control of Sonic hedgehog emanating from the zone of polarizing activity. Recently, Sonic hedgehog function in the limb bud has been shown to have a dual character controlling both growth and patterning of the digital field. This finding has prompted the proposal of new models of how these two functions are achieved, and this will be discussed in this review.     

Conformational states of a bacterial α2-macroglobulin resemble those of human complement C3

α(2) macroglobulins (α(2)Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α(2)Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α(2)M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α(2)M homolog (ECAM) was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS) techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials.     

Relative contributions of dectin-1 and complement to immune responses to particulate β-glucans

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.     

Why are higher plants green? Evolution of the higher plant photosynthetic pigment complement

The physiological reason that higher plants are green is unknown. Other photosynthetic organisms utilize pigments that strongly absorb green light; therefore, there must have been natural forces that ‘selected’ the photosynthetic pigments found in higher plants. Based on previously published data and our recent findings about green light and photosynthesis within leaves (Sun et al.), a specific functional role is described for the primary photosynthetic pigments of higher plants, that were derived from green algal progenitors. The particular absorptive characteristics of chlorophylls a and b appear to perform two contradictory, but necessary functions in higher plants. Firstly, chlorophylls a and b absorb light for maximum utilization under non‐saturating conditions, a function that is well understood. Secondly, they can act as protective pigments under over‐saturating light conditions, when absorbed light is dissipated as heat. Under such conditions, a significant portion of light can also be efficiently utilized, especially in the bottom portion of the leaf, that is mainly illuminated by green light and not down‐regulated. The second function may have been the selective force that gave rise to the extremely successful terrestrial plants, that evolved from green algae.     

Purification of complement components by hydrophobic affinity chromatography on phenyl—Sepharose: Purification of human C5

Human complement components C5 and C3 were purified with 41% and 20% yields, respectively, by euglobulin precipitation, DEAE—Sephacel ion-exchange chromatography and gel filtration. Phenyl—Sepharose chromatography allowed the complete separation of C3 and C5. C3 bound loosely on the resin whereas C5 bound firmly and was eluted with 50% glycerin solution. Gel filtration on Sephacryl S-300 allowed the depletion of C4bp and H that contaminated C5 preparations. Homogeneity of C5 and C3 preparations was demonstrated by SDS—PAGE and immunochemical analysis. C5 and C3 consisted of two chains (α, 110000; β, 75000) linked by disulfide bridges.     

Molecular analysis of complement component C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus

 The amino acid sequences of the human terminal complement components show extensive structural similarity to each other. In this study the C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus, were cloned and analyzed. The derived deduced amino acid sequences of the two terminal components were homologous to those of humans, in that the sequences of both species contained LDL receptor, EGF precursor, and two thrombospondin domains. Japanese flounder C9 was found to have a second thrombospondin region in the C-terminus, similar to that reported for rainbow trout and pufferfish. Moreover, these two complement component cDNAs of Japanese flounder had partial similarity to human perforin. These findings show that Japanese flounder C8β and C9 have similar structures, which supports the hypothesis that the terminal complement genes originated from the same ancestral gene. Collectively, these features emphasize the strong similarity among the members of the terminal complement family. Received: 23 March 1999 / Revised: 1 June 1999     

Genomic organization of human complement protein C8α and further examination of its linkage to C8β

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8, C8, C8) encoded by separate genes. C8 and C8 are linked on chromosome 1p32, whereas C8 is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 gene. The gene contains at least 11 exons spanning 70kb of DNA. When compared to C6, C8 and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 and C8 linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5kb) and in a 5- 3 orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.     

Interaction of human Waldenstr?m's IgM proteins with guinea pig and human complement.

The activity of purified human Waldenstr?m's IgM protein to fix complement of human and guinea pig origins was compared at different temperatures using the polystyrene latex particle-adsorption method. It was shown that the interaction of the IgM proteins with complement differed depending on the source of complement and that a pronounced heterogeneity in complement-fixing activity was observed among the IgM proteins when tested with guinea pig complement. Thus, by the use of guinea pig complement, six human IgM proteins examined were classified roughly into two groups, one having a high and the other a low activity at 3 C as well as at 37 degrees C. With human complement, five proteins showed a rather uniform activity at 37 degrees C. However, there was one protein with no detectable activity, suggesting the presence of non-complement-fixing protein in the IgM class. All the six proteins showed no significant activity with human complement at 3 C. No antigenic difference has been found as yet in the Fc or Cmu2 region among these IgM proteins examined.     

Interaction of two phagocytic host defense systems: Fcγ receptors and complement receptor 3

Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.     

Kinetics of conversion ofNeisseria gonorrhoeae to resistance to complement by cytidine 5′-monophospho-N-acetyl neuraminic acid

Freshly isolated gonococci upon subculture are readily lysed by normal human serum although a few strains remain inherently resistant to the complement activity. The sensitive gonococci can be converted to serum resistance by incubation with a host derived factor referred to as cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NANA). These gonococci resist complement mediated killing due to their sialylation of an epitope structure on a component of lipo-oligosaccharide (LOS). In the present study, the kinetics of conversion to serum resistance by the action of sialyltransferase (STase) inNeisseria gonorrhoeae was followed with very low concentrations of CMP-NANA. This conversion could not be perceived at 2×10^–3 nmol.ml^–1 but was fully attainable from 8×10^–3 to 2×10^–2 nmol.ml^–1 CMP-NANA. When pretreated up to 100 min in presence of the very low concentration of 2×10^–3 nmol.ml^–1, a potentiating effect on the conversion of gonococci by 2×10^–2 nmol.ml^–1 was observed in relation to the time of preincubation. This action was abolished after exposure to a subinhibitory concentration of chloramphenicol (0.5 µg.ml^–1). The gonococci recovered their ability to convert to serum resistance following adequate washing. The potential for increase in STase activity should be of interest for understanding the conversion from a serum sensitive to a serum resistance state.     

Molecular mechanisms of inflammation and tissue injury after major trauma-is complement the "bad guy"?

Trauma represents the leading cause of death among young people in industrialized countries. Recent clinical and experimental studies have brought increasing evidence for activation of the innate immune system in contributing to the pathogenesis of trauma-induced sequelae and adverse outcome. As the "first line of defense", the complement system represents a potent effector arm of innate immunity, and has been implicated in mediating the early posttraumatic inflammatory response. Despite its generic beneficial functions, including pathogen elimination and immediate response to danger signals, complement activation may exert detrimental effects after trauma, in terms of mounting an "innocent bystander" attack on host tissue. Posttraumatic ischemia/reperfusion injuries represent the classic entity of complement-mediated tissue damage, adding to the "antigenic load" by exacerbation of local and systemic inflammation and release of toxic mediators. These pathophysiological sequelae have been shown to sustain the systemic inflammatory response syndrome after major trauma, and can ultimately contribute to remote organ injury and death. Numerous experimental models have been designed in recent years with the aim of mimicking the inflammatory reaction after trauma and to allow the testing of new pharmacological approaches, including the emergent concept of site-targeted complement inhibition. The present review provides an overview on the current understanding of the cellular and molecular mechanisms of complement activation after major trauma, with an emphasis of emerging therapeutic concepts which may provide the rationale for a "bench-to-bedside" approach in the design of future pharmacological strategies.     

Interaction of non-adherent suspended neutrophils to complement opsonized pathogens： a new assay using optical traps

Phagocytosis of opsonized pathogens by circulating non-adherent neutrophils is an essential step in host defense, which when overwhelmed contributes to sepsis. To investigate the role played by ligation of complement receptors CR3 and CR4 in non-adherent neutrophils, we designed a novel assay system utilizing dual optical traps, respectively, holding a suspended unactivated cell and presenting a specific ligand-coated bead to the cell surface. We chose anti-CD 18 as an example ligand, mimicking the bacterial opsonizing complement fragment iC3b. Presentation of anti-CD 18-coated beads elicited both pseudopodial protrusion and subsequent phagocytosis. This is in sharp contrast to previously reported responses of adherent neutrophils, which phagocytize opsonized particles without pseudopod formation. We used this same new assay to probe actomyosin pathways in the neutrophil＇s pseudopodial and phagocytic response. Disruption of actin or inhibition of myosin light-chain kinase dose-dependently reduced pseudopod formation and phagocytosis rates. In summary, i） the new dual trap assay can be used to study the responses of suspended neutrophils to a variety of ligands, and ii） in a first application of this technique, we found that local ligation of CR3/4 in unactivated neutrophils in suspension induces pseudopod formation and phagocytosis at that site, and that these events occur via an actomyosindependent pathway.     

Genetic polymorphism of C′3 (β1C-globulin) component of complement in a German and a Spanish population

Summary The polymorphism of the major component of human complement (C3 or -globulin) at that time is interpreted formally by the genetic model 5 codominant alleles at one autosomal locus. Examination of this hypothesis has been performed examinating a sample of 482 unrelated persons in the two populations, 71 families with 127 children and 115 mother/child combinations in a German population. It could be stated that 4 alleles or more are involved in this polymorphism.