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PubMed1993

Structure of complement poly-C9 determined in projection by cryo-electron microscopy and single particle analysis.

The ring-like complement 'lesions' found on membranes of complement lysed cells comprise a complex of components C5b through C9 that coalesce to form hollow cylinders which penetrate the membrane bilayer and create lytic pores. Walls of these C5b-9 membrane attack complex cylinders may consist primarily of the C9 component, since samples of purified, isolated C9 can polymerize into cylindrical structures which appear identical with the fully assembled C5b-9 complex. The structure of these poly-C9 molecules has been investigated using the techniques of cryo-electron microscopy and single particle analysis. Sets of single poly-C9 particles viewed as rings were selected from cryo-EM images, then particles were aligned and treated by correspondence analysis to identify the principle interparticle similarities and variations. The highest ranking variation found was the presence or absence of a dense inner ring of protein density. Other important variations were interpreted as different types of particle tilt. These results were used in selecting a subgroup of untilted particles for averaging and symmetry analysis. The rotational power spectrum of the initial average suggested 13-fold symmetry. The 13-fold symmetry was used to select and group particles for further analysis. Individual particles were 13-fold rotational averaged and those with enhanced peripheral features were placed into either a right-handed subgroup or into a left-handed subgroup based on orientation of the peripheral features. Particles within each group were aligned and averaged, and a poly-C9 structure was produced which shows important structural details and from which the C9 monomer structure can be deduced. The poly-C9 structure contains a dense inner ring of diameter between 113-181 A and which is modulated into 13 discrete peaks with peak-to-peak separation of approx. 35 A. The dense inner ring is surrounded by a less dense, concentric outer rim extending to 254 A diameter. The outer rim contains projections that are contiguous with the inner peaks but are skewed relative to the ring radius to produce the appearance of a pin-wheel. These projections correspond with the peripheral features picked up in the rotationally averaged individual particles; the left- or right-handed orientation of projections may result from the up/down orientation of individual particles in ice. The C9 monomer structure within the cylinder is suggested by the density distribution. The monomer would be a rod with diameter of 35 A, oriented parallel to the cylinder axis and would be roughly perpendicular to a membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement C9↗
PubMed1995

Shedding of tyrosine and serine/threonine ecto-protein kinases from human leukemic cells.

Ecto-protein kinases (ecto-PK), primarily of the serine/threonine kinase type, have been previously described on the surface of various normal, transformed, and tumor cells. We have found that in the presence of ATP and Mg2+, exogenously added substrates such as phosvitin and poly(Glu4-Tyr) are phosphorylated by intact K562 erythroleukemia, HL60 promyelocytic leukemia, and U937 histiocytic leukemia human cells. Phosphoamino acid analysis indicated that phosvitin, histone H2B, casein, and protamine are phosphorylated on serine and threonine residues, whereas poly(Glu4-Tyr) is phosphorylated on tyrosine. We also present evidence showing that the C9 complement protein, a key component of the membranolytic protein complex of the complement system, is exclusively phosphorylated by the K562 cells on serine residues. Phosphorylation of poly(Glu4-Tyr) is markedly enhanced by Mn2+, whereas C9 phosphorylation is rather inhibited by Mn2+. It is concluded that human leukemic cells express on their surface two types of ecto-PK, one phosphorylating serines and threonines and one specific to tyrosines. The ecto-PKs are spontaneously shed from fully viable cells into the medium in a temperature-dependent manner. Upon sedimentation of cell supernatants at 100,000g, the ecto-PKs are found sedimented with small membrane vesicles. Treatment of intact K562 cells or of released membrane vesicles with bacterial phospholipase C, but not with trypsin or pronase, releases the two types of ecto-PK from the cell or vesicle membrane, respectively. This is accompanied by a marked increase in the released phosphorylating activity. It is, therefore, suggested that these ecto-PKs are either covalently linked to phospholipids or strongly attached to lipid-anchored molecules in the cell surface membrane. Several endogenous proteins in the released membranes are phosphorylated by the ecto-PKs on serines and to a lesser extend on threonines. Two proteins (PTP79 and PTP54) are phosphorylated in a manganese-dependent manner on tyrosines.

Adenosine Triphosphate↗
PubMed1987

Susceptibility of pathogenic and nonpathogenic Naegleria spp. to complement-mediated lysis.

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with [3H]uridine and visual observation with a compound microscope were used as indices of lysis. Highly pathogenic mouse-passaged N. fowleri was less susceptible to the lytic effects of NHS and NGPS than the weakly pathogenic, axenically grown N. fowleri or N. australiensis and the nonpathogenic amoebae N. gruberi and N. lovaniensis. However, both pathogenic and nonpathogenic Naegleria spp. depleted complement as assessed by total hemolytic activity. Treatment of serum with EDTA, heat (56 degrees C, 30 min), cobra venom factor, or antibody to C3 or C9 complement components decreased the amoebicidal activity of NHS. The presence of specific agglutinating antibody to N. fowleri enhanced the amoebicidal activity of NGPS for N. fowleri.

Amebiasis↗
PubMed1983

A simple immunoradiometric assay for the terminal SC5b-9 complex of human complement.

Complement activation in serum via classical or alternative pathway results in the formation of a water-soluble, macromolecular SC5b-9 protein complex consisting of non-covalently associated C5b-C9 complement components and serum S-protein. Characteristic neoantigens are expressed on this complex that permit its immunological differentiation from native complement proteins. The present paper describes a procedure for isolating radiolabelled, specific antibodies to C5b-9 neoantigens. With the use of these antibodies, a simple and rapid immunoradiometric assay for SC5b-9 has been developed that allows for detection of 3-4 micrograms SC5b-9 in 1 ml human serum or plasma. The method is suitable for routine screening analyses for SC5b-9 in patients' plasma, and for detection of SC5b-9 experimentally generated in vitro.

Complement Membrane Attack Complex↗
PubMed1987

Mechanisms of stable serum resistance of Neisseria gonorrhoeae.

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent sera from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by immunoglobulin G (IgG) or F(ab')2 isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, contained most of the blocking activity in IgG. In addition, immune convalescent DGI serum, which did not exhibit bactericidal activity, was restored to killing by selective immunodepletion of protein III antibodies. Blocking IgG or F(ab')2 prepared from IgG, partially inhibited binding of bactericidal antibody to N. gonorrhoeae. Also, binding of a monoclonal antibody recognizing N. gonorrhoeae outer membrane protein PIII was almost completely inhibited by blocking F(ab')2. Presensitization of N. gonorrhoeae with increasing concentrations of blocking IgG or F(ab')2 before incubation with bactericidal antibody and an antibody free source of complement, increased consumption and deposition of the third component of human complement (C3) and the ninth component of complement (C9) but inhibited killing in dose-related fashion.

Antibodies, Bacterial↗
PubMed1991

Assembly of macromolecular pores by immune defense systems.

Immune defence systems (complement, cytolytic lymphocytes) make use of transmembrane pores assembled from up to 20 soluble monomers in a highly regulated process to induce cell death. Inhibitors of pore formation have been found which protect blood, endothelial and epithelial cells from the destructive effect of complement lesions. Recently, a pore-forming protein showing immunological crossreactivity to complement C9 has been found in the protozoan parasite Trypanosoma cruzi, thereby extending this protein family and generalizing its means of generating non-selective membrane permeability.

Animals↗
PubMed1981

Familial C1q deficiency associated with renal and cutaneous disease.

A familial C1q deficiency of complement in three siblings has been established. The patients were two brothers and a sister (12, 11 and 9 years old) with clinical and pathological features of Rothmund-Thomson syndrome (Poikiloderma congenital) and mesangial proliferative glomerulonephritis with diffuse IgM deposits. Abnormality has been defined as a total lack of CH50 haemolytic activity, undetectable C1q, failure to correct the defect with functionally pure C2 to C9 complement components, normal values for C2, C3, C4 and C5 and restoration of CH50 haemolytic activity when purified human C1q was added to the assay.

Adult↗
PubMed1986

Mechanism of resistance to lysis by the alternative complement pathway in Trypanosoma cruzi trypomastigotes: effect of specific monoclonal antibody.

Trypanosoma cruzi G strain epimastigotes were lysed by normal human serum (NHS) through activation of the alternative complement pathway (ACP), whereas metacyclic trypomastigotes were resistant to lysis. Epimastigotes and metacyclics with equivalent amounts of C3b deposited on their surface bound factor B with similar affinities. In contrast, factor H bound with higher affinity to metacyclics than to epimastigotes. Both T. cruzi forms with bound C3b were extensively (60 to 80%) lysed after formation of surface C3-convertase and the addition of a C3-C9 complement source. In the presence of factors H and I, or incubation with NHS with EDTA, the percentage of lysis of metacyclics decreased faster than that of epimastigotes with increasing incubation times. These data suggest, as a possible mechanism of resistance to lysis in metacyclic trypomastigotes, the higher binding affinity of factor H to C3b and the inactivation of the latter by serum regulatory proteins. Metacyclics were lysed by NHS, through ACP, in the presence of human immune serum to T. cruzi or anti-T. cruzi monoclonal antibody, but not with the Fab fragment of the latter, which recognizes a 90,000 m.w. antigen from T. cruzi metacyclics. Protection of parasite-bound C3b from serum control proteins was observed when parasites were incubated, before C3 deposition, with the lytic monoclonal antibody but not with its Fab fragment or a nonrelated IgG control. When C3b was deposited on metacyclics before antibody binding, C3b inactivation occurred. In the lysis of metacyclics, through ACP activation, binding of antibody apparently creates new acceptor sites which prevent the activity of serum regulatory proteins.

Animals↗
PubMed1989

Secretion of the terminal complement proteins, C5-C9, by human platelets.

The terminal complement components, C8 and C9, and to a lesser extent C5, C6, and C7, but minimal amounts of C3, were shown to be associated with washed human platelets. In unactivated platelets, the complement components were detected in the platelet pellet by hemolytic assays after centrifugation and disruption of the platelets by freeze-thawing. However, after platelets had been activated by collagen, thrombin, or aggregated IgG to induce aggregation, the complement components were released into the supernatant. The rank order of hemolytic activity of C9, C8, C7, C6, and C5 detected in the supernatants of activated platelets was quite different from that found in serum from the same donors, in the same assays. In particular, the serum C7 hemolytic titer was more than twice the serum C9 hemolytic titer, whereas the activity of C9 detected from platelets was more than twice that of C7. This argues against a purely nonspecific uptake of these proteins by platelets from plasma. The functional role of terminal complement components released from platelets during activation is unknown, but it is tempting to speculate that these proteins may have a role in platelet-dependent immunological tissue injury. Because the C5b-9 membrane attack complex activates platelets, it is possible that release of terminal complement proteins serves to amplify platelet activation and may also play a role in diseases in which complement membrane attack complexes have been implicated.

Blood Platelets↗
PubMed1989

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.

Androgen-Binding Protein↗
PubMed2002

Binding of human complement C8 to C9: role of the N-terminal modules in the C8 alpha subunit.

Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.

Animals↗
PubMed2005

Complement inhibition attenuates brain edema and neurological deficits induced by thrombin.

The present study examined whether thrombin activates the complement cascade in the brain and whether N-acetylheparin, an inhibitor of complement activation, attenuates brain injury induced by thrombin. There were three sets of studies. In the first set, rats had an intracerebral infusion of either five-unit thrombin or a needle insertion. Brains were sampled at 24 hours for Western blot analysis and immuno-histochemistry. In the second set, rats received either five-unit thrombin+saline, five-unit thrombin+25 microg N-acetylheparin or five-unit thrombin+100 microg N-acetylheparin infusion. Brains were sampled 24 hours later for water content measurement. In the third set, rats received either five-unit thrombin+saline or five-unit thrombin+ 100 microg N-acetylheparin. Behavioral tests sensitive to unilateral striatal damage were carried out for two weeks. Western blotting demonstrated that complement C9 and clusterin levels increase 24 hours after thrombin infusion (P < 0.01). Both C9 and clusterin positive cells were found around the injection site. High-dose (100-microg) but not low-dose (25-microg) N-acetylheparin attenuated thrombin-induced brain edema (81.5 +/- 0.4% vs. 83.7 +/- 0.3% in the vehicle, P < 0.05). Behavior was also significantly improved by N-acetylheparin (P < 0.05). In conclusion, thrombin-induced edema formation and neurological deficits were both reduced by N-acetylheparin. This suggests that inhibition may be a novel treatment for the thrombin-induced brain injury that occurs in intracerebral hemorrhage.

Animals↗
PubMed1997

Enhanced sensitivity of P-glycoprotein-positive multidrug resistant tumor cells to complement-mediated lysis.

The interaction of KB-V1, a multidrug resistant (MDR) variant of the KB-3-1 human oral carcinoma, with human complement was investigated. KB-V1 cells were found to be more sensitive than KB-3-1 cells to complement-mediated lysis. Detailed analysis of the capacity of KB cells to activate human complement demonstrated that both C3b deposition and formation of the membrane attack complex (MAC) are higher on KB-V1 than on KB-3-1 cells. Furthermore, the MAC formed on KB-V1 cells, but not on KB-3-1 cells, was found to be resistant to trypsin treatment, i.e. more stably inserted into the plasma membrane. Immunofluorescence analysis by flow cytometry showed that KB-V1 cells express less decay-accelerating factor (DAF, CD55) than KB-3-1 cells. Two other complement regulatory proteins, membrane cofactor protein (MCP, CD46) and CD59 are expressed to a similar extent on both KB-V1 and KB-3-1 cells. Treatment of KB-V1 cells with neutralizing anti-P-glycoprotein (P-gp) monoclonal antibodies reduced their sensitivity to complement. In addition, KB-V1 revertants which cease to express P-gp become more resistant to complement. These results indicate that multiple factors, such as reduced expression of DAF, enhanced deposition of C3b and increased binding and stability of the MAC may contribute to the increased complement sensitivity of KB-V1 cells. It is suggested that P-gp is responsible for the complement-sensitive phenotype of KB-V1 cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗