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PubMed1991

Phosphatidylinositol-glycan linked proteins of the erythrocyte membrane.

The human erythrocyte bears a number of proteins anchored to the outer membrane surface via a phosphatidylinositol-glycan linkage. This class of proteins includes several complement regulatory proteins (including decay-accelerating factor, CD59 antigen (protectin), and C8 binding protein) as well as several enzymes and at least one protein important in cell-cell interaction. In addition, a number of blood group antigens have been identified to reside on proteins with phosphatidylinositol anchors. One blood group (Cromer) resides on DAF. Study of variants in this blood group system has led to interesting information about the function and expression of this protein. Several other blood groups, such as JMH and Holley/Gregory, appear to reside on as yet unidentified phosphatidylinositol-linked proteins. In paroxysmal nocturnal haemoglobinuria, a variable proportion of red cells fail to express or express weakly all phosphatidylinositol-linked proteins. The origin of this deficiency is now being worked out. In addition, individuals with inherited deficiency of DAF or CD59 (protectin) have been identified. Only the latter deficiency leads to a PNH-like syndrome.

Antigens↗

PubMed2004

[Interaction between the human and simian adenoviruses in human cells: complementation, transcapsidation and formation of defective adeno-adeno hybrids].

It was for the first time that complementation between the human and simian adenoviruses in human cells as well as the ability of the human adenovirus Ad2 (HADv2) genome to transform completely into the simian adenovirus SA7(C8) (SADv15) capsid (transcapsidation) was demonstrated. A defective adeno-adeno hybrid (recombinant) between the above viruses is described; the recombinant has the SA7(C8) capsid and Ad2 genome with a 10% insertion of SA7(C8) in the central region. Defective hybrid virions are able to replicate both in human and simian cells by using the SA7(C8) virus as helper. The hybrid virions help the above virus to replicate in human cells: they form a mutually complementing virion pair.

Adenoviruses, Human↗

PubMed1993

Stimulation of mononuclear cells by contact with cuprophan membranes: further increase of beta 2-microglobulin synthesis by activated late complement components.

Contact of mononuclear cells (MNC) with cuprophan membranes in vitro causes an increase in beta 2-microglobulin (beta 2m) synthesis. Since in vivo the dialyzer membrane is rapidly coated with plasma proteins, contact activation of MNC was tested in the presence of normal human serum (NHS). After contact with cuprophan, deposition of C5b-9 on the cells was seen, followed by an increase in beta 2m synthesis and cytokine release, exceeding that seen after contact activation in the absence of serum. Inactivated serum or serum deficient in C8 did not increase beta 2m production, indicating that the additional activation was due to complement C5b-9. The results suggest that there are two cuprophan-related mechanisms of cell activation: one by contact of cells with the membrane, the other by the complement activation products. Both might synergistically contribute to an increased beta 2m synthesis in hemodialysis patients.

Blotting, Northern↗

PubMed1984

The influence of electrochemical gradients of Na+ and K+ upon the membrane binding and pore forming activity of the terminal complement proteins.

The hemolytic activity of the terminal complement proteins (C5b-9) towards erythrocytes containing high potassium concentration has been reported to be dramatically increased when extracellular Na+ is substituted isotonically by K+ (Dalmasso, A.P., et al., 1975, J. Immunol. 115:63-68). This phenomenon was now further investigated using resealed human erythrocyte ghosts (ghosts), which can be maintained at a nonlytic osmotic steady state subsequent to C5b-9 binding: (1) The functional state of C5b-9-treated ghosts was studied from their ability to retain trapped [14C]-sucrose or [3H]-inulin when suspended either in the presence of Na+ or K+. A dramatic increase in the permeability of the ghost membrane to both nonelectrolytes - in the absence of significant hemoglobin release - was observed for C5b-9 assembly in the presence of external K+. (2) The physical binding of the individual 125I-labeled terminal complement proteins to ghost membranes was directly measured as a function of intra- and extracellular K+ and Na+. The uptake of 125I-C7, 125I-C8, and 125I-C9 into membrane C5b-9 was unaltered by substitution of Na+ by K+. (3) The binding of the terminal complement proteins to ghosts subjected to a transient membrane potential generated by the K+-ionophore valinomycin (in the presence of K+ concentration gradients) was measured. No significant change in membrane binding of any of the C5b-9 proteins was detected under the influence of both depolarizing and hyperpolarizing membrane potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane Permeability↗

PubMed2005

Recombinant C345C and factor I modules of complement components C5 and C7 inhibit C7 incorporation into the complement membrane attack complex.

Complement component C5 binds to components C6 and C7 in reversible reactions that are distinct from the essentially nonreversible associations that form during assembly of the complement membrane attack complex (MAC). We previously reported that the approximately 150-aa residue C345C domain (also known as NTR) of C5 mediates these reversible reactions, and that the corresponding recombinant module (rC5-C345C) binds directly to the tandem pair of approximately 75-residue factor I modules from C7 (C7-FIMs). We suggested from these and other observations that binding of the C345C module of C5 to the FIMs of C7, but not C6, is also essential for MAC assembly itself. The present report describes a novel method for assembling a complex that appears to closely resemble the MAC on the sensor chip of a surface plasmon resonance instrument using the complement-reactive lysis mechanism. This method provides the ability to monitor individually the incorporation of C7, C8, and C9 into the complex. Using this method, we found that C7 binds to surface-bound C5b,6 with a K(d) of approximately 3 pM, and that micromolar concentrations of either rC5-C345C or rC7-FIMs inhibit this early step in MAC formation. We also found that similar concentrations of either module inhibited complement-mediated erythrocyte lysis by both the reactive lysis and classical pathway mechanisms. These results demonstrate that the interaction between the C345C domain of C5 and the FIMs of C7, which mediates reversible binding of C5 to C7 in solution, also plays an essential role in MAC formation and complement lytic activity.

Amino Acid Motifs↗

PubMed1995

Immunohistochemical localization of C9 neoantigen and the terminal complement inhibitory protein CD59 in human endometrium.

PROBLEM: Human endometrium expresses complement components, receptors, and regulatory proteins, many of which appear to be expressed in a hormone-dependent manner. Whether terminal complement components are also present in the endometrium is unknown. CD59, a broadly expressed protein that blocks association of C9 with C8 in the membrane attack complex, is localized in reproductive tissue to human spermatozoa, seminal plasma, amniotic fluid, and placenta. The present study examines human endometrium for the presence of CD59 and terminal complement proteins. METHOD: Endometrial biopsies were obtained from six normal women from various phases of the menstrual cycle and analyzed by immunohistochemistry, using MEM-43 anti-human CD59 and anti-human SC5b-9 murine monoclonal antibodies and the immunoperoxidase technique. RESULTS: Both CD59 protein and SC5b-9 (C9 neoantigen) were demonstrated to be present in endometrial glandular epithelium throughout the menstrual cycle. No specific staining was demonstrated in the stromal compartment. CONCLUSION: CD59 protein and terminal complement proteins are expressed in glandular epithelial cells of normal human endometrium, in both proliferative and luteal phases, suggesting that expression is not hormonally dependent. These analyses further support the presence of a functionally active complement system in normal human endometrium.

Antigens↗

PubMed2000

Production and functional analysis of rat CD59 and chimeric CD59-Crry as active soluble proteins in Pichia pastoris.

Crry (CR1-related gene/protein) is a rodent complement regulator that inhibits C3 convertases. CD59 is a conserved protein inhibitor active towards C8 and C9. We have previously produced rat Crry as a recombinant soluble (rs) protein in Pichia pastoris. In this study we produced functionally active rat rsCD59 and a chimeric rsCD59-Crry protein in P. pastoris. The GPI anchor addition site of rat CD59 (Asn-79) was replaced either by a stop codon to produce rsCD59, or with the sequence of the first five short consensus repeats of Crry to produce rsCD59-Crry. Proteins were generated by fermentation and purified by affinity chromatography on an anti-CD59 column. In a standard classical pathway haemolysis assay, all three rs proteins had inhibitory activity, with 50% inhibition at 0.5 microM (rsCrry and rsCD59-Crry) and 4.4 microM (rsCD59). In an assay examining inhibition of C5b-9, in which C5b-7 was first formed, followed by purified C8 and C9, rsCD59 and rsCD59-Crry were active with 50% inhibition at 0.8 microM (rsCD59-Crry) and 1.3 microM (rsCD59). The degree of inhibition was independent of whether the C8 and C9 were of rat or human origin. Therefore, we have produced rsCD59 and rsCD59-Crry in P. pastoris. The rsCD59 retains its inhibitory activity towards C5b-9, while rsCD59-Crry appears to have the combined activities of Crry and CD59. In a haemolytic assay, the inclusion of CD59 to Crry is of no additional benefit to Crry, which may illustrate the overall importance of the C3 convertase step. Yet, inclusion of Crry to CD59 increases the potency of CD59 towards C5b-9.

Animals↗

PubMed1988

Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.

Glomerular injury in passive Heymann nephritis (PHN) in rats is mediated by the C5b-9 membrane attack complex (MAC) and is associated with morphologic changes in glomerular visceral epithelial cells (GEC). We determined if the nephritogenic antibody of PHN (gamma 1 sheep anti-Fx1A IgG) directs insertion of the MAC into GEC plasma membranes with consequent cytotoxicity. Antibody-sensitized GEC were exposed to various sera serving as sources of complement. Loss of cell viability was determined by trypan blue uptake and/or by release of cellular lactate dehydrogenase (LDH). Incubation of antibody-sensitized primary and passaged GEC in fresh human serum (FHS) resulted in sigmoidal relationships between cytotoxicity and complement dose (r = 0.97 and 0.94, respectively) such that cytolysis approached 100% with FHS (10% vol/vol). Cytotoxicity was not evident if C8-deficient (C8D) plasma was substituted for FHS, but was restored in a dose-dependent manner by reconstitution with purified rat C8. Sublytic injury was demonstrated by wide separation between simultaneous release curves of cell-incorporated biscarboxyethyl carboxyfluorescein (BCECF; mol wt approximately equal to 520) and LDH at limiting doses of complement (at 2% FHS, BCECF release was 51.1 +/- 0.6% of maximum vs. 3.2 +/- 1.3% for LDH; N = 3) and by blebbing of the plasma membrane on electron microscopy. Thus, the pathogenic antibody of PHN produces complement-mediated sublytic as well as lytic cytotoxicity of GEC.

Animals↗

PubMed1987

Interactions of purified Serratia marcescens metalloproteases with fresh human serum and with purified human serum proteins.

Exposure of fresh human serum to two purified metalloproteases of S. marcescens strains SF 178 and SH 186 (both of serotype O6/O14:H12) at 35 degrees C, 3 h, resulted in altered electrophoretic mobility of the protease inhibitors alpha 1-antitrypsin and alpha 2-macroglobulin, alpha 2-HS-glycoprotein, and complement (C) components C1q, C1s, C3a, C3c, C4, C5, and C9, but not C6, C7, C8, and properdin, as determined with electroimmunoassays (Laurell technique). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) documented the partial degradation of the heavy (H) chain of purified human immunoglobulin IgG by the metalloproteases of S. marcescens strains SF 178 and SV O1 (serotype O13/O19:H1), but not by the metalloproteases of strains SH 186 and SE 182 (serotype O9B:H11) following extended incubation (35 degrees C, 22 h). Human IgA and IgM were refractory. Purified human C components, C3, C4, C5, C6, C7, C8, and C9, as well as purified human alpha 1-antitrypsin, alpha 2-macroglobulin, transferrin, and lactoferrin were attacked by the S. marcescens metalloproteases SF 178 and SH 186 at 35 degrees C for 5 h, as demonstrated with the SDS-PAGE procedure; haptoglobin and C-reactive protein were resistant.

Acute-Phase Proteins↗

PubMed1991

[Altered metabolism of membrane glycosphingolipids in erythrocytes of paroxysmal nocturnal hemoglobinuria].

Paroxysmal nocturnal hemoglobinuria (PNH) is currently accepted to be a stem-cell disorder of a clonal nature with increased susceptibility to autologous complement attack. Consequent hemolytic feature has been partly explained by lack of complement regulatory membrane proteins such as decay-accelerating factor (DAF) or C8-binding protein that anchored to membrane via glycosyl-phosphatidyl inositol (GPI) lipids. Recent reports suggest essential PNH lesion is the synthetic defect of sugar moiety of the PI-anchor. In PNH, the abnormal expression of C3b/C4b receptor (CRI) glycoproteins, or glycophorin-alpha have been also pointed out. These altered expression of glycoproteins and glyceroglycolipids, especially in the carbohydrate structures, prompted us to analyze biochemically the membrane glycosphingolipids as one of major glycoconjugates in PNH. As results, PNH erythrocytes showed altered metabolism of gangliosides in comparison to control erythrocytes from healthy donors. IV6 NeuAc-nLc4 Cer and highly polar gangliosides variably disappeared in PNH erythrocytes, partly due to impaired sialylation of glycolipids. These results suggest metabolic disorder of carbohydrates of membrane glycoconjugates as a new aspect of PNH.

Adult↗

PubMed1993

Platelet activation and inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07.

The surface glycoprotein CD36 (GPIV) is known to mediate the adhesion of Plasmodium falciparum malaria-infected red blood cells and to be a receptor for extracellular matrix proteins such as collagen and thrombospondin. The murine monoclonal IgM antibody NL07, which is specific for CD36, has now been shown to also be a potent inhibitor of the adhesion of P falciparum malaria-infected red blood cells to C32 melanoma cells. Treatment of platelets with NL07 monoclonal antibody resulted in rapid degranulation, release of ATP and serotonin, increase in [Ca2+]i, and tyrosine phosphorylation of a substrate protein of 130 kD. In about one-half of the experiments, activation with NL07 resulted in the formation of small aggregates of 10 to 30 platelets, whereas in the other half of the experiments, large aggregates were seen similar to those induced by adenosine diphosphate (ADP) and these large aggregates could be converted to the small aggregates by ATP alpha S or by AP-2 or other antibodies against GPIIb and/or IIIa. Microaggregates of 2 to 5 platelets were seen with Glanzmann's platelets that constitutively lack GPIIb/IIIa. Aggregate formation was not seen with heat-treated serum, in the presence of anti C1q antibodies, or when using C5-, C8-, or C9-deficient human sera. Although activation of platelets with purified complement components results in a slow morphologic change without aggregation, involvement of CD36 results in rapid complement-mediated activation leading to formation of small aggregates that is largely independent of GPIIb/IIIa and that, under certain circumstances, proceeds to the formation of large ADP-dependent aggregates.

Adenosine Diphosphate↗

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↗

PubMed1998

Human heart generates complement proteins that are upregulated and activated after myocardial infarction.

In human heart, we detected mRNAs and proteins for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 with the use of reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical techniques. We found an upregulation of both mRNAs and proteins in areas of recent and old myocardial infarctions. In both situations, the classical complement pathway was activated, with C4d, C3d, and the membrane attack complex (C5b-9) being deposited on damaged cardiac myocytes. These activated complement components were also identified on Western blots of infarcted tissue. Complement mRNAs in infarcted heart tissue were higher than those in liver, and liver complement mRNAs were not upregulated in cases with infarcted hearts. Our results establish that (1) complement proteins are endogenously produced by human heart; (2) the classical complement pathway is fully activated after myocardial infarction; (3) complement activation is directly involved in myocardial damage after ischemic insults; and (4) damage from complement activation may be chronically sustained. These data suggest that inhibition of the complement system should be effective in treating myocardial infarction.

Adult↗

PubMed1996

Complement regulatory molecules on human myelin and glial cells: differential expression affects the deposition of activated complement proteins.

The expression of decay-accelerating factor CD55, membrane cofactor protein CD46, and CD59 was studied on Schwann cells cultured from human sural nerve and myelin membranes prepared from human cauda equina and spinal cord. These proteins are regulatory membrane molecules of the complement system. CD55 and CD46 are inhibitors of C3 and C5 convertases and CD59 inhibits C8 and C9 incorporation into C5b-9 complex and C9-C9 polymerization. The presence of these proteins was assessed by using antibodies to each of the proteins by fluorescent microscopy, fluorescence-activated cell sorter analysis, and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Schwann cells in culture expressed CD55, CD46, and CD59. It is interesting that only CD59 was detected on myelin from both central and peripheral nerve tissue. The ability of these proteins to limit C3 peptide deposition and C9 polymerization in myelin was studied by western blot analysis. C3b deposition was readily detected on antibody-sensitized myelin incubated with normal human serum used as a source of complement but not with EDTA-treated or heat-inactivated serum. C3b deposition was not affected by anti-CD55 antibody. On the other hand, poly-C9 formation in myelin, which was maximum when 50% normal human serum was used, was increased four-to fivefold when myelin was preincubated with anti-CD59. Our data suggest that complement activation on myelin is down-regulated at the step of the assembly of terminal complement complexes, including C5b-9, due to the presence of CD59.

Antigens, CD↗

PubMedDate not supplied

Participation of immunoglobulin and the alternative complement pathway in opsonization of Bacteroides fragilis and Bacteroides thetaiotaomicron.

Studies were conducted to determine the requirements for immunoglobulin and complement in opsonization of Bacteroides fragilis and Bacteroides thetaiotaomicron. The ability of human sera depleted of immunoglobulin or of components of complement to promote the phagocytosis and intracellular killing of the two strains of Bacteroides by human leukocytes was measured in vitro under anaerobic conditions. Neither hypogammaglobulinemic sera nor pooled normal human serum that was heated at 56 C for 30 min supported phagocytosis and killing of the two strains of Bacteroides. Neither sera depleted of terminal complement components by treatment with inulin or cobra venom factor nor human serum deficient in C8 supported phagocytosis of the tested strains. In addition, pooled normal human serum depleted of C3, factor B, or factor D did not support phagocytosis of either strain. Dose-dependent restoration of the opsonic activity of factor B-depleted serum was accomplished by purified human factor B but not by human C2. The results indicate that immunoglobulin and components of the alternative comed in this study.

Animals↗

PubMed1997

The complement membrane attack complex and the bystander effect in cerebral vasospasm.

Activation of complement results in formation of membrane attack complexes (MACs) that can insert themselves either into cells that initiate complement activation or into nearby ("innocent bystander") cells. The MACs form large-conductance, nonspecific ion channels that can cause lytic or sublytic cell damage. The authors used a highly sensitive patch clamp technique to assess the contribution of the bystander effect to the pathophysiology of cerebral vasospasm. They compared the effect of complement activation by autologous aged versus fresh erythrocytes on the membrane conductance of freshly isolated rat cerebral artery smooth-muscle cells. In the presence of autologous serum aged, but not fresh, erythrocytes caused a large increase in membrane conductance, an effect that was prevented by heat-inactivating the serum. Ethyleneglycol tetraacetic acid in the presence of Mg++ attenuated the effect, indicating that complement activation was taking place via the classic pathway. The effect was reproduced by zymosan-activated autologous serum, suggesting that such changes in conductance could result from insertion of MACs secondary to a bystander effect. Both C8- and C9-depleted heterologous sera produced minimal effects that were converted to full effect by addition of the missing complement component. Superoxide dismutase plus catalase did not attenuate the conductance changes produced by autologous serum plus aged erythrocytes. Autologous serum plus aged erythrocyte membrane ghosts that were free of lysate caused a typical increase in conductance. This study demonstrates that complement activation by aged erythrocytes can result in MAC insertion into innocent bystander smooth-muscle cell membranes and that this mechanism, heretofore undescribed, may contribute to development of vasospasm after subarachnoid hemorrhage.

Animals↗

PubMed1990

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.

Antibodies, Monoclonal↗

PubMed1991

Collagenase of Clostridium perfringens type A: degradation of human complement component C1q.

The semipurified collagenases from Clostridium perfringens type A strains 2-Cli and ATCC 13124, both characterized by molecular weights of 79.4 kilodaltons, partially degraded purified human complement (C) component C1q. The following purified human serum proteins were refractory: C components C3, C4, C5, C6, C7, C8, and C9; immunoglobulin (Ig)A (from colostrum), IgG, and IgM; alpha 2-macroglobulin, haptoglobin, and C-reactive protein.

Blotting, Western↗