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PubMed1994

Upregulation of human neutrophil CD59, a regulator of the membrane attack complex of complement, following cell activation.

CD59 is a membrane glycoprotein that regulates the membrane attack complex of complement and protects cells from autologous complement damage. Human polymorphonuclear leucocyte (PMN) expression of CD59 was confirmed by flow cytometry following staining with mAb 1F5, and western blotting revealed staining of a 19-23 kDa band. Warming of PMN from 4 to 37 degrees C resulted in spontaneous CD59 upregulation. A dose-dependent increase in expression following PMN stimulation with FMLP was observed and occurred within minutes, indicating that new protein synthesis was not required. Treatment of PMN with calcium ionophore A23187 resulted in similar increases in CD59 expression. This occurred in the presence or absence of extracellular calcium, indicating that upregulation was dependent on release of calcium from intracellular stores. Evidence for a mobilizable intracellular pool of CD59 was obtained by detection of increased binding of 1F5 following PMN permeabilization; CD59 could also be re-expressed after stripping by phosphatidylinositol specific phospholipase C (PI-PLC) by treatment with FMLP or A23187. There was a correlation between CD59 upregulation and lactoferrin release, suggesting that stores of CD59 may be associated with secondary granules. These studies indicate that PMN expression of CD59 is enhanced by cell activation and suggest the presence of an intracellular pool of CD59 which can be translocated to the cell membrane upon PMN stimulation.

Antibodies, Monoclonal↗
PubMed1985

The membrane attack complex of complement: lipid insertion of tubular and nontubular polymerized C9.

The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.

Animals↗
PubMed1984

Ultrastructure of the membrane attack complex of complement. Heterogeneity of the complex caused by different degree of C9 polymerization.

The membrane attack complex (MAC) of complement and its precursors, i.e. C5b-7 and C5b-8, were examined by electron microscopy. C5b-7 bound to lipid vesicles exhibits an extended structure of 25 nm connected to the lipid membrane via a 10-nm long, 3-nm wide stalk. Binding of C8 to vesicle-bound C5b-7 results in the disappearance of this stalk, whereas the overall length remains unchanged. Addition of 12 C9 molecules per C5b-8 induces C9 polymerization which is accompanied by the formation of C9 tubules and membrane lesions. By using biotinyl precursors and streptavidin -coated colloidal gold particles, C5b-6, C7, and C8 was found to be in the club-like part of the MAC; C9 was identified in the tubular moiety. Only one C5b-8 moiety was detected in an individual MAC complex thus excluding the proposed "dimeric" structure of the MAC. A membrane channel of 10 nm was formed by the MAC at a C9 to C5b-8 ratio equal or larger than 12 to 1, as suggested by the penetration of negative stain into the vesicle. In contrast, binding of an average of three C9 per C5b-8 caused formation of incomplete C9 tubules with apparent membrane channels of less than 10 nm diameter. The MAC isolated from red blood cells was ultrastructurally heterogenous . Although an excess of serum was used for the formation of the complexes, mostly incomplete poly C9 tubules were formed. It is proposed that the MAC is an ultrastructurally heterogenous complex that induces the formation of membrane channels of different sizes.

Animals↗
PubMed1985

The membrane attack complex of complement: relation of C7 to the metastable membrane binding site of the intermediate complex C5b-7.

Isolated C7 (m.w. 120,000) in 1% deoxycholate (DOC) forms dimers with an apparent m.w. of 230,000 and a DOC-binding capacity of 82 mol per mol of dimer. Dimerization of C7 also occurs in the presence of DOC-phospholipid mixed micelles and eventuates in the insertion of C7 dimers into the lipid bilayer upon the removal of the detergent. C5b-7 complex formation in the fluid phase or on lipid vesicles likewise involves polymerization. C5b-7 sedimented with 17-40S, which suggests a dimeric to hexameric composition. In avidin-biotin binding experiments in which two differentially labeled forms of C5b,6 (biotinyl 125I-C5b,6, and 131I-C5b,6) were used in equimolar amounts to assemble C5b-7, more than 50% of the biotinyl 125I-C5b,6-containing complexes also contained 131I label; again suggesting that C5b-7 consisted of oligomers rather than monomers. The conformation of C7 in C5b-7 and in dimeric C7 appeared similar by the following criteria. On formation of C5b-7 from C5b,6 and C7, a 20% increase in beta-pleated sheet structure was observed by circular dichroism spectroscopy, and a similar change occurred on dimerization of isolated C7. Tryptic and thermolytic digests of C5b-7 and C7 dimers containing 125I-C7 were analyzed by autoradiography after SDS-polyacrylamide gel electrophoresis and were found to contain similar peptides that were distinct from those in the digests of monomeric C7. Direct evidence showing that the metastable membrane binding site of the C5b-7 complex resides in the C7 subunit was obtained by using the conjugates of C5b,6 and colloidal gold. Viewed in the electron microscope, these conjugates were aggregated upon the addition of isolated C7. In contrast, when conjugates of C7 and colloidal gold were treated with soluble C5b,6, no such aggregates occurred, but instead, individual C5b-7 complexes were observed arranged around single gold particles, resulting in star-like structures. The results strongly suggest that structures of C7 are responsible for the expression of the membrane binding site of metastable C5b-7.

Binding Sites↗
PubMed1982

Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8.

The ultrastructure of the membrane attack complex (MAC) of complement had been described as representing a hollow cylinder of defined dimensions that is composed of the proteins C5b, C6, C7, C8, and C9. After the characteristic cylindrical structure was identified as polymerized C9 [poly(C9)], the question arose as to the ultrastructural identity and topology of the C9-polymerizing complex C5b-8. An electron microscopic analysis of isolated MAC revealed an asymmetry of individual complexes with respect to their length. Whereas the length of one boundary (+/- SEM) was always 16 +/- 1 nm, the length of the other varied between 16 and 32 nm. In contrast, poly(C9), formed spontaneously from isolated C9, had a uniform tubule length (+/- SEM) of 16 +/- 1 nm. On examination of MAC-phospholipid vesicle complexes, an elongated structure was detected that was closely associated with the poly(C9) tubule and that extended 16-18 nm beyond the torus of the tubule and 28-30 nm above the membrane surface. The width of this structure varied depending on its two-dimensional projection in the electron microscope. By using biotinyl C5b-6 in the formation of the MAC and avidin-coated colloidal gold particles for the ultrastructural analysis, this heretofore unrecognized subunit of the MAC could be identified as the tetramolecular C5b-8 complex. Identification also was achieved by using anti-C5 Fab-coated colloidal gold particles. A similar elongated structure of 25 nm length (above the surface of the membrane) was observed on single C5b-8-vesicle complexes. It is concluded that the C5b-8 complex, which catalyzes poly(C9) formation, constitutes a structure of discrete morphology that remains as such identifiable in the fully assembled MAC, in which it is closely associated with the poly(C9) tubule.

Complement C9↗
PubMed1985

Membrane attack complex of complement in dermatitis herpetiformis.

The assembly of membrane attack complex (MAC) of complement implies activation of complement to the attachment of C9 and the presence of MAC on tissue suggests a possible pathogenic role for complement in disease since MAC is able to damage membranes. We examined normal skin of five patients with dermatitis herpetiformis for the presence of MAC using a monoclonal antibody (poly C9-MA) that recognizes a neoantigen of C9 that is not present on monomeric C9 but is common to both isolated MAC and to polymerized C9. Granular deposits of polymerized C9 were found at the sites of IgA deposition in the dermal papillae of normal skin from all patients. The pathologic importance of this finding is uncertain.

Antibodies, Monoclonal↗
PubMed1994

The membrane attack complex of complement. Assembly, structure and cytotoxic activity.

The membrane attack complex of complement is formed by the molecular fusion of the five terminal complement proteins, C5, C6, C7, C8, and C9. While the assembly process on a target membrane and its modulation by restriction factors present on host cells is now quite well understood the molecular details of the architecture of the complex still need much further clarification. This is especially true for the interaction of the last acting protein C9, which provides the cytotoxic action of the complex, with the precursor C5b-8 complex. Because of this lack of structural details the molecular mechanisms that lead to complement-mediated cell death remain cryptic, however, it is hoped that recent advances in controlling the assembly process and in site-specific modification of the terminal complement proteins by recombinant DNA techniques should change this predicament quickly.

Bacteria↗
PubMed1988

Immunohistochemical study of the membrane attack complex of complement in IgA nephropathy.

The localization of the membrane attack complex of complement (MAC) was examined in the normal human kidneys and in biopsy specimens from patients with primary IgA nephropathy by immunofluorescent and immunoelectron microscopies. Immunofluorescent staining for MAC was significantly more intense than in the normal kidneys, and was observed in the mesangium and occasionally along the glomerular capillary walls of 22 of 30 patients with IgA nephropathy. By dual-staining, the MAC deposits were generally concordant with the deposits of IgA, C3, C5 and C9, or of IgG, when present. C1q or C4 was infrequently observed in the glomeruli. Immunoelectron microscopy revealed various staining patterns of glomerular MAC deposition; homogeneous fine-granular staining beneath the glomerular basement membrane (GBM) in the paramesangial zone, patchy staining within the mesangial electron dense deposits (EDD), and ring-shaped or ribbon-like staining, associated with the striated membrane structures (SMS), in the matrix of the mesangium, GBM and tubular basement membrane (TBM). This study suggests that the terminal complement system is activated, mainly by an alternative complement pathway mechanism, in the mesangium of IgA nephropathy, and is associated with the paramesangial lesion and EDD. MAC deposition in glomerular SMS may also result from in situ activation rather than trapping from the circulation. There was little correlation between glomerular MAC deposition and proteinuria or renal histology of patients with IgA nephropathy.

Complement Membrane Attack Complex↗
PubMed1984

Is the membrane attack complex of complement an enzyme?

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.

Animals↗
PubMed1983

The membrane attack complex of complement and its precursor proteins lack phospholipase activity.

The membrane attack complex of human complement and its highly purified precursor proteins have been analyzed for phospholipase activity. Using three different sensitive assays, phospholipase A1, A2, C or D activity could not be detected. Based on the sensitivity of the assays employed, these results indicate the complement-mediated membrane damage is not enhanced by covalent breakdown of membrane phospholipids, but is entirely caused by physical action of the membrane attack complex. The results also imply that the putative serine esterase sites of C6 and C7 are not acting on phospholipids.

Chromatography, Thin Layer↗
PubMed1984

Molecular composition of the tubular structure of the membrane attack complex of complement.

The composition of the tubular structure of the membrane attack complex of complement (MAC) which migrates as a high molecular weight band (Mr approximately 1.2- 1.3 X 10(6) upon sodium dodecyl sulfate, polyacrylamide gel electrophoresis under reducing conditions was analyzed and compared to the high molecular weight band (Mr approximately 1.1 X 10(6] of tubular poly(C9). The sodium dodecyl sulfate-resistant band of the MAC, designated MAC-poly(C9), is composed of C6, C7, C8 alpha-gamma, and poly(C9), in approximate molar ratios of the protomers of 1:1:1:10-18. This conclusion is based 1) on the results of the incorporation of labeled proteins into MAC-poly(C9); 2) on the immunostaining of MAC-poly(C9) with anti-C6, anti-C7, anti-C8 alpha-gamma, and anti-C9 and its lack of immunostaining with anti-C5 and anti-C8 beta; and 3) on the dissociation of MAC-poly(C9) to 1 mol of C6, C7, C8 alpha-gamma and 10 to 18 mol of C9 upon treatment with 8 M guanidine isothiocyanate. Ultrastructurally the sodium dodecyl sulfate-resistant poly(C9) tubule and MAC-poly(C9) tubule are indistinguishable, suggesting a similar ultrastructure of the C6, C7, C8 alpha-gamma, and C9 subunits in the MAC-poly(C9) tubule. Further analogies among these four proteins are their tendency to form disulfide-linked dimers. It is concluded that the transmembrane channel of the MAC is formed by a tubule in which C6, C7, C8 alpha-gamma are copolymerized with poly(C9), whereas the C5b and C8 beta subunits are not part of the tubule structure and may form the 170-A long appendage of the MAC. This appendage is dissociated upon boiling in sodium dodecyl sulfate whereas the tubule remains stable.

Complement Membrane Attack Complex↗
PubMed1999

Synergistic enhancement of chemokine generation and lung injury by C5a or the membrane attack complex of complement.

Complement plays an important role in many acute inflammatory responses. In the current studies it was demonstrated that, in the presence of either C5a or sublytic forms of the complement-derived membrane attack complex (MAC), rat alveolar macrophages costimulated with IgG immune complexes demonstrated synergistic production of C-X-C (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) and C-C (macrophage inflammatory protein-1alpha and monocyte chemoattractant-1) chemokines. In the absence of the costimulus, C5a or MAC did not induce chemokine generation. In in vivo studies, C5a and MAC alone caused limited or no intrapulmonary generation of chemokines, but in the presence of a costimulus (IgG immune complexes) C5a and MAC caused synergistic intrapulmonary generation of C-X-C and C-C chemokines but not of tumor necrosis factor alpha. Under these conditions increased neutrophil accumulation occurred, as did lung injury. These observations suggest that C5a and MAC function synergistically with a costimulus to enhance chemokine generation and the intensity of the lung inflammatory response.

Animals↗
PubMed1987

The membrane attack complex of complement induces permeability changes via thresholds in individual cells.

Flow cytometry was used to quantify the fluorescence of propidium iodide in rat polymorphonuclear leucocytes (PMN) attacked by the membrane attack complex (MAC) in order to establish the existence of permeability and lytic thresholds in individual cells, a 'threshold' being defined as a cellular event involving the rapid transition of cells from one state to another under physiological conditions. Activation of the complement pathway resulted in PMN being attacked by MAC within 5 min. Approximately 30-40% of the cell population subsequently became permeable to small molecules and macromolecules. Individual PMN passed through 'thresholds' of cell permeability and cell lysis, or recovered from complement attack at different times. In the flow cytometer, three distinct populations of PMN were identified: cells that had recovered before the permeability 'threshold', cells that had recovered after the permeability 'threshold' but before the lytic 'threshold', and cells that failed to recover from complement attack. Individual PMN attacked by MAC passed through permeability and lytic thresholds at different times after an initial lag of 7.5 +/- 2.5 min and 11.5 +/- 1.0 min, respectively. Adenosine, an activator of adenylate cyclase, inhibited removal of MAC from the cell surface. Consequently, more cells passed through the permeability and lytic 'thresholds', resulting in an increased percentage of lysed cells.

2-Chloroadenosine↗
PubMed1990

Comparison of channels formed by poly C9, C5b-8 and the membrane attack complex of complement.

The channels formed by poly C9, C5b-8 and C5b-9 were examined using the liposome swelling assay. By plotting the relative rate of swelling of C5b-8-containing liposomes vs the molecular weight of the sugar solute and by applying the Renkin equation, the size of the C5b-8 channel was estimated to be 1.5 mm radius. As increasing amounts of C9 were added during the formation of C5b-9, in C8:C9 ratios of 1:1, 1:2, 1:6 and 1:12, the size of the function channel increased. Poly C9 had a pore that was somewhat larger than C5b-9 at a C8:C9 ratio of 1:12. Using molecular sieving experiments with four different iodinated protein size markers, the channel diameter of poly C9 was estimated at between 90 and 100 A. Monoclonal antibodies to different complement proteins were added to the liposomes to see which might inhibit the channels. C5b-8 containing liposomes could be inhibited by antibodies to C8. Liposomes containing C5b-9 could be inhibited slightly by antibodies to C9 and most strongly by antibodies to the neoantigen of poly C9.

Antibodies, Monoclonal↗