Isolation of Membrane Attack Complex of Complement from Myelin Membranes Treated with Serum Complement

Abstract: The interaction between complement and myelin membranes and its possible role in myelin damage and in the disposal of damaged myelin in vivo is of interest because activation of complement generates both opsonin(s) and membrane attack complex of complement. In our studies on the role of complement in demyelin-ation, we have shown that isolated myelin activates serum complement in the absence of myelin-specific antibody and that membrane attack complex of complement is the required factor in antibody-mediated demyelination of mouse cerebellar expiant cultures. In the present study, we examined whether activation of serum complement by myelin is associated with the formation of membrane attack complex of complement in myelin membranes. Extracts of myelin-associated proteins following incubation of myelin with fresh serum were studied by ultracentrifugation on a sucrose density gradient for detection of C5b-9 neoantigen. The subunit structure of C5b-9 was determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, electroblotting, and immunostaining. Results indicate that the macromolecular complex consisting of late-acting complement components, C5-C9, was assembled in the target myelin membranes.     

Release of phospholipids from complement-mediated lesions on the surface structure of Escherichia coli.

When varying numbers of sensitized, 14C-labeled bacteria were treated with a certain amount of complement, in a fixed reaction volume, 14C compounds were liberated into the surrounding medium in proportion to the number of the bacteria, whereas the amount of the phospholipids liberated was constant regardless of the number of the bacteria even in the range of relative excess of complement. Since it is conceivable that a certain amount of complement might form a fixed number of lesions on the surface of all the sensitized bacteria, the amount of the liberated phospholipids seems to be proportional to the number of complement lesions. The 14C-materials released from complement-attacked bacteria were analyzed by isopycnic sucrose density gradient ultracentrifugation and they were mainly free phospholipids and other smaller molecules. A small amount of the smaller membrane proteins were also released as revealed by acid and SDS-polyacrylamide gel electrophoresis. These results suggest that the release of phospholipids is due to the displacement of membrane lipids by the complexes of the late acting complement components during their insertion into the membrane lipid bilayer.     

Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis

Nucleated cells can resist lysis by and recover from complement attack even after formation of the potentially cytolytic membrane attack complex on the cell surface. We have found that human neutrophils resist complement lysis by the physical removal of membrane attack complexes by both endocytic and exocytic process. The latter mechanism predominates, vesiculation being detectable within 60 sec of initiating the complement cascade. Sixty-five percent of the formed complexes are removed on plasma membrane vesicles, although only 2% of the cell surface is lost. Ultrastructural examination revealed that these vesicles were covered with ring-like "classical" complement lesions. Analysis of these vesicles by gel electrophoresis indicated that C9 was present exclusively in the form of a sodium dodecyl sulfate-resistant, high m.w. complex. In contrast, the 35% of C9 that remained associated with the cells was found to be inaccessible to a C9-specific monoclonal antibody, and was partly degraded, suggesting internalization of the membrane attack complex and proteolysis of some C9 molecules. The molar ratio of C9 to C8 was 12 to 1 on shed vesicles and on recovered cells.     

Induction of cellular procoagulant activity by the membrane attack complex of complement

In addition to their well-recognized role in immune defense, there is a growing recognition that the proteins of the complement system impact directly on vascular homeostatic mechanisms, evoking cellular responses that serve to both promote adherence of blood cells to the walls of blood vessels, and the formation of fibrin through the enzyme mechanisms of the coagulation system. This clot-promoting or ‘procoagulant’ activity initiated through the complement system entails both receptor-mediated as well as receptor-independent pathways of cell activation. In this review, I will focus specifically upon the role that is now thought to be played by the membrane attack complex of the complement system (MAC) in the induction of the procoagulant properties of human platelets and endothelium.     

Identification of P-57, a serine proteinase, from human erythrocyte membranes, which cleaves both chains of human third component (C3) of complement

A proteinase, which cleaves human third component of complement, was solubilized from erythrocyte membranes then purified by gel filtration chromatography, fluid phase electrophoresis, and hydroxylapatite chromatography. Labeling of the purified material by 125I or 3H-DFP and measurement of proteolytic activity subsequently isolated by SDS-polyacrylamide gel electrophoresis allowed to identify a 57 kDa single band, in non reducing conditions. Inhibition of this activity by PMSF supports covalent modification of an active serine residue. This membrane serine proteinase cleaved alpha and beta chains of human third component of complement, suggesting that p-57 is distinct from plasma serine proteinases.     

Expression of the complement regulatory protein CD59 on human spermatozoa: Characterization and role in gametic interaction

Protectin (CD59) is a complement regulatory protein which blocks the membrane attack complex during complement activation. CD59 was identifield on the human sperm surface by means of H19, an IgG₁ anti-protectin mouse monoclonal antibody. Using Indirect immunofluorescence, flow cytometry and immunoperoxidase, CD59 was found to be present on the whole plasma membrane including the head and tail of fresh ejaculated, capacitated and acrosome-reacted spermatozoa. Immunoperoxidase staining of normal testicular sections indicated that this protein was already present on intraluminal germ cells. Analysis of this sperm protein by gel electrophoresis and immunoblotting revealed that its molecular weight of 20 kDa was comparable to that of CD59 expressed on peripheral blood cells (erythrocytes, lymphocytes) and that it was bound to the membrane through a glycophospholipid tail which could be released after treatment with phosphatidylinositol-specific phospholipase C. Associated to membrane cofactor protein (CD46) and decay accelerating factor (CD55) located in the acrosomal membranes, CD59 may participate to the protection of male gametes against complement-mediated damage as they travel through the female genital tract. Moreover CD59, known as an adhesion molecule involved in lymphocyte rosettes, may also participate in cell to cell adhesion during gametic interaction since H19 inhibited sperm binding and reduced the penetration rate and index during the hamster egg penetration test. © 1994 Wiley-Liss, Inc.     

Physicochemical characterization of human S-protein and its function in the blood coagulation system.

S-protein, the main inhibitor of the assembly of the membrane attack complex of complement, was isolated from human plasma by a simple purification procedure, which includes barium citrate adsorption, ammonium sulphate precipitation, chromatography on DEAE-Sephacel and Blue Sepharose and gel filtration on Sephacryl S-200. The homogeneous protein (sedimentation coefficient 4.6 S) was obtained in approx. 5% yield relative to its concentration in plasma, which was found to be 0.3-0.5 mg/ml. The final product did not cross-react with antisera against complement proteins or other proteinase inhibitors of human plasma. On polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, S-protein migrated as a single-chain band with an apparent Mr of 74000 under non-reducing conditions and as a doublet of Mr 78000 and 65000 upon reduction. In plasma or serum S-protein also existed in two forms of corresponding Mr values, as was evidenced by an immunoblot enzyme-linked immunosorbent assay technique. S-protein was found to be an acidic glycoprotein with 10% (W/W) carbohydrate content and several isoelectric points in the range pH 4.75-5.25, and it contained one free thiol group per molecule of protein. The functional properties of S-protein in the complement system were demonstrated by its ability to inhibit complement-dependent cell lysis in a concentration-dependent manner (Ki 0.6 microM) and by its incorporation into the nascent SC5b-7 complex. A new function for S-protein could be revealed in the blood coagulation system. The slow progressive inhibition of thrombin by antithrombin III was not affected by S-protein, whereas the purified protein interfered with the fast inactivation of thrombin clotting as well as amidolytic activity by antithrombin III-heparin complex. The acceleration of this inhibition reaction by heparin was counteracted by S-protein, indicating the ability of S-protein to neutralize heparin activity.     

Avidin-induced lysis of biotinylated erythrocytes by homologous complement via the alternative pathway depends on avidin's ability of multipoint binding with biotinylated membrane.

It was reported that avidin and streptavidin induce lysis of prebiotinylated red blood cells via the alternative pathway of both homologous and heterologous complement. Both of these proteins have four biotin-binding sites, providing a polyvalent interaction with biotinylated components of the erythrocyte membrane. We have compared the effects of mono- and multipoint avidin attachment on the sensitivity of biotinylated erythrocytes to lysis by the complement system. In the presence of anti-avidin antibody, avidin-bearing biotinylated erythrocytes were rapidly lysed by heterologous serum. This lysis was independent from the mode of avidin attachment, implying that complement activation by the classical pathway triggered by interaction between C1 and avidin-bound antibody on the erythrocyte surface is independent from the avidin's ability of polyvalent (multipoint) binding with biotinylated membrane components. In the absence of anti-avidin antibody, biotinylated erythrocytes bearing polyvalently attached avidin were lysed by homologous complement better than cells bearing avidin, which possesses reduced ability for multipoint binding with biotinylated erythrocyte. Two independent approaches to reduce avidin's ability of multipoint binding were used: decrease in surface density of biotin on the erythrocyte membrane and blockage of biotin-binding sites of avidin. Both methods result in reduced lysis of avidin-bearing erythrocytes as compared with erythrocytes bearing an equal amount of polyvalent-bound avidin. Thus the activation of homologous complement via the alternative pathway depends on avidin's ability to 'cross-link' to the biotinylated components of the erythrocyte membrane.     

Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots

The polypeptide complement of enriched soybean (Glycine max [L.] Merr. cult. wells) root plasma membrane fractions was studied by two-dimensional gel electrophoresis. Good resolution was obtained when polypeptides were solubilized in sodium dodecyl sulfate and when butylated hydroxytoluene was included in the vesicle isolation and solubilization media. The pattern obtained on the two-dimensional slab gel for root plasma membrane was characteristic for that membrane. The polypeptide complements from mitochondrial membranes and from enriched fractions of three other endomembrane components were solubilized and electrophoresed for comparison. Each membrane preparation was identifiable on the basis of its characteristic electrophoretogram. Electrophoresis of protein solubilized from plasma membrane fractions isolated from meristematic and mature root tissue revealed both qualitative and quantitative differences in the respective protein complements.     

Complement Regulatory Molecules on Human Myelin and Glial Cells: Differential Expression Affects the Deposition of Activated Complement Proteins

Abstract: 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.     

Proteins and glycoproteins of the erythrocyte membrane

Erythrocyte membranes from several species were prepared by three different methods of hypotonic hemolysis and examined for variations in protein and glycoprotein content by acrylamide gel electrophoresis in sodium dodecyl sulfate. Significant variations were noted in morphology of the membranes prepared by the different methods without attendant variations in protein patterns of the major membrane proteins for most cases observed, which show a similar pattern of nine common bands for all of the species observed. The significant difference in protein pattern which was noted was attributed to proteolytic digestion of membranes which were fragmented during preparation. Failure to remove white blood cells from membrane preparations was shown to be a significant source of the problem with proteolytic digestion. Glycoproteins were analyzed by acrylamide gel electrophoresis or by column chromatography. Each species appears to have a different major glycoprotein (or group of closely related glycoproteins). Molecular weights of glycoproteins calculated from acrylamide gel electrophoresis were found to vary with the percentage of acrylamide in the gel, indicating that these proteins do not behave in a normal fashion in this electrophoresis system. The molecular weight calculated from gel filtration data for the human membrane glycoproteins (26,000) was quite disparate from those calculated from gel electrophoresis (88,000 to 62,000 in 5 to 10% gels).     

Attachment of human polymorphonuclear leukocytes to herpes simplex virus-infected fibroblasts mediated by antibody-independent complement activation

Herpes simplex virus (HSV)-infected cells can activate the human complement system without interference of specific anti-HSV antibodies. Analysis by flow cytometry showed that C3-like molecules were deposited on the membrane of the infected cell when incubated with human serum without specific antibodies. Depletion of calcium to block the classical pathway of the complement system had no effect on fluorescence intensity. The complement activation could be blocked by chelating both calcium and magnesium or by heating the serum. Furthermore, in the fluid phase C3 was converted to C3b by infected cells and not by uninfected cells. The antibody-independent activation did not lead to lysis of the virus-infected fibroblasts, indicating that the complement cascade is abrogated before formation of the membrane attack complex. This was also confirmed by measurement of the 50% hemolytic complement activities for total and alternative pathways. Polymorphonuclear leukocytes attached to infected fibroblasts after incubation of these fibroblasts with intact complement. This is most probably mediated by complement receptor binding of C3b and C3bi which is deposited on the membrane of the HSV-infected cell. Both type 1 and type 2 HSVs showed the same characteristics in complement activation and thereby mediated polymorphonuclear leukocyte adherence.     

Activation of Complement by Myelin: Identification of C1-Binding Proteins of Human Myelin from Central Nervous Tissue

Myelin isolated from central nervous tissue activates the classic pathway of complement by directly activating C1. Activation of C1 can proceed to form membrane attack complex, C5b-9, in the myelin. Such an interaction between myelin and complement may be important in diseases involving myelin damage, in view of the role of complement in membrane attack and inflammation. To identify the C1-activating protein, myelin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The blots were incubated with C1 or with whole serum complement, followed by immunostaining for C1 or C3, respectively. A duplicate strip was stained with amido black or anti-myelin antibody to visualize the myelin proteins. The results showed that two major protein bands were capable of activating C1. An approximately 56-58-kilodalton band comigrated with the W2 protein and an approximately 45-47-kilodalton band migrated along with, but slightly behind, the W1 Wolfgram doublet.     

Homologous species restriction in lysis of human erythrocytes: a membrane-derived protein with C8-binding capacity functions as an inhibitor

An intrinsic membrane protein with a m.w. of 65,000 that can bind human C8 has been identified after separation of human erythrocyte membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransfer to nitrocellulose sheets. The protein, tentatively designated as the C8-binding protein (C8bp) could be isolated from papain-treated erythrocyte (E) membranes by phenol-water extraction and isoelectric focusing. In a functional assay, with chicken (ch) E as target cells, C8bp inhibited the lysis of ch E C5b67 intermediates by human C8 and C9, whereas the lysis by rabbit C8 and C9 was not affected. Because the decay accelerating factor (DAF) from human erythrocyte membranes also inhibits the activity of C3/C5 convertases in an homologous system, we tested whether or not a DAF activity was present in C8bp. C8bp, however, did not accelerate the decay of the classic C3 convertases. Thus, it appears that C8bp and DAF are two different factors of E membranes with a similar molecular size inhibiting different sites of the activation cascade of complement while they can function synergistically to minimize the self-inflicted damage by complement.     

Complement mediated Klebsiella pneumoniae capsule changes

The Gram-negative bacterium Klebsiella pneumoniae is an opportunistic pathogen, which can cause life-threatening infections such as sepsis. Worldwide, emerging multidrug resistant K. pneumoniae infections are challenging to treat, hence leading to increased mortality. Therefore, understanding the interactions between K. pneumoniae and the immune system is important to develop new treatment options.We characterized ten clinical K. pneumoniae isolates obtained from blood of bacteremia patients. The interaction of the isolates with human serum was investigated to elucidate how K. pneumoniae escapes the host immune system, and how complement activation by K. pneumoniae changed the capsule structure. All K. pneumoniae isolates activated the alternative complement pathway despite serum resistance of seven isolates. One serum sensitive isolate activated two or all three pathways, and this isolate was lysed and had numerous membrane attack complexes in the outer membrane. However, we also found deposition of complement components in the capsule of serum resistant isolates resulting in morphological capsule changes and capsule shedding. These bacteria did not lyse, and no membrane attack complex was observed despite deposition of C5b-9 within the capsule, indicating that the capsule of serum resistant K. pneumoniae isolates is a defense mechanism against complement-mediated lysis.     

Identification and subcellular distribution of adipocyte peptides and phosphopeptides.

Subcellular fractions of high purity (including plasma membrane, endoplasmic reticulum, mitochondria, nuclei, and cytoplasm) were prepared from isolated adipocytes, and the peptide components were examined by detergent gel electrophoresis. Each fraction except the endoplasmic reticulum exhibited a unique and reproducible complement of major peptides. Although the endoplasmic reticulum was distinctive in its enzymic markers, its peptide components showed striking homologies with certain species in the plasma membrane and cytoplasm. The two major adipocyte glycopeptides appear to be contained in the plasma membrane, inasmuch as they followed the distribution of 5'-nucleotidase. Incubation of adipocytes with extracellular 32Pi led to a uniform rate of incorporation of 32P into cellular peptides, with steady-state incorporation reached by 2 hours. Plasma membrane, mitochondria, nuclei, and cytoplasm all contained a distinctive complement of from two to five major phosphopeptides of different molecular weights. The majority of endoplasmic reticulum phosphopeptides exhibited molecular weights closely similar to those of certain species in the plasma membrane and cytoplasm. The phosphopeptides of the plasma membrane exhibited the highest absolute 32P incorporation of all phosphopeptides, next was the single major mitochondiral phosphopeptide. All fractions except the mitochondria contained, in addition to the few major phosphopeptides, numerous minor 32P-labeled phosphopeptides.     

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.     

Pore formation by complement in the outer membrane of Gram-negative bacteria studied with asymmetric planar lipopolysaccharide/phospholipid bilayers

Summary The interaction of complement with an asymmetric planar lipopolysaccharide/phospholipid bilayer system as a model for the lipid matrix of the outer membrane of Gram-negative bacteria has been studied. The addition of whole human serum to the aqueous solution at the lipopolysaccharide side of the asymmetric membrane resulted in a rapid increase of the bilayer conductance in discrete steps, indicating the formation of transmembrane pores, which were not observed in the case of pure phospholipid membranes. The amplitudes of the discrete conductance steps varied over a range of more than one order of magnitude. The mean single step conductance was (0.39±0.24) nS for a subphase containing (inmm): 100 KCl, 5 MgCl₂ and 5 HEPES buffer. The steps were grouped into bursts of typically 9±3 events per burst and the conductance change within one burst was (8.25±4.00) nS.The pore-forming activity of serum at the asymmetric membrane system was independent of the presence of specific antibodies against the lipopolysaccharide but was dependent on calcium ions. Furthermore, the pore-forming activity required complement component C9.A model for the mode of pore formation by complement is proposed: The complement pore is generated in discrete steps by insertion of C9 monomers into the membrane and their irreversible aggregation to water-filled channels with a diameter of approximately 7 nm assuming a circular geometry.     

Roles of the complement system in human neurodegenerative disorders

Complement is an important component of the innate immune response with the capacity to recognize and clear infectious challenges that invade the CNS through a damaged blood brain barrier. For instance, the membrane attack complex is involved in cytotoxic and cytolytic activities while other smaller fragments lead to cell activation (chemotaxis) and phagocytosis of the intruders. It is noteworthy that there is a growing body of evidence that uncontrolled complement biosynthesis and activation in the CNS can contribute to exacerbate the neuronal loss in several neurodegenerative disorders. We provide here an insightful review of the double-edged sword activities of the local innate complement system in the CNS and discuss further the potential therapeutic avenues of delivering complement inhibitors to control brain inflammation.