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We describe the first case of systemic lupus erythematosus (SLE) showing the 9th component of complement (C9) deficiency. In the serum of this patient, a low level of serum total hemolytic complement (CH50) and the absence of C9 was demonstrated. The patient's sister, with normal level of C9, was also found to have an autoimmune disorder.
A sensitive sandwich ELISA was applied to the measurement of the terminal component of complement C9 in CSF and plasma from 40 tension headache patients (reference group), 33 affected by clinically definite MS and 10 by aseptic meningitis. The levels of C9 in plasma were increased in aseptic meningitis. The determinations of CSF/plasma C9 ratio and C9 index, equal to (CSF C9/plasma C9): (CSF albumin/plasma albumin), thus accounting for changes of plasma C9 levels as well as damaged blood brain barrier, documented the existence of local consumption of C9 in aseptic meningitis. In contrast, only borderline alterations were evident in MS. The results indicate that local consumption of total C9 in CSF is an additional variable reflecting an acute inflammation within the CNS, but not demonstrable in MS, a chronic inflammatory CNS disorder.
Complement component C9 undergoes a major conformational change during its insertion into a biological membrane from a globular to an extended form. At 0 degrees C a single C9 binds but a membrane attack complex (MAC) is not formed. We show that the C9 bound at 0 degrees is accessible to the intracellular space and sensitive to trypsin digestion, suggesting that C9 inserts in its globular state and requires an elevated temperature in order to change conformation.
Cytolytic lymphocytes are endowed with a pore-forming protein called perforin. Recently, a cytolytic domain was located in the first 34 residues of the perforin N-terminus. It has been proposed that the first 19 residues are composed of a 3-domain structure including a putative amphipathic beta-sheet and that the 19 residues are sufficient for cytolytic activity. This model has now been tested by synthesizing peptides covering different portions of the N-terminus, and testing their ability to lyse lipid vesicles or increase the conductance of lipid bilayers or plasma membranes. It was found that the putative beta-sheet is indispensable for lytic activity and that the first 19 residues of the N-terminus are required for optimal lytic activity but that shorter peptides, containing only 16 residues, can form pores in lipid bilayers and cell membranes. A putative amphipathic alpha-helix from the central portion of perforin, homologous to complement C9, is nonlytic to lipid vesicles, but it can form pores in lipid bilayers. Taken together, these results support the model that the perforin N-terminus is important in initial pore formation and that the putative alpha-helical domain may be involved in subsequent perforin polymerization into large pores.
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The domain structure of human complement protein C9 was investigated by determining the functional activities of the NH2-terminal (C9a) and COOH-terminal (C9b) fragments obtained by cleavage of C9 with alpha-thrombin. The two fragments were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured by dialysis against buffers containing zwitterionic detergents. The C9b fragment produced membranolytic activities in three independent assays. First, it produced single, ion-conducting channels of varying conductances in planar lipid membranes. Most of the channels had an average conductance of 11 picoSiemens and an average lifetime of about 30 s. The channels showed lipid specificity and a 3-fold preference for conducting K+ over Na+. Second, the fragment also caused specific marker release from liposomes which was inhibitable by a C9b-specific monoclonal antibody, and third, it lysed erythrocytes in the absence of a fully assembled C5b-8 complex. The isolated C9a fragment did not produce single channels in planar lipid membranes but was also effective in releasing markers from liposomes and in lysing erythrocytes. Secondary structure predictions indicate the presence of several amphiphilic, "surface-seeking" segments in the primary structure of C9 which are mainly alpha-helices in C9b and beta-sheets in C9a. These results may indicate the presence of surface-binding domains in the NH2-terminal half and channel-forming domains in the COOH-terminal portion of native, monomeric C9.
By hybridizing a cloned cDNA coding for human complement factor C9 to hybrid cells containing subsets of human chromosomes on a rodent background, we have determined that the human gene for C9 is localized on chromosome 5.
Ten monoclonal antibodies (mAb) against native human C9 exhibiting various inhibitory effects on the hemolytic activity of C9 (Bausback, J., Kontermann, R. and Rauterberg, E. W., Immunobiology 1988. 178: 58) were further analyzed regarding their reactivities with monomeric C9 (mC9), polymerized C9 (pC9), and the non-lytic SC5b-9 complex in enzyme-linked immunosorbent assay and with the membrane attack complex (MAC) generated on rabbit erythrocytes analyzed by flow cytometry. In addition, the inhibitory effects of mAb on zinc-induced C9 polymerization were investigated. One epitope of the C-terminal half of C9b exposed on the surface of pC9 and the MAC seems not to participate directly in lytic function or polymerization since no inhibitory effect of the respective mAb was observed. The nine other mAb directed against epitopes of the C9a part exhibit various inhibitory potentials. The mAb inhibit either hemolysis or polymerization, or both processes. Due to the reactivity with the tested antigens the mAb can be divided into two groups. mAb of the first group bind with nearly the same affinity to all four antigens, whereas mAb of the second group react preferentially with mC9 while their affinity to pC9, SC5b-9 and the MAC is reduced. Comparison of reaction patterns and inhibitory effects strongly suggest that different epitopes on the surface of native C9 are involved in interaction of C9 with C5b-8 and/or in C9-C9 interaction. The finding that mAb inhibiting polymerization of C9 in vitro have no inhibitory effect on hemolysis confirms that C9 polymers are no prerequisite for lysis.
A large amount of human C9 was purified from plasma by the following procedures: 1) Polyethylene glycol precipitation; 2) Depletion of plasminogen by passing over an L-lys-sepharose column; 3) DEAE-sephadex A-50 chromatography; and 4) Hydroxylapatite (HA) chromatography. The method of C9 purification was improved by altering the column-elution conditions and by the establishment of a novel method for preparing high-flow-rate HA. As a result, the rate of recovery of C9 was high (28.2%) and no impurities were detected either on gel electrophoretic or immunochemical examination. The hemolytic activity of purified C9 was retained.
We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal polymorphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [3H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B4 (LTB4), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB4 was rapid; the initial release occurred within 4-6 min and a second rise in release coincided with cell death. Virtually all the LTB4 produced was released as we found no evidence of retention of intracellular LTB4 at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB4 was completely abolished and the release of C20:4 and PGs was drastically reduced. [3H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation of C9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.
We measured the concentration of C9 in the CSF and plasma of 93 consecutive patients referred for CSF examination in an outpatient multispecialty clinic. We noted no differences in CSF C9 or C9 index between patients with multiple sclerosis and neurologic controls.
Complement component C9 is a globular serum protein which can insert and polymerise in a target membrane to form a large membrane channel. The ability to insert in the membrane is conferred by amphipathetic elements of secondary structure in the central part of the molecule. Towards each end high cysteine domains are found, one of which is homologous to the apoprotein binding domains of the LDL receptor. From the sequence and topological data for C9 we present a model for its structure and insertion into the membrane.
Complement component C9 contains two distinct cysteine-rich domains exhibiting high sequence resemblance to a domain present in the low density lipoprotein (LDL) receptor and epidermal growth factor precursor, respectively. Antibodies were raised against a peptide corresponding to the most conserved region of the LDL receptor/C9-homology segment. The antibodies were shown by immunoblotting to bind specifically to C9 but also to crossreact with C8 alpha, the alpha subunit of complement component C8. Moreover, a monoclonal antibody to a neoantigen present in polymerized C9 bound to an epitope exposed on C8 within the C5b-8 complex but buried in monomeric C8, suggesting that C8 and C9 undergo similar conformational changes during membrane-attack-complex assembly. Isolated C8 alpha-gamma exhibited the propensity to polymerize in the presence of Zn2+ and urea, as already demonstrated for C9. These data indicate that C8 alpha is closely related, both structurally and functionally, to C9.
Human complement protein C9 is a hydrophilic serum glycoprotein responsible for efficient expression of the cytotoxic and cytolytic functions of complement. It assembles on the surface of a target cell together with C5, C6, C7 and C8 to form the membrane attack complex (MAC) and therefore has to change structure to become an integral membrane protein. As the protein assumes a stable structure in an aqueous environment, the question arises as to how it can enter the hydrophobic interior of a membrane. During MAC assembly C9 polymerizes into a circular structure, termed poly(C9) (ref. 8), which is responsible for the cylindrical electron microscopic appearance of the MAC. The suggestion has been made that C9 must at least partly unfold in order to enter a membrane and also that polymerization of the molecule is intimately linked to insertion and cytotoxicity. The extent of unfolding and the mechanism of polymerization are not understood, nor is it known precisely which parts of the molecule participate in the proposed structural changes. We have been able to capture refolding C9 conformers during membrane insertion with the help of sequence-specific anti-peptide antibodies. Some of these antibodies inhibit C9-mediated haemolysis but not C9 polymerization, while others have the opposite effect. This suggests that the two processes are independent.
The deposition of terminal complement component C9 within the central nervous system (CNS) has been studied immunohistochemically in three models of experimental allergic encephalomyelitis (EAE) in the rat; inflammatory EAE induced by the passive transfer of myelin basic protein-specific T cells (tEAE), antibody-mediated, demyelinating tEAE and a subacute/chronic model induced by active immunisation with guinea pig spinal cord tissue in adjuvant. Two distinct patterns of C9 reactivity were observed, a diffuse staining of the tissue adjacent to inflammatory lesions, similar to that seen for other extra-vasculated serum proteins, and also granular, sometimes fibrillar C9 deposits around some inflammed vessels and in areas of active demyelination. The latter staining pattern was most pronounced in animals with acute antibody-mediated demyelinating tEAE, in which extensive, but transient, subpial and perivascular granular deposits of C9 were associated with regions of acute demyelination. A similar pattern of granular C9 reactivity was also associated with demyelinating lesions in animals with actively induced chronic progressive EAE. However, these C9 deposits were not observed in rats with purely inflammatory, clinically mild tEAE, although C9 deposition was occasionally observed around a small number of inflammed vessels in animals with hyperacute, lethal tEAE. These observations demonstrate that deposition of C9, the major component of the cytolytic membrane attack complex, in EAE is related to myelin injury rather than CNS inflammation.
Protozoan parasite T. cruzi invades cells within acidic vacuoles, but shortly afterward escapes into the cytosol. Exit from the phagosome is blocked by raising the pH of acidic compartments, suggesting that a previously described acid-active hemolysin secreted by T. cruzi might be involved in the membrane disruption process. Here we show that T. cruzi supernatants are cytotoxic for nucleated cells at pH 5.5 and contain a protein reactive with antibodies against reduced and alkylated human C9 (the ninth component of complement). The C9 cross-reactive protein (TC-TOX) copurified with the cytolytic activity, and the active fractions induced conductance steps characteristic of transmembrane ion channels in planar phospholipid bilayers. Immunocytochemical studies using antibodies against purified TC-TOX showed that the protein was localized to the luminal space of parasite-containing phagosomes. We postulate that TC-TOX, when secreted into the acidic environment of the phagosome, forms pores in the membrane, which contribute to its disruption.
A sensitive, haemolytic microassay of human complement component C9 was developed. The assay is based on the principle of reactive (C5b6-initiated) haemolysis and uses commercially available C9-depleted serum as reagent for C9. The specificity of the assay was verified by rapid, activity-guided isolation of the haemolytic component from human serum using high-performance liquid chromatography (HPLC) on a system for fast protein liquid chromatography. This isolation yielded a single component with characteristics of C9. The results suggest that rapid, activity-guided isolation as a new application of HPLC can be a useful tool to demonstrate the specificity of a functional assay.
We describe the first cases, to our knowledge, of C9 deficiency in Europe that were detected in a Swiss family, of which two members--one with a complete deficiency and the other with approximately half-normal C9 levels--experienced bacterial meningitis. The index patient, a 56-year-old white man with a history of purulent meningitis at the age of 23 years, presented with an acute meningococcal meningitis. No impairment of cellular immunity or immunoglobulin deficiency could be found. Complement assays showed a complete deficiency of the C9 component, while the other individual component levels were normal and the hemolytic activity (measured using the CH50 assay) was only slightly reduced. A family study revealed complete C9 deficiency in the patient's healthy brother and half-normal C9 concentrations in his sister, his son (who also had experienced an episode of bacterial meningitis), and his niece, consistent with an inherited C9 deficiency. This first case of recurrent meningitis in a white patient with complete C9 deficiency suggests that this complement defect may also be a risk factor for bacterial, especially neisserial, infections.