Immunology 1979 37 901
Proteolytic transformation of SC5b-9 into an amphiphilic macromolecule resembling the C5b-9 membrane attack complex of complement
S. BHAKDI, BIRGIT BHAKDI-LEHNEN & J. TRANU M-JENSEN* Institute of Medical Microbiology, Giefen, W. Germany and tAnatomy Institute C. University of Copenhagen, Copenhagen N, Denmark
Acceptedfor publication 1 March 1979
(Borsos, Dourmashkin & Humphrey, 1964; Humphrey & Dourmashkin, 1969; Tranum-Jensen et al., 1978). The complex possesses detergent- and lipidbinding surfaces at one terminus which penetrate into and possibly through the lipid matrix of the target membrane (Bhakdi, Bjerrum, Bhakdi-Lehnen & Tranum-Jensen, 1978; Bhakdi & Tranum-Jensen, 1978). Current evidence indicates that a hydrophilic, trans-membrane channel is thus created (Michaels, Abramovitz, Hammer & Mayer, 1976; Bhakdi & Tranum-Jensen, 1978). In basic agreement with Mayer's doughnut hypothesis (Mayer, 1972), we have proposed that this may represent the mechanism of cytolysis by complement (Bhakdi & Tranum-Jensen, 1978). Complement can also be activated in a hydrophilic environment, causing formation of a fluid-phase C5b-9 complex (Muller-Eberhard, 1975). This macromolecule, however, differs from its membrane counterpart in several basic respects. It is hydrophilic, lytically inactive (Muller-Eberhard 1975), possesses an overall globular structure, and is also antigenically distinct from membrane C5b-9 (Bhakdi, BhakdiLehnen, Bjerrum & Tranum-Jensen, 1979). SDS polyacrylamide gel electrophoresis of purified, fluidphase C5b-9 reveals the presence of an additional protein band other than C5b-9 (Kolb & Muller-Eberhard, 1975a; Podack, Kolb & Muller-Eberhard, 1976; Bhakdi et al., 1979). Podack et al. refer to this as the 'S-protein'. They have presented evidence that the
Summary. Proteolysis of fluid-phase SC5b-9 left a major part of the macromolecule intact and caused transition of the molecule from a hydrophilic to an amphiphilic state. The transformed complex exhibited neoantigens characteristic of the C5b-9 membrane attack complex of the complement. It yielded an SDS gel electrophoresis pattern that was similar, but not identical to that of the proteolysed, membrane attack complex. The proteolytically altered SC5b-9 complex bound lipid and incorporated into artificial lipid vesicles to yield a membrane-bound structure resembling the C5b-9 complement lesion. INTRODUCTION Membrane perturbation by complement is caused by the assembly of the five terminal complement components C5-C9 on the target-membrane (Thompson & Lachmann, 1970; Lachmann & Thompson, 1970; Muller-Eberhard, 1975). These serum proteins associate with each other to form an apparently hollow, cylindrical macromolecule (Tranum-Jensen, Bhakdi, Bhakdi-Lehnen, Bjerrum & Speth, 1978), which is identical with the classical complement lesion observed on membranes after electron microscopy Correspondence: Dr S. Bhakdi, Institute for Medical Microbiology, Schuberstrasse 1,6300 Giefien, W. Germany.
0019-2805/79/0800-0901 $02.00 ©) 1979 Blackwell Scientific Publications
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protein functions as an inhibitor/inactivator of nascent C5b-7 (Podack, Kolb & Muller-Eberhard, 1977; Podack, Halverson, Esser, Kolb & Muller-Eberhard, 1978; Podack & Muller-Eberhard, 1978). According to this concept, the S-protein attaches itself to C5b-7 during complex generation in serum, thereby abrogating the potential of the trimolecular complex to bind to bystander lipid bilayers (Podack et al., 1977). Fluidphase C5b-9 is now designated 'SC5b-9' to denote the presence of the S-protein (Podack et al., 1977; Podack et al., 1978; Podack & Muller-Eberhard, 1978). As in previous communications (Bhakdi et al., 1978; Bhakdi & Tranum-Jensen, 1978), we will refer to the membrane C5b-9 complex as 'C5b-9(m)' to denote its membrane origin. Podack & Miiller-Eberhard have recently presented data suggesting that the S-protein may be selectively removed from SC5b-9 through the action of deoxycholate (DOC) (Podack et al., 1978; Podack & MullerEberhard, 1978). The DOC-treated SC5b-9 complex purportedly regains its ability to bind detergent (Podack et al., 1978; Podack & Muller-Eberhard, 1978). In this communication, we do not confirm such a dissociation of the S-protein, with concomitant alteration of biochemical or ultrastructural properties of SC5b-9, through the action of DOC. We wish, however, to present evidence that SC5b-9 can be proteolytically transformed into an amphiphilic macromolecule whose antigenic, ultrastructural, and lipid-binding properties closely resemble those of the C5b-9(m) membrane attack complex.
MATERIALS AND METHODS Sources of reagents and antisera have been given previously (Bhakdi, Bjerrum, Rother, Knufmann & Wallach, 1975; Tranum-Jensen et al., 1978) Native and proteolysed C5b-9(m), and SC5b-9 were isolated as described (Bhakdi, Ey & Bhakdi-Lehnen, 1976; Tranum-Jensen et al., 1978; Bhakdi et al., 1979). Antisera to SC5b-9 were raised in three rabbits following procedures described by Harboe & Ingild (1973). Antiserum to SC5b-9 neoantigens (Kolb & Muller-Eberhard, 1975b) was prepared by absorption of anti-SC5b-9 antiserum with 1-5 volumes of human serum in the presence of 10 mm EDTA (final concentration) (see also Bhakdi et al., 1978a).
DOC-treatment Samples of SC5b-9 (I mg/ml in 10 mM Tris, 50 mM,
NaCI, 15 mm NaN3, pH 8-2) were made 2% (w/v) in DOC and allowed to stand at room temperature (25°) for 2-6 h. Trypsin inhibitor (20 ug/ml) was added to ensure than no proteolytic degradation took place during this period of incubation. Samples were subsequently subjected either to ultracentrifugation through linear 10-50% sucrose density gradients in the same buffer containing 0.10% DOC, or to descending chromatography over Sepharose 6B equilibrated with 0.10% DOC. Proteolysis of SC5b-9 Experiments were performed following two protocols. (a) Samples were made 10% in Triton X-100+ 10% in DOC, respectively. They were treated with a combination of trypsin + a-chymotrypsin at final individual enzyme concentrations of 20 ug/ml for 6-18 h at 37°. Thereafter, proteolysed SC5b-9 was recovered through centrifugation through linear sucrose density gradients containing 0 1o% Triton X-100 +0 1o% DOC, or through chromatography over Sepharose 6B containing the same detergents. Fractions containing reisolated, proteolysed SC5b-9 were directly analysed in SDS polyacrylamide gel electrophoresis, quantitative immunoelectrophoresis, and utilized in membrane reconstitution experiments. (b) Alternatively, samples of SC5b-9 were made 0.1% in Triton X-100 (for immunoelectrophoretic analyses) or 1I% in DOC (for membrane reconstitution studies), proteolysed, and directly utilized without repurification of the proteolysed complex. Membrane reconstitition DOC-solubilized sheep erythrocyte membrane lipids (Bhakdi & Tranum-Jensen, 1978) were used in all experiments. Approximately 3 mg of lipids in 3 ml DOC solution were added to 0 5 mg SC5b-9 in 1 ml buffer solution. The protein samples employed in different experiments were the following: (1) purified SC5b-9, not pre-treated with DOC; (2) DOC-treated SC5b-9, re-isolated by sucrose density gradient centrifugation or Sepharose chromatography after pretreatment with this detergent; (3) proteolysed SC5b-9, re-isolated after protease treatment through sucrose density gradient centrifugation or Sepharose chromatography in the presence of 0-1% Triton X-100+0-1% Doc; (4) SC5b-9 which was added to lipid in DOC and directly proteolysed in detergent/lipid solution. All samples were dialysed against 400 volumes of 4 mM Tris, 8 mm NaN3, pH 8 0 for 2 h at 250. MgC12 (3 mM final concentration) was than added and dialysis
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continued for 36-45 h. Vesicle preparations were concentrated and the vesicles floated through sucrose solution as described (Bhakdi & Tranum-Jensen, 1978). Electron microscopy was performed on dialysed aliquots after each step in the vesicle preparations. Lipid analyses Extensively dialysed fractions from sucrose density gradients were lyophilized, extracted with chloroform/methanol (2:1), and analysed in conventional thin-layer chromatography on silica gels using a chloroform/methanol/water (65:25:4) solvent system. Electron microscopy Negative staining and electron microscopy was performed as described (Tranum-Jensen et al., 1978; Bhakdi & Tranum-Jensen, 1978) using hydrophilized, carbon-coated formvar films carried on copper grids and 2% silicotungstate as staining solution.
SDS-Polyacrylamide gel electrophoresis A discontinuous slab gel electrophoresis system based on the Laemmli procedure (Laemmli, 1970) was utilized. Modifications were as detailed by Bosch, Orlich, Klenk & Rott (1979). Running gels were 10% acrylamide, 0-18% N,N-methylenebisacrylamide, in 400 mM Tris, 2 5 M urea, pH 8-9. Stacking gels were 5% acrylamide, 0-15% N,N-methylenebisacrylamide, 60 mm Tris, 5 M urea, pH 6-8. The electrophoresis buffer was 50 mM Tris, 385 mM glycin, pH 8-3. The cathodal buffer contained 0 3% (w/v) SDS. Gels were calibrated with RNA polymerase, bovine serum albumin, trypsin inhibitor and cytochrome c (Boehringer). Electrophoresis was performed at 100 for I h at 70 V followed by another 5 h at 100 V.
Quantitative immunoelectrophoresis, charge-shift crossed immunoelectrophoresis, and autoradiographical analyses of Triton-binding in crossed immunoelectrophoresis were performed as described (Bhakdi et al., 1975; Bhakdi, Bhakdi-Lehnen & Bjerrum, 1977; Bjerrum & Bhakdi, 1977). RESULTS Effect of proteolysis on the antigenicity of SC5b-9 SC5b-9 was proteolysed and subsequently directly analysed in double-diffusion. The proteolysed complex still reacted with antisera specific for native C5, C6 and C9 to yield coalescing immunoprecipitates
Figure 1. (A) Rocket immunoelectrophoresis of native and proteolysed SC5b-9 using an antiserum to C5b-9(m) neoantigens (7 MI/cm2). Samples applied were (1) purified SC5b-9; (2) identical sample of SC5b-9 which was proteolysed with trypsin + a-chymotrypsin prior to immunoelectrophoresis; (3) SC5b-9 which had been treated with DOC and re-isolated in the presence of DOC; (4) proteolysed and re-isolated SC5b-9. Proteolysis exposes neoantigenic determinants characteristic of the membrane attack complex on SC5b-9. (B) The same samples were analysed using an unabsorbed antiserum to C5b-9(m). Note the analogeous, stronger precipitation reaction of this antiserum with the proteolysed SC5b-9 complex.
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(not shown). This indicated that these terminal complement components remain complexed to each other after proteolytic attack, as is also the case for the membrane C5b-9(m) complex (Tranum-Jense et al., 1978; Bhakdi et al., 1978). Proteolysis induced a striking antigenic modification of SC5b-9, which was detectable with an antiserum directed against the neoantigens of C5b-9(m). Native or DOC-treated SC5b-9 cross-react weakly with this antiserum. The very faint immunoprecipitates obtained at the given antiserum concentration are not visible on the reproduction of Fig. IA (wells 1 and 3). By contrast, the proteolysed complex was well precipitated with the same antiserum under identical experimental conditions (Fig. IA, wells 2 and 4). Analogously the unabsorbed antiserum to C5b-9(m) reacted more strongly with proteolysed SC5b-9 than with the native SC5b-9 complex (Fig. 1 B: the stronger reaction of anti-C5b-9(m) with proteolysed SC5b-9 is apparent from the lower heights of the rockets above wells 2 and 4, compared to the heights of the rockets above wells 1 and 3. The same amount of protein was applied in all wells). Thus, proteolysis of SC5b-9 exposed neoantigenic determinants on the complex which are characteristic of the C5b-9(m) membrane attack complex, and which are not originally exposed on the native SC5b-9 molecule. This antigenic modification was not observed after treatment of SC5b-9 with DOC alone (Fig. 1, well 3), or with a combination of Triton + DOC (not shown).
Unusual reaction of anti-SC5b-9 antiserum Figure 2 depicts a double-diffusion analysis of C5b-9(m) and SC5b-9 using an antiserum raised SC5b-9. This unabsorbed antiserum (well 1) precipitated both the membrane (m) and the fluid-phase (s) complex. After absorption with EDTA-serum, the antiserum was still capable of precipitating both C5b-9(m) and SC5b-9(m) (Fig. 2, well 2). This finding confirms the presence of neoantigens present on both C5b-9(m) and SC5b-9 detectable with such antisera (Kolb & MullerEberhard, 1975b). Anti-SC5b-9 was absorbed with 1-5 volumes of unfractionated, inulin-activated serum in 10 mM EDTA. The absorbed antiserum (well 3) then no longer precipitated native SC5b-9. This antiserum, however, was still capable of precipitating the membrane C5b-9 complex, as well as the proteolysed SC5b-9 complex
Figure 2. Double diffusion analyses of purified membrane C5b-9 (wells labelled 'm') and fluid-phase SC5b-9 (wells labelled 's'), using an antiserum raised against SC5b-9. Well I contained anti-SC5b-9; the unabsorbed antiserum precipitated both C5b-9(m) and SC5b-9. Well 2 contained the same antiserum which had been absorbed with EDTA-serum. This antiserum, directed against SC5b-9 neoantigens, no longer precipitated any native serum protein (Kolb & Muller- Eberhard, 1975b; Bhakdi et al., 1978), but still precipitated both C5b-9(m) and SC5b-9. Well 3 contained purified and concentrated immunoglobulins of an anti-SC5b-9 antiserum which had been absorbed with inulin-activated serum. This antibody preparation no longer precipitated SC5b-9, but still precipitated the membrane C5b-9 complex. the anti-SC5b-9 antiserum contains antibodies directed against neoantigens of the actual membrane attack complex which are not primarily exposed on the native SC5b-9 molecule.
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(the latter reaction is not shown in Fig. 2). Thus, the antiserum raised against SC5b-9 exhibits similar specificity as an antiserum raised against the membrane complex. It contains antibodies directed against neoantigens of the actual membrane attack complex C5b-9(m), which are not primarily exposed on the native SC5b-9 molecule. When anti-SC5b-9 was absorbed with an excess of washed, complement-lysed sheep erythrocyte membranes, or with isolated C5b-9(m), the antiserum subsequently failed to precipitate either C5b-9(m) or SCSb-9. It also did not precipitate any single serum protein. Thus, we have not yet succeeded in producing an antiserum that is entirely specific for SC5b-9, or in obtaining antibodies against the S-protein. Behaviour of proteolysed SC5b-9 in sucrose density gradient ultracentrifugation and Sepharose chromatography Native SC5b-9 sediments as a 22 5S protein in a sucrose density gradient (Kolb & Miuller-Eberhard, 1973; Podack et al., 1976; Bhakdi et al., 1979), and elutes in a molecular region comparable to that of IgM on Sepharose 6B (Kolb & Muller-Eberhard, 1975a; Bhakdi et al., 1979). Following proteolysis, the complex no longer yielded a symmetrical sedimenting peak in a sucrose density gradient, but smeared out over a region corresponding to > 28S (Fig. 3B). On a Sepharose 6B column the protein eluted with and directly after the void volume (not shown). The re-isolated, proteolysed SC5b-9 complex yielded a single immunoprecipitate of f-electrophoretic mobility when tested in a crossed immunoelectrophoresis using a polyspecific antiserum against human serum proteins (not shown). In the electron microscope, markedly aggregated ring and rectangular structures, reminiscent of the C5b-9(m) complex viewed en face and in profile, were observed in preparations of purified, proteolysed SC5b-9 re-isolated by Sepharose chromatography. The aggregates persisted despite the presence of Triton+ DOC, impeding ultrastructural image interpretation. Data on the ultrastructure of the complex, however, became available through membrane reconstitution experiments to be described below. No alteration in the sedimentation behaviour of the SC5b-9 complex was observed following treatment with DOC alone. DOC-treated SC5b-9 also eluted on Sepharose at an identical position as did native
SC5b-9 (not shown).
Figure 3. Sedimentation behaviour of native and proteolysed SC5b-9 in a sucrose density gradient. SC5b-9 was treated with DOC at a final concentration of 2%. An unproteolysed sample (A) was centrifuged through a linear 10-50% sucrose density gradient containing 0 1% DOC at 150,000 g for 13 h (5 ml gradient, rotor SW 50 1). A proteolysed sample (B) was centrifuged in parallel. The positions of the protein complexes in the gradients were determined by fused rocket immunoelectrophoresis of the collected fractions using an antiserum to C5b-9(m). Direction of sedimentation; right to left. Arrows point to the sedimentation positions of haemoglobin (Hb), IgG and IgM in the same gradients. native or DOC-treated SC5b-9 sediments as 22 5S protein; after proteolytic attack, the sedimentation coefficient of the complex increases to > 28S.
SDS polyacrylamide gel electrophoresis The gel electrophoresis patterns of SC5b-9 (a), DOCtreated SC5b-9 (b), proteolysed and re-isolated SC5b-9 (c), and proteolysed C5b-9(m) (d) are shown in Fig. 4. No differences in the protein banding patterns were observed between DOC-treated and native
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Figure 4. SDS polyacrylamide gel electrophoresis of SC5b-9 (gel a), DOC-treated SC5b-9 (gel b), proteolysed and reisolated SC5b-9 (gel c), proteolysed, isolated C5b-9(m) (gel d). Complement components are labelled according to Kolb & Muller-Eberhard (1975a) and Podack et al. (1976), after correlation of the protein bands (Bhakdi et al., 1976; Bhakdi et al., 1979)*. The C5b band persists in both SC5b-9 and C5b-9(m) following proteolytic attack. C6 and the S-protein are degraded. The residual protein bands in the molecular weight region of 50 K and 75-100 K have not been identified. The protein banding patterns of proteolysed SC5b-9 and C5b-9(m) are not identical. Direction of electrophoresis; top to bottom. Coomassie brilliant blue.
SC5b-9 (gels a and b). Discontinuous gel electrophoresis provides superior resolution of the protein bands compared to the continuous system previously employed (Bhakdi et al., 1979). In the region of C6, C7 and anodal to C9, additional minor bands are observed. Of these, the band cathodal to C6 is inconstant; the other bands are constantly present. As the latter are also observed in gel electrophoresis patterns of membrane C5b-9, they probably represent molecular derivatives of the C5b-C9 complement components. Alternatively, they may reflect the presence of microheterogeneous species of C6-C9. Proteolysis removed the protein bands corresponding to C6 and the S-protein. C5b was not affected. Five protein bands were constantly visible in the region of 90 K, 85 K, 80 K, 75 K and 50 K (gel c); an inconstant * The designation of the C8# band is preliminary.
band was observed at 100 K. The individual bands appearing after proteolysis of SC5b-9 have not been identified. Proteolysed C5b-9(m) exhibited a similar protein banding pattern (gel d), but slight deviations were observed in the region of 80-90 K. This suggests some differences in the suspectibility of peptide bonds toward proteolysis in SC5b-9 and C5b-9(m).
Binding of detergent by proteolysed SC5-9 Native SC5b-9 is hydrophilic according to the criterion of charge-shift crossed immunoelectrophoresis; no indication of Triton-binding has been obtained using the method of autoradiography in crossed immunoelectrophoresis (Bhakdi et al., 1979). Following proteolysis, the fluid-phase complex exhibited a significant charge-shift in DOC and a borderline charge-shift
Proteolytic transformation of SC5b-9 in CTAB; a faintly radioactive immunoprecipitate was obtained in crossed immunoelectrophoresis after incorporation of radioactive Triton in the agarose gel (not shown). The detergent-binding studies thus yielded an indication that proteolysis exposes apolar regions on the complex which may bind Triton. The contention that proteolysed SC5b-9 is amphiphilic received support from lipid-binding studies, as demonstrable by membrane reconstitutions.
Membrane reconstitution Figure 5A depicts an electron micrograph of an unfractionated preparation of vesicles formed in the presence of DOC-treated SC5b-9. There is no visible interaction between the lipid vesicles (Li) and the pro-
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tein complexes; the latter are seen as fuzzy, globular structures with irregular contours (Bhakdi et al., 1979). Blurred ring and rectangular profiles, reminiscent of the structure of C5b-9(m), are sporadically found (inset, Fig. 5A). Figure 6A shows that following flotation of the vesicles, virtually all of the DOCtreated SC5b-9 remains at the bottom of the sucrose density gradient. Thus, DOC-treated SC5b-9 does not associate with lipid under our experimental conditions. As verified by thin-layer chromatography, all lipid was recovered in the top fractions of such sucrose gradients. Figure 5B depicts a lipid vesicle preparation prepared by using the same SC5b-9 sample which was, however, proteolysed in the presence of DOC and lipid
5B
Figure 5 (A) Electron micrograph of a negatively stained preparation of lipid vesicles formed in the presence of native SC5b-9. The lipid vesicles (Li) appear as smoothly contoured structures of a uniform, low density. The complement complexes (s) appear as uniformly dispersed, irregular structures with an approximate diameter of 20 nm. Single complexes are sporadically observed as fuzzy, ring-shaped or rectangular profiles (inset at lower left), reminiscent of the structure of the C5b-9(m) membrane attack complex (compare with Fig. 6F). No specific association between lipid vesicles and the native SC5b-9 complex could be detected in such preparations. (B) When lipid vesicles were formed in the presence of proteolysed SC5b-9, massive aggregates resulted. Lipid vesicles are seen to carry structures (p) similar to the membrane-derived C5b-9 complexes. Nearly all lipid vesicles exhibit central stain deposit, indicating permeability to the stain molecules. A single vesicle which escaped incorporation of the complex is seen at lower right (asterisk). Scale bars indicate 100 nm.
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Figure i. (A) Tbe veside preparation of Fig. 5A was applied to the bottom of a sucrose density gradient. Following flotation of the lipid vesicesvirtualy all the protein was recovered at the bottom of the gradient. Thus, native SC5b-9 does not significantly bind to lipid. (B) lhe vesicd preparation of Fig. 5B was treated similarly. Approximately 75% of proteolysed SC5b-9 floated in association with the lipid vesies through sucrose. The direction of the sucrose gradients is given. Detection and relative quantification of protein in the fractions collected after centrifugation was done using rocket immunoelectrophoresis and an antiserum to C5b-9(m). Agarose gels contained 0- 5% Triton X-100.
prior to detergent removal. Here a structural modification of the SC5b-9 molecules, which appear bound to the lipid vesicles, becomes apparent. Flotation of such samples reveals that a fraction (50-80%) of the protein is lipid-bound (Fig. 6B), and is recovered in association with the vesicles. Similar results were obtained with proteolysed SC5b-9 re-isolated from sucrose density gradients or Sepharose columns, but the degree of protein incorporation into the vesicles
was reduced compared to that obtained by the former method. This may be due to the increase in molecular aggregation of proteolysed SC5b-9 during the procedure of re-isolation. Marked ultrastructural changes accompany proteolysis of the SC5b-9 complex, with the appearance of numerous ring-shaped profiles of approximately 20 nm outer diameter and with a central stain filling, as can be discerned in large numbers in Fig. 5B. The
difficulties of analysing individual complexes in their aggregated form was circumvented by examination of the top fractions of sucrose density gradients which contained vesicles that had incorporated only small numbers of complexes. The incorporated complexes covered a range of shapes, of which distinct cylindrical structures, morphologically indistinguishable from reincorporated C5b-9(m) complexes, dominated (Figs 5B and 7D-F. More foliaceous, branching structures were also observed, however, and representative examples of these are shown in Fig. 7A-C. Lipid vesicles which escaped incorporation of complexes are characteristically empty of stain, as can be seen in the preparations of Figs 5A, B and 7A, F. Incorporation of proteolytically transformed SC5b-9 complexes renders the vesicles permeable to the silicotungstate stain, as indicated by a central stain deposit, found in all vesicles carrying a complex (Figs 5B and 7A-E). Thus, incorporation of a proteolysed SC5b-9 complex into a lipid vesicle appears to create a transmembrane pore at the complex attachment site, as is also the case with the membrane attack complex (Bhakdi & Tranum-Jensen, 1978, and Fig. 7F).
DISCUSSION
Figure 7. (A-E) These illustrate the appearance of lipid vesicles recovered in the top fraction of a sucrose density gradient following flotation of the vesicle preparation illustrated in Figs 5B and 6B. The proteolytically transformed SC5b-9 complexes incorporated into lipid vesicles exhibit somewhat heterogeneous shapes ranging from foliaceous structures (A-C) to distinct cylindrical structures. The latter dominate and are morphologically indistinguishable from the membrane-derived C5b-9 complexes (D and E). An unfractionated preparation of lipid vesicles reconstituted with the membrane complex is shown for comparison in F. Vesicles which escape incorporation of complement complexes (A and F) are empty of stain. Scale bars indicate 100 nm.
The assembly of C5b-C9 into the membrane C5b-9 complex constitutes the only known case of a molecular transition of hydrophilic serum proteins into a protein complex that is amphiphilic, and that can associate with membrane lipids in a manner akin to that of an integral membrane protein. We found that the exposure of lipid-binding regions in the complex was, somewhat unexpectedly, not confined to the situation where complex assembly occurred in immediate contact with a lipid bilayer. Apolar molecular regions could be exposed through proteolytic attack of the pre-formed SC5b-9 complex in solution. This resulted in a transformation of SC5b-9 into an amphiphilic macromolecule that structurally resembled the membrane attack complex of complement. In disagreement with previous data of Podack et al. (Podack et al., 1978; Podack & Muller-Eberhard, 1978), we did not find that treatment of SC5b-9 with DOC alone altered the detergent- or lipid-binding properties of the
complex. Proteolytic attack appeared to leave the 'core' of the SC5b-9 complex intact. This was evidenced by (a) the persistence of C5, C6 and C9 antigenic determinants on the proteolysed complex; (b) the persistence of C5b-9 neoantigens; (c) the apparent large molecular
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size of the proteolysed complex as judged by sucrose density gradient ultracentrifugation and Sepharose chromatography (these findings, however, are not compelling since the complex is aggregated; (d) the persistence of macromolecular structure as evaluated by electron microscopy; (e) the persistence of high molecular weight protein bands in SDS-polyacrylamide gel electrophoresis. Several observations indicated that proteolysed SC5b-9, although similar, is not entirely identical to its membrane counterpart. The SDS gel electrophoresis patterns of proteolysed SC5b-O(m) and C5b-9(m) were not identical. In both cases, C5b was retained in high molecular weight form. The persistence of C5b, as well as of the other residual, high molecular weight peptides may be due to stabilizing inter- and intrachain disulphide bonds within these molecules. The present gel electrophoresis data cannot yet be correlated to previous data of Hammer et al. (Hammer, Nicholson & Mayer, 1975; Hammer, Shin, Abramovitz & Mayer, 1977). These investigators reported 40-50% removal of radiolabelled C5 from membranebound guinea-pig C5b-7 complexes, but C5 removal from the C5b-9 complex was not studied (Hammer et al., 1975). In a later communication (Hammer et al., 1977), tryptic degradation of guinea-pig C9 in the membrane C5b-9 complex to an 18,000 mol. wt product was described. Our data need not conflict with these results as we deal with the human C5b-9 complex and also have not yet identified the proteolysis products observed in SDS gel electrophoresis. Another indication that proteolysed SC5b-9 is not entirely identical to its membrane counterpart derives from its aggregation behaviour in detergent solution. Whereas monomerized, cylindrical structures are obtained by chromatography of proteolysed C5b-9(m) in the presence of Triton+ DOC, the proteolysed SC5b-9 complex remains markedly aggregated under the same conditions. Aggregation may also be responsible for the smaller charge-shifts found for proteolysed SC5b-9, compared to those of the membrane complex (Bhakdi et al., 1978). Electron microscopy of proteolysed SC5b-9 complexes following their incorporation into artificial lipid vesicles indicated that they possess a similar ultrastructure as C5b-9(m). Proteolysis appeared to remove the fuzzy 'coat' of SC5b-9, revealing what we interpreted to be a cylindrical structure obviously inherent, but masked in the native SC5b-9 molecule. In the majority of cases the vesicle-bound complexes were seen as cylindrical structures projecting approximately
10 nm from the lipid bilayer and rimmed by 20 nm wide annuli. These structures were morphologically identical to the C5b-9(m) complement lesions. Other more foliaceous structures are occasionally observed; these may be related to the structures described by Dourmashkin after assembly of the C5b-8 complex on membranes (Dourmashkin, 1978). The molecular weights of the individual protein subunits comprising the SC5b-9 monomer total approximately 1,000,000 (Kolb & Muller-Eberhard, 1973; Kolb & Muller-Eberhard, 1975a). Hydrodynamic data to be discussed elsewhere are in agreement with this value. The dimensions of native SC5b-9 are comparable to that of C5b-9(m), the diameter of the globule being in the order of 20 nm. The structure observed after proteolysis of SC5b-9 possesses the same size as C5b-9(m) in the electron microscope. A hollow protein cylinder, 10 nm in diameter, 15 nm in height, with walls 1 nm thick, rimmed by an annulus of 4 nm diameter (Bhakdi & Tranum-Jensen, 1978b) would possess a volume of approximately 970 nm3. Assuming a partial specific volume of 0 75, this implies a molecular weight of approximately 800,000, a value which would be in good agreement with the sum molecular weights of the C5b-C9 subunits contained in the
C5b-9(m) complex. The immunochemical reactions of C5b-9(m), SC5b-9 and their respective antisera indicated that antisera raised against native SC5b-9 contained antibodies directed against neoantigens of the actual membrane attack complex, which were not primarily exposed on SC5b-9. This finding suggests that proteolysis of SC5b-9 occurred during molecular processing of SC5b-9 in the immunized animal, leading to exposure of the concealed C5b-9(m) neoantigenic determinants. The possibility of immunochemically distinguishing between SC5b-9 and C5b-9(m) through the use of appropriately absorbed antisera will be of use for investigations into the occurrence of C5b-9 neoantigens on cell surfaces (Sundsmo, Kolb & Miuller-Eberhard, 1978a; Sundsmo, Curd, Kolb & MullerEberhard, 1978b) and in diseased tissues. Proteolytic exposure of apolar, lipid-binding regions on a primarily hydrophilic molecule is a novel observation in serum protein chemistry. The possible biological significance of the SC5b-9 transformation, and the molecular events associated with the hydrophilic-amphiphilic transition await further investigation. Exposure of lipid-binding regions on the macromolecule may be directly due to proteolytic removal of the S-protein, as might be anticipated from
Proteolytic transformation of SCSb-9 data of Podack et al. (1977). Other conformational changes accompanying proteolysis may, however, additionally be responsible for the observed molecular transformation.
ACKNOWLEDGMENTS We are grateful to Ortrud Klump for excellent technical assistance, and to Christine Reitz and Ilona Vogel for photographical and secretarial help. We thank Dr Ari Helenius (European Molecular Biology Laboratory, Heidelberg) for critical discussions, and Dr H.-J. Wellensiek for his continued interest in this work. This study was supported by the Deutsche Forschungsgemeinschaft. REFERENCES BHAKDI S., BJERRUM O.J., ROTHER U., KNUFERMANN H. & WALLACH D.F.H. (1975) Immunochemical analyses of membrane-bound complement: detection of the terminal complement complex and its similarity to 'intrinsic' erythrocyte membrane proteins. Biochim. Biophys. Acta, 406, 21. BHAKDI S., EY P. & BHAKDI-LEHNEN B. (1976) Isolation of the terminal complement complex from target sheep erythrocyte membranes. Biochim. Biophys. Acta, 419, 445. BHAKDI S., BHAKDI-LEHNEN B. & BJERRUM O.J. (1977) Detection of amphiphilic proteins and peptides in complex mixtures: charge-shift crossed immunoelectrophoresis and two-dimensional charge-shift electrophoresis. Biochim. Biophys. Acta 470, 35. BHAKDI S., BJERRUM O.J., BHAKDI-LEHNEN B. & TRANUMJENSEN J. (1978) Complement lysis: evidence for an amphiphilic nature of the terminal membrane CSb-9 complex of human complement. J. Immunol. 121, 2526. BHAKDI S. & TRANUM-JENSEN J. (1978) Molecular nature of the complement lesion. Proc. natn. Acad. Sci. U.S.A. 75, 5655. BHAKDI S., BHAKDI-LEHNEN B., BJERRUM O.J. & TRANUMJENSEN J. (1979) Difference in antigenic reactivity and ultrastructure between fluid-phase SCSb-9 and the C5b-9 membrane attack complex of human complement. FEBS Letts. 99, 15. BJERRUM O.J. & BHAKDI S. (1977) Demonstration of Triton X-100 binding to amphiphilic proteins in crossed immunoelectrophoresis. FEBS Letts, 81, 56. BoRsos T., DOURMASHKIN R.R. & HUMPHREY J.H. (1964) Lesions in erythrocyte membranes caused by immune haemolysis. Nature (Lond.), 202, 251. BOSCH F., ORLICH M., KLENK H.-D. & Rorr R. (1979) The structure of the hemagglutinin, a determinant for the pathogenicity of influenza virus. Virology (In press.) DOURMASHKIN R.R. (1978) The structural events associated with the attachment of complement components to cell membranes in reactive lysis. Immunology, 35, 205.
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HAMMER C.H., NICHOLSON A. & MAYER M.M. (1975) On the mechanism of cytolysis by complement: evidence on insertion of C5b and C7 subunits of the C5b, 6, 7 complex into the phopholipid bilayer of erythrocyte membranes. Proc. natn. Acad. Sci. U.S.A. 72, 5076. HAMMER C.H., SHIN M.L., ABRAMOVITZ A.S. & MAYER M.M. (1977) On the mechanism of cell membrane damage by complement: evidence on insertion of polypeptide chains from C8 and C9 into the lipid bilayer of erythrocytes. J. Immunol. 119, 1. HARBOE N. & INGILD A (1973) Immunization, isolation of immunoglobulins. Estimation and antibody titer. Scand. J. Immunol. 2 (Suppl. 1), 161. HUMPHREY J.H. & DOURMASHKIN R.R. (1969) The lesions in cell membranes caused by complement. Adv. Immunol. 11, 75. KOLB W.P. & MuLLER-EBERHARD H.J. (1973) The membrane attack mechanism of complement. Verification of a stable C5-C9 complex in free solution. J. exp. Med. 138,438. KOLB W.P. & MuLLER-EBERHARD H.J. (1975a) The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex. J. exp. Med. 141, 724. KOLB W.P. & MuLLER-EBERHARD H.J. (1975b) Neoantigens of the membrane attack complex of human complement. Proc. natn. Acad. Sci. U.S.A. 72, 1687. LACHMANN P.J. & THOMPSON R.A. (1970) Reactive lysis: the complement-mediated lysis of unsensitized cells. 11. The characterization of activated reactor as C5b and the participation of C8 and C9. J. exp. Med. 131, 644. LAEMMLI U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophase T4. Nature (Lond.), 227, 680. MAYER M.M. (1972) Mechanism ofcytolysis by complement. Proc. natn. Acad. Sci. U.S.A. 69, 2954. MICHAELs D.W., ABRAMOVITZ A.S., HAMMER C.H. & MAYER M.M. (1976) Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement. Proc. nain. Acad. Sci. U.S.A. 73, 2852. MuJLLER-EBERHARD H.J. (1975) Complement. Ann. Rev. Biochem. 44, 697. PODACK E.R., KOLB W.P. & MuLLER-EBERHARD H.J. (1976) The C5b-9 complex: subunit composition of the classical and alternative pathway-generated complex. J. Immunol. 116, 1431. PODACK, E.R., KOLB W.P. & MULLER-EBERHARD H.J. (1977) The SC5b-7 complex: formation, isolation, properties, and subunit composition. J. Immunol. 119, 2024. PODACK E.R., HALVERSON C., ESSER A.F., KOLB W.P. & MuLLER-EBERHARD H.J. (1978) C5b-9: Regeneration of the ability to interact with lipid by selective removal of the S-protein. J. Immunol. 120, 1792 (Abstr.) PODACK E.R. & MOLLER-EBERHARD H.J. (1978) Binding of deoxycholate, phosphatidylcholine vesicles, lipoprotein and of the S-protein to complexes of terminal complement components. J. Immunol. 121, 1025. SUNDSMO J.S., KOLB W.P. & MuLLER-EBERHARD H.-J. (1 978a) Leukocyte complement: neoantigens of the membrane attack complex on the surface of human leukocytes prepared from defibrinated blood. J. Immunol. 120, 850. SUNDSMO J.S., CURD J.G., KOLB W.P. & MULLER-EBERHARD
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H.J. (1978b) Leukocyte complement: assembly of the membrane attack complex by human peripheral blood leukocytes in the presence and absence of serum. J. Immunol. 120, 855. THOMPSON R.A. & LACHMANN P.J. (1970) Reactive lysis: the complement-mediated lysis of unsensitized cells. I. The
characterization of the indicator factors and its identification as C7. J. exp. Med. 131, 629. TRANUM-JENSEN, J., BHAKDI, S., BHAKDI-LEHNEN, B., BJERRUM, O.J. & SPETH, V. (1978) Complement lysis: the ultrastructure and orientation of the C5b-9 complex on target sheep erythrocyte membranes. Scand. J. Immunol.
7,45.
Proteolytic transformation of SC5b-9 into an amphiphilic macromolecule resembling the C5b-9 membrane attack complex of complement
S. BHAKDI, BIRGIT BHAKDI-LEHNEN & J. TRANU M-JENSEN* Institute of Medical Microbiology, Giefen, W. Germany and tAnatomy Institute C. University of Copenhagen, Copenhagen N, Denmark
Acceptedfor publication 1 March 1979
(Borsos, Dourmashkin & Humphrey, 1964; Humphrey & Dourmashkin, 1969; Tranum-Jensen et al., 1978). The complex possesses detergent- and lipidbinding surfaces at one terminus which penetrate into and possibly through the lipid matrix of the target membrane (Bhakdi, Bjerrum, Bhakdi-Lehnen & Tranum-Jensen, 1978; Bhakdi & Tranum-Jensen, 1978). Current evidence indicates that a hydrophilic, trans-membrane channel is thus created (Michaels, Abramovitz, Hammer & Mayer, 1976; Bhakdi & Tranum-Jensen, 1978). In basic agreement with Mayer's doughnut hypothesis (Mayer, 1972), we have proposed that this may represent the mechanism of cytolysis by complement (Bhakdi & Tranum-Jensen, 1978). Complement can also be activated in a hydrophilic environment, causing formation of a fluid-phase C5b-9 complex (Muller-Eberhard, 1975). This macromolecule, however, differs from its membrane counterpart in several basic respects. It is hydrophilic, lytically inactive (Muller-Eberhard 1975), possesses an overall globular structure, and is also antigenically distinct from membrane C5b-9 (Bhakdi, BhakdiLehnen, Bjerrum & Tranum-Jensen, 1979). SDS polyacrylamide gel electrophoresis of purified, fluidphase C5b-9 reveals the presence of an additional protein band other than C5b-9 (Kolb & Muller-Eberhard, 1975a; Podack, Kolb & Muller-Eberhard, 1976; Bhakdi et al., 1979). Podack et al. refer to this as the 'S-protein'. They have presented evidence that the
Summary. Proteolysis of fluid-phase SC5b-9 left a major part of the macromolecule intact and caused transition of the molecule from a hydrophilic to an amphiphilic state. The transformed complex exhibited neoantigens characteristic of the C5b-9 membrane attack complex of the complement. It yielded an SDS gel electrophoresis pattern that was similar, but not identical to that of the proteolysed, membrane attack complex. The proteolytically altered SC5b-9 complex bound lipid and incorporated into artificial lipid vesicles to yield a membrane-bound structure resembling the C5b-9 complement lesion. INTRODUCTION Membrane perturbation by complement is caused by the assembly of the five terminal complement components C5-C9 on the target-membrane (Thompson & Lachmann, 1970; Lachmann & Thompson, 1970; Muller-Eberhard, 1975). These serum proteins associate with each other to form an apparently hollow, cylindrical macromolecule (Tranum-Jensen, Bhakdi, Bhakdi-Lehnen, Bjerrum & Speth, 1978), which is identical with the classical complement lesion observed on membranes after electron microscopy Correspondence: Dr S. Bhakdi, Institute for Medical Microbiology, Schuberstrasse 1,6300 Giefien, W. Germany.
0019-2805/79/0800-0901 $02.00 ©) 1979 Blackwell Scientific Publications
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protein functions as an inhibitor/inactivator of nascent C5b-7 (Podack, Kolb & Muller-Eberhard, 1977; Podack, Halverson, Esser, Kolb & Muller-Eberhard, 1978; Podack & Muller-Eberhard, 1978). According to this concept, the S-protein attaches itself to C5b-7 during complex generation in serum, thereby abrogating the potential of the trimolecular complex to bind to bystander lipid bilayers (Podack et al., 1977). Fluidphase C5b-9 is now designated 'SC5b-9' to denote the presence of the S-protein (Podack et al., 1977; Podack et al., 1978; Podack & Muller-Eberhard, 1978). As in previous communications (Bhakdi et al., 1978; Bhakdi & Tranum-Jensen, 1978), we will refer to the membrane C5b-9 complex as 'C5b-9(m)' to denote its membrane origin. Podack & Miiller-Eberhard have recently presented data suggesting that the S-protein may be selectively removed from SC5b-9 through the action of deoxycholate (DOC) (Podack et al., 1978; Podack & MullerEberhard, 1978). The DOC-treated SC5b-9 complex purportedly regains its ability to bind detergent (Podack et al., 1978; Podack & Muller-Eberhard, 1978). In this communication, we do not confirm such a dissociation of the S-protein, with concomitant alteration of biochemical or ultrastructural properties of SC5b-9, through the action of DOC. We wish, however, to present evidence that SC5b-9 can be proteolytically transformed into an amphiphilic macromolecule whose antigenic, ultrastructural, and lipid-binding properties closely resemble those of the C5b-9(m) membrane attack complex.
MATERIALS AND METHODS Sources of reagents and antisera have been given previously (Bhakdi, Bjerrum, Rother, Knufmann & Wallach, 1975; Tranum-Jensen et al., 1978) Native and proteolysed C5b-9(m), and SC5b-9 were isolated as described (Bhakdi, Ey & Bhakdi-Lehnen, 1976; Tranum-Jensen et al., 1978; Bhakdi et al., 1979). Antisera to SC5b-9 were raised in three rabbits following procedures described by Harboe & Ingild (1973). Antiserum to SC5b-9 neoantigens (Kolb & Muller-Eberhard, 1975b) was prepared by absorption of anti-SC5b-9 antiserum with 1-5 volumes of human serum in the presence of 10 mm EDTA (final concentration) (see also Bhakdi et al., 1978a).
DOC-treatment Samples of SC5b-9 (I mg/ml in 10 mM Tris, 50 mM,
NaCI, 15 mm NaN3, pH 8-2) were made 2% (w/v) in DOC and allowed to stand at room temperature (25°) for 2-6 h. Trypsin inhibitor (20 ug/ml) was added to ensure than no proteolytic degradation took place during this period of incubation. Samples were subsequently subjected either to ultracentrifugation through linear 10-50% sucrose density gradients in the same buffer containing 0.10% DOC, or to descending chromatography over Sepharose 6B equilibrated with 0.10% DOC. Proteolysis of SC5b-9 Experiments were performed following two protocols. (a) Samples were made 10% in Triton X-100+ 10% in DOC, respectively. They were treated with a combination of trypsin + a-chymotrypsin at final individual enzyme concentrations of 20 ug/ml for 6-18 h at 37°. Thereafter, proteolysed SC5b-9 was recovered through centrifugation through linear sucrose density gradients containing 0 1o% Triton X-100 +0 1o% DOC, or through chromatography over Sepharose 6B containing the same detergents. Fractions containing reisolated, proteolysed SC5b-9 were directly analysed in SDS polyacrylamide gel electrophoresis, quantitative immunoelectrophoresis, and utilized in membrane reconstitution experiments. (b) Alternatively, samples of SC5b-9 were made 0.1% in Triton X-100 (for immunoelectrophoretic analyses) or 1I% in DOC (for membrane reconstitution studies), proteolysed, and directly utilized without repurification of the proteolysed complex. Membrane reconstitition DOC-solubilized sheep erythrocyte membrane lipids (Bhakdi & Tranum-Jensen, 1978) were used in all experiments. Approximately 3 mg of lipids in 3 ml DOC solution were added to 0 5 mg SC5b-9 in 1 ml buffer solution. The protein samples employed in different experiments were the following: (1) purified SC5b-9, not pre-treated with DOC; (2) DOC-treated SC5b-9, re-isolated by sucrose density gradient centrifugation or Sepharose chromatography after pretreatment with this detergent; (3) proteolysed SC5b-9, re-isolated after protease treatment through sucrose density gradient centrifugation or Sepharose chromatography in the presence of 0-1% Triton X-100+0-1% Doc; (4) SC5b-9 which was added to lipid in DOC and directly proteolysed in detergent/lipid solution. All samples were dialysed against 400 volumes of 4 mM Tris, 8 mm NaN3, pH 8 0 for 2 h at 250. MgC12 (3 mM final concentration) was than added and dialysis
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continued for 36-45 h. Vesicle preparations were concentrated and the vesicles floated through sucrose solution as described (Bhakdi & Tranum-Jensen, 1978). Electron microscopy was performed on dialysed aliquots after each step in the vesicle preparations. Lipid analyses Extensively dialysed fractions from sucrose density gradients were lyophilized, extracted with chloroform/methanol (2:1), and analysed in conventional thin-layer chromatography on silica gels using a chloroform/methanol/water (65:25:4) solvent system. Electron microscopy Negative staining and electron microscopy was performed as described (Tranum-Jensen et al., 1978; Bhakdi & Tranum-Jensen, 1978) using hydrophilized, carbon-coated formvar films carried on copper grids and 2% silicotungstate as staining solution.
SDS-Polyacrylamide gel electrophoresis A discontinuous slab gel electrophoresis system based on the Laemmli procedure (Laemmli, 1970) was utilized. Modifications were as detailed by Bosch, Orlich, Klenk & Rott (1979). Running gels were 10% acrylamide, 0-18% N,N-methylenebisacrylamide, in 400 mM Tris, 2 5 M urea, pH 8-9. Stacking gels were 5% acrylamide, 0-15% N,N-methylenebisacrylamide, 60 mm Tris, 5 M urea, pH 6-8. The electrophoresis buffer was 50 mM Tris, 385 mM glycin, pH 8-3. The cathodal buffer contained 0 3% (w/v) SDS. Gels were calibrated with RNA polymerase, bovine serum albumin, trypsin inhibitor and cytochrome c (Boehringer). Electrophoresis was performed at 100 for I h at 70 V followed by another 5 h at 100 V.
Quantitative immunoelectrophoresis, charge-shift crossed immunoelectrophoresis, and autoradiographical analyses of Triton-binding in crossed immunoelectrophoresis were performed as described (Bhakdi et al., 1975; Bhakdi, Bhakdi-Lehnen & Bjerrum, 1977; Bjerrum & Bhakdi, 1977). RESULTS Effect of proteolysis on the antigenicity of SC5b-9 SC5b-9 was proteolysed and subsequently directly analysed in double-diffusion. The proteolysed complex still reacted with antisera specific for native C5, C6 and C9 to yield coalescing immunoprecipitates
Figure 1. (A) Rocket immunoelectrophoresis of native and proteolysed SC5b-9 using an antiserum to C5b-9(m) neoantigens (7 MI/cm2). Samples applied were (1) purified SC5b-9; (2) identical sample of SC5b-9 which was proteolysed with trypsin + a-chymotrypsin prior to immunoelectrophoresis; (3) SC5b-9 which had been treated with DOC and re-isolated in the presence of DOC; (4) proteolysed and re-isolated SC5b-9. Proteolysis exposes neoantigenic determinants characteristic of the membrane attack complex on SC5b-9. (B) The same samples were analysed using an unabsorbed antiserum to C5b-9(m). Note the analogeous, stronger precipitation reaction of this antiserum with the proteolysed SC5b-9 complex.
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(not shown). This indicated that these terminal complement components remain complexed to each other after proteolytic attack, as is also the case for the membrane C5b-9(m) complex (Tranum-Jense et al., 1978; Bhakdi et al., 1978). Proteolysis induced a striking antigenic modification of SC5b-9, which was detectable with an antiserum directed against the neoantigens of C5b-9(m). Native or DOC-treated SC5b-9 cross-react weakly with this antiserum. The very faint immunoprecipitates obtained at the given antiserum concentration are not visible on the reproduction of Fig. IA (wells 1 and 3). By contrast, the proteolysed complex was well precipitated with the same antiserum under identical experimental conditions (Fig. IA, wells 2 and 4). Analogously the unabsorbed antiserum to C5b-9(m) reacted more strongly with proteolysed SC5b-9 than with the native SC5b-9 complex (Fig. 1 B: the stronger reaction of anti-C5b-9(m) with proteolysed SC5b-9 is apparent from the lower heights of the rockets above wells 2 and 4, compared to the heights of the rockets above wells 1 and 3. The same amount of protein was applied in all wells). Thus, proteolysis of SC5b-9 exposed neoantigenic determinants on the complex which are characteristic of the C5b-9(m) membrane attack complex, and which are not originally exposed on the native SC5b-9 molecule. This antigenic modification was not observed after treatment of SC5b-9 with DOC alone (Fig. 1, well 3), or with a combination of Triton + DOC (not shown).
Unusual reaction of anti-SC5b-9 antiserum Figure 2 depicts a double-diffusion analysis of C5b-9(m) and SC5b-9 using an antiserum raised SC5b-9. This unabsorbed antiserum (well 1) precipitated both the membrane (m) and the fluid-phase (s) complex. After absorption with EDTA-serum, the antiserum was still capable of precipitating both C5b-9(m) and SC5b-9(m) (Fig. 2, well 2). This finding confirms the presence of neoantigens present on both C5b-9(m) and SC5b-9 detectable with such antisera (Kolb & MullerEberhard, 1975b). Anti-SC5b-9 was absorbed with 1-5 volumes of unfractionated, inulin-activated serum in 10 mM EDTA. The absorbed antiserum (well 3) then no longer precipitated native SC5b-9. This antiserum, however, was still capable of precipitating the membrane C5b-9 complex, as well as the proteolysed SC5b-9 complex
Figure 2. Double diffusion analyses of purified membrane C5b-9 (wells labelled 'm') and fluid-phase SC5b-9 (wells labelled 's'), using an antiserum raised against SC5b-9. Well I contained anti-SC5b-9; the unabsorbed antiserum precipitated both C5b-9(m) and SC5b-9. Well 2 contained the same antiserum which had been absorbed with EDTA-serum. This antiserum, directed against SC5b-9 neoantigens, no longer precipitated any native serum protein (Kolb & Muller- Eberhard, 1975b; Bhakdi et al., 1978), but still precipitated both C5b-9(m) and SC5b-9. Well 3 contained purified and concentrated immunoglobulins of an anti-SC5b-9 antiserum which had been absorbed with inulin-activated serum. This antibody preparation no longer precipitated SC5b-9, but still precipitated the membrane C5b-9 complex. the anti-SC5b-9 antiserum contains antibodies directed against neoantigens of the actual membrane attack complex which are not primarily exposed on the native SC5b-9 molecule.
Proteolytic transformation of SCSb-9
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(the latter reaction is not shown in Fig. 2). Thus, the antiserum raised against SC5b-9 exhibits similar specificity as an antiserum raised against the membrane complex. It contains antibodies directed against neoantigens of the actual membrane attack complex C5b-9(m), which are not primarily exposed on the native SC5b-9 molecule. When anti-SC5b-9 was absorbed with an excess of washed, complement-lysed sheep erythrocyte membranes, or with isolated C5b-9(m), the antiserum subsequently failed to precipitate either C5b-9(m) or SCSb-9. It also did not precipitate any single serum protein. Thus, we have not yet succeeded in producing an antiserum that is entirely specific for SC5b-9, or in obtaining antibodies against the S-protein. Behaviour of proteolysed SC5b-9 in sucrose density gradient ultracentrifugation and Sepharose chromatography Native SC5b-9 sediments as a 22 5S protein in a sucrose density gradient (Kolb & Miuller-Eberhard, 1973; Podack et al., 1976; Bhakdi et al., 1979), and elutes in a molecular region comparable to that of IgM on Sepharose 6B (Kolb & Muller-Eberhard, 1975a; Bhakdi et al., 1979). Following proteolysis, the complex no longer yielded a symmetrical sedimenting peak in a sucrose density gradient, but smeared out over a region corresponding to > 28S (Fig. 3B). On a Sepharose 6B column the protein eluted with and directly after the void volume (not shown). The re-isolated, proteolysed SC5b-9 complex yielded a single immunoprecipitate of f-electrophoretic mobility when tested in a crossed immunoelectrophoresis using a polyspecific antiserum against human serum proteins (not shown). In the electron microscope, markedly aggregated ring and rectangular structures, reminiscent of the C5b-9(m) complex viewed en face and in profile, were observed in preparations of purified, proteolysed SC5b-9 re-isolated by Sepharose chromatography. The aggregates persisted despite the presence of Triton+ DOC, impeding ultrastructural image interpretation. Data on the ultrastructure of the complex, however, became available through membrane reconstitution experiments to be described below. No alteration in the sedimentation behaviour of the SC5b-9 complex was observed following treatment with DOC alone. DOC-treated SC5b-9 also eluted on Sepharose at an identical position as did native
SC5b-9 (not shown).
Figure 3. Sedimentation behaviour of native and proteolysed SC5b-9 in a sucrose density gradient. SC5b-9 was treated with DOC at a final concentration of 2%. An unproteolysed sample (A) was centrifuged through a linear 10-50% sucrose density gradient containing 0 1% DOC at 150,000 g for 13 h (5 ml gradient, rotor SW 50 1). A proteolysed sample (B) was centrifuged in parallel. The positions of the protein complexes in the gradients were determined by fused rocket immunoelectrophoresis of the collected fractions using an antiserum to C5b-9(m). Direction of sedimentation; right to left. Arrows point to the sedimentation positions of haemoglobin (Hb), IgG and IgM in the same gradients. native or DOC-treated SC5b-9 sediments as 22 5S protein; after proteolytic attack, the sedimentation coefficient of the complex increases to > 28S.
SDS polyacrylamide gel electrophoresis The gel electrophoresis patterns of SC5b-9 (a), DOCtreated SC5b-9 (b), proteolysed and re-isolated SC5b-9 (c), and proteolysed C5b-9(m) (d) are shown in Fig. 4. No differences in the protein banding patterns were observed between DOC-treated and native
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Figure 4. SDS polyacrylamide gel electrophoresis of SC5b-9 (gel a), DOC-treated SC5b-9 (gel b), proteolysed and reisolated SC5b-9 (gel c), proteolysed, isolated C5b-9(m) (gel d). Complement components are labelled according to Kolb & Muller-Eberhard (1975a) and Podack et al. (1976), after correlation of the protein bands (Bhakdi et al., 1976; Bhakdi et al., 1979)*. The C5b band persists in both SC5b-9 and C5b-9(m) following proteolytic attack. C6 and the S-protein are degraded. The residual protein bands in the molecular weight region of 50 K and 75-100 K have not been identified. The protein banding patterns of proteolysed SC5b-9 and C5b-9(m) are not identical. Direction of electrophoresis; top to bottom. Coomassie brilliant blue.
SC5b-9 (gels a and b). Discontinuous gel electrophoresis provides superior resolution of the protein bands compared to the continuous system previously employed (Bhakdi et al., 1979). In the region of C6, C7 and anodal to C9, additional minor bands are observed. Of these, the band cathodal to C6 is inconstant; the other bands are constantly present. As the latter are also observed in gel electrophoresis patterns of membrane C5b-9, they probably represent molecular derivatives of the C5b-C9 complement components. Alternatively, they may reflect the presence of microheterogeneous species of C6-C9. Proteolysis removed the protein bands corresponding to C6 and the S-protein. C5b was not affected. Five protein bands were constantly visible in the region of 90 K, 85 K, 80 K, 75 K and 50 K (gel c); an inconstant * The designation of the C8# band is preliminary.
band was observed at 100 K. The individual bands appearing after proteolysis of SC5b-9 have not been identified. Proteolysed C5b-9(m) exhibited a similar protein banding pattern (gel d), but slight deviations were observed in the region of 80-90 K. This suggests some differences in the suspectibility of peptide bonds toward proteolysis in SC5b-9 and C5b-9(m).
Binding of detergent by proteolysed SC5-9 Native SC5b-9 is hydrophilic according to the criterion of charge-shift crossed immunoelectrophoresis; no indication of Triton-binding has been obtained using the method of autoradiography in crossed immunoelectrophoresis (Bhakdi et al., 1979). Following proteolysis, the fluid-phase complex exhibited a significant charge-shift in DOC and a borderline charge-shift
Proteolytic transformation of SC5b-9 in CTAB; a faintly radioactive immunoprecipitate was obtained in crossed immunoelectrophoresis after incorporation of radioactive Triton in the agarose gel (not shown). The detergent-binding studies thus yielded an indication that proteolysis exposes apolar regions on the complex which may bind Triton. The contention that proteolysed SC5b-9 is amphiphilic received support from lipid-binding studies, as demonstrable by membrane reconstitutions.
Membrane reconstitution Figure 5A depicts an electron micrograph of an unfractionated preparation of vesicles formed in the presence of DOC-treated SC5b-9. There is no visible interaction between the lipid vesicles (Li) and the pro-
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tein complexes; the latter are seen as fuzzy, globular structures with irregular contours (Bhakdi et al., 1979). Blurred ring and rectangular profiles, reminiscent of the structure of C5b-9(m), are sporadically found (inset, Fig. 5A). Figure 6A shows that following flotation of the vesicles, virtually all of the DOCtreated SC5b-9 remains at the bottom of the sucrose density gradient. Thus, DOC-treated SC5b-9 does not associate with lipid under our experimental conditions. As verified by thin-layer chromatography, all lipid was recovered in the top fractions of such sucrose gradients. Figure 5B depicts a lipid vesicle preparation prepared by using the same SC5b-9 sample which was, however, proteolysed in the presence of DOC and lipid
5B
Figure 5 (A) Electron micrograph of a negatively stained preparation of lipid vesicles formed in the presence of native SC5b-9. The lipid vesicles (Li) appear as smoothly contoured structures of a uniform, low density. The complement complexes (s) appear as uniformly dispersed, irregular structures with an approximate diameter of 20 nm. Single complexes are sporadically observed as fuzzy, ring-shaped or rectangular profiles (inset at lower left), reminiscent of the structure of the C5b-9(m) membrane attack complex (compare with Fig. 6F). No specific association between lipid vesicles and the native SC5b-9 complex could be detected in such preparations. (B) When lipid vesicles were formed in the presence of proteolysed SC5b-9, massive aggregates resulted. Lipid vesicles are seen to carry structures (p) similar to the membrane-derived C5b-9 complexes. Nearly all lipid vesicles exhibit central stain deposit, indicating permeability to the stain molecules. A single vesicle which escaped incorporation of the complex is seen at lower right (asterisk). Scale bars indicate 100 nm.
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Figure i. (A) Tbe veside preparation of Fig. 5A was applied to the bottom of a sucrose density gradient. Following flotation of the lipid vesicesvirtualy all the protein was recovered at the bottom of the gradient. Thus, native SC5b-9 does not significantly bind to lipid. (B) lhe vesicd preparation of Fig. 5B was treated similarly. Approximately 75% of proteolysed SC5b-9 floated in association with the lipid vesies through sucrose. The direction of the sucrose gradients is given. Detection and relative quantification of protein in the fractions collected after centrifugation was done using rocket immunoelectrophoresis and an antiserum to C5b-9(m). Agarose gels contained 0- 5% Triton X-100.
prior to detergent removal. Here a structural modification of the SC5b-9 molecules, which appear bound to the lipid vesicles, becomes apparent. Flotation of such samples reveals that a fraction (50-80%) of the protein is lipid-bound (Fig. 6B), and is recovered in association with the vesicles. Similar results were obtained with proteolysed SC5b-9 re-isolated from sucrose density gradients or Sepharose columns, but the degree of protein incorporation into the vesicles
was reduced compared to that obtained by the former method. This may be due to the increase in molecular aggregation of proteolysed SC5b-9 during the procedure of re-isolation. Marked ultrastructural changes accompany proteolysis of the SC5b-9 complex, with the appearance of numerous ring-shaped profiles of approximately 20 nm outer diameter and with a central stain filling, as can be discerned in large numbers in Fig. 5B. The
difficulties of analysing individual complexes in their aggregated form was circumvented by examination of the top fractions of sucrose density gradients which contained vesicles that had incorporated only small numbers of complexes. The incorporated complexes covered a range of shapes, of which distinct cylindrical structures, morphologically indistinguishable from reincorporated C5b-9(m) complexes, dominated (Figs 5B and 7D-F. More foliaceous, branching structures were also observed, however, and representative examples of these are shown in Fig. 7A-C. Lipid vesicles which escaped incorporation of complexes are characteristically empty of stain, as can be seen in the preparations of Figs 5A, B and 7A, F. Incorporation of proteolytically transformed SC5b-9 complexes renders the vesicles permeable to the silicotungstate stain, as indicated by a central stain deposit, found in all vesicles carrying a complex (Figs 5B and 7A-E). Thus, incorporation of a proteolysed SC5b-9 complex into a lipid vesicle appears to create a transmembrane pore at the complex attachment site, as is also the case with the membrane attack complex (Bhakdi & Tranum-Jensen, 1978, and Fig. 7F).
DISCUSSION
Figure 7. (A-E) These illustrate the appearance of lipid vesicles recovered in the top fraction of a sucrose density gradient following flotation of the vesicle preparation illustrated in Figs 5B and 6B. The proteolytically transformed SC5b-9 complexes incorporated into lipid vesicles exhibit somewhat heterogeneous shapes ranging from foliaceous structures (A-C) to distinct cylindrical structures. The latter dominate and are morphologically indistinguishable from the membrane-derived C5b-9 complexes (D and E). An unfractionated preparation of lipid vesicles reconstituted with the membrane complex is shown for comparison in F. Vesicles which escape incorporation of complement complexes (A and F) are empty of stain. Scale bars indicate 100 nm.
The assembly of C5b-C9 into the membrane C5b-9 complex constitutes the only known case of a molecular transition of hydrophilic serum proteins into a protein complex that is amphiphilic, and that can associate with membrane lipids in a manner akin to that of an integral membrane protein. We found that the exposure of lipid-binding regions in the complex was, somewhat unexpectedly, not confined to the situation where complex assembly occurred in immediate contact with a lipid bilayer. Apolar molecular regions could be exposed through proteolytic attack of the pre-formed SC5b-9 complex in solution. This resulted in a transformation of SC5b-9 into an amphiphilic macromolecule that structurally resembled the membrane attack complex of complement. In disagreement with previous data of Podack et al. (Podack et al., 1978; Podack & Muller-Eberhard, 1978), we did not find that treatment of SC5b-9 with DOC alone altered the detergent- or lipid-binding properties of the
complex. Proteolytic attack appeared to leave the 'core' of the SC5b-9 complex intact. This was evidenced by (a) the persistence of C5, C6 and C9 antigenic determinants on the proteolysed complex; (b) the persistence of C5b-9 neoantigens; (c) the apparent large molecular
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size of the proteolysed complex as judged by sucrose density gradient ultracentrifugation and Sepharose chromatography (these findings, however, are not compelling since the complex is aggregated; (d) the persistence of macromolecular structure as evaluated by electron microscopy; (e) the persistence of high molecular weight protein bands in SDS-polyacrylamide gel electrophoresis. Several observations indicated that proteolysed SC5b-9, although similar, is not entirely identical to its membrane counterpart. The SDS gel electrophoresis patterns of proteolysed SC5b-O(m) and C5b-9(m) were not identical. In both cases, C5b was retained in high molecular weight form. The persistence of C5b, as well as of the other residual, high molecular weight peptides may be due to stabilizing inter- and intrachain disulphide bonds within these molecules. The present gel electrophoresis data cannot yet be correlated to previous data of Hammer et al. (Hammer, Nicholson & Mayer, 1975; Hammer, Shin, Abramovitz & Mayer, 1977). These investigators reported 40-50% removal of radiolabelled C5 from membranebound guinea-pig C5b-7 complexes, but C5 removal from the C5b-9 complex was not studied (Hammer et al., 1975). In a later communication (Hammer et al., 1977), tryptic degradation of guinea-pig C9 in the membrane C5b-9 complex to an 18,000 mol. wt product was described. Our data need not conflict with these results as we deal with the human C5b-9 complex and also have not yet identified the proteolysis products observed in SDS gel electrophoresis. Another indication that proteolysed SC5b-9 is not entirely identical to its membrane counterpart derives from its aggregation behaviour in detergent solution. Whereas monomerized, cylindrical structures are obtained by chromatography of proteolysed C5b-9(m) in the presence of Triton+ DOC, the proteolysed SC5b-9 complex remains markedly aggregated under the same conditions. Aggregation may also be responsible for the smaller charge-shifts found for proteolysed SC5b-9, compared to those of the membrane complex (Bhakdi et al., 1978). Electron microscopy of proteolysed SC5b-9 complexes following their incorporation into artificial lipid vesicles indicated that they possess a similar ultrastructure as C5b-9(m). Proteolysis appeared to remove the fuzzy 'coat' of SC5b-9, revealing what we interpreted to be a cylindrical structure obviously inherent, but masked in the native SC5b-9 molecule. In the majority of cases the vesicle-bound complexes were seen as cylindrical structures projecting approximately
10 nm from the lipid bilayer and rimmed by 20 nm wide annuli. These structures were morphologically identical to the C5b-9(m) complement lesions. Other more foliaceous structures are occasionally observed; these may be related to the structures described by Dourmashkin after assembly of the C5b-8 complex on membranes (Dourmashkin, 1978). The molecular weights of the individual protein subunits comprising the SC5b-9 monomer total approximately 1,000,000 (Kolb & Muller-Eberhard, 1973; Kolb & Muller-Eberhard, 1975a). Hydrodynamic data to be discussed elsewhere are in agreement with this value. The dimensions of native SC5b-9 are comparable to that of C5b-9(m), the diameter of the globule being in the order of 20 nm. The structure observed after proteolysis of SC5b-9 possesses the same size as C5b-9(m) in the electron microscope. A hollow protein cylinder, 10 nm in diameter, 15 nm in height, with walls 1 nm thick, rimmed by an annulus of 4 nm diameter (Bhakdi & Tranum-Jensen, 1978b) would possess a volume of approximately 970 nm3. Assuming a partial specific volume of 0 75, this implies a molecular weight of approximately 800,000, a value which would be in good agreement with the sum molecular weights of the C5b-C9 subunits contained in the
C5b-9(m) complex. The immunochemical reactions of C5b-9(m), SC5b-9 and their respective antisera indicated that antisera raised against native SC5b-9 contained antibodies directed against neoantigens of the actual membrane attack complex, which were not primarily exposed on SC5b-9. This finding suggests that proteolysis of SC5b-9 occurred during molecular processing of SC5b-9 in the immunized animal, leading to exposure of the concealed C5b-9(m) neoantigenic determinants. The possibility of immunochemically distinguishing between SC5b-9 and C5b-9(m) through the use of appropriately absorbed antisera will be of use for investigations into the occurrence of C5b-9 neoantigens on cell surfaces (Sundsmo, Kolb & Miuller-Eberhard, 1978a; Sundsmo, Curd, Kolb & MullerEberhard, 1978b) and in diseased tissues. Proteolytic exposure of apolar, lipid-binding regions on a primarily hydrophilic molecule is a novel observation in serum protein chemistry. The possible biological significance of the SC5b-9 transformation, and the molecular events associated with the hydrophilic-amphiphilic transition await further investigation. Exposure of lipid-binding regions on the macromolecule may be directly due to proteolytic removal of the S-protein, as might be anticipated from
Proteolytic transformation of SCSb-9 data of Podack et al. (1977). Other conformational changes accompanying proteolysis may, however, additionally be responsible for the observed molecular transformation.
ACKNOWLEDGMENTS We are grateful to Ortrud Klump for excellent technical assistance, and to Christine Reitz and Ilona Vogel for photographical and secretarial help. We thank Dr Ari Helenius (European Molecular Biology Laboratory, Heidelberg) for critical discussions, and Dr H.-J. Wellensiek for his continued interest in this work. This study was supported by the Deutsche Forschungsgemeinschaft. REFERENCES BHAKDI S., BJERRUM O.J., ROTHER U., KNUFERMANN H. & WALLACH D.F.H. (1975) Immunochemical analyses of membrane-bound complement: detection of the terminal complement complex and its similarity to 'intrinsic' erythrocyte membrane proteins. Biochim. Biophys. Acta, 406, 21. BHAKDI S., EY P. & BHAKDI-LEHNEN B. (1976) Isolation of the terminal complement complex from target sheep erythrocyte membranes. Biochim. Biophys. Acta, 419, 445. BHAKDI S., BHAKDI-LEHNEN B. & BJERRUM O.J. (1977) Detection of amphiphilic proteins and peptides in complex mixtures: charge-shift crossed immunoelectrophoresis and two-dimensional charge-shift electrophoresis. Biochim. Biophys. Acta 470, 35. BHAKDI S., BJERRUM O.J., BHAKDI-LEHNEN B. & TRANUMJENSEN J. (1978) Complement lysis: evidence for an amphiphilic nature of the terminal membrane CSb-9 complex of human complement. J. Immunol. 121, 2526. BHAKDI S. & TRANUM-JENSEN J. (1978) Molecular nature of the complement lesion. Proc. natn. Acad. Sci. U.S.A. 75, 5655. BHAKDI S., BHAKDI-LEHNEN B., BJERRUM O.J. & TRANUMJENSEN J. (1979) Difference in antigenic reactivity and ultrastructure between fluid-phase SCSb-9 and the C5b-9 membrane attack complex of human complement. FEBS Letts. 99, 15. BJERRUM O.J. & BHAKDI S. (1977) Demonstration of Triton X-100 binding to amphiphilic proteins in crossed immunoelectrophoresis. FEBS Letts, 81, 56. BoRsos T., DOURMASHKIN R.R. & HUMPHREY J.H. (1964) Lesions in erythrocyte membranes caused by immune haemolysis. Nature (Lond.), 202, 251. BOSCH F., ORLICH M., KLENK H.-D. & Rorr R. (1979) The structure of the hemagglutinin, a determinant for the pathogenicity of influenza virus. Virology (In press.) DOURMASHKIN R.R. (1978) The structural events associated with the attachment of complement components to cell membranes in reactive lysis. Immunology, 35, 205.
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HAMMER C.H., NICHOLSON A. & MAYER M.M. (1975) On the mechanism of cytolysis by complement: evidence on insertion of C5b and C7 subunits of the C5b, 6, 7 complex into the phopholipid bilayer of erythrocyte membranes. Proc. natn. Acad. Sci. U.S.A. 72, 5076. HAMMER C.H., SHIN M.L., ABRAMOVITZ A.S. & MAYER M.M. (1977) On the mechanism of cell membrane damage by complement: evidence on insertion of polypeptide chains from C8 and C9 into the lipid bilayer of erythrocytes. J. Immunol. 119, 1. HARBOE N. & INGILD A (1973) Immunization, isolation of immunoglobulins. Estimation and antibody titer. Scand. J. Immunol. 2 (Suppl. 1), 161. HUMPHREY J.H. & DOURMASHKIN R.R. (1969) The lesions in cell membranes caused by complement. Adv. Immunol. 11, 75. KOLB W.P. & MuLLER-EBERHARD H.J. (1973) The membrane attack mechanism of complement. Verification of a stable C5-C9 complex in free solution. J. exp. Med. 138,438. KOLB W.P. & MuLLER-EBERHARD H.J. (1975a) The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex. J. exp. Med. 141, 724. KOLB W.P. & MuLLER-EBERHARD H.J. (1975b) Neoantigens of the membrane attack complex of human complement. Proc. natn. Acad. Sci. U.S.A. 72, 1687. LACHMANN P.J. & THOMPSON R.A. (1970) Reactive lysis: the complement-mediated lysis of unsensitized cells. 11. The characterization of activated reactor as C5b and the participation of C8 and C9. J. exp. Med. 131, 644. LAEMMLI U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophase T4. Nature (Lond.), 227, 680. MAYER M.M. (1972) Mechanism ofcytolysis by complement. Proc. natn. Acad. Sci. U.S.A. 69, 2954. MICHAELs D.W., ABRAMOVITZ A.S., HAMMER C.H. & MAYER M.M. (1976) Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement. Proc. nain. Acad. Sci. U.S.A. 73, 2852. MuJLLER-EBERHARD H.J. (1975) Complement. Ann. Rev. Biochem. 44, 697. PODACK E.R., KOLB W.P. & MuLLER-EBERHARD H.J. (1976) The C5b-9 complex: subunit composition of the classical and alternative pathway-generated complex. J. Immunol. 116, 1431. PODACK, E.R., KOLB W.P. & MULLER-EBERHARD H.J. (1977) The SC5b-7 complex: formation, isolation, properties, and subunit composition. J. Immunol. 119, 2024. PODACK E.R., HALVERSON C., ESSER A.F., KOLB W.P. & MuLLER-EBERHARD H.J. (1978) C5b-9: Regeneration of the ability to interact with lipid by selective removal of the S-protein. J. Immunol. 120, 1792 (Abstr.) PODACK E.R. & MOLLER-EBERHARD H.J. (1978) Binding of deoxycholate, phosphatidylcholine vesicles, lipoprotein and of the S-protein to complexes of terminal complement components. J. Immunol. 121, 1025. SUNDSMO J.S., KOLB W.P. & MuLLER-EBERHARD H.-J. (1 978a) Leukocyte complement: neoantigens of the membrane attack complex on the surface of human leukocytes prepared from defibrinated blood. J. Immunol. 120, 850. SUNDSMO J.S., CURD J.G., KOLB W.P. & MULLER-EBERHARD
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H.J. (1978b) Leukocyte complement: assembly of the membrane attack complex by human peripheral blood leukocytes in the presence and absence of serum. J. Immunol. 120, 855. THOMPSON R.A. & LACHMANN P.J. (1970) Reactive lysis: the complement-mediated lysis of unsensitized cells. I. The
characterization of the indicator factors and its identification as C7. J. exp. Med. 131, 629. TRANUM-JENSEN, J., BHAKDI, S., BHAKDI-LEHNEN, B., BJERRUM, O.J. & SPETH, V. (1978) Complement lysis: the ultrastructure and orientation of the C5b-9 complex on target sheep erythrocyte membranes. Scand. J. Immunol.
7,45.
