Proc. Nat. Acad. Sci. USA Vol. 72, No. 5 pp. 1687-1689, May 1975
Neoantigens of the Membrane Attack Complex of Human Complement (complement 5b-9 complex/multimolecular assembly/membrane damage/ complement activation detection)
WILLIAM P. KOLB AND HANS J. MULLER-EBERHARD Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037
Contributed by Hans J. Muller-Eberhard, February 14, 1976 The membrane attack complex of compleABSTRACT ment is a fusion product of five complement proteins: C5b, C6, C7, C8, and C9. The complex causes complementdependent cell membrane damage. It is assembled following complement activation both on the target cell surface and in the fluid phase. The isolated soluble complex, which has a molecular weight of one million, exhibited reduced expression of the antigenic determinants of the native precursor proteins. Antisera produced to the intact complex contained antibodies to neoantigens which were not detectable on the five precursor proteins. Antisera were rendered neoantigen-specific by adsorption with fresh human serum. Since the adsorbed antisera precipitated the complex, the complex must contain multiple neoantigenic sites. The complex-specific antibodies not only reacted with the soluble complex, but also with the target cell-bound membrane attack complex.
The capacity of complement to impair the structure and function of biological membranes resides in a stable association product of five serum proteins (1, 2). In the course of the complement reaction these five proteins undergo self-assembly to a multimolecular complex which in its nascent state has the ability to bind to the surface of biological membranes. The self-assembling process is initiated through limited enzymatic proteolysis of component C5 by C5 convertase of either the classical complement sequence or the properdin pathway. Fusion of the C5 cleavage product, C5b, with C6, C7, C8, and C9 is the result of multiple adsorptive reactions and is probably associated with conformational changes of these proteins. The unique assembly of the membrane attack complex raised the possibility of formation of complex-specific antigenic sites that are not expressed by the unassembled precursors. The purpose of this communication is to document the occurrence of neoantigens on the cell-bound and on the soluble complex. MATERIALS AND METHODS
The stable association product of C5b, C6, C7, C8, and C9 is referred to as C5b-9 complex. It was isolated from whole human serum after activation of the complement system with particulate inulin as described (1, 2). C5 (3), C6 (4), C7 (5), C8 (6), and C9 (7) were isolated from human serum by previously published procedures. Antisera to the isolated C5b-9 complex and to each of the five precursor proteins were raised in rabbits by injecting 50-100 ,g of protein in complete Freund's adjuvant into the popliteal lymph nodes (8). The anti-C5b-9 antiserum was rendered specific for the neoantigens by adsorption with a 6-fold excess of fresh human Abbreviations: C, complement; E, sheep erythrocytes; A, antibody. 1687
serum in the presence of 10 mM EDTA at 40 for 12 hr. The immunoglobulin fraction of the serum mixture was obtained by precipitation with 50% saturated ammonium sulfate. The precipitate was dialyzed against 0.02 M sodium phosphate buffer, pH 8, and the dissolved protein was applied to a DE32 DEAE-cellulose column, equilibrated with the same buffer. The protein fraction corresponding to -y-globulin was recovered and concentrated. Immunochemical doublediffusion-in-gel analyses were performed in 1.5% agarose according to Ouchterlony (9). Hemagglutination was carried out in microtitration plates with various intermediate complexes of sheep erythrocytes (E), antibody (A) to E, and isolated human complement proteins (10). Agglutination of these complexes was determined with doubling dilutions of the antiC5b-9 immunoglobulin fraction. To examine the ability of isolated C5b-9 to inhibit the agglutinating activity, 50 M1 of various dilutions of isolated C5b-9 (0.25-250 ,ug/ml) was incubated with 50 ul of a 1:10 dilution of complex-specific antibody. After 60 min incubation at 370 the solutions were diluted in microtitration plates (Cooke Engineering Co., Alexandria, Va.) and 25 ,u1 of 1 X 108 EAC1-7 was added to each well.
RESULTS Comparative analysis of the C5b-9 complex and whole human serum with antisera to the precursor proteins revealed antigenic deficiencies of the complex compared to native C5, C6, and C8 (Fig. 1A, B, and D). This conclusion is based on the observation that the precipitin line formed with whole human serum spurs over the precipitin line produced by the complex. The antigenic determinants of C7 and C9, as detected by the antisera employed, were fully expressed by the complex, as evidenced by the pattern of antigenic identity (Fig. 1C and E). In contrast, the antiserum produced to the isolated C5b-9 complex detected antigenic determinants on the complex that were not expressed in whole human serum. As shown in Fig. 2A, the precipitin line produced by the complex formed a pronounced spur over the precipitin lines formed with whole human serum. The anti-complex antiserum was rendered complex-specific by adsorption with fresh whole human serum. Fig. 2B demonstrates the ability of the adsorbed antiserum to enter into a precipitin reaction with the isolated complex in complete absence of a detectable reaction with fresh human serum. Further, the antiserum readily detected the complex after its formation in serum following complement activation. A pattern of antigenic identity was obtained with the isolated complex and activated serum.
1688
Immunology: Kolb and Muller-Eberhard A
B
.
C5b-9
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.00f.1
An>,
C
WH
C5b-9
I.
£
w
HS
.:2,
Anti ^ C7
MAti C6
D
(1975)
.I.
tb-91
WHS
Proc. Nat. Acad. Sci. USA 729
4 F --,
WHS
WIHS
C5b-9
C5b-9
H
WHS
C...
An
Anti
J9
. :C3;
*1.
FIG. 1. Comparative antigenic expression of isolated C5b-9 complex and its precursor proteins. Double diffusion in 1.5% agarose gel conducted by reacting C5b-9 and whole human serum (WHS) with antiserum to the following terminal complement proteins for 16-48 hr at 220: (A) C5, (B) C6, (C) C7, (D) C8, (E) C9, and (F) C3. All slides were washed in 0.15 M NaCl, dried, and stained with 0.05% amido black.
was
The neoantigenic complex-specific properties were also expressed by the target cell-bound, partially assembled membrane attack complex. Antibody-coated sheep erythrocytes were reacted with complement components C1 to C7 to form the non-lytic intermediate complex EAC1-7 (10). A portion of this preparation was converted to the complex EAC1-8 with isolated C8. As summarized in Table 1, both intermediate complexes could be agglutinated with the C5b-9-neoantigenspecific antiserum. EAC1-9 could not be examined conclusively because these cells undergo rapid lysis. In addition, agglutination of EAC1-7 by the neoantigen-specific antibody could be completely inhibited by preincubating the antibody with isolated C5Sb-9 complex. As little as 250 nanograms of A
B N
WHS
C5b-9
Anti C5b-9
(Table 2). DISCUSSION In recent years we have expended considerable effort to elucidate the chemical nature and the mode of action of the membrane attack unit of complement because it constitutes the only known mechanism of blood plasma that is capable of impairing biological membranes. The proteins C5, C6, C7, C8, and C9 could be distinguished with respect to their function from earlier acting components and defined as the functional unit that is solely responsible for complementdependent membrane damage (11, 12). After isolation of the five proteins was accomplished (3-7), earlier indications for functionally relevant molecular interactions between these proteins (13-15) were corroborated and the C5b-9 complex was postulated (10), demonstrated (1) and isolated (2). Dissociation of the complex with sodium dodecyl sulfate and analysis of the subunit composition confirmed our earlier postulate that C5 is incorporated in the form of C5b and that the other components are recruited apparently without undergoing chemical change. Enzymatic cleavage of C5 thus constitutes the biochemical signal for self-assembly by adsorption of the C5Sb-9 complex. It is likely that the quaternary
TABLE 1. Neoantigens on target cell-bound membrane attack components
~WHS
Inulin
C5b-9 per ml caused detectable agglutination inhibition
WHS'
Inulln
FIG. 2. Neoantigen expression by the C5b-9. complex. (A) C5b-9 and whole human serum reacted with antiserum raised in rabbit to the isolated, undissociated complex. (B) This antiserum, subsequent to adsorption with whole human serum, was reacted with C5b-9 either in isolated form or as it occurs in whole human serum after incubation with inulin.
Cell-complement complex EAC1-3 EAC1-5 EAC1-6 EAC1-7 EAC1-8
Agglutination with antisera to: C6 C7 C8 C5b9* C5 Neg. Pos. Neg. Pos. Pos. Pos.
Pos. Pos. Pos.
-
Pos.
-
-
Pos. Pos. Pos.
* Rendered neoantigen-specific by adsorption with fresh whole human serum.
Proc. Nat. Acad. Sci. USA 72
Neoantigens of Membrane Attack Complex of Complement
(1975)
TABLE 2. Agglutination of EAC1-7 by C5b-9-neoantigenspecific antibody: inhibition by isolated C5b-9 C5b-9 (,sg/ml)*: Reciprocal of minimum agglutinating dilution of antibody:
0
640
0.25 320
2.5
160
25
250
Neoantigens of the Membrane Attack Complex of Human Complement (complement 5b-9 complex/multimolecular assembly/membrane damage/ complement activation detection)
WILLIAM P. KOLB AND HANS J. MULLER-EBERHARD Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037
Contributed by Hans J. Muller-Eberhard, February 14, 1976 The membrane attack complex of compleABSTRACT ment is a fusion product of five complement proteins: C5b, C6, C7, C8, and C9. The complex causes complementdependent cell membrane damage. It is assembled following complement activation both on the target cell surface and in the fluid phase. The isolated soluble complex, which has a molecular weight of one million, exhibited reduced expression of the antigenic determinants of the native precursor proteins. Antisera produced to the intact complex contained antibodies to neoantigens which were not detectable on the five precursor proteins. Antisera were rendered neoantigen-specific by adsorption with fresh human serum. Since the adsorbed antisera precipitated the complex, the complex must contain multiple neoantigenic sites. The complex-specific antibodies not only reacted with the soluble complex, but also with the target cell-bound membrane attack complex.
The capacity of complement to impair the structure and function of biological membranes resides in a stable association product of five serum proteins (1, 2). In the course of the complement reaction these five proteins undergo self-assembly to a multimolecular complex which in its nascent state has the ability to bind to the surface of biological membranes. The self-assembling process is initiated through limited enzymatic proteolysis of component C5 by C5 convertase of either the classical complement sequence or the properdin pathway. Fusion of the C5 cleavage product, C5b, with C6, C7, C8, and C9 is the result of multiple adsorptive reactions and is probably associated with conformational changes of these proteins. The unique assembly of the membrane attack complex raised the possibility of formation of complex-specific antigenic sites that are not expressed by the unassembled precursors. The purpose of this communication is to document the occurrence of neoantigens on the cell-bound and on the soluble complex. MATERIALS AND METHODS
The stable association product of C5b, C6, C7, C8, and C9 is referred to as C5b-9 complex. It was isolated from whole human serum after activation of the complement system with particulate inulin as described (1, 2). C5 (3), C6 (4), C7 (5), C8 (6), and C9 (7) were isolated from human serum by previously published procedures. Antisera to the isolated C5b-9 complex and to each of the five precursor proteins were raised in rabbits by injecting 50-100 ,g of protein in complete Freund's adjuvant into the popliteal lymph nodes (8). The anti-C5b-9 antiserum was rendered specific for the neoantigens by adsorption with a 6-fold excess of fresh human Abbreviations: C, complement; E, sheep erythrocytes; A, antibody. 1687
serum in the presence of 10 mM EDTA at 40 for 12 hr. The immunoglobulin fraction of the serum mixture was obtained by precipitation with 50% saturated ammonium sulfate. The precipitate was dialyzed against 0.02 M sodium phosphate buffer, pH 8, and the dissolved protein was applied to a DE32 DEAE-cellulose column, equilibrated with the same buffer. The protein fraction corresponding to -y-globulin was recovered and concentrated. Immunochemical doublediffusion-in-gel analyses were performed in 1.5% agarose according to Ouchterlony (9). Hemagglutination was carried out in microtitration plates with various intermediate complexes of sheep erythrocytes (E), antibody (A) to E, and isolated human complement proteins (10). Agglutination of these complexes was determined with doubling dilutions of the antiC5b-9 immunoglobulin fraction. To examine the ability of isolated C5b-9 to inhibit the agglutinating activity, 50 M1 of various dilutions of isolated C5b-9 (0.25-250 ,ug/ml) was incubated with 50 ul of a 1:10 dilution of complex-specific antibody. After 60 min incubation at 370 the solutions were diluted in microtitration plates (Cooke Engineering Co., Alexandria, Va.) and 25 ,u1 of 1 X 108 EAC1-7 was added to each well.
RESULTS Comparative analysis of the C5b-9 complex and whole human serum with antisera to the precursor proteins revealed antigenic deficiencies of the complex compared to native C5, C6, and C8 (Fig. 1A, B, and D). This conclusion is based on the observation that the precipitin line formed with whole human serum spurs over the precipitin line produced by the complex. The antigenic determinants of C7 and C9, as detected by the antisera employed, were fully expressed by the complex, as evidenced by the pattern of antigenic identity (Fig. 1C and E). In contrast, the antiserum produced to the isolated C5b-9 complex detected antigenic determinants on the complex that were not expressed in whole human serum. As shown in Fig. 2A, the precipitin line produced by the complex formed a pronounced spur over the precipitin lines formed with whole human serum. The anti-complex antiserum was rendered complex-specific by adsorption with fresh whole human serum. Fig. 2B demonstrates the ability of the adsorbed antiserum to enter into a precipitin reaction with the isolated complex in complete absence of a detectable reaction with fresh human serum. Further, the antiserum readily detected the complex after its formation in serum following complement activation. A pattern of antigenic identity was obtained with the isolated complex and activated serum.
1688
Immunology: Kolb and Muller-Eberhard A
B
.
C5b-9
-~ ~
.00f.1
An>,
C
WH
C5b-9
I.
£
w
HS
.:2,
Anti ^ C7
MAti C6
D
(1975)
.I.
tb-91
WHS
Proc. Nat. Acad. Sci. USA 729
4 F --,
WHS
WIHS
C5b-9
C5b-9
H
WHS
C...
An
Anti
J9
. :C3;
*1.
FIG. 1. Comparative antigenic expression of isolated C5b-9 complex and its precursor proteins. Double diffusion in 1.5% agarose gel conducted by reacting C5b-9 and whole human serum (WHS) with antiserum to the following terminal complement proteins for 16-48 hr at 220: (A) C5, (B) C6, (C) C7, (D) C8, (E) C9, and (F) C3. All slides were washed in 0.15 M NaCl, dried, and stained with 0.05% amido black.
was
The neoantigenic complex-specific properties were also expressed by the target cell-bound, partially assembled membrane attack complex. Antibody-coated sheep erythrocytes were reacted with complement components C1 to C7 to form the non-lytic intermediate complex EAC1-7 (10). A portion of this preparation was converted to the complex EAC1-8 with isolated C8. As summarized in Table 1, both intermediate complexes could be agglutinated with the C5b-9-neoantigenspecific antiserum. EAC1-9 could not be examined conclusively because these cells undergo rapid lysis. In addition, agglutination of EAC1-7 by the neoantigen-specific antibody could be completely inhibited by preincubating the antibody with isolated C5Sb-9 complex. As little as 250 nanograms of A
B N
WHS
C5b-9
Anti C5b-9
(Table 2). DISCUSSION In recent years we have expended considerable effort to elucidate the chemical nature and the mode of action of the membrane attack unit of complement because it constitutes the only known mechanism of blood plasma that is capable of impairing biological membranes. The proteins C5, C6, C7, C8, and C9 could be distinguished with respect to their function from earlier acting components and defined as the functional unit that is solely responsible for complementdependent membrane damage (11, 12). After isolation of the five proteins was accomplished (3-7), earlier indications for functionally relevant molecular interactions between these proteins (13-15) were corroborated and the C5b-9 complex was postulated (10), demonstrated (1) and isolated (2). Dissociation of the complex with sodium dodecyl sulfate and analysis of the subunit composition confirmed our earlier postulate that C5 is incorporated in the form of C5b and that the other components are recruited apparently without undergoing chemical change. Enzymatic cleavage of C5 thus constitutes the biochemical signal for self-assembly by adsorption of the C5Sb-9 complex. It is likely that the quaternary
TABLE 1. Neoantigens on target cell-bound membrane attack components
~WHS
Inulin
C5b-9 per ml caused detectable agglutination inhibition
WHS'
Inulln
FIG. 2. Neoantigen expression by the C5b-9. complex. (A) C5b-9 and whole human serum reacted with antiserum raised in rabbit to the isolated, undissociated complex. (B) This antiserum, subsequent to adsorption with whole human serum, was reacted with C5b-9 either in isolated form or as it occurs in whole human serum after incubation with inulin.
Cell-complement complex EAC1-3 EAC1-5 EAC1-6 EAC1-7 EAC1-8
Agglutination with antisera to: C6 C7 C8 C5b9* C5 Neg. Pos. Neg. Pos. Pos. Pos.
Pos. Pos. Pos.
-
Pos.
-
-
Pos. Pos. Pos.
* Rendered neoantigen-specific by adsorption with fresh whole human serum.
Proc. Nat. Acad. Sci. USA 72
Neoantigens of Membrane Attack Complex of Complement
(1975)
TABLE 2. Agglutination of EAC1-7 by C5b-9-neoantigenspecific antibody: inhibition by isolated C5b-9 C5b-9 (,sg/ml)*: Reciprocal of minimum agglutinating dilution of antibody:
0
640
0.25 320
2.5
160
25
250
