THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc

Vol. 259, No. 16, Issue of August 25. pp. 10569-10573,1984 Printed in U.S.A.

Circular Polymerization of the Membranolytic Ninth Component of Complement DEPENDENCE ON METAL IONS*

(Received for publication, October 31, 1983)

Jiirg TschoppS From the Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

The ninth component of complement (C9) polymer- izes to tubules either spontaneously at a very slow rate (3 days at 37 "C) or within minutes if induced by C5b- 8 as part of the membrane attack assembly (Tschopp, J., Podack, E. R., and Muller-Eberhard, H. J. (1984) J. Immunol., in press).

The spontaneous formation of tubular sodium dode- cy1 sulfate-resistant C9 polymers was completely in- hibited in the presence of 1 mM EDTA. In contrast, the presence of Cu2+, Zn2+, and Cd2+ accelerated C9 polym- erization. Zn2+ was the most efficient metal ion. At a molar ratio of one Zn2+ per monomeric C9, all C9 polymerized within 2 h. At Zn2+ concentrations above

M, tubular C9 formation was inhibited due to nonspecific aggregation of C9.

It is concluded that the presence of metal ions is required for the formation of tubular poly C9 and that Zn2+ in particular catalyzes C9 polymerization.

Expression of the cytolytic activity of complement occurs after the five terminal proteins C5, C6, C7, C8, and C9 have assembled on the target membrane (1). The membrane attack proceeds in two steps: 1) insertion of the assembled C5b-7 complex into the membrane after cleavage of C5 t o C5b by the respective convertases of the classical or the alternative pathway and binding of C8; and 2) binding and circular polymerization of u p to 12-16 molecules of C9 to form a functional membrane lesion (2, 3). In the assembled C5b-9 complex (MAC'), the C9 polymer (poly C9) constitutes the major lipid-binding site as evidenced by photoactivatable membrane-restricted probes (4-6).

Polymerization of C9 may also occur in the absence of C5b- 8, resulting in the formation of tubular complexes with am- phiphilic properties (7-9). Tubular poly C9 has a length of approximately 16 nm and an internal diameter of 10 nm. During polymerization the length of individual C9 molecules increases from 8 t o 16 nm, and this change in structure is accompanied by an increase in &pleated sheet structure (10).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

and 3.049-0.99. $ Supported by Swiss National Science Foundation Grants 3.267.82

The abbreviations used are: MAC, membrane attack complex of complement; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; tubular poly C9, cylindrical C9 polymer that is re- sistant to dissociation by SDS; C5b-8, molecular complex comprising the complement proteins C5b, C6, C7, and C8; TBS, Tris-buffered saline (10 mM Tris-HCI, pH 7.4, 150 mM NaCI; GVB2+, veronal- buffered saline containing Ca2+, M P , and gelatin; EA, antibody- sensitized erythrocytes.

Tubular poly C9 is resistant to dissociation by SDS and to proteolytic digestion by trypsin (11).

Although poly C9 induced by C5b-8 and spontaneously formed poly C9 share biochemical and ultrastructural char- acteristics, the rate of formation varies enormously. Whereas polymerization of isolated C9 requires incubation at 37 "C for at least 3 days, C9 polymerization triggered by C5b-8 occurs within minutes (3).

Recent studies of Yamamoto and Migita (12) suggest that formation of inter-C9 disulfide bonds occurs and that free sulfhydryl groups appear during C9 polymerization. They report that certain sulfhydryl reagents slightly accelerate spontaneous C9 polymerization. We now report that the ad- dition of Zn2+ ions to monomeric C9 at a molar ratio of 1:l accelerates spontaneous C9 polymerization, leading to reac- tion times which are comparable to those induced by C5b-8.

EXPERIMENTAL PROCEDURES

Isolation and Polymerization of C9-Human C9 was isolated from outdated human plasma by polyethylene glycol precipitation, DEAE- Sephacel chromatography, and hydroxylapatite chromatography as described by Biesecker and Muller-Eberhard (13). Polymerization of C9 was achieved by incubating C9 for different lengths of time in a buffer containing 10 mM Tris-HCI, 0.15 M NaCI, pH 7.4 (TBS) containing 0.02% NaN3. An appropriate amount of metal ions or chelator was included when needed. Metal salts added were of the highest purity available.

Assessment of Progress of C9 Polymerization-The fraction of polymerized C9 was determined in two ways. 1) Remaining C9 he- molytic activity was assayed by quantitation of the degree of lysis obtained after a 1-h incubation of C9 samples with 500 ~1 of EACl.s cells (3 X lo7 cells/ml) in GVB". EAC1.s were prepared by incubating 0.2 ml of C8-depleted serum that had been dialyzed against Ca2+ and M P containing TBS and had been supplemented with purified C1 (14) (20 pg/ml of C8-depleted serum), with 1 ml of EA (5 X 10' cells/ ml) for 20 min at 30 "C. Cells were washed 4 times in GVB" and diluted to 1.5 X 10' cells/ml. 100 ~1 of EAC'.? were then incubated with an excess of C8 and the resulting EAC1.8 washed 3 times in GVB". 2) Remaining monomeric C9 (4.7 S) was determined by ultracentrifugation using a Beckman L-65 centrifuge equipped with a Prep UV scanner. Sedimentation velocity experiments were per- formed at 20,000 rpm for the analysis of C9 polymers and at 50,000 rpm for the determination of monomeric C9. An AN-F &hole rotor was used, and centrifugation was performed at 4 "C. Absorption profiles were recorded at 6-min intervals at a wavelength of 280 nm.

SDS-Polyacrylamide Slab Gel Analysis-After polymerization of C9, samples were incubated in the presence of 2% SDS and subjected to SDS-PAGE analysis. Electrophoresis was carried out on 2.5-10 % polyacrylamide gels as outlined elsewhere (11). Quantitation of tu- bular poly C9 was achieved by scanning the Coomassie Blue-stained gels by means of a Zeiss KM gel scanner. The bands were analyzed using a computer program.

Electron Microscopy-Poly C9 samples were diluted to approxi- mately 0.2 mg/ml with a buffer containing 100 mM ammonium acetate, 50 mM ammonium bicarbonate, pH 7.4. Samples were applied to carbon-coated grids, washed twice with the same buffer, and

10569

10570 Metal Ion Dependence of C9 Polymerization

negatively stained with 2% uranyl formate. Samples were imaged in a Philips EM 400 electron microscope operating at 80 kV with a 200- pm C2 aperture and a 30-pm objective aperture.

RESULTS

C9 Polymerization: Metal Ion Dependence-13 PM mono- meric C9 (1 mg/ml) was incubated in the presence of different metal ions at a molar ratio of 1:l for 2 h at 37 "C, and the degree of polymeriation quantitated in two ways, as described under "Experimental Procedures" (residual C9 activity and sedimentation analysis). In addition, the amount of SDS- resistant tubular C9 polymers was measured by SDS-polya- crylamide gel electrophoresis. Among the metal ions tested, only Cu2+, Zn2+, and Cd2+ mediated a noticeable acceleration of C9 polymerization (see Table I and Fig. 1). In the presence of 13 PM Zn2+ all the C9 polymerized within 2 h, as determined by ultracentrifugation. The amounts of C9 polymers formed were 25% and 22% in the presence of 13 PM Cu2+ or Cd", respectively. These results were entirely compatible with the remaining hemolytic activity; C9 activity was completely abol- ished in the presence of 13 p~ Zn2+, whereas 72 and 76% of the C9 activity was found after a 2-h incubation with Cu2+ and Cd", respectively. Znz+-mediated C9 polymerization was irreversible, since addition of 10 mM EDTA, after C9 polym- erization had occurred, neither reduced the quantity of tubular poly C9 (Fig. 1, Track 7) nor reversed the loss of C9 activity that occurred after Zn2+-induced C9 polymerization.

Other metal ions, i.e. Ca2+, MP, and the remaining diva- lent cations from the first transition period showed no signif- icant accelerating effect at a concentration of 13 pM. The same metal ion dependence was observed when the amount of SDS-resistant tubular poly C9 was determined (Table I and Fig. 1). Zn2+ ions induced the formation of a considerable amount of tubular poly C9 (38%). Of the 25% of polymers

TABLE I Influence of metal ions on C9 polymerization

Residual Residual Tubular poly C9 activity monomeric C96 CY

Metal ion (metal ion (metal ion concentration, concentration, 13 pMd 65 pMd

% % % 13 pM)' 13 cM)

Mi? >85 ND' <5 ND Ca2+ >85 ND c5 ND Mn2+ >85 ND <5 ND Fez+ >85 ND <5 ND co2+ >85 ND <5 ND Ni2+ >85 ND (5 ND cu2+ 72 75 8 18 Zn2+ <5 <5 38 46 Cd2+ 76 78 <5 6 Hg2+ >85 82 <5 <5

TBs' >85 TBS' 37

>85 35

<5 35

EDTA (1 mM)' >85 >95 <5 EDTA (1 mM)' 58 45 <5

NaCl (1.15 M)' 37 ND <5 NaCl (50 mM)' <5 ND 70 ' 13 pM corresponds to 1 metal ion per C9 molecule.

Measured by sedimentation velocity analysis. Measured by SDS-PAGE tubular poly C9 is resistant to disso-

Metal ion concentration. ciation by SDS.

e ND, not determined. 'Sample incubated 2 h at 37 "C.

Sample incubated 3 days at 37 "C.

- Poly-CQ

- CQ """"

0

1 2 3 4 5 6 7 8 FIG. 1. Tubular poly C9 formation in the presence of differ-

ent metal ions. C9 (1 mg/ml, 13 pM) was incubated in the presence of 13 p~ metal ions for 2 h at 37 "C. Track I, Cu2+; 2, Zn2+; 3, Cd2+; 4, Hz+; 5, 10 mM EDTA; 6, TBS; 7, C9 was incubated 2 h in the presence of 13 PM Zn2S04. 10 mM EDTA was then added and incu- bation was continued for an additional hour. 8, molecular weight markers (from top to bottom): myosin, 200,000, @-galactosidase, 116,000; phosphorylase b, 97,000; bovine serum albumin, 66,000; egg albumin, 45,000. The samples were electrophoresed on a 2.5-10% polyacrylamide gradient slab gel.

found in the presence of Cu2+, 32% were in their SDS-resistant forms (8% of total C9). Tubular poly C9 formation was minimal in the presence of TBS and was completely prevented in the presence of 1 mM EDTA, indicating that TBS may be contaminated with traces of metal ions. Indeed, when C9 was incubated in the presence of 1 mM EDTA-containing buffer for 3 days at 37 "C, spontaneous tubular poly C9 formation dropped to 3% of total C9 input, as compared to 24% in a buffer control. Nevertheless, C9 lost 40% of its activity under these conditions. The ultrastructure of C9 exposed to 1 mM EDTA for 3 days was examined in the electron microscope (Fig. 2). The typical tubular ultrastructure of poly C9 was almost completely absent; instead globular spheres arranged in necklace-like chains were observed. The diameter of the spheres was not constant and varied from approximately 10 to 20 nm. Occasionally, substructures could be discerned in a single spherical complex, suggesting an oligomeric structure of an individual sphere.

The rate of C9 polymerization was also influenced by other parameters, such as ionic strength and pH of the polymerizing buffer. At 1 mM NaCl concentration, no SDS-resistant poly C9 was formed, whereas in the presence of 50 mM NaCl, 70% of the poly C9 was observed in its tubular form, a quantity never achieved in buffers of normal ionic strength. At low pH in the absence of metal ions (pH < 6), C9 lost its activity within 2 h (see Fig. 3). The same observation was made at pH values above 9. At low pH, C9 formed undefined aggregates, whereas polymerization at high pH resulted in tubular C9.

Tubular Poly C9 Formation: Dependence on Zinc Concen- tration-Tubular poly C9 formation in the presence of varying concentrations of Zn2+ was measured by SDS-PAGE analysis. As shown in Fig. 4, A and B, tubular poly C9 formation occurred optimally at Zn2+ concentrations between 50 and 100 pM. u p to 60% of the total polymeric c 9 was resistant to dissociation by SDS. At higher Zn2+ input, the amount of tubular poly C9 dropped gradually and tubular poly C9 was completely absent at a Zn2+ concentration of 10 mM, although complete absence of hemolytically active C9 was still ob- served. On the other hand, at Zn2+ concentration below 3 x

M the activity of C9 increased due to unpolymerized hemolytically active C9 (followed by ultracentrifugation, data not shown). In parallel, a decrease of tubular poly C9 was observed. The inhibition of tubular poly C9 formation due to high concentrations of Zn2+ ions was further examined. Sedi-

Metal Ion Dependence of C9 Polymerization 10571

FIG. 2. Ultrastructure of C9 incubated in the presence of EDTA. C9 was incubated at 37 "C for 3 days in the presence of 1 mM EDTA. Spheres of different diameters (3-20 nm) are formed (black open arrow) which exhibit a propensity to form chain-like aggregates. The black arrow points to occasionally found tubular poly C9. The bar represents 100 nm.

2ot 0 "0 I 5 6 7 8 9

PH FIG. 3. Influence of pH on C9 polymerization. C9 (1 mg/ml)

was incubated for 2 h in buffers of different pH values and the remaining hemolytic activity tested. Loss of hemolytic activity was due to C9 polymerization. At low pH undefined C9 aggregates were formed, whereas at high pH, tubular poly C9 was assembled.

mentation analysis indicated the presence of large polymers, since all C9 sedimented at 20,000 rpm within minutes. There- fore, C9 polymers were inspected in the electron microscope. Fig. 5b shows poly C9 which was polymerized for 2 h at 37 "C in the presence of 1 mM Zn2+. Tubular C9 polymers (black arrows) occur less frequently than C9 aggregates of different size and shape. The C9 aggregates often occur in long bundles

with a diameter and length of up to 40 and 200 nm, respec- tively (open black arrow). No regular repetitive ultrastructure can be discerned. At a hundred times smaller Zn2+ concentra- tion (10 p ~ ; Fig. 5a), poly C9 is imaged as the typical structure of 16 nm length in side views (black arrowhead) and as a ring structure (arrow) with an internal diameter of 10 nm in top views. Poly C9 is rimmed at one end with a torus having a cross-sectional diameter of 3 nm. Tubular poly C9 often occurs in staggered aggregates, which may be related to its amphi- philic nature.

Kinetics of C9 Polymerization in the Presence of Zinc-The kinetics of C9 polymerization was measured at 37 "C in the presence of 13 pM Zn2+, corresponding to one ion per individ- ual C9 molecule. C9 polymerization was essentially complete after 2 h as determined by the loss of C9 activity (Fig. 6). Half of the C9 had polymerized within 40 min. At a five times higher Zn2+ concentration (66 PM), complete C9 polymeriza- tion was achieved after 30 min (Fig. 6). C9 polymerization was also examined by sedimentation velocity analysis. If the C9 sample was subjected to ultracentrifugation after 20-min incubation at 37 "C in the presence of 13 PM Zn2+, 70% were still in the monomeric form as indicated by the 4.7 S sedi- menting component. The remaining C9 sedimented as a dis- tinct peak at 27 S. After 1-h incubation the amount of monomeric C9 was reduced to 20%. The C9 polymers now sedimented at 27 and 37 S to 90 S.

DISCUSSION

Incubation of C9 for a prolonged period of time results in spontaneous polymerization of C9 to tubular complexes (7, 8). Tubular poly C9 is resistant to dissociation by boiling in SDS and to proteolytic digestion (1 1). Polymerization of C9 as part of the MAC is induced by C5b-8 and results in the same tubular complexes with the same characteristics as described for spontaneous C9 polymerization. However, po-

10572 Metal Ion Dependence of C9 Polymerization

logZn2* concentration

FIG. 4. Dependence of C9 polymerization on Zn" concen- tration. C9 (13 PM) was incubated at 37 "C for 2 h in the presence of varying concentrations of Zn2+ ions. A, the fraction of C9 polym- erized to SDS-resistant tubular poly C9 was assessed by SDS-PAGE. B, quantitative representation of the results shown in A including the determination of the remaining C9 hemolytic activity of the samples.

/. ,

FIG. 5. Ultrastructure of C9 polymerized in the presence of ZnZ*. a, C9 was polymerized in the presence of 10 p~ ZnZ+. The typical tubular ultrastructure of poly C9 is visualized in a top view (black arrow) and in a side view (black arrowhad). b, C9 polymers assembled in the presence of 1 mM Zn2+. Besides the tubular poly C9 structure (black arrow), morphologically irregular bundles can be observed (black open arrow). The bar corresponds to 100 nm.

I I I I

-* ** \ t ia0

1 ' /+ 0 10 2 0 30 40 50 60 120

Tlmo (min 1 FIG. 6. Kinetics of Zn"*-mediated C9 polymerization.

(13 PM) was polymerized by Zn2+ (13 PM, 65 PM, M) and the loss of hemolytic activity and the extent of tubular poly C9 formation determined after different periods of time.

lymerization of isolated C9 at 37 "C requires high C9 concen- tration (approximately 1 mg/ml) and a prolonged incubation (3 days) (3). In contrast, C9 polymerization by C5b-8 is complete within minutes even at low C9 concentrations (50 pglml). Our interest as to how C5b-8 exerts its catalytic effect arose from these observations. We have found that the "cat- alytic" function of C5b-8 can partly be mimicked by the metal ion Zn2+. Maximal rate of C9 polymerization occurred in the presence of 30-100 pM Zn2+; at a c 9 concentration of 1 mg/ ml (13 p ~ ) , polymerization was complete within 2 h. Even at physiological serum concentrations of Zn2+, i.e. 10 p ~ , 70% of the C9 molecules polymerized within 2 h. Why continuous C9 polymerization in serum is not observed is not known. At ion concentrations above 100 p ~ , tubular poly C9 formation was inhibited. Sedimentation and electron microscopic anal- ysis indicated that inhibition resulted from "nonspecific" C9 aggregation. Compared to Zn2+ no other metal ions tested were as effective in mediating C9 polymerization. Only Cu2+, and to a smaller extent Cd2+, showed a slight effect.

The mechanism of catalysis of C9 polymerization by Zn2+ and its relation to that of C5b-8-mediated polymerization are unknown at present. In a recent report of Yamamoto and Migita (12), the authors provided evidence that Hg2+ en- hanced "slightly" with formation of SDS-resistant C9 poly- mers after a 3-day incubation at 37 "C. They interpreted these results to mean that Hg2+ ions react with spontaneously formed free sulfhydryl groups within poly C9. However, the accelerating effect of Hg2+ compared to Zn2+ is negli 'ble (Table I); this may be due to its different radius (0.74 f f o r Zn and 1.10 A for Hg) or to different reactivities toward free sulfhydryl groups. Interestingly, Cd2+ is the only other metal ion, besides Cu2+, mediating a significant effect on C9 polym- erization. As far as its chemical reactivity and its radius (0.97 A) is concerned, Cd2+ is located beteen Zn2+ and H e . On the other hand, Zn2+ may react with groups other than free sulfhydryls, e.g. histidyl residues in the case of many Zn2+- dependent enzymes (15).

The presence of EDTA abrogates the spontaneous forma- tion of tubular poly C9. Even at 64-h incubation at 37 "C, C9 does not polymerize to SDS-resistant tubules, indicating that this arbitrary incubation time may be only sufficiently high in the presence of contaminating metal ions, e.g. 5 X M Zn2+. A contamination of distilled water with traces of metal ions might explain why other groups have reported different amounts of C9 polymers after this period of incubation (as

Metal Ion Dependence of C9 Polymerization 10573

little as 55%, (12) or “at most” 15% (Shiver et al. (16)). In addition to metal ions, low ionic strength buffers mediated C9 polymerization; during C9 polymerization, hydrophobic amino acids are exposed to the hydrophilic environment, a process which is favored in the absence of highly charged salts.

In 1979, Boyle et al. (17) reported the inhibition of C9- induced red blood cell lysis by Zn2+. The concentration at which Zn2+ exhibited this inhibitory effect was similar to that determined for mediating spontaneous C9 polymerization. I t could be hypothesized that Zn2+ occupies the binding site in C9 where C5b-8 normally exerts its accelerating function, thereby inhibiting cell lysis and mediating C9 polymerization in the fluid phase.

In conclusion, we have shown that C9 polymerization can be mediated by low concentration of ZnZ+ almost as efficiently as by C5b-8. This finding will facilitate the study of the mechanism of C9 polymerization as well as the elucidation of the role played by zinc in the inhibition of C5b-9-induced hemolysis.

Acknowledgments-I am grateful to Dr. Henri Isliker for his con- tinuing support and interest and to Dr. Tibor Borsos for his help concerning the C9 activation test. I would like to thank Christine Estrade and Florella Penea for expert technical assistance and Edith Burnier for typing the manuscript.

REFERENCES 1. Lachmann, P. J., Munn, E. A., and Weissmann, G . (1970) Im-

munology 19,983-986

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

Podack, E. R., Tschopp, J., and Muller-Eberhard, H. J. (1982) J.

Tschopp, J., Podack, E. R., and Muller-Eberhard, H. J. (1984) J.

Hu, V. W., Esser, A. F., Podack, E. R., and Wisnieski, B. J. (1981)

Podack, E. R., Stoffel, W., Esser, A. F., and Mlller-Eberhard, H.

Steckel, E. W., Welbaum, B. E., and Sodetz, J. M. (1983) J . Biol.

Tschopp, J., and Podack, E. R. (1981) Biochem. Biophys. Res.

Podack, E. R., and Tschopp, J. (1982) J. Biol. Chem. 257,15204-

Yamamoto, K., Kawashima, T., and Migita, S. (1982) J. Biol.

Tschopp, J., Muller-Eberhard, H. J., and Podack, E. R. (1982)

Podack, E. R., and Tschopp, J. (1982) J. Biol. Chem. 257,15204-

Yamamoto, K., and Migita, S. (1983) J. Biol. Chem. 258, 7887-

Biesecker, G., and Muller-Eberhard, H. J. (1980) J. Immunol.

Medicus, R. G., and Chapuis, R. M. (1980) J. Immunol. 125 ,

McCracken, S., and Meighen, E. A. (1981) J. Biol. Chem. 256,

Shiver, J. W., Dankert, J. R., and Esser, A. F. (1983) Immuno-

Boyle, M. D. P., Langone, J. J., and Borsos, T. (1979) J. Immunol.

Exp. Med. 156,268-282

Immunol., in press

J. Zmmunol. 127,380-386

J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,4544-4548

Chem. 258,4318-4324

Commun. 16,1409-1414

15212

Chem. 257,8573-8576

Nature (Lond.) 298,534-538

15212

7889

124,1291-1296

390-395

3945-3950

biology 164, 237

122,1209-1216