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The Journal of Immunology, 2000, 165: 2048-2051.
Copyright © 00 by The American Association of Immunologists

The Cleavage of Two C1s Subunits by a Single Active C1r Reveals Substantial Flexibility of the C1s-C1r-C1r-C1s Tetramer in the C1 Complex1

Zsolt Lörincz, Péter Gál, József Dobó, Sándor Cseh, Katalin Szilágyi, Géza Ambrus and Péter Závodszky2

Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The activation of the C1s-C1r-C1r-C1s tetramer in the C1 complex, which involves the cleavage of an Arg-Ile bond in the catalytic domains of the subcomponents, is a two-step process. First, the autolytic activation of C1r takes place, then activated C1r cleaves zymogen C1s. The Arg463Gln mutant of C1r (C1rQI) is stabilized in the zymogen form. This mutant was used to form a C1q-(C1s-C1rQI-C1r-C1s) heteropentamer to study the relative position of the C1r and C1s subunits in the C1 complex. After triggering the C1 by IgG-Sepharose, both C1s subunits are cleaved by the single proteolytically active C1r subunit in the C1s-C1rQI-C1r-C1s tetramer. This finding indicates that the tetramer is flexible enough to adopt different conformations within the C1 complex during the activation process, enabling the single active C1r to cleave both C1s, the neighboring and the sequentially distant one.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The first component of the complement system, C1,3 is a heteropentameric complex that triggers the classical pathway of complement activation after binding to immune complexes or nonimmune activators (1, 2). The recognition subunit of C1 is the C1q molecule, which binds to the activator structures through its C-terminal globular heads while its N-terminal collagen-like arms provide a framework for the catalytic unit. The catalytic unit itself is a tetrameric complex of two different serine protease proenzymes, C1r and C1s (3). Two C1r and two C1s molecules form a calcium-dependent tetramer that associates with C1q to yield the C1 complex (4). The activation of C1 is a multistep process involving recognition of the activator structure by C1q, transferring the activation signal from the C1q heads to the C1s-C1r-C1r-C1s zymogen tetramer, autoactivation of C1r, and subsequent activation of zymogen C1s by the active C1r. The protein-protein interactions between the subcomponents, which regulate the activation of C1, have been extensively studied. The roles of the individual domains of the serine-protease subcomponents have been investigated by limited proteolysis (5, 6, 7, 8, 9), recombinant protein expression (10, 11, 12) and chemical synthesis (13, 14, 15). These experiments provided important information about the structure and function of C1, but our knowledge about the mechanism of the activation process is far from complete.

Previously, we reported the construction and expression of a recombinant mutant C1r, which was trapped in the zymogen state due to the replacement of Arg463 by Gln (C1rQI) (16). This stable zymogen C1r mutant was able to form a C1r - C1rQI heterodimer, and, after adding C1s and C1q, the C1 complex could be reconstituted. In this way, we demonstrated that one active C1r unit in the C1 complex is sufficient for the biological activity of the C1 complex. The aim of the present work is to reveal the details of the activation process by means of this mutant, C1rQI. In particular, we were interested in finding out whether the single active C1r unit can activate both C1s zymogen in the tetramer or only one of them.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cells and viruses

Spodoptera frugiperda 9 cells and wild-type (wt) Autographa californica nuclear polyhedrosis virus were provided by Max Summers (Texas A&M University, College Station, TX). Cells were grown at 27°C in Grace’s insect medium with 10% FCS (Serva, Heidelberg, Germany), 3.3 g/L lactalbumin hydrolysate (Sigma, St. Louis, MO), and 3.3 g/L yeastolate (Oxoid, Basingstoke, England). Recombinant viruses were produced as described by Summers and Smith (17).

Expression of the recombinant proteins

The expression was done essentially as described earlier (18). A total of 2 x 107 Spodoptera frugiperda 9 cells were infected in 175-cm2 flasks at a multiplicity of infection of 10. After incubation at 27°C for 1 h the medium was changed to 50 ml Sf900 serum-free medium (Life Technologies, Grand Island, NY). The cell culture supernatant was harvested 72 h later and was concentrated 20-fold on a PM10 ultrafiltration membrane (Amicon, Beverly, MA).

Reconstitution of C1s-C1rQI-C1r-C1s tetramer and the C1 complex

To reconstitute the C1 containing C1r-C1rQI heterodimer, samples were prepared as described earlier by Dobó et al. (16). Fixed amounts of concentrated supernatant of insect cells that produce wt C1r (0.4 µg in 5 µl) were mixed with increasing amounts of concentrated supernatant containing C1rQI (0.4, 0.8, 1.2, 1.6, and 2.4 µg in 6, 12, 18, 24, and 36 µl, respectively) to obtain heterodimer. One sample was prepared with 5 µl of wt C1r alone and one with 36 µl C1rQI alone (Table IGo) to serve as 100 and 0% references, respectively. Samples were diluted to 65 µl with TBS buffer containing 1 mM final concentration of CaCl2 and were incubated for 3 h at 4°C. Each sample was used for the reconstitution of C1 by adding recombinant C1s (3 µg in 20 µl concentrated supernatant) and 8.5 µg of C1q (Calbiochem, La Jolla, CA) in 15 µl, followed by incubation in the presence of 1 mM Ca2+ at 4°C for 20 min. Activation of C1s during this step was checked by immunoblotting, and no detectable activation was observed.


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  		Table I. Calculated amounts of C1s-C1r-C1r-C1s, C1s-C1rQI-C1r-C1s, and C1s-C1rQI-C1rQI-C1s tetramer variants in the reconstituted C1 complex at equilibrium, as a function of the initial concentration of the subcomponents1

 
Activation of C1 by IgG-Sepharose

IgG-Sepharose was prepared from CNBr-activated Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) and human IgG (Humán RT, Gödöllö, Hungary). For the coupling reaction, we followed the procedure recommended by the manufacturer. Small columns containing 40 µl IgG-Sepharose were equilibrated with TBS containing 10 mM CaCl2. After binding of C1 samples for 10 min, the columns were washed twice with 80 µl TBS containing 10 mM CaCl2. The columns were incubated at 37°C for 1 h to activate C1. C1r and C1s were eluted using 100 µl TBS containing 25 mM EDTA. Fifteen microliters of each sample was analyzed by SDS-PAGE-Western blot under reducing conditions.

Western blot analysis

Protein samples were loaded onto a 10% SDS-polyacrylamide gel prepared as described by Laemmli (19). For the immunoblotting, the method of Towbin et al. (20) was followed. Proteins were transferred to a nitrocellulose sheet (0.45 µm; Schleicher & Schüll, Dassel, Germany) in a semidry apparatus (Pharmacia Biotech). Nitrocellulose strips were incubated with goat anti-human C1s Ab (1:1000; Incstar, Stillwater, MN), then later with alkaline phosphatase-labeled rabbit anti-goat IgG conjugate as secondary Ab (1:3000; Sigma). Visualization was performed by the commercially available substrates of alkaline phosphatase: 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. For densitometry we used the GEL DOC 1000 instrument and Molecular Analyst Software (both obtained from Bio-Rad, Hercules, CA).


   Results and Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The aim of this study was to assess the flexibility of the C1r2C1s2 and to test the accessibility of C1s by C1r in C1, a complex reconstituted with the composition (C1s- C1rQI-C1r-C1s)-C1q and activated on IgG-Sepharose. By adding increasing amounts of C1rQI to fixed amounts of wt C1r, the proportion of wt C1r2 homodimers was shifted from 100% to a negligible value, i.e., practically all enzymatically active C1r subunits were transferred into C1r-C1rQI heterodimer as described earlier (16). The molar ratio of C1rQI:C1r varied from 0:1 to 6:1 (see Materials and Methods and Table IGo). After heterodimer formation, C1s and C1q were added at large molar excesses to consume all C1r, and C1 was reconstituted by incubating the mixtures for 20 min at 4°C. The C1 samples were absorbed onto IgG-Sepharose columns, and excess C1s was washed away. Samples were then incubated at 37°C for 1 h to trigger C1 activation. After activation, C1r and C1s were eluted using an EDTA-containing buffer and the components were separated by SDS-PAGE under reducing conditions. Control experiments verified that under the conditions of elution (concentration, temperature, time) no detectable activation of C1s by C1r occurs in the fluid phase. The samples were blotted to nitrocellulose membranes, and C1s was made visible using anti-C1s Abs (Fig. 1Go).



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  		FIGURE 1. Western blot detection of C1s activation. Samples were prepared using components as shown in Table IGo to reconstitute C1. After incubation at 4°C, the samples were applied to IgG-Sepharose columns. After binding, the columns were washed extensively with buffer containing Ca2+. IgG-Sepharose-bound C1 was activated at 37°C for 1 h. C1r and C1s were then eluted with buffer containing EDTA. The eluates were analyzed by Western blotting using anti-C1s Abs (more details in Materials and Methods).

 
The relative amounts of uncleaved and cleaved C1s were assessed by densitometry of the membranes (see Figs. 2Go and 3Go). Simple statistical considerations predict the composition of the C1 complex. A value n moles of wt C1r2 homodimers bind and activate 2n moles of C1s in the complex. If large molar excesses of C1rQI, C1s, and C1q are added, 2n moles of C1 containing 2n moles of C1r- C1rQI and 4n moles of C1s are formed; an excess of C1 containing inactive C1rQI homodimers and C1s will also be present. The calculated amounts of the different tetramer variants in the C1 samples as a function of the initial concentration of the subcomponents are shown in Table IGo. Increasing total amounts of C1s are seen eluted by EDTA as the C1rQI:C1r ratio increases due to the increase in total amount of macromolecular C1 assembled. However, as the C1rQI2 homodimers even in the assembled C1 do not activate C1s, the amount of uncleaved C1s (in the C1 bound to IgG-Sepharose) increases. The question to be answered was: are one or two C1s subunits cleaved in the C1 complex with C1s-C1rQI-C1r-C1s tetramer composition? If the single wt C1r cleaves two C1s subunits in C1, then we expect 2 moles of cleaved C1s per mole of wt C1r in the eluted samples, that is, the amount of activated C1s doubles as compared with the reference signal (sample 1 in Table IGo). As seen in Fig. 2Go, the signal for the uncleaved C1s increased with an increasing C1rQI:C1r molar ratio, while the signal of the activated C1s (the C1s A chain, Fig. 3Go) practically doubled when reaching the plateau level. This experiment clearly demonstrates that the wt C1r in the C1s-C1rQI-C1r-C1s tetramer cleaves both C1s subunits: the one binding directly to its N-terminal {alpha} region and the other one at the distant end of the tetramer, adjacent to the inactive C1rQI. Two obvious explanations arise at first glance: 1) exchange among the subunits via dissociation and reassociation of the C1 complex or 2) conformational flexibility. Under our experimental conditions, the dissociation and reassociation of the C1 complex is negligible because the dissociation constant of unactivated C1 is in the picomolar range (21). It is plausible to assume that the Kd of the half-activated C1 is not greater than that of the fully activated complex (i.e., 6.5 nM) (21), reflecting a fairly tight complex. If there were significant exchanges of subunits between the C1 complexes, the signal for activated C1s would increase continuously instead of reaching a plateau of two activated C1s per C1r.



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  		FIGURE 2. Densitometric analysis of the Western blot bands corresponding to uncleaved C1s. The amounts of uncleaved C1s increases along with the increasing amounts of C1rQI2 homodimers added to the different samples. The C1 complex was prepared as shown in Table IGo and treated as summarized in Fig. 1Go. Blots were scanned and the intensity of the bands corresponding to uncleaved C1s in each sample were normalized to sample 7. Each column represents the average of five experiments. SE bars are indicated.

 


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  		FIGURE 3. Densitometric analysis of the Western blot bands corresponding to activated C1s. The amount of activated C1s reaches a plateau as increasing amounts of C1rQI are added to the samples. Experimental conditions were shown in Table IGo and treated as summarized in Fig. 1Go. Blots were scanned, and the intensity of the bands corresponding to the C1s A chain in each sample were normalized to sample 1. Each column represents the average of five experiments. SE bars are indicated.

 
There are two models for the structure of C1 that are consistent with the electron microscopic images; both models use symmetry and functional considerations. In both models, the C1r2 portion of the tetramer is located inside the cone formed by the spreading C1q arms, while the protruding arms of the tetramer (dominantly the two C1s subunits) wrap around the collagenous arms of C1q. In the Los Angeles model (22), the entire C1s is placed outside of the cone formed by the C1q arms, having free access to the substrates (complement components C2, C4). This arrangement maximizes the number of identical contact surfaces with C1q, because the C1s-C1r-C1r-C1s tetramer interacts with four collagen-like C1q arms. The Grenoble model (4) assumes that the C1s repenetrates the C1q cone so that the catalytic domains of the C1s zymogen can have access to the catalytic domains of C1r. In this model, the C1s-C1r-C1r-C1s tetramer interacts with only two C1q arms. Probing the structure of C1 with an anti-C1s mAb led to the conclusion that these configurations are interconvertible and both can exist in solution (23). The conversion of the two forms of C1 requires considerable conformational flexibility of the pentameric structure, in particular the flexibility of the tetramer inside the C1q arms (Fig. 4Go). Our results support this view by demonstrating that the C1s-C1r-C1r-C1s tetramer has indeed significant conformational flexibility within the C1 complex. In the case of the Grenoble model, this implies that the catalytic domains of C1r and C1s can change their relative position to each other inside the cone formed by the C1q arms during the activation process.



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  		FIGURE 4. Eclectic model of C1. Illustration of the cleavage of two C1s by one active C1r in the complex, without dissociation. The flexibility of the necklace-like C1s-C1rQI-C1r-C1s tetramer provides access to both C1s by both C1r.

 
Models based on chemical cross-linking and homology modeling (9) suggest that the second complement control protein (CCP) module in C1r and C1s is attached to the serine protease domain while significant flexibility is assumed at the CCP1-CCP2 domain-domain interface. This assumption is in accord with experimentally (nuclear magnetic resonance) observed flexibility between two contiguous CCP modules in a related molecule, human factor H (24). The rotation of one CCP module about the longitudinal axis could provide sufficient conformational freedom to allow the suggested interconversion between the two forms of C1 complex together with some obvious flexibility (e.g., in the {alpha} region) of these extended multidomain serine protease subunits.

The finding that the C1 molecule is flexible enough to adopt a set of conformations allowing one of the two C1r subunits to cleave both C1s proenzymes is in accord with independent structural observations of C1r and C1s and points to the significance of conformational dynamics in the mechanism of activation of the first component of complement.


   Acknowledgments
 
We thank Dr. András Szilágyi for his suggestions by reading the manuscript and Júlia Balczer for her skillful technical assistance.


   Footnotes
 
1 This work was supported by the Hungarian National Science Foundation (OTKA) Grant T030588 and the Foundation for the Technological Progress of Industry. Financial support from Chemical Works of Gedeon Richter is gratefully acknowledged. Back

2 Address correspondence and reprint requests to Dr. Péter Závodszky, Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest 1518, P.O. Box 7, Hungary. Back

3 Abbreviations used in this paper: C1, the first component of complement; C1q, C1r, and C1s, subcomponents of C1; C1rQI, Arg463Gln mutant of C1r; wt, wild-type; CCP, complement control protein. Back

Received for publication April 14, 2000. Accepted for publication May 30, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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