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The Journal of Immunology, 1999, 162: 1108-1112.
Copyright © 1999 by The American Association of Immunologists

One Active C1r Subunit Is Sufficient for the Activity of the Complement C1 Complex: Stabilization of C1r in the Zymogen Form by Point Mutations1

József Dobó*, Péter Gál*, Katalin Szilágyi*, Sándor Cseh*, Zsolt Lörincz*, Verne N. Schumaker{dagger} and Péter Závodszky2,*

* Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary; and {dagger} Molecular Biology Institute, and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The binding of C1 (the first component of complement) to immune complexes leads to the autoactivation of C1r through the cleavage of the Arg463-Ile464 bond in the catalytic domain. Spontaneous activation of C1r (and C1) also occurs in the fluid phase, preventing the characterization of the zymogen form of C1r. To overcome this difficulty, the zymogen form of human C1r was stabilized by mutating the Arg in the Arg463-Ile464 bond to Gln. This mutant was designated as mutant QI. Recombinant C1r (wild type (wt) or mutant) was expressed in insect cells using serum-free medium in functionally pure form; therefore, the cell culture supernatant was suitable to reconstruct C1 for the hemolytic assay. Mutant QI was a stable, nonactivable zymogen and showed no hemolytic activity in reconstituted C1. However, this stable zymogen C1r mutant could form an active mixed dimer with the wt C1r, indicating that one active C1r subunit in the C1 complex is sufficient for the full activity of the entire complex. Our experiments also showed that the exchange of C1r monomers between the C1r dimers is completed in less than 16 h even at pH 7 and 4°C. Two other mutants were also constructed by changing Arg463 to Lys, or Ile464 to Phe, and were designated as mutants KI and RF, respectively. Although these substitutions did increase the stability of the proenzyme in the cell culture supernatant, the mutant proteins retained their ability to autoactivate, and both had a wt-like hemolytic activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The classical pathway of complement activation is initiated by the activation of C1,3 the first component of the complement system. C1, a complex heteropentameric protease, is composed of one C1q, two C1r, and two C1s subunits. The serine protease subunits C1r and C1s form a Ca2+-dependent C1s-C1r-C1r-C1s tetramer that is thought to be bent around the collageneous arms of the nonenzymatic C1q. Upon activation the C1q, heads bind to an immune complex and an activation signal is transmitted to the C1r2C1s2 tetramer via the C1q arms (for reviews, see Refs. 1 and 2). Autoactivation of the C1r zymogen follows receipt of this signal, resulting in the cleavage of an Arg-Ile bond (3) in the catalytic part of the molecule. ActivatedC1r then cleaves the corresponding Arg-Ile bond in the C1s zymogen (4), converting it into an active-serine protease that will activate subsequent complement components (C4 and C2) of the classical pathway (5).

C1r is a single-chain glycoprotein containing 705 amino acid residues (6). It serves as a precursor that is converted into a 688 long serum form (7) after the cleavage of the signal peptide. Like many other plasma proteases, C1r has a mosaic structure. The domain structure of C1r is homologous to that of C1s (8) and (mannan-binding lectin)-associated serine protease (MASPs) (9, 10). The function of the individual domains was examined using both limited proteolysis (for review, see ref. 11) and genetic engineering approach (12). C1r is a dimer at neutral pH (both in the presence or absence of Ca2+) that is dissociated to monomers at pH 5 (13). Highly purified C1r has the capacity to autoactivate (14), presumably via an intramolecular autocatalytic mechanism. This mechanism i.e., the cleavage of an Arg-Ile bond during the activation, is widespread among serine proteases. For example, it is found in complement proteases: C1r (3), C1s (4), MASP-1 (9), MASP-2 (10), factor D (15), factor I (16), and in other plasma proteases such as factor VII (17), factor XI (18), prekallikrein (19), and in digestive enzymes such as chymotrypsinogen (20). The activation of the two other complement serine proteases (i.e., C2, factor B) also requires the cleavage of an arginyl bond (21, 22), whereas the corresponding bond in trypsinogen is Lys-Ile (23).

Previously, we have expressed cDNA clones for human C1r (24, 25) and C1s (26) in insect cells using baculovirus vectors. The posttranslational modifications (27) and the biological activity of the recombinant proteins were checked. The objective of the present work was to obtain a stabile zymogen by mutating the very conserved Arg463-Ile464 bond that allows the examination of pure, proenzymic C1r and its behavior in the C1 complex.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
C1r. cDNA, and DNA constructions

The full-length cDNA coding sequence for human C1r (HC1r2200) in pUC8 was generously provided by Prof. Earl Davie (University of Washington, Seattle, WA) and was described previously (6). The BamHI fragment of the cDNA coding for human C1r was subcloned into the M13 mp9 vector in the proper orientation. Mutagenesis was conducted by the Kunkel method (28). The following primers were used: 5'-CCCTCCGATTATCTTCTGCCTCTGT-3' for mutant KI, 5'-CCCTCCGATTATCTGCTGCCTCTGT-3' for mutant QI, and 5'-GCCCTCCGATGAAGCGCTGCCTCT-3' for mutant RF. The mutations were checked by DNA sequencing, then the BamHI fragments were placed into the full-length mutant C1r genes. The mutant cDNAs were inserted into the pBlueBac transfer vector (Invitrogen, San Diego, CA) under the control of the strong polyhedrin promoter. Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, MA). DNA manipulations were conducted essentially as summarized (29). Components of the site-directed mutagenesis system were purchased from Bio-Rad (Richmond, CA). Primers were obtained from SzBK (Szeged, Hungary). DNA was sequenced by the dideoxy method (30) using Sequenase 2.0 (United States Biochemical, Cleveland, OH).

The baculovirus-insect cell expression system

Spodoptera Frugiperda 9 cell line (Sf9) insect cells and wild type (wt) Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) were provided by Max Summers (A&M University, College Station, TX) and maintained as summarized (31). Cells were grown at 27°C in TNM-FH (supplemented Grace’s insect cell medium with 10% FCS (Serva, Heidelberg, Germany), 3.3 g/l lactalbumin hydrolysate (Sigma, St. Louis, MO) and 3.3 g/l yeast extract (Oxoid, Basingstoke, U.K.). After cotransfection of Sf9 cells with the recombinant transfer plasmids and wt AcMNPV DNA, recombinant virus clones were isolated by blue-white selection and amplified. Sf9 cells (8 x 107) were infected in a 175 cm2 flask at a multiplicity of infection 10. After 1 h at 27°C the medium was changed to serum-free Graces’s medium. The cell culture supernatant was harvested 72 h later and analyzed for the presence of the recombinant proteins. Samples were concentrated 50-fold by a PM10 ultrafiltration membrane (Amicon, Beverly, MA), and used freshly or aliquoted, frozen in liquid N2 and stored at -20°C.

ELISA

The C1r content of the samples was determined by a solid-phase ELISA "sandwich" system. The assay was conducted essentially as described previously (32). Goat anti-human C1r Ab was purchased from Atlantic Abs (Stillwater, MN; now Incstar) and rabbit anti-human C1r Ab was obtained from Calbiochem (La Jolla, CA). Goat anti-rabbit IgG Ab conjugated to horseradish peroxidase was a Sigma (St. Louis, MO) product. Purified, activated C1r (Calbiochem) was used as a standard to determine concentration.

Western blot

Samples were analyzed by SDS-PAGE (33) on 12.5% gels followed by transfer to nitrocellulose sheets (0.45 µm; Millipore, Bedford, MA) (34). The membranes were first incubated with goat anti-C1r Ab overnight, then with rabbit anti-goat IgG Ab conjugated to alkaline phosphatase for 1 h. Color was developed using substrates the 5-bromo-4-chloro-3-indolyl phosphate and the nitro blue tetrazolium (both from Sigma).

Reconstitution of the C1 complex

C1 was reconstituted from its subcomponents as follows: a solution of 20 µg C1q, 30 µl 50-fold concentrated serum-free supernatant containing ~15 µg C1s and 30 µl 50-fold concentrated serum-free supernatant of C1r (~1.5 µg) were mixed and incubated in the presence of 1 mM Ca2+ at 30°C for 20 min. The mixture was made up to 300 µl (10 x dilution for C1r) with veronal-buffered saline (VBS) sucrose-G-M++ (5 mM Na-barbital, 85 mM NaCl, 114 mM sucrose, 0.1% gelatin, 1 mM MgCl2, 0.15 mM CaCl2, pH 7.35), then further diluted and used in the hemolytic assay. C1q was purified from human serum (35), and recombinant-proenzymic C1s was produced in Sf9 cells (26) as before.

For the reconstitution of C1 containing mixed (wt + mutant QI) dimers, samples were prepared in the following way: fixed amounts of concentrated supernatant of insect cells producing wt C1r (~0.4 µg in 15 µl) were mixed with increasing amounts of concentrated supernatant containing mutant QI (~0.4, 0.8, 1.2, 1.6, 2.4 µg in 5, 10, 15, 20, 30 µl, respectively). Increasing amounts of concentrated supernatant of wt AcMNPV-infected insect cells were added to fixed amounts of wt C1r in a similar fashion to serve as controls. One sample was prepared with 15 µl wt C1r alone, and one with 30 µl mutant QI alone. Samples were made up to 75 µl with buffer A (150 mM NaCl, 30 mM Na-acetate, pH 5.0) and dialyzed 8 h at 4°C against buffer A (pH 5.0) to dissociate the dimers. Then they were dialyzed 8 h at 4°C with buffer B (150 mM NaCl, 30 mM Na-acetate, pH 7.0) to form mixed dimers (wt + QI) besides homodimers of wt C1r and of the mutant C1r. Another set of samples were prepared in a similar way, differing in that they were made with buffer B (pH 7.0), and instead of dialysis, they were incubated 16 h at 4°C. Thirty microliters of each sample was used for the reconstitution of C1.

Hemolytic assay

Buffers, solutions, and sheep red cells were prepared and the assay was conducted essentially as described (36) with a little modification as follows. The amount of components used was reduced to fit the mixture into a 1.5 ml microcentrifuge tube. To 100 µl sheep red cells (1.5 x 108/ml) sensitized with IgM hemolysin, 100 µl of diluted sample containing C1 was added. After 1 h incubation at 30°C, 100 µl of C4 solution (50 CH50 U/ml) and then 100 µl of C2 solution (50 CH50 U/ml) were added, followed by incubation for 8 or 10 min at 30°C. Finally, samples were mixed with 1 ml C-EDTA (1 part of guinea pig serum mixed with 39 parts of 5 mM Na-barbital, 128 mM NaCl, 10 mM Na2EDTA, 0.1% gelatin) and kept at 37°C for 30 min. After centrifugation the absorbance of the supernatants was measured at 412 nm. Dilutions (5,000x to 10,0000x) of normal human serum were always measured in parallel with the reconstituted samples to test the assay components. Samples and assay components were diluted in VBS-sucrose-G-M++. Rabbit IgM directed against sheep red cells, C2, and C4 were purchased from Diamedix (Miami, FL). Sheep blood and guinea pig serum were obtained from Human Rt. (Gödöllô, Hungary). Normal human serum was drawn from a healthy laboratory person.

C1 activation experiments

Three microliters of concentrated cell supernatant containing ~0.15 µg mutant QI and 6 µl of concentrated cell supernatant containing ~0.15 µg wt C1r were mixed and made up with 120 mM NaCl, 20 mM Tris (pH 7.4) buffer to 100 µl. The mixture was incubated 16 h at 4°C. Then recombinant C1s (~1 µg in 6 µl concentrated supernatant), 2 µg of C1q, CaCl2 solution (1 mM final), and the previous buffer were added. The final volume was 200 µl. The mixture was incubated 20 min at 4°C. One-half of the mixture was mixed with 100 µl SDS-PAGE gel loading buffer (33) and heated for 3 min at 95°C. To the other half, 20 µg of heat-aggregated IgG was added in a small volume (2 µl). The mixture was incubated for 60 min at 30°C, then treated as the first half. As a control, C1 was reconstituted from 0.3 µg of serum proenzymic C1r2s2 tetramer and 2 µg of C1q. A total of 15 µl of concentrated supernatant of wt AcMNPV-infected cells, CaCl2 solution (1 mM final), and 120 mM NaCl, 20 mM Tris (pH 7.4) were added to make the final volume of 200 µl. The mixture was incubated for 20 min at 4°C. Half of the mixture was mixed with 100 µl SDS-PAGE gel loading buffer and heated for 3 min at 95°C. To the other half, 20 µg of heat-aggregated IgG was added in a small volume. The mixture was incubated for 60 min at 30°C, then treated as the first half. Equal amounts of the samples were analyzed by SDS-PAGE Western blot analysis under reducing conditions as above. Heat-aggregated human IgG was prepared according to Cooper and Ziccardi (37), and proenzymic C1r2s2 tetramer was purified from human serum (38).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of point mutant C1r cDNAs and protein expression

The peptide bond Arg463-Ile464, cleaved during the activation of C1r, was altered using site-directed mutagenesis. (Numbers indicate the amino acid positions in the entire coding sequence in which 1 is the initiator Met.) Three mutants were constructed: Arg463 was replaced by Lys or Gln, respectively, and Ile464 was changed to Phe. Using the one-letter code of amino acids, the constructs were designated conveniently as mutants KI, QI, and RF in which the two letters refer to the altered scissile RI bond in the wt C1r (Fig. 1Go). The mutant proteins were expressed in the baculovirus-insect cell expression system. The concentrations of the mutant proteins were estimated by ELISA. The yields of the mutant proteins were similar to that of the wt C1r (up to 3 µg/ml). SDS-PAGE and Western blot analysis were also used to detect the point mutants. They had the same apparent m.w. as the recombinant wt protein.



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  		FIGURE 1. Schematic representation of the active form of C1r. Only the disulfide bridge that joins the two chains is indicated. The A-chain carries the regulatory modules of C1r, whereas the B-chain corresponds to the catalytic domain. The scissile RI bond cleaved during the autoactivation was altered to KI, QI, and RF bonds at the DNA level using site specific mutagenesis.

 
The point mutants are activated to a different extent as detected by Western blot analysis

Wild-type C1r was found mostly in the active, two-chain form in the cell culture supernatant 3 days postinfection (Fig. 2GoA), although a significant amount of zymogen was also present. The proenzyme, one-chain form appeared as a weaker band whereas the A-chain had a stronger intensity under reducing conditions on the blot. The B-chain band was less intense, probably due to poor reactivity with the Ab. Point mutants KI and RF were predominantly in the zymogen form, with mutant KI having more activated enzyme. Point mutant QI appeared as a single band under reducing as well as under nonreducing conditions, showing that it was a stable zymogen. On nonreducing gels (Fig. 2GoB), the proenzyme had a slightly greater mobility than the active form. The results obtained under nonreducing conditions were in accordance with those under reducing conditions (Fig. 2Go).



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  		FIGURE 2. Western blot analysis of wt and point mutant C1r recombinant proteins under reducing (A) and nonreducing (B) conditions. Insect cells producing the recombinant proteins were grown at 27°C. The cell culture supernatants were harvested 3 days postinfection, and the concentrated supernatants were analyzed by SDS-PAGE Western blot analysis.

 
Hemolytic activity of the mutant proteins

Hemolytic assays were performed with C1 reconstituted from wt or point mutant recombinant C1r, an excess of C1q isolated from human blood, and an excess of recombinant C1s also produced in insect cells (see Materials and Methods). The reconstitution mixtures were further diluted for use in the hemolytic assay. Mutants KI and RF had a hemolytic activity similar to that of the wt enzyme, whereas mutant QI showed no significant signal (Fig. 3Go). It is important to note that the curves (in case of the active proteins) had a maximum that indicated that the cell culture medium might have had an inhibitory effect for the hemolytic assay. This inhibition could be eliminated by diluting the samples at least 1000-fold (Fig. 3Go).



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  		FIGURE 3. Hemolytic activity of wt C1r and mutants KI, RF, and QI. Dilution factors are given for concentrated wt or point mutant C1r, containing cell supernatants used for the reconstitution of C1 as described in Materials and Methods.

 
Hemolytic activity of a mixed dimer consisting of wt and QI mutant C1r

Fixed amounts of recombinant wt C1r were mixed with increasing amounts of the QI mutant. The ratio of mutant QI/wt was increased from 0 to 6, and one sample was prepared with the applied largest amount of mutant QI alone. The formation of mixed dimers was facilitated by a sequence of pH shifts: from pH 7.0 to pH 5.0, then back to pH 7.0. Another set of samples were prepared in a similar way, but without the pH shift and they were incubated for the same total time as the dialyzed samples. The hemolytic activity of the sample containing merely wt C1r was measured at many dilutions (data not shown) and an appropriate dilution well beyond the maximum of the curve (see previous section) was chosen to make sure that any increase in the activity was detected. All samples were then measured at the selected dilution. Dialyzing wt C1r with mutant QI increased its hemolytic activity compared with the sample containing the same amount of wt C1r alone. When a large quantity of mutant QI was used, the activity was practically doubled within the error of hemolytic measurements. Adding various amounts of wt AcMNPV-infected cell culture supernatant to wt C1r left the hemolytic activity unchanged. The sample containing solely mutant QI had no significant activity (Fig. 4Go). Surprisingly in the case of the undialyzed samples, practically the same results were obtained (data not shown) and that reflects that the exchange of C1r molecules between the dimers is rapid even at pH 7.0 and 4°C.



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  		FIGURE 4. Hemolytic activity of C1 containing mixed (wt + mutant QI) dimers of C1r. Sample 1 (filled column) contained wt C1r alone (~0.4 µg in concentrated cell supernatant). Samples 2–6 (filled columns) contained the same amount of wt C1r and increasing amounts of mutant QI in a molar ratio (QI/wt) of 1, 2, 3, 4, and 6, respectively. Sample 7 (filled column) was prepared like sample 6, but without wt C1r. Control samples 2–6 (open columns) were prepared with wt C1r (the same amount as in sample 1) and increasing amounts of concentrated supernatant of wt AcMNPV-infected insect cells. The same volumes of "empty" supernatant were applied as mutant QI containing supernatant in the respective samples. All samples were made up to the same final volume and dialyzed as described in the text. Then the samples were used for C1 reconstitution and further diluted for the hemolytic assay, which means a total dilution factor of 2000 for these samples and 10,000-fold dilution for the original wt C1r containing supernatant (in samples 1–6). Columns represent averages from three parallel measurements with SD bars.

 
Although cleavage by C1r after Gln or at a point other then the activation site is unlikely, to rule out this possibility, cleavage of mutant QI by wt C1r within the C1 complex was checked by Western blot analysis (Fig. 5Go). No decrease in the intensity of the band corresponding to mutant QI was observed. As a control, C1 was reconstituted from serum proenzymic C1r2s2 (because recombinant wt C1r was almost completely activated) and C1q. Under similar conditions, practically complete activation of serum C1r was achieved.



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  		FIGURE 5. Western blot analysis demonstrating that mutant QI is not cleavable by wt (active) C1r. Recombinant wt C1r and mutant QI were preincubated in equimolar amounts to form mixed dimers. C1 was reconstituted using this mixture and an excess of recombinant C1s and (serum) C1q. The recombinant proteins were applied as cell culture supernatant. As a control, C1 was reconstituted from proenzymic serum C1r2s2 and C1q in the presence of supernatant of wt AcMNPV-infected cells. Lane 1 is the reconstitution mixture. Lane 2 is the same mixture after incubation for 60 min at 30°C with heat-aggregated IgG (added in a negligible volume). Lane 3 is the control reconstitution mixture. Lane 4 is the control mixture after activation with aggregated IgG (60 min, 30°C). Equal amounts were analyzed by SDS-PAGE under reducing conditions. Western blot analysis detection was performed using anti-human C1r Ab. (Recombinant C1r has a smaller apparent m.w. than the serum form probably due to incomplete glycosylation by insect cells.) The composition of the mixtures is described in more detail in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During the activation of C1r, an Arg-Ile bond (3) is cleaved in the catalytic domain. This mechanism of activation through the cleavage of an Arg-Ile bond is quite common among serine proteases (3, 4, 9, 10, 15, 16, 17, 18, 19, 20). Our aim was to stabilize the proenzymic form of C1r by mutating this very conserved bond. Mutations were designed using isomorphic replacements (39), so they would not disturb the folding of the proteins. Because isolated C1r and C1r within the C1 complex have the capacity to autoactivate, (to cleave themselves), the mutants also provide us with information on the specificity of the C1r zymogen.

The recombinant proteins were expressed in the baculovirus-insect cell expression system, and cell culture supernatant was the source of recombinant C1r throughout the work. The activity of the mutants or wt C1r was tested using the very sensitive and specific hemolytic assay. Because serum-free medium was used for the expression, C1r was functionally pure, and cell culture supernatant was appropriate to reconstruct C1 for the hemolytic assay with carefully selected controls.

With mutants KI and RF, our aim was to obtain C1r that cannot autoactivate, but that can be activated by another serine protease like trypsin. The stability of the zymogen form was increased substantially by both mutations. Although wt C1r was mostly activated in the cell culture supernatant, the two mutants were found predominantly in their proenzyme form, although some activation still occurred. We believe that the rate of autoactivation is reduced in the mutant proteins, though increased resistance to cleavage by an extrinsic protease is also possible. Our study revealed that the presence of the Arg-Ile bond was not essential for the activity of C1r in vitro. Changing Arg463 to Lys or Ile464 to Phe did not impair the hemolytic activity of these proteins. Because these mutants are predominantly proenzymic in the cell supernatant, their activity in the hemolytic assay implies that they are able to autoactivate within the C1 complex.

In the third mutant, designated as mutant QI, the Arg in question was replaced by Gln. This substitution led to the complete stabilization of the zymogen form. This mutant, like wt C1r, was a dimer at pH 7.0, dissociated to monomers at pH 5.0, and formed a tetramer with C1s that could associate with C1q. (Verified by gel filtration. The fractions were analyzed by ELISA (data not shown).) However, mutant QI had no hemolytic activity if reconstituted to form C1. To test whether a single wt C1r molecule in the C1qC1r2C1s2 complex is enough for the biological activity, mixed dimers were formed between mutant QI and wt C1r. First, the pH was lowered to dissociate homodimers, then increased to neutral to form mixed dimers besides homodimers. To fixed amounts of wt C1r, increasing amounts of mutant QI were added. When a large amount of mutant QI was applied, the mixture was practically depleted of wt C1r homodimers and only mixed dimers were present beside the mutant QI homodimers. Mathematical analysis of this experiment predicts that if the mixed dimers were inactive, then the original activity of pure wt C1r would be reduced approaching zero if a large excess of mutant QI was used. If no mixed dimers were formed, then no change would be observed in the activity. If the mixed dimers were active, then the addition of mutant QI to wt C1r would increase the activity up to a maximum of twice of the original activity when a large excess of mutant QI was applied. Our results were consistent with the latter case, so we concluded that the mixed dimers were active. (Actually the plateau was already reached when an ~3-fold excess of mutant QI was used.) Our data imply that one active C1r subunit within C1 is sufficient for the full activity of the entire complex. Surprisingly, when mutant QI and wt C1r were simply mixed and incubated overnight at neutral pH and 4°C, the same results were observed indicating that the dimers were dissociated and reformed at a high rate even at pH 7.0. We have shown that mutant QI is not cleaved by wt C1r under conditions similar to that applied during the hemolytic assay. This observation solidifies our conclusion that one active C1r subunit in C1 is sufficient for the activity of the complex.

Additional experiments are required to reveal whether a single active C1r subunit activates only one or both C1s subunits. C1, containing one wt (active) C1r subunit and one inactive mutant QI subunit, is fully active. This can mean that both C1s subunits are accessible to one active C1r or that one active C1s subunit is sufficient for cleaving enough C4 and C2 to sustain the cascade.

When proenzymic C1r is purified from serum, the final product always contains some activated C1r. Our mutant QI is a nonactivable zymogen that allows the examination of pure proenzymic C1r and provides homogenous nonactivated material for purification and crystallization.


   Acknowledgments
 
We thank Dr. Earl Davie for providing us with the plasmid containing the full-length C1r cDNA, Dr. Max Summers for sending us the baculovirus expression system, and Júlia Balczer for her skillful technical assistance.


   Footnotes
 
1 This work was supported by Hungarian National Science Foundation (OTKA) Grants T017478 and F017316, by the Hungarian Academy of Sciences Research Grant AKP-96/2–684, and by the National Science Foundation Grant DMB 9120491. Back

2 Address correspondence and reprint requests to Péter Závodszky, Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest 1518, Pf. 7, Hungary. E-mail: Back

3 Abbreviations used in this paper: C1, the first component of complement; C1q, C1r, and C1s, subcomponents of C1; MASP, (mannan-binding lectin)-associated serine protease; Sf9, Spodoptera Frugiperda 9 cell line; AcMNPV, Autographa californica multiple nuclear polyhedrosis virus; VBS, veronal-buffered saline; C2 and C4, complement components; C-EDTA, serum (usually from guinea pig) diluted with EDTA-containing buffer so that only the late complement components are active; wt, wild-type Back

Received for publication January 1, 1998. Accepted for publication October 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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