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Factor D in the alternative pathway of complement activation: Purification, physicochemical characterization and functional role
lmmunochemistry, 1974, Vol. 11, pp. 527 532. Pergamon Press. Printed in Great l~ritain
FACTOR D IN THE ALTERNATE PATHWAY OF COMPLEMENT ACTIVATION: PURIFICATION, PHYSICOCHEMICAL CHARACTERIZATION A N D F U N C T I O N A L ROLE M. P. DIERICH,* U. H A D D I N G , W. K O N I G , M. L I M B E R T , H.-U. S C H O R L E M M E R a n d D. B I T T E R - S U E R M A N N Institut flir Medizinische Mikrobiologie, Johannes Gutenberg-Universit/it, Mainz, Germany (First received 20 November 1973; in revised form 22 February 1974) Abstract--A protein was isolated from guinea-pig serum which enabled cobra venom factor (VF), in
combination with purified C3 proactivator (C3PA) and Mg ÷+, to induce turnover of isolated C3. To parallel the terminology for human serum, the protein was designated as factor D of the alternate pathway of complement activation. Purified factor D has a mol. wt of 22,000 and an s-rate of 2.6. The isoelectric point was determined to be at pH 9.5. Factor D, although resistant to trypsin and hydrazine treatment, was relatively heat labile. It was found that the enzymatic activity directed against C3 was exerted by a trimolecular complex consisting of VF, C3PA and factor D. The existence of such a complex was demonstrated by gel filtration; additional evidence was provided by assembling an active complex on Sepharose-VF. Anti factor D serum blocked the C3 cleaving activity of the complex. Factor D itself was required for the C3 shunt activation induced by inulin, Dextran sulfate, Zymosan or endotoxin.
In earlier publications (Miiller-Eberhard et al., 1966; Dierich et al., 1971; MiJller-Eberhard a n d Fjellstr/Sm, 1971) it was reported that cobra v e n o m factor (VF) interacted with C3 proactivator (C3PA) in the presence of M g ÷÷, a n d lead to a complex which enzymatically activated C3. Hunsicker et al. (1973) have added to those findings by their discovery that an additional c o m p o n e n t of the alternate pathway, termed factor D, was necessary for the induction of C3 turnover by VF. In reexamining our own data (Bitter-Suermann et al., 1972) it became a p p a r e n t that since the C3PA preparations were always tested by addition of VF, Mg +÷ and purified C3 as substrate, they contained factor D associated to the proactivator. To date the literature dealing with physicochemical data or details of the functional behavior of factor D has not in our opinion been adequate; therefore it is the purpose of this paper to (1) describe a procedure for the preparation of purified factor D from guinea pig serum, and (2) to describe some of factor D's physicochemical characters and reaction mechanisms. Part of the results have been presented at the Fifth International C o m p l e m e n t W o r k s h o p (Hadding et al., 1973). MATERIALS AND METHODS
Chemicals The following chemicals were used: Zymosan (Nutritional Biochemicals Corporation, Cleveland, Ohio, U.S.A.); Sepharose 2B (Pharmacia, Uppsala, Sweden); trypsin (Schuchardt, Mfinchen, Germany); soybean trypsin inhibitor * Present address: Scripps Clinic and Research Foundation. 476 Prospect Street, La Jolla. California 92037, U.S.A. IMM Vol. [I. No,9
A
527
(Serva, Heidelberg, Germany); hydrazinium-hydroxide (Merck, Darmstadt, Germany); 2-mercaptoethanol, jodacetamide (Fluka A. G., Buchs S. W., Switzerland); human serum albumin (HSA), human IgG (Beriglobin) and Anti HSA (Behringwerke A. G., Marburg, Germany); catalase (Boehringer, Mannheim). Chromatography materials Whatman CM 52 cellulose, Whatman DE52 cellulose (Balston, Maidstone, England); CaOH-apatite, prepared as described earlier (Bitter-Suermann et al., 1970); Sephadex G 200, 100, 50 (Pharmacia, Uppsala, Sweden). Isolated proteins C3 (Bitter-Suermann et al., 1970) and VF (BitterSuermann et al., 1972) were purified as outlined previously. For some experiments VF was coupled to sepharose by using the bromcyanide method (Edelman et al., 1971). C3PA was purified as follows: EDTA was added to guineapig serum to a 0.01 M final concentration, after which it was diluted 1 : 10 in distilled HzO and the pH was corrected to 6.2 with HC1. This pool was stirred at 4°C for 30 min. The pseudoglobulins were put on CM cellulose (5 × 20cm, 0.01 M KPO 4 at pH 6.0) and eluted on a NaC1 gradient towards 15mS: activity was maximal at 7mS. The pool was dialysed and put on a DEAE cellulose (5 × 20cm, 0.01 M K P O 4 at pH 8-0); elution was performed by a NaC1 gradient towards 15 mS: activity was maximal at 4 mS. For next step the pool was put on a CaOH-apatite column (5 × 20 cm, 0.01 M KPO 4 at pH 8.6); activity could be eluted at 7 mS by a gradient towards 15 mS KPO4. The pool was rechromatographed on DEAE cellulose and then subjected to preparative polyacrylamide gel electrophoresis (7'5~o gel, Tris-HC1 buffer at pH8.6, 18hr run at a constant 30mA on ultraphor apparatus. Colora, Lorch, Germany). Activity could be eluted from a single band. The protein showed a homogeneous peak on a Sephadex G 200 column; its position corresponded to a mol. wt of 110,000.
528
M . P . DIERICH et al.
Anti factor D serum One millilitre of a concentrated factor D pool (2 mg/ml) from a Sephadex G 50 column was emulsified in 1 ml of complete Freund's adjuvant (CFA) Difco Laboratories, Detroit, Michigan, U.S.A.). Equal volumes of this mixture (0.5 ml) were injected into the both rear footpads of each of two rabbits. Fourteen days later a similar injection was given. After the same interval the rabbits were given a booster without CFA. A week later the animals were bled. For functional blocking of factor D an IgG fraction of anti factor D serum was obtained by precipitation with 50~o saturated ammonium sulfate and chromatography on DEAE cellulose (0'005 M sodium phosphate, pH 7.5). Test system An isotonic veronal buffer (VBS) containing Mg ÷+ in a concentration of I x 10-3M (VBS-Mg) was used as general diluent.
Test system for C3 Hemolytic activity of C3 was determined as described (Bitter-Suermann etal., 1972)and given either as O.D. values or as site forming units (SFU). Test system for C3PA Test system for kinetic experiments: 0-1 ml VF solution (50 ,ug/ml VBS-Mg) + 0.1 ml of a factor D solution + 0.1 ml C3 solution (containing about 20 x t08 site forming units/ml VBS) + 0'1 ml MgC12 solution (5 × 10 3 M/ml H 2 0 ) + 0.l ml sample were incubated, after which the test for C3 activity was done. The mentioned concentrations of VF, C3 and MgCI2 were used in all further test systems. Screening test: 0.1 ml heated (30 min at 56°C) guinea-pig serum + 0.1 ml VF solution + 0.1 ml sample were incubated at 37°C for 60 min, after which 0.1 ml of the mixture in appropriate dilutions was tested for C3. Test system for VF 0"1 ml C3PA solution, 0.1 ml factor D solution, 0.1 ml Mg solution (as above) and 0.1 ml sample were incubated at 37°C for 15 min, after which 0.1 ml C3 solution containing EDTA were added. The final concentration of EDTA in the mixture was 0.01 M. After a further incubation at 37°C for 60min, 0.1 ml of the mixture was tested for C3. Test system for factor D For kinetic experiments (and during the earlier work of purification) the following test system was used: 0.1 ml C3PA solution + 0-1 ml VF solution + 0.1 ml C3 solution + 0.1 ml MgCI2 solution + 0.1 ml test sample were incubated at 37°C for 60 min. Next, 0.1 ml of the mixture was tested for C3 (the loss of C3 is directly proportional to factor D activity). In the controls, C3PA or VF were replaced by VBS. Since guinea-pig serum can be deprived of factor D by molecular sieving because of its low mol. wt (see under results), the following screening method was used: Guinea-pig serum was first passed through a Sephadex G 100 column and then through a second Sephadex column with three consecutive segments (G 100, 2.5 x 40cm; G 75, 35 cm; and G 50, 40cm) after which factor D negative fractions were pooled and concentrated to the original volume. Such a serum is called D-free serum (Dfs). Addition of VF to 1:10 diluted Dfs does not lead to C3 turnover; however, addition of VF and factor D gives rise to marked C3 consumption. Dfs was used routinely in a 1:80 dilution to prepare Dfs-VF (a mixture of equal volumes of Dfs, C3 in VBS, and VF
in VBS and MgCI2). For controls, VF was replaced by VBS; this mixture was called Dfs-VBS. To test for factor D. 0.1 ml Dfs VF + 0.05 ml sample were incubated at 37 C for 60min, tested for C3 activity and compared to the control (0.1 ml Dfs-VBS + 0.05 ml sample). The amount of factor D which gave 50 per cent inactivation of C3 was defined as one unit (U) of factor D activity expressed on a 'per ml' basis.
Physicochemical characterization Except otherwise stated factor D material after the last purification step was taken for physicochemical characterization. The isoelectric point of factor D was determined by using LKB 8100 ampholine electrofocusing equipment and carrier ampholytes of a pH range from 3 to l0 and from 8 to 10. Estimation of factor D's molecular size was done using sephadex G 100 columns (2.5 x40cm). Markers (protein calibration kit, Boehringer, Mannheim, Germany) were human serum albumin (HSA, tool. wt 67,000), ovalbumin (mol. wt 45,000), chymotrypsinogen (tool. wt 25,000) and cytochrome c (mol. wt 13,500), all of which were located immunochemically or spectrophotometrically. Determination of the sedimentation rate was done at 30,000 rev/min for 18hr in a Beckman L2/65B ultracentrifuge with a SW 40 Ti rotor, using 5-20~o linear sucrose gradients in VBS containing 0.001 M Mg ++ and 0-00015 M Ca ++. H SA (4,6s), ovalbumin (3,0s), chymotrypsinogen (2, 6 s) and cytochrome e ( 1,7 s) served as markers. Sensitivity to trypsin digestion was checked by incubating 0.1 ml trypsin solution (0.3mg/ml VBS-Mg + C a * * 5 x 10- 4 M) with 0.1 ml factor D solution for 30 rain at 37"C, and then adding 0.1 ml soybean trypsin inhibitor (I mg/ml VBS-Mg) and incubating again for 10 min at 37°C. At this concentration the inhibitor showed no interference in the C3 test, but completely blocked the trypsin activity. The whole mixture was then examined for factor D activity. Sensitivity to hydrazine was tested by incubating guineapig serum with an equal volume of hydrazine (final concentration, 0.125 M) at 37°C for 45 min. This mixture was dialysed and tested for factor D. For dialysis of factor D pools cellophane bags (Kalle A. G., Wiesbaden, Germany) were used, since in comparison to other materials the loss of factor D in these bags was small. Reduction and alkylation of factor D was done by incubating it with 2-mercaptoethanol (0.1 M final concentration) at 25"C for 60min and then adding jodacetamide (final concentration 0.2 M) and incubating the mixture for another 10 min. The sample was then extensively dialysed and tested for factor D. Acid polyacrylamide gel electrophoresis was performed in 7"5Yo gels in /~-alanine-acetic acid buffer according to Reisfeld (1962). The gels were stained by a mixture of amidoblack (0"25yo) and coomassie blue R 250 (0"25Yo) in trichloracetic acid. The amino acid composition of factor D was determined after acid hydrolysis (6 N HC1, 110 ° C, 24 hr, vacuum) with an automatic analyser (Biotronik), essentially according to Hamilton (1963). Protein was estimated according to Lowry et al. (19511. RESULTS
Purification procedure The purification of factor D was started by preparing eu- a n d p s e u d o g l o b u l i n s from 600 ml guinea-pig s e r u m
Factor D in the Alternate Pathway of Complement Activation
529
Table 1. Purification procedure for factor D Column CM cellulose (5 × 20 cm) DEAE cellulose (6 x 20cm) CaOH apatite (6 × 20 cm) Sephadex G 100 (2"5 × 90 cm) Sephadex G 50 (2"5 x 90 cm)
Starting buffer
Elution of factor D at:
Gradient
0.02 M KPO 4 pH 6"0
Towards 30 mS NaCI
18-20 mS
0'02 M KPO 4 pH 8"0
Towards 15 mS NaCI
5-6 mS
0.02 M KPO4 pH 8.0
Towards 25 mS KPO4
8-10 mS
0.02 M Tris-HCl pH 7.5 NaC10.2 M 0.02 M Tris-HCl pH 7.5 NaCI 0"2 M
at pH 6"2 as described for the C3PA purification in Materials and Methods. The pseudoglobulins containing the bulk of factor D activity were further processed on three different ion exchange and two different molecular sieving columns in the sequence shown in Table 1. To prepare the sample for Sephadex G 100 and G 50 columns, dialysis was performed against distilled water and followed by lyophilization. The average yield of a preparation was 2-4 mg after dialysis against H 2 0 and lyophilization. Use of the Folin method showed that only about 10 per cent of this material was protein. According to preliminary experiments the major part of the material seemed to be polysaccharide. The data for a typical preparation related to the protein content were as follows: The final product contained 385#g protein with 66U of factor D per #g. The starting material (6000 ml pseudoglobulins with 7'5 mg protein/ml) had an activity of 2080U/ml or 0"28 U/#g protein. Thus the yield with regard to units was 0"2Vo and the increase in specific activity 234-fold. To demonstrate the purity of the material after the last isolation step we performed PAA gel electrophoresis at pH 8.6; factor D activity could not be detected in the gels. At pH 4'3 a component with relatively poor factor D activity was present in the gel; a faint band corresponding to factor D activity could be detected by staining; no other contaminating proteins were present.
'X•A
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VE/Vo Fig. 1. Determination of the mol. wt of factor D on a Sephadex G100 column, using HSA, ovalbumin, chymotrypsinogen and cytochrome c as markers. Factor D elutes shortly behind chymotrypsinogen (mol. wt 25,000), which indicates a mol. wt of about 22,000. D in guinea-pig serum, a marked reduction of activity occurred at 50°C. At 56°C, purified factor D showed an activity loss of 90-95 per cent and factor D activity in guinea-pig serum decreased about 70 per cent. Thus, the activity remaining at 56°C should be
Physicochemical characterization Factor D's isoelectric point was determined in several electrofocusing analyses. Using ampholines from p H 3"0 to 10"0, the peak of factor D activity was found between pH 9'34 and 9-76; with ampholines from pH 8-0 to 10'0 the maximal activity was located at pH 9.47 to 9.51. The mol. wt of factor D was 22,000 (Fig. 1) and the sedimentation rate 2'6 (Fig. 2). To find out whether the sedimentation rate was influenced by pH we prepared the gradient in buffers of pH 4"5 to 8'0. The sedimentation rate turned out to be independent of the pH. The activity was markedly reduced at pH 4'5 and stable at pH 5'5 to 8-0. Heat stability was determined by exposure of purified factor D or guinea-pig serum to various temperatures for 30 min. For purified factor D as well as for factor
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Fig. 2. Determination of the s-rate of factor D using sucrose gradient ultracentrifugation an.d HSA, ovalbumin and cytochrome c as markers. The s-rate of factor D was found to be 2.6 s.
530
M. P. DIERICH et al. Table 2. Physicochemical characteristics of factor D Mol.wt s-rate Isoelectric point Sensitivity to: Hydrazine (0-125M) Heat (30min 56~C)
22,000 2"6s pH 9"5
Trypsin (0' 15 mg/ml) Reduction and carboxamidomethylation H+ ion concn (18 hr)
taken into consideration when using heated sera in assays of C3PA. For both preparations the activity was completely lost at 65°C. Another remarkable feature of factor D is its resistance to trypsin digestion. An incubation at 37°C for 30rain with trypsin at a final concentration of0'15 mg/ ml did not even slightly reduce the activity of factor D (concentration 0.07 mg protein/ml). On the other hand, in a parallel experiment, 0.005 mg trypsin/ml reduced C3 (concentration 0.2 mg/ml) to 10 per cent of its original activity within 10 min. A trypsin-treated factor D pool when subjected to gel filtration on a Sephadex G 100 column turned out to be unaltered with respect to its elution behavior, which indicates that no change of mol. wt had occurred. Factor D's hydrazine sensitivity was assessed by incubating guinea-pig serum in the presence of 0-125 M hydrazine. After dialysis, factor D activity turned out to be unimpaired. However, factor D activity could be destroyed completely by reduction and carboxamidomethylation when exposed to 2-mercaptoethanol andjodacetamide. The physicochemical parameters are summarized in Table 2. To determine the amino acid composition of factor D a pool of purified material was analysed. The amino acid composition is shown in Table 3. Binding of factor D to macromolecules At low NaC1 concentration factor D tends to bind to proteins and to Sephadex particles. Therefore, 20mS NaC1 was always applied during preparation of Dfs and during the final preparation step on Sephadex. We investigated the ability of factor D to adsorb Table 3. Amino acid composition of factor D CySO3H Asp Thr Ser Glu Pro Gly Ala Val
1.0" 5"5 3.4 6"1 9.9 3.4 6.1 7.9 3.8
"All values in mole %.
lleu Leu Tyr Phe Unidentified NH 3 Lys His Arg
2.7 5-1 1.4 2.4 4.4 27.0 5-5 2-1 2.4
No loss of activity 90~-950/oloss of activity for purified factor D, 7070 loss for factor D in serum No loss of activity, no change of mol.wt Complete loss of activity pH 5.5-8.0: no reduction of activity pH 4.5: 950,', reduction of activity
to different substances like Zymosan, Sepharose 2B and Sephadex and found that those substances were able to bind factor D. The binding process is very rapid and proceeds equally well at 4°C and at 37°C. Binding was demonstrated both by loss of activity from the supernatant and appearance of factor D activity on the surface of Zymosan or Sepharose 2B particles. The fixation of factor D on a surface increased its potency as cofactor in the bypass system, since much more activity was found on the particles than was lost from the supernatant. After adsorption factor D remained in its native state, exhibiting an activity only after incubation in a standard test system (Dfs-VF) or by interaction with purified VF, C3PA and Mg ++. Purified factor D adsorbed on Sepharose particles could be functionally blocked by the IgG fraction of an anti factor D serum. After treating the particles with these antibodies and washing them, no further factor D activity could be demonstrated. An unrelated IgG preparation did not show this effect. Functional role of factor D The functional role of factor D was investigated in a system consisting of purified VF, C3PA, factor D and Mg +÷. Only in the presence of all factors could a C3 cleaving enzyme be generated. As shown in Fig. 3, the enzyme formation was strictly dose dependent for each of the four reactants involved. Mg ++ ions are needed only for generation of the enzyme and not for its action, since C3 cleavage proceeds in the presence of EDTA (Bitter-Suermann et al., 1972). We therefore used EDTA to stop the enzyme formation at different time intervals. The amount of enzyme formed up to the respective time was determined by its ability to turn over C3 within 90rain at 37°C. As shown in Fig. 4, the kinetics of C3 cleaving enzyme formation revealed a time and temperature dependence typical of enzymatic processes. To elucidate the sequential interaction of VF, C3PA and factor D, we preincubated two of the three proteins at 3T'C for 20 min in.the presence of Mg ++. Afterward, the mixtures were colled to 25°C, the third reactant was added, and the mixture was incubated up to 20min. The 25°C temperature was chosen to slow down the reaction velocity of complex formation. At
Factor D in the Alternate Pathway of Complement Activation
531
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600
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400
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,
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Fig. 3. Dose dependence of the formation of the C3 cleaving enzyme from purified VF (A--A), C3PA (r-l--r-q), factor D ( O - - - O ) and Mg + + (x . . . x). The three partners in the reaction are kept constant and one is varied in its concentration by dilution (abscissa). The respective mixtures contained 0.05 ml VF (0.25 mg/ml VBS), 0'1 ml C3PA in VBS, 0.1 ml factor D in VBS and 0"05 ml 1 x 10 -2 M MgC12 in H20. The original concentrations of C3PA and factor D were chosen to yield an optimal C3 cleaving activity with the mentioned VF concentration. After 30 rain at 37°C samples were taken and were incubated with C3 in the presence of 0.015 M EDTA for another 30 min at 37°C in order to determine the amount of C3 cleaving enzyme formed. Large amounts of remaining C3 indicate poor enzyme formation and small amounts of remaining C3 indicate optimal enzyme formation. various times during the 25°C incubation period samples were taken, E D T A was added to them and they were tested for C3 turnover at 37°C for 9 0 m i n (Fig. 5). N o one c o m b i n a t i o n had a significant advantage over the others, which indicated that none of the ~--
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~ o
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,
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rain
Fig. 4. Time and temperature dependence of the formation of C3 cleaving enzyme from VF, C3PA, factor D and Mg ÷ +. The mixtures were kept at 4°C ( O - - - O ) , 25°C (/x--/x) and 37°C ( 1 2 - - - I-q)for 5 sec to 10 min. At various time intervals samples were taken and were incubated with C3 in the presence of 0.015M EDTA for 30min at 37°C to test for C3 cleaving activity (ordinate as in Fig. 3). The symbols i , • and • represent the controls for the three temperature curves. In these samples VF was replaced by VBS.
I0 min
15
20
Fig. 5. Influence of preincubating various combinations of VF, C3PA and factor D on the generation of a stable enzyme. C3PA and factor D ( © - - - O ) , VF and C3PA (/X A), and VF and factor D ( C ] - - - D). The time on the abscissa represents the incubation period at 25°C prior to the final C3 test. combinations decayed or were activated during the first incubation at 37 ° C. Therefore, it was t h o u g h t possible to find out which interaction needed M g ÷+. In order to find this out, either V F + C 3 P A or V F + f a c t o r D or C3PA + factor D were preincubated in the presence of M g ÷÷, after which E D T A was added, followed by factor D, C3PA or VF, respectively. However, in n o case was any C3 cleaving enzyme detectable. It seems, then, that VF, C3PA and factor D have to be present along with M g ÷÷ in order to give rise to an enzyme acting on C3. The participation of factor D in enzyme formation was also evident from the fact that blocking of factor D by the IgG fraction of the anti factor D serum prevented the generation of enzyme activity. To assess the binding capacity of factor D to V F or other proteins we u n d e r t o o k the following experiments. Factor D was incubated with either VF, C3PA, HSA or h u m a n IgG (Beriglobin) for 3 0 m i n at 37°C a n d then c h r o m a t o g r a p h e d on Sephadex G 100 columns (0.02 M Tris-HCl, pH 7.5). As a result of this treatment the four proteins eluted at their expected positions, and all carried factor D activity with them; C3PA, HSA and IgG carried small amounts of activity and V F carried a fairly large a m o u n t of activity. At this stage of the experiments the question again was raised, of whether a complex formation between two or more of the reaction partners was a prerequisite for the generation of enzymatic activity from VF, C3PA, Mg ÷+ and factor D. The question was answered by the following experiment. V F and C3PA were incubated in the presence of Mg ++ a n d then applied to a Sephadex G 200 column. Besides the two isolated proteins in their proper elution position, fractions were detected which on mere addition of factor D + M g ÷+ were capable of turning over C3. These fractions were located in front of V F (which indicated a mol. wt of a b o u t 200,000) and so are t h o u g h t to contain V F - C 3 P A complexes lacking enzymatic activity against C3 (Fig. 6). Consequently, V F apparently reacts equally well with C3PA a n d factor D. For the generation of a n active V F - C 3 P A - f a c t o r D complex it did not matter which
532
M. P. DIERICH et al. ~0 6
--
VF-C3PA-D ',
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PA C3PA
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II
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v~/vo Fig. 6. Demonstration of complex formation between VF and C3PA or VF, C3PA and factor D by gelfiltration on a Sephadex G200 column (90 × 1.5 cm). The position of the VF C3PA-D complex was determined in a separate run under exactly the same conditions. As markers served catalase, IgG and human Hb. reactant was offered first to VF. When such complexes were chromatographed on Sephadex G 200 columns they eluted slightly prior to the position of V F - C 3 P A complexes (Fig. 6). Furthermore, an active complex could be built on Sepharose particles to which VF was first coupled. This Sepharose-VF was later incubated with C3PA, factor D and Mg ++. The supposition that factor D is incorporated into the V F - C 3 P A complex was confirmed by the use of anti factor D serum. This antiserum completely blocked the activity of the C3 cleaving enzyme. Factor D, then, is not only needed for C3 turnover by VF but also for induction of C3 activation by other subs~;ances. Using inulin, endotoxin, Zymosan or Dextran sulfate, typical dose response curves were obtained with Dfs (diluted 1:10) and factor D (in various concentrations) (Hadding et al., 1973). Factor D (guinea-pig) was also able to substitute for human factor D and induce a C3 turnover in a system consisting of VF or DS and D free human serum used in the guinea-pig system described above. DISCUSSION Since the reaction mechanism of the whole bypass sequence are very complex, we studied that part which is believed to represent the final steps of the sequence and which can be initiated by VF. It has been demonstrated in this paper that factor D was incorporated into a V F - C 3 P A complex and that the incorporation of factor D in the presence of Mg ++ rendered the final trimolecular complex active against C3. During our investigations described here we did not obtain any evidence that additional factors were needed for cleavage of C3. In so far our data are in agreement with those of Cooper (1973) and Hunsicker et aL (1973), but they are at variance with results of Hunsicker, who could not find a complex consisting of VF, C3PA and factor D even though participation of these three factors in cleaving C3 is well accepted. Our various kinetic experiments failed to elucidate a reaction sequence for the three factors because it
was found that all three and Mg +÷ had to be present simultaneously for the generation of enzymatic activity. In addition, no conclusion can be drawn from our data as to which factor carries the enzymatically active center for C3 conversion. This active site could be formed by factor D, by C3PA or by a C3PA-factor D complex analogous to the C42 enzyme. A remarkable feature of factor D is its tendency to bind to various substances even at low (4°C) temperatures, e.g. to particles of Sephadex, Sepharose 2B and Zymosan. When using a zymosan absorbed serum and testing for properdin it has to be taken into consideration that the serum's factor D content will be reduced. In this report we preferred to call the isolated protein factor D and not C3PAase (MiJller-Eberhard and G6tze, 1972), since we had no direct evidence for cleavage of C3PA by factor D. A possibility which seems likely is that, because of their similar molecular weights, the factors may prove to be identical. Acknowledyements--This work was supported by grants
from the DFG. We are indebted to Miss M. C. Brand and S. Risi, Ph.D., for performing the amino acid analysis of factor D at the Institut r0r Biochemie, Johannes GutenbergUniversitfit, Mainz, Germany. The authors also wish to thank Miss U. Aulbach, Mrs. M. Liider and Miss R Schwarz for their competent technical assistance. REFERENCES
Alper Ch. A., Goodkofsky J. and Lepow I. H. (1973) J. exp. Med. 137, 424. Bitter-Suermann D., Hadding U., Melchert F. and Wellensiek H. J. (1970) lmmunochemistry 7, 955. Bitter-Suermann D., Dierich M. P., K6nig W. and Hadding U. (1972) Immunology 23, 267. Cooper N. (1973) J. exp. Med. 137, 451. Dierich M. P., Bitter-Suermann D., K6nig W. and Hadding U. (1971) Europ. J. lmmun. 1, 309. Edelman G. M., Rutishauser U. and Millette C. F. (1971) Proc. natn. Acad. Sci. U.S.A. 68, 2153. G/Stze O. and Miiller-Eberhard J. J. (1972) New Engl. J. Med. 286. 180.
Hadding U., Dierich M. P., KSnig W., Limbert M., Schorlemmer U. und Bitter-Suermann D. (1973) J. Immun. 111,286. Hadding U., Dierich M., K6nig W., Limbert M., Schorlemmer H. U. and Bitter-Suermann D. (1973) Europ. J. lmmun. 3, 527. Hamilton P. B. (1963) Analyt. Chem. 35, 2055. Hunsicker L. G., Ruddy S. and Austen K. F. (1973) J. lmmun. 110, 128. Lowry O. H., Rosebrough N. Y., Farr A. L. and Randall R. J. (1951) d. biol. Chem. 193, 265. Mtiller-Eberhard H. J., Nilsson U. R., Dalmasso A. P., Polley M. J. and Calcott M. A. (1966) Archs Path. 82, 205. Mi.iller-Eberhard J. J. and Fjellstr6m K. E. (1971) J. lmmun. 107, 1666. Mtiller-Eberhard J. J. and GiStze O. (1972) J. exp. Med. 135, 1003. Reisfeld R. A., Lewis U. J. and Williams D. E. (1962) Nature, Lond. 195. 281.
FACTOR D IN THE ALTERNATE PATHWAY OF COMPLEMENT ACTIVATION: PURIFICATION, PHYSICOCHEMICAL CHARACTERIZATION A N D F U N C T I O N A L ROLE M. P. DIERICH,* U. H A D D I N G , W. K O N I G , M. L I M B E R T , H.-U. S C H O R L E M M E R a n d D. B I T T E R - S U E R M A N N Institut flir Medizinische Mikrobiologie, Johannes Gutenberg-Universit/it, Mainz, Germany (First received 20 November 1973; in revised form 22 February 1974) Abstract--A protein was isolated from guinea-pig serum which enabled cobra venom factor (VF), in
combination with purified C3 proactivator (C3PA) and Mg ÷+, to induce turnover of isolated C3. To parallel the terminology for human serum, the protein was designated as factor D of the alternate pathway of complement activation. Purified factor D has a mol. wt of 22,000 and an s-rate of 2.6. The isoelectric point was determined to be at pH 9.5. Factor D, although resistant to trypsin and hydrazine treatment, was relatively heat labile. It was found that the enzymatic activity directed against C3 was exerted by a trimolecular complex consisting of VF, C3PA and factor D. The existence of such a complex was demonstrated by gel filtration; additional evidence was provided by assembling an active complex on Sepharose-VF. Anti factor D serum blocked the C3 cleaving activity of the complex. Factor D itself was required for the C3 shunt activation induced by inulin, Dextran sulfate, Zymosan or endotoxin.
In earlier publications (Miiller-Eberhard et al., 1966; Dierich et al., 1971; MiJller-Eberhard a n d Fjellstr/Sm, 1971) it was reported that cobra v e n o m factor (VF) interacted with C3 proactivator (C3PA) in the presence of M g ÷÷, a n d lead to a complex which enzymatically activated C3. Hunsicker et al. (1973) have added to those findings by their discovery that an additional c o m p o n e n t of the alternate pathway, termed factor D, was necessary for the induction of C3 turnover by VF. In reexamining our own data (Bitter-Suermann et al., 1972) it became a p p a r e n t that since the C3PA preparations were always tested by addition of VF, Mg +÷ and purified C3 as substrate, they contained factor D associated to the proactivator. To date the literature dealing with physicochemical data or details of the functional behavior of factor D has not in our opinion been adequate; therefore it is the purpose of this paper to (1) describe a procedure for the preparation of purified factor D from guinea pig serum, and (2) to describe some of factor D's physicochemical characters and reaction mechanisms. Part of the results have been presented at the Fifth International C o m p l e m e n t W o r k s h o p (Hadding et al., 1973). MATERIALS AND METHODS
Chemicals The following chemicals were used: Zymosan (Nutritional Biochemicals Corporation, Cleveland, Ohio, U.S.A.); Sepharose 2B (Pharmacia, Uppsala, Sweden); trypsin (Schuchardt, Mfinchen, Germany); soybean trypsin inhibitor * Present address: Scripps Clinic and Research Foundation. 476 Prospect Street, La Jolla. California 92037, U.S.A. IMM Vol. [I. No,9
A
527
(Serva, Heidelberg, Germany); hydrazinium-hydroxide (Merck, Darmstadt, Germany); 2-mercaptoethanol, jodacetamide (Fluka A. G., Buchs S. W., Switzerland); human serum albumin (HSA), human IgG (Beriglobin) and Anti HSA (Behringwerke A. G., Marburg, Germany); catalase (Boehringer, Mannheim). Chromatography materials Whatman CM 52 cellulose, Whatman DE52 cellulose (Balston, Maidstone, England); CaOH-apatite, prepared as described earlier (Bitter-Suermann et al., 1970); Sephadex G 200, 100, 50 (Pharmacia, Uppsala, Sweden). Isolated proteins C3 (Bitter-Suermann et al., 1970) and VF (BitterSuermann et al., 1972) were purified as outlined previously. For some experiments VF was coupled to sepharose by using the bromcyanide method (Edelman et al., 1971). C3PA was purified as follows: EDTA was added to guineapig serum to a 0.01 M final concentration, after which it was diluted 1 : 10 in distilled HzO and the pH was corrected to 6.2 with HC1. This pool was stirred at 4°C for 30 min. The pseudoglobulins were put on CM cellulose (5 × 20cm, 0.01 M KPO 4 at pH 6.0) and eluted on a NaC1 gradient towards 15mS: activity was maximal at 7mS. The pool was dialysed and put on a DEAE cellulose (5 × 20cm, 0.01 M K P O 4 at pH 8-0); elution was performed by a NaC1 gradient towards 15 mS: activity was maximal at 4 mS. For next step the pool was put on a CaOH-apatite column (5 × 20 cm, 0.01 M KPO 4 at pH 8.6); activity could be eluted at 7 mS by a gradient towards 15 mS KPO4. The pool was rechromatographed on DEAE cellulose and then subjected to preparative polyacrylamide gel electrophoresis (7'5~o gel, Tris-HC1 buffer at pH8.6, 18hr run at a constant 30mA on ultraphor apparatus. Colora, Lorch, Germany). Activity could be eluted from a single band. The protein showed a homogeneous peak on a Sephadex G 200 column; its position corresponded to a mol. wt of 110,000.
528
M . P . DIERICH et al.
Anti factor D serum One millilitre of a concentrated factor D pool (2 mg/ml) from a Sephadex G 50 column was emulsified in 1 ml of complete Freund's adjuvant (CFA) Difco Laboratories, Detroit, Michigan, U.S.A.). Equal volumes of this mixture (0.5 ml) were injected into the both rear footpads of each of two rabbits. Fourteen days later a similar injection was given. After the same interval the rabbits were given a booster without CFA. A week later the animals were bled. For functional blocking of factor D an IgG fraction of anti factor D serum was obtained by precipitation with 50~o saturated ammonium sulfate and chromatography on DEAE cellulose (0'005 M sodium phosphate, pH 7.5). Test system An isotonic veronal buffer (VBS) containing Mg ÷+ in a concentration of I x 10-3M (VBS-Mg) was used as general diluent.
Test system for C3 Hemolytic activity of C3 was determined as described (Bitter-Suermann etal., 1972)and given either as O.D. values or as site forming units (SFU). Test system for C3PA Test system for kinetic experiments: 0-1 ml VF solution (50 ,ug/ml VBS-Mg) + 0.1 ml of a factor D solution + 0.1 ml C3 solution (containing about 20 x t08 site forming units/ml VBS) + 0'1 ml MgC12 solution (5 × 10 3 M/ml H 2 0 ) + 0.l ml sample were incubated, after which the test for C3 activity was done. The mentioned concentrations of VF, C3 and MgCI2 were used in all further test systems. Screening test: 0.1 ml heated (30 min at 56°C) guinea-pig serum + 0.1 ml VF solution + 0.1 ml sample were incubated at 37°C for 60 min, after which 0.1 ml of the mixture in appropriate dilutions was tested for C3. Test system for VF 0"1 ml C3PA solution, 0.1 ml factor D solution, 0.1 ml Mg solution (as above) and 0.1 ml sample were incubated at 37°C for 15 min, after which 0.1 ml C3 solution containing EDTA were added. The final concentration of EDTA in the mixture was 0.01 M. After a further incubation at 37°C for 60min, 0.1 ml of the mixture was tested for C3. Test system for factor D For kinetic experiments (and during the earlier work of purification) the following test system was used: 0.1 ml C3PA solution + 0-1 ml VF solution + 0.1 ml C3 solution + 0.1 ml MgCI2 solution + 0.1 ml test sample were incubated at 37°C for 60 min. Next, 0.1 ml of the mixture was tested for C3 (the loss of C3 is directly proportional to factor D activity). In the controls, C3PA or VF were replaced by VBS. Since guinea-pig serum can be deprived of factor D by molecular sieving because of its low mol. wt (see under results), the following screening method was used: Guinea-pig serum was first passed through a Sephadex G 100 column and then through a second Sephadex column with three consecutive segments (G 100, 2.5 x 40cm; G 75, 35 cm; and G 50, 40cm) after which factor D negative fractions were pooled and concentrated to the original volume. Such a serum is called D-free serum (Dfs). Addition of VF to 1:10 diluted Dfs does not lead to C3 turnover; however, addition of VF and factor D gives rise to marked C3 consumption. Dfs was used routinely in a 1:80 dilution to prepare Dfs-VF (a mixture of equal volumes of Dfs, C3 in VBS, and VF
in VBS and MgCI2). For controls, VF was replaced by VBS; this mixture was called Dfs-VBS. To test for factor D. 0.1 ml Dfs VF + 0.05 ml sample were incubated at 37 C for 60min, tested for C3 activity and compared to the control (0.1 ml Dfs-VBS + 0.05 ml sample). The amount of factor D which gave 50 per cent inactivation of C3 was defined as one unit (U) of factor D activity expressed on a 'per ml' basis.
Physicochemical characterization Except otherwise stated factor D material after the last purification step was taken for physicochemical characterization. The isoelectric point of factor D was determined by using LKB 8100 ampholine electrofocusing equipment and carrier ampholytes of a pH range from 3 to l0 and from 8 to 10. Estimation of factor D's molecular size was done using sephadex G 100 columns (2.5 x40cm). Markers (protein calibration kit, Boehringer, Mannheim, Germany) were human serum albumin (HSA, tool. wt 67,000), ovalbumin (mol. wt 45,000), chymotrypsinogen (tool. wt 25,000) and cytochrome c (mol. wt 13,500), all of which were located immunochemically or spectrophotometrically. Determination of the sedimentation rate was done at 30,000 rev/min for 18hr in a Beckman L2/65B ultracentrifuge with a SW 40 Ti rotor, using 5-20~o linear sucrose gradients in VBS containing 0.001 M Mg ++ and 0-00015 M Ca ++. H SA (4,6s), ovalbumin (3,0s), chymotrypsinogen (2, 6 s) and cytochrome e ( 1,7 s) served as markers. Sensitivity to trypsin digestion was checked by incubating 0.1 ml trypsin solution (0.3mg/ml VBS-Mg + C a * * 5 x 10- 4 M) with 0.1 ml factor D solution for 30 rain at 37"C, and then adding 0.1 ml soybean trypsin inhibitor (I mg/ml VBS-Mg) and incubating again for 10 min at 37°C. At this concentration the inhibitor showed no interference in the C3 test, but completely blocked the trypsin activity. The whole mixture was then examined for factor D activity. Sensitivity to hydrazine was tested by incubating guineapig serum with an equal volume of hydrazine (final concentration, 0.125 M) at 37°C for 45 min. This mixture was dialysed and tested for factor D. For dialysis of factor D pools cellophane bags (Kalle A. G., Wiesbaden, Germany) were used, since in comparison to other materials the loss of factor D in these bags was small. Reduction and alkylation of factor D was done by incubating it with 2-mercaptoethanol (0.1 M final concentration) at 25"C for 60min and then adding jodacetamide (final concentration 0.2 M) and incubating the mixture for another 10 min. The sample was then extensively dialysed and tested for factor D. Acid polyacrylamide gel electrophoresis was performed in 7"5Yo gels in /~-alanine-acetic acid buffer according to Reisfeld (1962). The gels were stained by a mixture of amidoblack (0"25yo) and coomassie blue R 250 (0"25Yo) in trichloracetic acid. The amino acid composition of factor D was determined after acid hydrolysis (6 N HC1, 110 ° C, 24 hr, vacuum) with an automatic analyser (Biotronik), essentially according to Hamilton (1963). Protein was estimated according to Lowry et al. (19511. RESULTS
Purification procedure The purification of factor D was started by preparing eu- a n d p s e u d o g l o b u l i n s from 600 ml guinea-pig s e r u m
Factor D in the Alternate Pathway of Complement Activation
529
Table 1. Purification procedure for factor D Column CM cellulose (5 × 20 cm) DEAE cellulose (6 x 20cm) CaOH apatite (6 × 20 cm) Sephadex G 100 (2"5 × 90 cm) Sephadex G 50 (2"5 x 90 cm)
Starting buffer
Elution of factor D at:
Gradient
0.02 M KPO 4 pH 6"0
Towards 30 mS NaCI
18-20 mS
0'02 M KPO 4 pH 8"0
Towards 15 mS NaCI
5-6 mS
0.02 M KPO4 pH 8.0
Towards 25 mS KPO4
8-10 mS
0.02 M Tris-HCl pH 7.5 NaC10.2 M 0.02 M Tris-HCl pH 7.5 NaCI 0"2 M
at pH 6"2 as described for the C3PA purification in Materials and Methods. The pseudoglobulins containing the bulk of factor D activity were further processed on three different ion exchange and two different molecular sieving columns in the sequence shown in Table 1. To prepare the sample for Sephadex G 100 and G 50 columns, dialysis was performed against distilled water and followed by lyophilization. The average yield of a preparation was 2-4 mg after dialysis against H 2 0 and lyophilization. Use of the Folin method showed that only about 10 per cent of this material was protein. According to preliminary experiments the major part of the material seemed to be polysaccharide. The data for a typical preparation related to the protein content were as follows: The final product contained 385#g protein with 66U of factor D per #g. The starting material (6000 ml pseudoglobulins with 7'5 mg protein/ml) had an activity of 2080U/ml or 0"28 U/#g protein. Thus the yield with regard to units was 0"2Vo and the increase in specific activity 234-fold. To demonstrate the purity of the material after the last isolation step we performed PAA gel electrophoresis at pH 8.6; factor D activity could not be detected in the gels. At pH 4'3 a component with relatively poor factor D activity was present in the gel; a faint band corresponding to factor D activity could be detected by staining; no other contaminating proteins were present.
'X•A
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VE/Vo Fig. 1. Determination of the mol. wt of factor D on a Sephadex G100 column, using HSA, ovalbumin, chymotrypsinogen and cytochrome c as markers. Factor D elutes shortly behind chymotrypsinogen (mol. wt 25,000), which indicates a mol. wt of about 22,000. D in guinea-pig serum, a marked reduction of activity occurred at 50°C. At 56°C, purified factor D showed an activity loss of 90-95 per cent and factor D activity in guinea-pig serum decreased about 70 per cent. Thus, the activity remaining at 56°C should be
Physicochemical characterization Factor D's isoelectric point was determined in several electrofocusing analyses. Using ampholines from p H 3"0 to 10"0, the peak of factor D activity was found between pH 9'34 and 9-76; with ampholines from pH 8-0 to 10'0 the maximal activity was located at pH 9.47 to 9.51. The mol. wt of factor D was 22,000 (Fig. 1) and the sedimentation rate 2'6 (Fig. 2). To find out whether the sedimentation rate was influenced by pH we prepared the gradient in buffers of pH 4"5 to 8'0. The sedimentation rate turned out to be independent of the pH. The activity was markedly reduced at pH 4'5 and stable at pH 5'5 to 8-0. Heat stability was determined by exposure of purified factor D or guinea-pig serum to various temperatures for 30 min. For purified factor D as well as for factor
--
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Fig. 2. Determination of the s-rate of factor D using sucrose gradient ultracentrifugation an.d HSA, ovalbumin and cytochrome c as markers. The s-rate of factor D was found to be 2.6 s.
530
M. P. DIERICH et al. Table 2. Physicochemical characteristics of factor D Mol.wt s-rate Isoelectric point Sensitivity to: Hydrazine (0-125M) Heat (30min 56~C)
22,000 2"6s pH 9"5
Trypsin (0' 15 mg/ml) Reduction and carboxamidomethylation H+ ion concn (18 hr)
taken into consideration when using heated sera in assays of C3PA. For both preparations the activity was completely lost at 65°C. Another remarkable feature of factor D is its resistance to trypsin digestion. An incubation at 37°C for 30rain with trypsin at a final concentration of0'15 mg/ ml did not even slightly reduce the activity of factor D (concentration 0.07 mg protein/ml). On the other hand, in a parallel experiment, 0.005 mg trypsin/ml reduced C3 (concentration 0.2 mg/ml) to 10 per cent of its original activity within 10 min. A trypsin-treated factor D pool when subjected to gel filtration on a Sephadex G 100 column turned out to be unaltered with respect to its elution behavior, which indicates that no change of mol. wt had occurred. Factor D's hydrazine sensitivity was assessed by incubating guinea-pig serum in the presence of 0-125 M hydrazine. After dialysis, factor D activity turned out to be unimpaired. However, factor D activity could be destroyed completely by reduction and carboxamidomethylation when exposed to 2-mercaptoethanol andjodacetamide. The physicochemical parameters are summarized in Table 2. To determine the amino acid composition of factor D a pool of purified material was analysed. The amino acid composition is shown in Table 3. Binding of factor D to macromolecules At low NaC1 concentration factor D tends to bind to proteins and to Sephadex particles. Therefore, 20mS NaC1 was always applied during preparation of Dfs and during the final preparation step on Sephadex. We investigated the ability of factor D to adsorb Table 3. Amino acid composition of factor D CySO3H Asp Thr Ser Glu Pro Gly Ala Val
1.0" 5"5 3.4 6"1 9.9 3.4 6.1 7.9 3.8
"All values in mole %.
lleu Leu Tyr Phe Unidentified NH 3 Lys His Arg
2.7 5-1 1.4 2.4 4.4 27.0 5-5 2-1 2.4
No loss of activity 90~-950/oloss of activity for purified factor D, 7070 loss for factor D in serum No loss of activity, no change of mol.wt Complete loss of activity pH 5.5-8.0: no reduction of activity pH 4.5: 950,', reduction of activity
to different substances like Zymosan, Sepharose 2B and Sephadex and found that those substances were able to bind factor D. The binding process is very rapid and proceeds equally well at 4°C and at 37°C. Binding was demonstrated both by loss of activity from the supernatant and appearance of factor D activity on the surface of Zymosan or Sepharose 2B particles. The fixation of factor D on a surface increased its potency as cofactor in the bypass system, since much more activity was found on the particles than was lost from the supernatant. After adsorption factor D remained in its native state, exhibiting an activity only after incubation in a standard test system (Dfs-VF) or by interaction with purified VF, C3PA and Mg ++. Purified factor D adsorbed on Sepharose particles could be functionally blocked by the IgG fraction of an anti factor D serum. After treating the particles with these antibodies and washing them, no further factor D activity could be demonstrated. An unrelated IgG preparation did not show this effect. Functional role of factor D The functional role of factor D was investigated in a system consisting of purified VF, C3PA, factor D and Mg +÷. Only in the presence of all factors could a C3 cleaving enzyme be generated. As shown in Fig. 3, the enzyme formation was strictly dose dependent for each of the four reactants involved. Mg ++ ions are needed only for generation of the enzyme and not for its action, since C3 cleavage proceeds in the presence of EDTA (Bitter-Suermann et al., 1972). We therefore used EDTA to stop the enzyme formation at different time intervals. The amount of enzyme formed up to the respective time was determined by its ability to turn over C3 within 90rain at 37°C. As shown in Fig. 4, the kinetics of C3 cleaving enzyme formation revealed a time and temperature dependence typical of enzymatic processes. To elucidate the sequential interaction of VF, C3PA and factor D, we preincubated two of the three proteins at 3T'C for 20 min in.the presence of Mg ++. Afterward, the mixtures were colled to 25°C, the third reactant was added, and the mixture was incubated up to 20min. The 25°C temperature was chosen to slow down the reaction velocity of complex formation. At
Factor D in the Alternate Pathway of Complement Activation
531
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600
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Fig. 3. Dose dependence of the formation of the C3 cleaving enzyme from purified VF (A--A), C3PA (r-l--r-q), factor D ( O - - - O ) and Mg + + (x . . . x). The three partners in the reaction are kept constant and one is varied in its concentration by dilution (abscissa). The respective mixtures contained 0.05 ml VF (0.25 mg/ml VBS), 0'1 ml C3PA in VBS, 0.1 ml factor D in VBS and 0"05 ml 1 x 10 -2 M MgC12 in H20. The original concentrations of C3PA and factor D were chosen to yield an optimal C3 cleaving activity with the mentioned VF concentration. After 30 rain at 37°C samples were taken and were incubated with C3 in the presence of 0.015 M EDTA for another 30 min at 37°C in order to determine the amount of C3 cleaving enzyme formed. Large amounts of remaining C3 indicate poor enzyme formation and small amounts of remaining C3 indicate optimal enzyme formation. various times during the 25°C incubation period samples were taken, E D T A was added to them and they were tested for C3 turnover at 37°C for 9 0 m i n (Fig. 5). N o one c o m b i n a t i o n had a significant advantage over the others, which indicated that none of the ~--
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,
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,
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Fig. 4. Time and temperature dependence of the formation of C3 cleaving enzyme from VF, C3PA, factor D and Mg ÷ +. The mixtures were kept at 4°C ( O - - - O ) , 25°C (/x--/x) and 37°C ( 1 2 - - - I-q)for 5 sec to 10 min. At various time intervals samples were taken and were incubated with C3 in the presence of 0.015M EDTA for 30min at 37°C to test for C3 cleaving activity (ordinate as in Fig. 3). The symbols i , • and • represent the controls for the three temperature curves. In these samples VF was replaced by VBS.
I0 min
15
20
Fig. 5. Influence of preincubating various combinations of VF, C3PA and factor D on the generation of a stable enzyme. C3PA and factor D ( © - - - O ) , VF and C3PA (/X A), and VF and factor D ( C ] - - - D). The time on the abscissa represents the incubation period at 25°C prior to the final C3 test. combinations decayed or were activated during the first incubation at 37 ° C. Therefore, it was t h o u g h t possible to find out which interaction needed M g ÷+. In order to find this out, either V F + C 3 P A or V F + f a c t o r D or C3PA + factor D were preincubated in the presence of M g ÷÷, after which E D T A was added, followed by factor D, C3PA or VF, respectively. However, in n o case was any C3 cleaving enzyme detectable. It seems, then, that VF, C3PA and factor D have to be present along with M g ÷÷ in order to give rise to an enzyme acting on C3. The participation of factor D in enzyme formation was also evident from the fact that blocking of factor D by the IgG fraction of the anti factor D serum prevented the generation of enzyme activity. To assess the binding capacity of factor D to V F or other proteins we u n d e r t o o k the following experiments. Factor D was incubated with either VF, C3PA, HSA or h u m a n IgG (Beriglobin) for 3 0 m i n at 37°C a n d then c h r o m a t o g r a p h e d on Sephadex G 100 columns (0.02 M Tris-HCl, pH 7.5). As a result of this treatment the four proteins eluted at their expected positions, and all carried factor D activity with them; C3PA, HSA and IgG carried small amounts of activity and V F carried a fairly large a m o u n t of activity. At this stage of the experiments the question again was raised, of whether a complex formation between two or more of the reaction partners was a prerequisite for the generation of enzymatic activity from VF, C3PA, Mg ÷+ and factor D. The question was answered by the following experiment. V F and C3PA were incubated in the presence of Mg ++ a n d then applied to a Sephadex G 200 column. Besides the two isolated proteins in their proper elution position, fractions were detected which on mere addition of factor D + M g ÷+ were capable of turning over C3. These fractions were located in front of V F (which indicated a mol. wt of a b o u t 200,000) and so are t h o u g h t to contain V F - C 3 P A complexes lacking enzymatic activity against C3 (Fig. 6). Consequently, V F apparently reacts equally well with C3PA a n d factor D. For the generation of a n active V F - C 3 P A - f a c t o r D complex it did not matter which
532
M. P. DIERICH et al. ~0 6
--
VF-C3PA-D ',
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v~/vo Fig. 6. Demonstration of complex formation between VF and C3PA or VF, C3PA and factor D by gelfiltration on a Sephadex G200 column (90 × 1.5 cm). The position of the VF C3PA-D complex was determined in a separate run under exactly the same conditions. As markers served catalase, IgG and human Hb. reactant was offered first to VF. When such complexes were chromatographed on Sephadex G 200 columns they eluted slightly prior to the position of V F - C 3 P A complexes (Fig. 6). Furthermore, an active complex could be built on Sepharose particles to which VF was first coupled. This Sepharose-VF was later incubated with C3PA, factor D and Mg ++. The supposition that factor D is incorporated into the V F - C 3 P A complex was confirmed by the use of anti factor D serum. This antiserum completely blocked the activity of the C3 cleaving enzyme. Factor D, then, is not only needed for C3 turnover by VF but also for induction of C3 activation by other subs~;ances. Using inulin, endotoxin, Zymosan or Dextran sulfate, typical dose response curves were obtained with Dfs (diluted 1:10) and factor D (in various concentrations) (Hadding et al., 1973). Factor D (guinea-pig) was also able to substitute for human factor D and induce a C3 turnover in a system consisting of VF or DS and D free human serum used in the guinea-pig system described above. DISCUSSION Since the reaction mechanism of the whole bypass sequence are very complex, we studied that part which is believed to represent the final steps of the sequence and which can be initiated by VF. It has been demonstrated in this paper that factor D was incorporated into a V F - C 3 P A complex and that the incorporation of factor D in the presence of Mg ++ rendered the final trimolecular complex active against C3. During our investigations described here we did not obtain any evidence that additional factors were needed for cleavage of C3. In so far our data are in agreement with those of Cooper (1973) and Hunsicker et aL (1973), but they are at variance with results of Hunsicker, who could not find a complex consisting of VF, C3PA and factor D even though participation of these three factors in cleaving C3 is well accepted. Our various kinetic experiments failed to elucidate a reaction sequence for the three factors because it
was found that all three and Mg +÷ had to be present simultaneously for the generation of enzymatic activity. In addition, no conclusion can be drawn from our data as to which factor carries the enzymatically active center for C3 conversion. This active site could be formed by factor D, by C3PA or by a C3PA-factor D complex analogous to the C42 enzyme. A remarkable feature of factor D is its tendency to bind to various substances even at low (4°C) temperatures, e.g. to particles of Sephadex, Sepharose 2B and Zymosan. When using a zymosan absorbed serum and testing for properdin it has to be taken into consideration that the serum's factor D content will be reduced. In this report we preferred to call the isolated protein factor D and not C3PAase (MiJller-Eberhard and G6tze, 1972), since we had no direct evidence for cleavage of C3PA by factor D. A possibility which seems likely is that, because of their similar molecular weights, the factors may prove to be identical. Acknowledyements--This work was supported by grants
from the DFG. We are indebted to Miss M. C. Brand and S. Risi, Ph.D., for performing the amino acid analysis of factor D at the Institut r0r Biochemie, Johannes GutenbergUniversitfit, Mainz, Germany. The authors also wish to thank Miss U. Aulbach, Mrs. M. Liider and Miss R Schwarz for their competent technical assistance. REFERENCES
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