THE JOURNAL OF BIOLOGICAL CHEMISTRY Vnl 258 No. 12 Issue of June 25, pp. 7411-7415,1983 printed L u . s . ~ .

C3 Convertase of the Alternative Complement Pathway DEMONSTRATION OF AN ACTIVE, STABLE C3b,Bb(Ni) COMPLEX*

(Received for publication, January 31,1983)

Zvi Fishelson$, Michael K. Pangburna, and Hans J. Muller-EberhardlI From the Department of Immumbgy, Research Institute of Scripps Clinic, La JoUa, California 92037

The purposes of this study were to demonstrate the C3 convertase complex, C3b,Bb (EC 3.4.21.47), of the alternative pathway of complement by ultracentrifu- gation and to determine whether the metal ion required for enzyme formation is present in the active enzyme complex. It has been shown previously that Clb,Bb formed with Nia+ rather than M e + exhibits enhanced stability. Using sucrose density gradient ultracentri- fugation, an enzymatically active C3b,Bb(Ni) complex could be demonstrated which has a sedimentation coef- ficient of 10.7 S and which is stable in 10 mM EDTA. Upon formation of the enzyme with the radioisotope esNia+, the ultracentrifugal distribution of the metal correlated with that of the enzyme complex. The molar ratio of Ni to C3b,Bb was 1:l. Displacement of Ni by Mg during formation of the enzyme indicated that both metals may bind to the same site in the enzyme. Binding of "Ni to the catalytic site bearing fragment Bb was significantly stronger than its binding to C3b or to the zymogen, Factor B. It is proposed that there is one metal-binding site in the Clb,Bb enzyme which is not susceptible to chelation by EDTA and which is located in the Bb subunit.

C3b,Bb is the C3 convertase (EC 3.4.21.47) of the alterna- tive pathway of complement.' The enzyme is responsible for amplification of pathway activation and for deposition on target cells of C3b and the membrane attack complex (1-6). The enzyme is controlled by the serum proteins Factor H, Factor I (EC 3.4.21.45), and properdin (1, 7-14). The forma- tion of the enzyme requires C3b, Factor B, Factor D (EC 3.4.21.46), and Mg2+ (2-6, 15). After formation of the revers- ible, bimolecular complex C3b,B(Mg), Factor D cleaves Factor B, releases the activation fragment Ba, and generates the active enzyme C3b,Bb, which has a calculated molecular weight of 239,000 (C3b, M, = 176,000; Bb, M, = 63,000). The enzyme is a serine protease (16-18) whose catalytic site resides in the Bb subunit. The enzyme is inherently labile, and the spontaneous dissociation of its subunits results in irreversible loss of enzymatic activity. Due to this lability, previous at-

* This work was supported by United States Public Health Service Grants AI 17354 and HL 16411. This is Publication 2900 from the Research Institute of Scripps Clinic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a Chaim Weizmann postdoctoral fellowship. 3 Recipient of American Heart Association Established Investiga-

ll Cecil H. and Ida M. Green Investigator in Medical Research, torship 81-225.

Research Institute of Scripps Clinic.

(1981) J. Zmmunol. 127,1261. * For nomenclature of the alternative pathway of complement, see

tempts to directly demonstrate the enzyme by ultracentrifu- gation, electrophoresis, or molecular sieve chromatography have failed. Since the spontaneous decay dissociation of C3b,Bb is not accelerated by EDTA, it was uncertain whether the metal ion required for enzyme formation remained asso- ciated with the active enzyme. We have recently found that Ni2+ can replace Mg in C3b,Bb formation and that the enzyme formed with Ni (CSb,Bb(Ni)) is 6 to 10 times more stable than that formed with Mg (C3b,Bb(Mg)) (19). Utilizing Ni and its radioisotope 63Ni, it was possible in the present study to demonstrate the active C3b,Bb complex by ultracentrifu- gation and to detect the metal ion in the complex.

MATERIALS AND METHODS

Buffers and Reagents-The following buffers were used: VBS? VBS containing 0.1% gelatin (w/v), and VBS containing 0.1% gelatin (w/v) and 20 mM EDTA (Sigma). MgC12 and NiClz of analytical grade were purchased from Fisher.

Preparation and Radiolabeling of Purified Complement Proteins- C3 (20), Factor B (21), and Factor D (22) were isolated from human serum as previously described. C3b and Bb were generated by incu- bating 10 mg of C3, 1 mg of Factor B, and 50 pg of Factor D in 2 ml of VBS containing 4 mM Mg for 60 min a t 37 "C. EDTA (7.5 mM) was then added, and the components were gel-filtered through a Sephadex G-200 column (2.5 X 90 cm) (Pharmacia Fine Chemicals, Piscataway, NJ). Trace amounts of 1261-labeled C3 and '311-labeled Factor B were included in the reaction mixture to allow detection of C3b and Bb. The proteins were pure 88 judged by SDS-PAGE (Fig. 1). Bovine thyroglobulin was purchased from Sigma. Proteins were radiolabeled with '%I or I3'I (Amersham Corp.) by the Iodogen tech- nique (Pierce Chemical Co.) (23). '961-labeled Clq was kindly provided by Dr. Andrea Tenner of this department. The labeled proteins had a specific activity of 0.7-1.8 mCi/mg.

Radioactivity Assay of a7Ni-a7Ni2+ was obtained from New England Nuclear a t a concentration of 0.28 M (11.277 mCi/mg) in 0.5 M HCl solution. A 0.01 M stock solution was made in distilled water. Addition of B3Ni to reaction mixtures did not lower the pH below 7.0. "Ni radioactivity (@-emission) was measured using polypropylene liquid scintillation vials (Kimble, Toledo, OH), ACS-I1 counting scintillant (Amersham Corp.), and a Beckman LS8OOO liquid scintillation coun- ter. Since '%I and '''I also activate the scintillant, samples containing =Ni and radioiodinated proteins were fvst analyzed for ?-radiation 90 that appropriate corrections for iodine contribution could be made.

Electrophoresis-Slab gel electrophoresis in presence of SDS was performed using the Canalco PAGE system from MiIes Laboratories, Inc., Elkhart, IN. The 5-12% gradient polyacrylamide gel contained 0.01% SDS.

Sucrose Density Gradient Ultracentri,bgatiun-Ultracentrifugation was performed in 5-20% sucrose (Fisher) density gradients in VBS containing 6 mM EDTA. Five-ml gradients were formed in Ultra- Clear tubes (% X 2 inch) (Beckman) using a Buchler Auto-Densi- Flow I1 C. The tubes were subjected to centrifugation in an SW 50.1

The abbreviations used are: VBS, veronal (5 mM)-buffered sa- line (0.15 M), pH 7.2, containing 0.02% (w/v) sodium azide; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CBb,Bb(Mg) and CBb,Bb(Ni), the C3 convertase formed with Mg and Ni, respectively.

741 1

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7412 C3 Convertase of the Alternative Complement Pathway

C3b B Bb D

200k -

92k -

45k - 21k-

FIG. 1. Demonstration by SDS-polyacrylamide eleetropho- resia of the proteins wed for C3 convertase formation. Elec- trophoresis of 7 pg of C3b, Factor B, or Bb or 10 pg of Factor D was performed in 5-12% gradient gels (see "Materials and Methods"). 200k represents M, = 200,000, for example.

rotor and a Beckman W-65 Ultracentrifuge for 12 h at 43,000 rpm and 2 "C. Eight-drop fractions were collected from the top of the tube using a Gilson Microfractionator (Gilson Medical Electronics, Inc., Middleton. WI).

Separation of Free and Protein-bound aNi on Bio-Gel P-6-Plastic tubes (10 X 75 mm; Falcon, Oxnard, CA) were pierced at their bottom with a needle, plugged with scrubbed nylon fibers (Fenwal Labs, Deerfield, IL), and packed with Bio-Gel P-6 (100-200 mesh; BioRad) in VBS. A small piece of Parafilm was wrapped around the middle of each tube which was then placed inside a plastic tube (12 X 75 mm; Falcon). To remove void volume buffer, these small Bio-Gel P-6 columns were centrifuged for 2 min at 1000 rpm and 4 'C in a CRU- 5000 centrifuge (IEC, Needham Heights, MA), and the inside tubes were transferred to clean tubes (12 X 75 mm). The samples containing =Ni (100-150 pl) were then layered on top of the Bio-Gel P-6, and the tubes were centrifuged at once as above. Only protein-bound "Ni was eluted from the column by this procedure and was collected at the bottom of the tubes (12 X 75 mm). When 10' cpm of =Ni in absence of protein was applied to such a Bio-Gel P-6 column, lese than 50 cpm were eluted. The association constant of a Ni-protein complex was derived from the slope of a Scatchard plot (24) in which bound/free Ni/mol of protein (I"') was plotted uersus bound Ni (mol/mol of protein).

Hemolytic Assays-Sheep erythrocytes bearing rabbit hemolysin antibodies and human C1, C4b, and C2a were prepared as described (25,26). Titration of effective C3 molecules was performed as previ- ously described (26).

RESULTS

Demonstration of C3b,Bb(Ni) by Sucrose Density Gradient Ultracentrifugation-The C3b,Bb enzyme was formed with purified C3b, Factor B, and Factor D. An SDS-PAGE analysis of the proteins employed in this study is presented in Fig. 1. To facilitate detection of the C3b,Bb complex, 1261-labeled Factor B was included in the reaction mixture which was incubated for 5 min at 24 "C prior to ultracentrifugation. When the enzyme was formed with Mg, only a slowly sedi- menting peak of radioactivity was observed, indicating com- plete decay dissociation of C3b,Bb(Mg) during sedimentation. However, when the enzyme was formed with Ni (Fig. 2), an additional faster sedimenting peak was observed which con- tained C3 convertase activity. Fifteen-microliter aliquots of the ultracentrifugal fractions were mixed with 5 pl of 1.4 mg/ ml of C3 and incubated for 30 min at 37 "C, after which residual C3 hemolytic activity was determined. The distribu- tion of C3 convertase activity correlated closely with that of the faster sedimenting peak of radioactivity (Fig. 2). When differentially radiolabeled C3b and Factor B were employed, C3b was also detected in the faster sedimentingpeak, although

it was not completely separated from free C3b. These results indicate that the fast sedimenting peak of radioactivity and enzyme activity represents the C3b,Bb(Ni) complex. To de- termine the sedimentation coefficient of C3b,Bb, its velocity was compared with that of Factor B (5.9 S ) , C3 (9.5 S ) , Clq (11 S ) , and thyroglobulin (12 and 19 S ) . Relative to these four proteins, C3b,Bb(Ni) had an apparent sedimentation coeffi- cient of 10.7 S (Fig. 3).

Demonstration of -Ni in the Enzyme Complex-The effi- ciency of =Ni (atomic weight, 58.7) and its radioactive isotope =Ni in forming C3b,Bb was compared and found identical. When the enzyme was formed with C3b, Factors B and D, and 63Ni and then subjected to sucrose density gradient ultra- centrifugation, the large excess of free 63Ni interfered with the detection of the protein-bound -Ni. Therefore, free -Ni was removed before ultracentrifugation by filtration of the reaction mixture through a Bio-Gel P-6 column. Residual proenzyme complex, CSb,B(Ni), which had not been activated by Factor D was dissociated by addition of 10 mM EDTA to the reaction mixture. As shown in Fig. 4, the distribution of

I I 12004 A

1 -80 '

L - -60

.- 0 CT .d

5 -40 2 3

-20 c3

d

1 0 20 30 Fraction Number

FIG. 2. Demonstration of the CSb,Bb(Ni) enzyme by s u c m density gradient ultracentrifugation. 81 pg of C3b, 38 pg of '=I- labeled Factor B, and 3 pg of Factor D were incubated in 0.14 ml of VBS containing 0.1 mM NiClz for 5 min at 24 "C. 10 mM EDTA was added, and ultracentrifugation was performed in a 5-20% sucrose density gradient for 12 h at 43,000 rpm and 2 'C. To assay for C3 convertase activity 15-p1 aliquots of selected fractions were incubated with 5 pl of C3 (1.4 mg/ml) for 30 min at 37 'C. 180 pl of cold VBS containing 0.1% gelatin (w/v) and 20 mM EDTA was then added to stop the reaction. Percentage of C3 consumed was determined by effective molecule titration. Fraction 1 is at the top of the gradient.

- 12-

E 11-

.- s 9- q 8-

._ E 7- I b

10- a

c

5-

Ib i 2 1'4 16 18 20 Fraction Numb

FIG. 3. Determination of the sedimentation rate of C3b,Bb(Ni). '%labeled Factor B (5.9 S). '261-labeled C3 (9.5 S), '"I-labeled Clq (11 S), and '"I-labeled thyroglobulin ( T c ) (12 S) were used as reference substances and submitted to 5-20% sucrose density gradient ultracentrifugation together with C3b,Bb(Ni) as described under "Materials and Methods." The arrow indicates the position of Clb,'=I-Bb(Ni) in the gradient.

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C3 Convertase of the Alternative Complement Pathway 7413 -

5.9s 9.5s 12s - 1 6000

-12000 1 c - -8000 E

f

-4000

10 20 30 Fractlon Number

FIG. 4. Demonstration of "Ni in the CBb,Bb(Ni) enzyme using sucrose density gradient ultracentrifugation. 73 pg of C3b, 37 pg of '=I-labeled Factor B, and 2 gg of Factor D were incubated in 0.15 ml of VBS containing 0.1 mM B3Ni for 5 min at 24 "C. 10 mM EDTA was then added, and the reaction mixture was filtered through a Bio-Gel P-6 column. The reaction mixture was subjected to 5-20% sucrose density gradient ultracentrifugation for 12 h at 43,000 rpm and 2 "C. The fractions were analyzed for '=I and for "Ni. Arrows point at the position of the markers: '=I-labeled Factor B (5.9 S), '"I-labeled C3 (9.5 S), and "'I-labeled thyroglobulin (12 S).

TABLE I Quantitatwn of "Ni in C3b.Bb(Ni)

Experi- Fraction ~~~~

ment No. No."

1 21 22

2 20 21

3 18 19

4 18 19

5 16 17

"Nib

w 0.12 0.14 0.31 0.25 0.083 0.068 0.62 0.58 0.043 0.045

'?61"abe'ed Ni/CBb,Bb Factor B"'

w mol /ml 171 1.04 158 1.31 311 1.47 259 1.42 127 0.96 111 0.90 653 1.40 639 1.34 61 1.04 56 1.19

fi = 1.21 * 0.21 aB3Ni was quantitated in two fractions of the 10.7 S peak after

sucrose density gradient ultracentrifugation. *Total mass (nanogram)/fraction was determined from the total

counts/min and the specific activity of =Ni or '2sI-labeled Factor B. Total '=I-labeled Factor B mass was corrected after determining

that the Bb fragment contains 62% of '%I-labeled Factor B counts.

63Ni correlated with the 10.7 S peak of 1251-Fa~tor B which contained C3 convertase activity (Fig. 2). Since the sucrose density gradient contained 6 mM EDTA, it is apparent that the 10.7 S enzyme complex retained =Ni that was not suscep- tible to chelation by EDTA. The stoichiometry of Ni to C3b,Bb was determined from the specific activities of 63Ni and '251-Fa~tor B, and the results show (Table I) that over a wide range of enzyme concentration (56-653 ng of 1251-Fa~tor B/fraction), the molar ratio of 63Ni to C3b,Bb varied from 0.9 to 1.47. Those results strongly suggest that there is only one Ni ion/C3b,Bb which is resistant to chelation by EDTA.

Further evidence for the identity of the 10.7 S material came from two additional experiments. The 10.7 S peak was not observed when Factor D, which is essential for C3b,Bb formation, was omitted from the reaction mixture (Fig. 5), and it was also missing when, prior to ultracentrifugation, the enzyme was incubated for 80 min at 37 "C, resulting in decay of its activity (not shown). Ni. Protein Interaction-To determine the ability of Mg to

displace Ni in C3b,Bb formation, a mixture of C3b, '251-Fa~tor B and Factor D was incubated for 5 min at 24 "C with 0.1 mM Ni and increasing concentrations of Mg. The reaction mixture was then subjected to sucrose density gradient ultracentrifu- gation, and the radioactivity in the 10.7 S C3b,Bb peak was

1 n -800

A . Wlthout 0 1200- - 600

800- - 400

1 '0 20 30 40 Fractlon Numbel

FIG. 5. Lack of C3b,Bb("Ni) complex formation in absence of Factor D as detected by sucrose density gradient ultracen- trifugation. 36 pg of C3b and 18 pg of I3'I-labeled Factor B were incubated for 5 min at 24 "C in 0.1 ml of VBS containing 0.1 mM "Ni in presence or absence of 1 pg of Factor D. 10 mM EDTA was added, and the reaction mixtures were filtered through Bio-Gel P-6 columns and subjected to sucrose density gradient ultracentrifugation as de- scribed under "Materials and Methods." Fraction l is at the top of the gradient.

Molar Excess of Mg Over Ni

FIG. 6. Displacement of Ni by Mg during C3b,Bb formation as measured by decrease in ultracentrifugally detectable com- plex. 18 pg of C3b, 9 pg of I3lI-labeled Factor B, and 1 pg of Factor D were incubated for 5 min at 24 "C in 0.1 ml of VBS containing 0.1 mM NiC12 and 0, 0.2, 0.6, 2.0, or 6.0 mM MgC12. 10 mM EDTA was added, and the reaction mixtures were subjected to sucrose density gradient ultracentrifugation as described under "Materials and Meth- ods." Amount of the C3b,'311-Bb(Ni) complex was determined by '''1 quantitation in the fast sedimenting radioactivity peak. Results are expressed as per cent of control (no MgC12).

measured. At 60-fold molar excess of Mg over Ni (Fig. 6), most of the Ni was displaced by Mg, as evidenced by the loss of the 10.7 S peak (Fig. 2). These results suggest that Ni and Mg compete for the same binding site in C3b,Bb.

The relative affinity of Ni for C3b, Factor B, or Bb was assessed from the amount of 83Ni bound to these proteins at

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7414 C3 Convertase of the Alternative Complement Pathway

," 400

2 .I 3004

\

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Bound Ni hole per mole protein)

FIG. 7. Scatchard plots of Ni binding to Bb, Factor B, and C3b. 15 gg of Bb, 18 pg of Factor B, or 36 r g of C3b were incubated for 10 min at 24 "C in 0.1 ml of VBS containing 0.1 DM =Ni and increasing concentrations of NiClz up to 2 mM Ni. Proteins were radiolabeled with '%I. Free Ni was then removed by a rapid gel filtration through Bio-Gel P-6, and the protein yield and amount of Ni bound were determined.

different Ni concentrations. The proteins were incubated at a concentration of 2-3 X M in 0.1 ml of VBS for 10 min at 4 "C with 0.1 mM @Ni and increasing amounts of NiC12. To remove the free Ni, the reaction mixture was filtered through Bio-Gel P-6 and the amount of protein-bound Ni was deter- mined from the specific radioactivity. The proteins were used in lZ5I-labeled form to allow their quantitation after gel filtra- tion. Scatchard plots (24) of Ni binding to C3b, Factor B, and Bb (Fig. 7) are based on saturation curves. The association constant of Ni binding to Bb was 8.3 X lo3 M-'. The associa- tion constants for Factor B and C3b were too low to be determined by the method used. However, it is apparent that the affinity of =Ni for Bb is markedly higher than its affinity for either C3b or Factor B. Upon addition of 20 mM EDTA to the reaction mixtures, more than 90% of the bound Ni was rapidly removed from the proteins (not shown).

DISCUSSION

Formation of the alternative pathway C3 convertase, C3b,Bb, is Mg-dependent (15). Direct ultracentrifugal and electrophoretic demonstration of the inactive but stable proenzyme complex C3b,B(Mg) has been reported (1, 5). In contrast, C3b,Bb(Mg) is a labile enzyme with a half-life of 1.5 min at 37 "C and 3 h at 4 "C, and attempts to demonstrate the complex by physical methods were unsuccessful. However, using the autoantibody nephritic factor which increases the half-life of the enzyme to 40 min at 37 "C, a trimolecular complex of C3b,Bb(Mg)-nephritic factor could be demon- strated by ultracentrifugation (27, 28). Whereas the proen- zyme complex C3b,B(Mg) is dissociated by EDTA, the active enzyme C3b,Bb(Mg) is refractory to EDTA, which raises the question as to whether the metal ion is present in the active enzyme complex. We have recently found (19) that Ni can replace Mg in C3b,Bb formation. Enzyme formation with Ni is more efficient than with Mg, and in addition C3b,Bb(Ni) is more stable than C3b,Bb(Mg). In spite of these differences, C3b,Bb(Ni) and C3b,Bb(Mg) resemble each other with respect to kinetic characteristics, stabilization by properdin, acceler- ation of decay by Factor H, and resistance to EDTA (19). A stable and active C3b,Bb(Ni) complex with a sedimentation coefficient of 10.7 S has now been demonstrated by sucrose density gradient ultracentrifugation. This sedimentation coef- ficient is compatible with a complex composition of one

molecule of C3b (9.1 S ) and one molecule of Bb (4.5 S). Each C3b,Bb(Ni) complex contains only one Ni ion which is not susceptible to chelation by EDTA. Since Ni and Mg have similar ionic radii (29) and since Mg can displace Ni in enzyme formation, both metals probably bind to the same site in C3b,Bb. The stoichiometry of C3bBb:metal ion in the enzyme complex is proposed to be 1:l:l.

That Ni binds to Bb more strongly than to C3b or Factor B suggests that in C3b,Bb the Ni ion is bound to the catalytic Bb subunit. The apparent association constant of the Ni . Bb complex at 4 "C, as calculated from the Scatchard plot in Fig. 7, is 8.3 X lo3 M" (log K = 3.9). In contrast, as quoted in Ref. 30, the association constant of the NieEDTA complex at 20 "C (log K ) is 18.56. It is probable therefore that in C3b,Bb the metal ion is so tightly bound that all its coordination positions are occupied and not available for interaction with EDTA. The much tighter binding of Ni to the complex compared to its subunits may be a function of a critical conformational change affecting the metal-binding site in Bb. Since upon decay dissociation of the enzyme, C3b is capable of new enzyme formation while Bb is not, it is probable that dissociated Bb loses the critical conformation necessary for high affinity metal binding. In fact, it has been observed in this study that 63Ni is readily released from CBb,Bb(Ni) upon decay of the enzyme. This event may account for lack of reassociation of the subunits to an active complex.

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C3 Convertme of the Alternative Complement Pathway 7415

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Z Fishelson, M K Pangburn and H J Müller-Eberhardactive, stable C3b, Bb (Ni) complex.

C3 convertase of the alternative complement pathway. Demonstration of an

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