(Received for publication, February 27, 1995; and in revised form, June 8, 1995)
From the
Human factor B is required for the initiation and propagation of
the complement alternative pathway. It also participates in the
amplification of the complement classical pathway. Alone, factor B is a
zymogen with little known biochemical activity, but in the context of
the alternative pathway convertases, the factor B serine protease is
activated in a process that first involves the association with C3b and
subsequently the cleavage of factor B into two fragments, Ba and Bb.
Ba, the NH-terminal fragment, is composed mainly of three
tandem short consensus repeats, globular domains found in other
complement proteins. It dissociates from the convertase during
assembly, leaving the active C3 convertase, C3bBb. Previous reports
suggest that the Ba region may be instrumental in convertase assembly.
This hypothesis was tested using site-directed mutagenesis of
recombinant factor B and monoclonal antibody epitope mapping to
evaluate the relative importance of specific short consensus repeat
amino acid residues. Three sites of interest were identified. Site 1 is
a stretch of 19 contiguous amino acids in short consensus repeat 1 that
form the epitope of a monoclonal antibody that effectively blocks
factor B function. Site 2, composed of 6 contiguous amino acids in
short consensus repeat 2, and site 3, consisting of 7 contiguous amino
acids in short consensus repeat 3, were defined by mutations that
reduce factor B hemolytic activity to 3% or less. Further analyses
indicated that sites 2 and 3 contribute to factor B-C3b interactions.
The complement system consists of about 30 proteins that function in the identification and removal of foreign substances and immune complexes and in the stimulation of inflammatory responses (reviewed in (1) ). There are two major pathways of complement activation, the classical pathway and the alternative pathway. Activation of the classical pathway is induced primarily by antibody-antigen complexes, while the alternative pathway is initiated by the binding of C3b to activating surfaces, frequently microbial in nature. A third pathway induced by lectins has recently been described(2, 3) . In each case sequential activation of a series of serine proteases occurs, each protease amplifying the effects of the previous one. Key constituents of both pathways are the C3 and C5 convertases, which are assembled on target surfaces and produce biologically active fragments through the cleavage of the circulating complement components C3 and C5.
The first step in the assembly of the alternative pathway C3 convertase is the association of factor B with C3b(1, 4) . In this context factor B can be cleaved by factor D, resulting in Ba and Bb, a process that requires a divalent cation. Ba then dissociates from the complex while Bb remains bound to C3b. C3bBb can be partially stabilized by association with properdin. C3bBb and C3bBbP (where P represents properdin) are active enzymes that cleave C3 at a single point, generating more C3b and ultimately more convertases. Alternative pathway C5 convertase activity occurs through the association of C3 convertase and additional C3b. In all cases, dissociation of Bb from the convertases is inevitable, irreversible, and followed by inactivation of proteolytic function(5, 6) .
Factor B is a 90-kDa single-chain
glycoprotein composed of five protein domains(7) . The
amino-terminal region (Ba) consists predominantly of three short
consensus repeats (SCRs), ()domains found in complement
regulatory proteins(8) . The carboxyl-terminal region (Bb)
consists of a type A domain found in von Willebrand factor and
complement receptors(9) , followed by a trypsin-like serine
protease domain(10) . Examination of factor B by electron
microscopy reveals three globular regions of about equal size, while
the Bb fragment features two globular regions connected by a short
linker(6, 11) .
Since C3b binding is mediated by SCR domains in a number of complement proteins(8) , the Ba fragment has affinity for C3b(12) , and some monoclonal antibodies directed against Ba block factor B-C3b interactions(6) , it appears that the association of the factor B SCRs with C3b could be instrumental in the earliest stages of convertase assembly. We tested this hypothesis using site-directed mutagenesis and anti-Ba mAb epitope mapping to evaluate the relative roles of specific SCR amino acid residues in factor B function.
In the second method (transformer
site-directed mutagenesis; (14) ; Clontech, Palo Alto, CA),
simultaneous mutagenesis of a factor B site with a mutagenic primer and
a unique XbaI vector site with a selection primer
(5`-monophosphate-GGAAGCGGAAGAGTCGCGAGTCGACCAGACATG-3`) formed the
basis of efficient selection of mutant plasmids: Parental plasmids
(grown in E. coli strain BMH 71-18 mutS) were
denatured by alkaline treatment and neutralized, and mutant strands
were synthesized by T4 polymerase and T4 ligase utilizing mutagenic
primers (Table 1) and the selection primer. DNA was digested with XbaI and used to transform competent BMH 71-18 mutS. A mixed population of DNA was isolated from the
transformant pool and cut with XbaI. DNA was used to transform
competent E. coli strain DH5 (Life Technologies, Inc.),
and transformants were grown on LB plates supplemented with ampicillin.
The B(+) template was used for all mutagenic procedures. Transformants were screened by DNA sequencing(15) . In general, 5-10 candidates were sufficient to obtain at least one desired mutant.
Biosynthetic labeling was begun 48
h after transfection (16) with
[S]cysteine (1075 Ci/mmol, 10 mCi/ml; DuPont)
and allowed to continue for 4-16 h. For immunoprecipitation,
samples were first precleared with protein A-agarose (Boehringer
Mannheim) and then incubated with goat anti-factor B polyclonal
antibody (IgG fraction, 12.9 mg/ml, Incstar) or normal goat serum
(Sigma). Immune complexes adsorbed to protein A-agarose were washed
twice with PBS (8.1 mM Na
HPO
, 1.8
mM NaH
PO
, 145 mM NaCl, pH
7.4) containing 360 mM NaCl, 5 mM Na
EDTA,
1% Nonidet P-40, 0.1% sodium deoxycholate, 0.25% SDS, and twice with
PBS containing 1% Nonidet P-40. Samples were eluted in an
SDS/glycerol/glycine dissociation buffer and analyzed by SDS-PAGE (10%; (18) ). Signal was enhanced utilizing Amplify (Amersham Corp.)
followed by autoradiography.
Standard curves were determined using a 2-fold dilution series of purified factor B between 50 ng/ml and 1.56 ng/ml. The optical density was graphed versus the log of antigen concentration, yielding a straight line between 2 and 25 ng/ml, and used to determine the concentration of the unknowns. Standard sera samples were measured along with mutant and wild type recombinant factor B at four different dilutions.
Purified factor
B (Quidel) was used as a standard. COS supernatants were diluted in
Mg-EGTA buffer to equivalent levels (20-80
ng/ml) as predetermined by ELISA. For each determination, 100 µl of
prepared (C3b-coated) sheep erythrocytes, 50 µl of purified factor
D (5 ng in Mg
-EGTA buffer; Quidel), 50 µl of
properdin (45 ng in Mg
-EGTA buffer; Quidel), and 50
µl of factor B source or standard were mixed together and incubated
at 30 °C for 30 min. A negative control substituted 50 µl of
DGVB
buffer for the factor B source. Additional
controls included complete cell lysis and cells mixed with buffer only.
All points were determined in triplicate.
Alternative pathway C3
convertase sites were developed with 300 µl of a 1:40 dilution of
guinea pig serum (Colorado Serum Co., Denver, CO) in 40 mM EDTA buffer (40 mM NaEDTA, 0.1% gelatin, 85
mM NaCl, 0.061%, Na-5`-5`'-diethyl barbiturate, pH 7.35)
(except for the 450 µl of distilled water and the 450 µl of
DGVB
buffer controls), samples were centrifuged, and A
of the supernatants was determined.
Z values (average lethal hits/cell) were calculated for each sample (see (19) ). Z values for wild type factor B were linear between 10 and 160 ng/ml. The activity of our recombinant factor B was similar to that of commercially purified factor B. In most cases, COS supernatants were diluted to 80 ng/ml, which resulted in a Z value between 0.5 and 1.00 for recombinant wild type factor B. Hemolytic activity levels for the factor B mutants were expressed as percentage of the average Z value obtained for the wild type recombinant protein determined in parallel. Wild type recombinant factor B preparations, separately measured by ELISA to determine factor B concentration, varied up to 20% in specific hemolytic activity (i.e. in one experiment that compared 5 preparations isolated over the course of 6 months, average of Z = 1.04, S.D.= 0.167).
In a
second experimental design, C3 convertase assembly was divided into two
steps: 50 µl of pure factor B (500 ng/ml in
Mg-EGTA buffer) was mixed with 100 µl of
C3b-coated cells and 100 µl of Mg
-EGTA buffer and
incubated for 30 min at 30 °C. Cells were subjected to
centrifugation, washed with 5 ml of Mg
-EGTA, and
resuspended in 200 µl of buffer. Cells were treated with factor D
and properdin (to 250 µl) and incubated for 30 min at 30 °C.
The alternative pathway C3 convertases were detected as described
above. mAb was preincubated (30 min at 4 °C) at a 5:1 molar ratio
with factor B before addition to cells, or mAb was incubated with
washed C3bB cells (30 min at 4 °C) prior to factor D +
properdin treatment. The mAb treatments and the nontreated controls
included both preincubation steps, with or without mAb.
Figure 1: Expression of recombinant factor B. Radiolabeled COS supernatants were immunoprecipitated and subjected to SDS-PAGE as described under ``Materials and Methods.'' Lanes1 and 2 were derived from COS cells transfected with the B(+) plasmid while lanes3 and 4 were transfected with the B(-) plasmid. Lanes2 and 4 were immunoprecipitated with goat polyclonal anti-human factor B antibody, while in lanes1 and 3 normal goat serum was used in place of antibody. Positions of molecular mass markers, measured in kilodaltons, are shown at the left.
Supernatants derived from the transfection of unlabeled cells were
assayed by ELISA for the presence of human factor B protein. By this
criterion, the B(+) cells produced 500-2000 ng/ml factor B in 72 h
while the B(-) cells produced 1-10% of the B(+) value.
Supernatants were assayed for factor B-dependent hemolytic activity.
The recombinant factor B was comparable in activity with the purified
factor B (data not shown), while the B(-) activity was negligible
(1% activity for an equivalent volume of supernatant).
Figure 2: Analysis of the factor B SCRs by mutagenesis. Factor B was mutagenized and assayed as described under ``Materials and Methods.'' Substitutions are indicated by boxes, with identical residues indicated by periods and deleted residues indicated by dashes. Percentage of hemolytic activity was determined for each mutant as a percentage of the Z value obtained with wild type recombinant factor B in parallel determinations. Similarly, C3b binding was determined by ELISA, and values obtained for each mutant were compared with values obtained with wild type recombinant factor B. All substitutions were derived from the human C2 sequence (24) except for 7L, 12L, and 13L, which were all derived from the lamprey factor B/C2 sequence(25) . The factor B sequence begins with residue 11 of the secreted protein(7) .
Mutant factor B proteins were analyzed by immunoprecipitation
followed by PAGE and by ELISA (data not shown). Those that produced
sufficient full-length factor B forms were assayed for hemolytic
activity (Fig. 2). Most mutants retained hemolytic capacity
similar to the recombinant factor B control. In contrast, two mutants
derived by substitution of human C2 sequence resulted in 5% activity or
less. In one case substitution of SGQTAI
DGETAV in
SCR-2 (mutant 16) reduced hemolytic activity to 3 ± 2% of wild
type recombinant levels. At a second site, substitution of
P
IGTRKV
SLGAVRT in SCR-3 (mutant 18) reduced
activity to less than 3%.
The Bmut16 and Bmut18 regions were mutated
one amino acid at a time. In the case of Bmut16, single substitutions
resulted in relatively modest reductions in hemolytic activity (Table 2). In contrast, in the case of the Bmut18 region,
substitution of Pro with Ser resulted in 11% activity,
while substitution of Val
with Thr left no more than 3%
activity.
Binding assays were performed on the SCR mutations ( Fig. 2and Table 2). Normal hemolytic activity was accompanied by at least normal binding levels. Of the mutations that reduced hemolytic activity severely, Bmut16 retained full binding levels while Bmut18 and its related single amino acid substitutions reduced binding substantially.
Binding of wild type recombinant factor B to immobilized C3b could be inhibited by preincubation with fluid phase C3b (Table 3). Selected factor B mutants were preincubated with fluid phase C3b (Table 3). In the case of wild type recombinant factor B, as well as mutants Bmut7L and Bmut9, fluid phase C3b was an effective inhibitor of C3b binding at molar ratios no greater than 5:1. Fluid phase C3b was ineffective as an inhibitor with Bmut16 at 5:1 and 20:1.
Figure 3: Characterization of the anti-Ba mAb 14-III-33 epitope. Mutations that did not bind mAb 14-III-33 are shaded. Percentage of recognition was calculated as the percentage of each mutant bound to 14-III-33 on a microtiter plate, as compared with wild type recombinant factor B.
In two variations of the hemolytic assay, mAb 014-III-33 was either preincubated with factor B prior to C3b association and subsequently washed away before treatment with C3b-coated cells or incubated with C3bB cells prior to treatment with factor D and properdin. mAb 014-III-33 blocked hemolysis in both cases (Table 5). In addition, factor B preincubated with mAb 014-III-33 failed to bind immobilized C3b (Table 6).
Complement activation can account for substantial tissue damage in a wide variety of autoimmune/immune complex-mediated syndromes such as systemic lupus erythematosus, rheumatoid arthritis, hemolytic anemias, and myasthenia gravis(28) . It mediates the hyperacute rejection of xenografts (29, 30) and contributes to tissue damage brought about by vascular injury such as myocardial infarction(31) , cerebral vascular accidents, and acute shock lung syndrome(28) . Thus, the clinical regulation of complement would be potentially useful for many therapeutic purposes. An essential step to the therapeutic control of complement is a detailed understanding of complement activation. In this report we focus on the SCR domains of factor B, a complement protease that mediates the initiation and propagation of the alternative pathway and the amplification of the classical pathway.
Full-length human factor B cDNA was isolated from an acute phase liver library, sequenced, subcloned, and expressed in COS cell cultures. Recombinant factor B produced was similar to natural factor B as determined by PAGE, ELISA, and hemolytic assay. A panel of factor B SCR mutants was constructed; each mutation replaced several factor B amino acids with those derived from the corresponding region of a structural homolog. Analysis of the panel revealed two factor B regions essential to hemolytic capacity: Bmut16, near the carboxyl terminus of SCR 2, and Bmut18, near the amino terminus of SCR 3. Each resulted in hemolytic activity levels of no more than 3%. Moreover, mutation of two different amino acids in the Bmut18 region led to activity levels of 11 and 2%.
A C3b binding
assay was used to analyze the SCR mutations further. Binding of factor
B to immobilized C3b was dependent on Mg, consistent
with hemolytic activity. Moreover, fluid phase C3b preincubated with
factor B inhibited subsequent binding to immobilized C3b. While most
SCR mutants, including Bmut16, could bind immobilized C3b at least as
effectively as wild type recombinant factor B, Bmut18 and Bmut18F,
substitutions that abrogate hemolytic activity, also suffered
substantially reduced binding capacity (10-25% of wild type).
Interestingly, fluid phase C3b was not an effective inhibitor of the
binding of Bmut16 to immobilized C3b, although it can inhibit
C3b-binding of wild type factor B and other mutants (Table 3).
The mutant panel was also used to map the epitope of the anti-Ba mAb 14-III-33, an agent that blocks factor B hemolytic activity; of 26 mutant proteins, only three failed to be recognized by the anti-human factor B mAb 014-III-33. Those mutations define 19 contiguous amino acids that lie at the COOH terminus of SCR-1, including the amino acids that link SCR-1 with SCR-2. Two of those three mutants (Bmut7L and Bmut8) retain full hemolytic capacity, but neither Bmut7L nor Bmut8 is blocked by 14-III-33. This result demonstrates that 14-III-33 blocks hemolysis through interaction at the mapped epitope. mAb 14-III-33 appears to interfere with the normal binding of factor B to C3b; preincubation of factor B with 14-III-33 precludes its binding to immobilized C3b (Table 6). Additional experiments showed that 14-III-33 can block factor B-dependent hemolytic activity before or after the association of factor B with C3b has occurred (Table 5).
These studies were initiated to test the hypothesis that early steps in the assembly of the alternative pathway convertases require interactions between C3b and the SCR region of factor B. Three sites of interest have been identified in the SCR region (Fig. 4): site 1, TLKTQDQKTVRKAECRAIH in SCR-1, is recognized by a mAb that inhibits hemolytic activity. Of the three multiple substitutions that lie in this region, two result in little change in hemolytic function or capacity to interact with C3b (Bmut7L and Bmut8), and one results in 25% activity (Bmut9) but, again, with little change in observed C3b interactions ( Fig. 2and Table 3). The mutants that retain hemolytic capacity are not conservative and include most of the amino acids at this site. Comparison of this region of human factor B with related homologs reveals substantial sequence divergence (Fig. 4).
Figure 4: Active site candidates in factor B SCRs 1-3. Regions implicated in hemolytic function are shaded (above) and compared with evolutionary homologs (below). Only diverging amino acids are indicated; deletions are shown by a dash. HUB, human factor B(7) ; MOB, mouse factor B(32) ; PGB, pig factor B(33) ; XEB, Xenopus laevis factor B(34) ; LAB/C2, lamprey factor B/C2(25) ; HUC2, human C2(24) .
Site 2, SGQTAI in SCR-2, is defined by the dramatic loss of activity found in Bmut16 (3% activity), a substitution derived from the human C2 sequence. The greatest loss in activity seen in single amino acid changes was in the substitution of Gln by Glu (32% activity). The site 2 sequence is more conserved than site 1 (Fig. 4), with only a single divergent residue in both mouse and pig(32, 33) , a Ser to Asp substitution. This substitution (Bmut16A) resulted in 59% activity (Table 2). None of these mutants appeared to disrupt binding to immobilized C3b, but fluid phase C3b fails to effectively inhibit the binding of Bmut16 to immobilized C3b (Table 3).
Site 3, PIGTRKV in SCR-3, is
also defined by dramatic functional loss; Bmut18, derived from human
C2, results in less than 3% activity. In addition, these effects were
seen in the individual substitutions of Ser for Pro (Bmut18A, 11%
activity) and Val for Thr (Bmut18F, 2% activity). These three
mutants bound immobilized C3b substantially less than did control
factor B. The site 3 sequence is relatively conserved (Fig. 4)
and is identical to human in both mouse and
pig(32, 33) .
Although one or two of the amino acids in site 1 could be of direct functional importance, given the substantial evolutionary divergence that has taken place in this region and the ability of these site 1 mutants to interact with both immobilized and native C3b, the deleterious effects of mAb 14-III-33 could be more simply attributed to steric effects that interfere with factor B-C3b interactions.
In contrast, based on the dramatic effects of several Bmut18 mutants on hemolytic activity and interactions with immobilized C3b, and given the relatively high level of evolutionary conservation, site 3 appears to encompass elements essential to factor B-C3b interactions.
It also appears that site 2 is of functional importance, given the great reduction of hemolytic activity associated with Bmut16. Although Bmut16 binds to immobilized C3b, fluid phase C3b is not an effective inhibitor of the binding of Bmut16 to immobilized C3b. We conclude that the Bmut16 region also contributes significantly to the factor B-C3b interaction. Interestingly, the homologous region of complement receptor 1 SCR-9 is also involved in C3b interactions(16, 23) .
We have used site-directed mutagenesis and mAb epitope-mapping to analyze the three SCRs of factor B. Previous work suggests the importance of the Ba region in the assembly of the C3 convertase(6, 12) . The present report describes two regions in the factor B SCRs that contribute significantly to hemolytic capacity; mutations in each region have been shown to affect factor B-C3b interactions. Both regions are highly variable within the family of SCRs (35) and do not appear to determine the SCR inner core in the cases where structural models are available(36, 37, 38) . Thus, in principle, one or both sites could mediate direct intermolecular contacts with C3b. Alternatively, one or both sites could promote the C3b binding mediated by the Bb region. Further work will be directed to understanding the roles played by each site and to identifying amino acids that mediate intermolecular contacts with C3b.