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Volume 270, Number 34, Issue of August 25, pp. 19716-19722, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Analysis of the Short Consensus Repeats of Human Complement Factor B by Site-directed Mutagenesis (*)

(Received for publication, February 27, 1995; and in revised form, June 8, 1995)

Dennis E. Hourcade (§),  ,  Lynne M. Wagner ,  Teresa J. Oglesby

From the Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2)-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.

INTRODUCTION

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), (^1)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.

MATERIALS AND METHODS

Isolation of a Full-length Factor B cDNA

A full-length human factor B cDNA was isolated by screening a size-selected oligo(dT)-primed human acute phase liver cDNA ZAP II (Stratagene, La Jolla, CA) library (a gift of Dr. Rick Wetsel, St. Louis Children's Hospital) with a P-labeled partial human factor B cDNA insert derived from pBfA28 ((7) ; also a gift of Dr. Wetsel). The partial factor B probe hybridized to about of the plaques. Selected isolates were subjected to Southern blot analysis with the pBfA28 probe. Isolates containing 1900- and 600-bp EcoRI fragments homologous to factor B underwent in vivo excision, performed according to the instructions of the manufacturer. The insert of one of the resulting plasmids, A14, was sequenced completely in both directions using a collection of primers derived from the published partial cDNA sequence(7) .

Subcloning Factor B cDNA into an Expression Vector

The complete A14 factor B cDNA insert was excised from its plasmid by partial digestion with EcoRI and inserted into the EcoRI site of expression vector pSG5 ((13) ; Stratagene) and used to transform Escherichia coli strain DH5alpha (Life Technologies, Inc.). Clones were isolated that carried the factor B cDNA in the sense (B(+)) and the antisense (B(-)) directions.

Site-directed Mutagenesis

Two oligonucleotide-directed methods were employed to mutagenize factor B/pSG5 constructs directly. In the first method (double take double-stranded mutagenesis; Stratagene), a unique vector XmnI restriction site was utilized to cut parental plasmids. The resulting 3` ends were biotinylated and bound to avidin-coated beads. Mutant strands were synthesized by T7 polymerase and T4 ligase utilizing mutagenic primers (Table 1), extension primers, and bead-bound parental template. DNA was dissociated, and the mutagenic strands were purified from the parental strands by centrifugation. Mutant plasmids were completed by T7 polymerase and T4 ligase utilizing a bridging primer. DNA was used to transform competent E. coli strain DH5alpha (Life Technologies, Inc.), and transformants were grown on LB plates supplemented with ampicillin. This method was curtailed when the beads were no longer available.

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 DH5alpha (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.

Expression and Biosynthetic Labeling of Recombinant Factor B

Plasmid DNA was isolated using the Wizard Mini-Prep DNA isolation kit (Promega, Madison, WI). SV40-transformed green monkey kidney cells (COS-7) were maintained as described(16) . Transfections were performed with Lipofectin Reagent (Life Technologies, Inc.; (17) ) according to the manufacturer's directions.

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(2)HPO(4), 1.8 mM NaH(2)PO(4), 145 mM NaCl, pH 7.4) containing 360 mM NaCl, 5 mM Na(2)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.

Measurement of Factor B by ELISA

Nunc MaxiSorp microtiter plates (VWR Scientific; Chicago, IL) were coated with murine anti-human factor Bb monoclonal antibody (catalog number 227; Quidel, San Diego, CA) at 2 µg/ml in PBS overnight at 4 °C, blocked with 1% BSA, 0.1% Tween 20 in PBS (1 h, 37 °C), and washed with PBS, 0.05% Tween 20. Factor B standards were dilutions of commercially purified factor B protein (Quidel). Standards, controls, and samples, diluted in 4% BSA and 0.25% Nonidet P-40 in PBS, were incubated for 2 h at 37 °C. After washing, wells were treated with a 1:10,000 dilution of goat anti-human factor B polyclonal antibody (see above) and incubated for 1 h at 37 °C. Wells were washed, treated with a 1:15,000 dilution of peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch Laboratories, West Grove, PA), and incubated for 1 h at 37 °C. Color was developed using a 0.2% orthophenylenediamine and H(2)O(2) solution in a citrate/phosphate buffer at pH 6.5. Absorbance at 414 nm was read in a microplate reader (Dynatech, Chantilly, VA). Standards, samples, and controls were assayed in duplicate.

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.

C3b Binding Assay

In a variation of the factor B quantitation ELISA assay, microtiter plates were coated with 5 µg/ml C3b (19) in PBS overnight at 4 °C, blocked with 1% BSA, 0.1% Tween 20 in PBS (1 h, 37 °C), and washed with in PB (8.1 mM Na(2)HPO(4), 1.8 mM NaH(2)PO(4)) supplemented with 0.05% Tween 20, 25 mM NaCl, 10 mM MgCl(2). Standards, controls, and samples were diluted in PB supplemented with 4% BSA, 0.05% Tween 20, 75 mM NaCl, 10 mM MgCl(2). Subsequent antibodies were diluted in PB supplemented with 4% BSA, 0.05% Tween 20, 25 mM NaCl, 10 mM MgCl(2). Factor B samples and controls were incubated in triplicate in the microtiter wells for 2 h at 37 °C. After washing, wells were treated with a 1:10,000 dilution of goat anti-human factor B polyclonal antibody (see above) and incubated for 1 h at 37 °C. Wells were washed, treated with a 1:15,000 dilution of peroxidase-conjugated rabbit anti-goat IgG (see above), and incubated for 1 h at 37 °C. Color was developed and absorbance was determined as above. A near linear signal was detected with recombinant wild type factor B from 25 to 400 ng/ml. No signal was detected with equivalent volumes of control A14E COS supernatants or with BSA-coated microtiter wells. Factor B mutants were assayed at 200 ng/ml. All COS supernatants were first dialyzed into 25 mM NaCl PB, factor B concentrations were determined by ELISA, and aliquots were brought to 75 mM NaCl, 10 mM MgCl(2) prior to the C3b binding assay. Results are representative of three or more separate determinations. In the experiments shown in Table 3and Table 6, factor B forms at 200 ng/ml were preincubated with C3b or mAb at various molar ratios in 75 mM NaCl, 10 mM MgCl(2) PB overnight at 4 °C.

Mapping the mAb 14-III-33 Epitope

In a variation of the protocol above, anti-Ba mAb 14-III-33 (catalog number A225; Quidel) was used as capture antibody. After treatment with blocking buffer and subsequent washing, factor B mutants or wild type recombinant factor B (diluted to 20 µg/ml as determined using an anti-Bb mAb, above) was added to each well, and plates were incubated for 2 h at 37 °C. Detection was as above. mAb recognition was determined for each mutant and expressed as (calculated concentration of mutant)/(calculated concentration of wild type recombinant factor B) 100.

Factor B-dependent Cell Lysis Assay

A variation of the assays described by Whaley (19) and of Borsos and Rapp (20) was utilized. Sheep erythrocytes were obtained precoated with antibody, guinea pig C1, and human C4b (10^9/ml; catalog number 789-053; Diamedix Corp., Miami, FL). 0.5 ml of cells were washed twice (5 ml of DGVB (2.5% dextrose, 0.1% gelatin, 1.0 mM MgCl(2), 0.15 mM CaCl(2), 71 mM NaCl, 0.051% sodium 5`,5"-diethyl barbiturate, pH 7.35), 4 °C), resuspended to 0.5 ml, prewarmed at 30 °C for 5 min, and treated with C2 and C3 (50 µl of purified human C3 (1 mg/ml, Quidel) and 50 µl of purified human C2 (0.58 µg/ml, a gift of Dr. Paul Higgins of CytoMed, Inc., Cambridge, MA), 400 µl of DGVB preincubated for 5 min at 30 °C). The mixture was incubated at 30 °C for 30 min with gentle mixing every 5 min to allow the assembly of active convertases (C2aC4b) and the coating of the cells with C3b. The mixture was centrifuged, and the pellet was resuspended in 0.5 ml of 10 mM EDTA buffer (10 mM Na(2)EDTA, 0.1% gelatin, 128 mM NaCl, 0.092%, sodium 5`,5"-diethyl barbiturate, pH 7.35) and incubated at 37 °C for 2 h to allow dissociation of the active convertases and inhibit the production of new convertases. The cells were washed twice in 10 mM EDTA buffer, washed twice in 10 mM Mg-EGTA buffer (10 mM Na(2)EGTA, 10 mM MgCl(2), 2.075% dextrose, 0.083% gelatin, 59 mM NaCl, 0.042% sodium 5`,5"-diethyl barbiturate, pH 7.3-7.6), and resuspended to a calculated final concentration of 10^8/ml.

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 Na(2)EDTA, 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).

Effects of Anti-factor B mAbs on Hemolytic Activity

In one experimental design, 80 ng/ml recombinant factor B (or mutant factor B) was preincubated with mAb at various molar ratios in Mg-EGTA buffer for 30 min at 4 °C. Aliquots were taken and used to assay hemolytic activity. Percentage of activity was determined as percentage of the average hemolytic activity of the recombinant factor B form preincubated in buffer alone.

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.

RESULTS

Isolation of Factor B cDNA

A full-length human factor B cDNA (designated A14) was isolated from an acute phase liver cDNA library. The A14 sequence is 2483 bp, composed of a 115-bp 5`-untranslated region, a 2292-bp open reading frame, and a 3`-untranslated region of 76 bp, including a 20-bp poly(A) tail. The open reading frame encodes a polypeptide of 764 amino acids. The carboxyl-terminal 739 amino acids are identical to the published sequence of secreted factor B (7) with only one exception, a Q rather than R at position 7 of the mature protein. Since there are two methionines in the first 12 amino acids of the open reading frame, it is not clear whether one or both is utilized as the start of translation. In either case, the NH(2)-terminal region appears to be a standard signal peptide, rich in hydrophobic residues (21

Expression of Active Recombinant Factor B

COS-7 cells were transfected with A14 cDNA oriented in expression vector pSG5 (13) in the forward (B(+)) or reverse (B(-)) direction. Proteins were recovered from the supernatants of biosynthetically labeled and transfected cells, isolated by immunoprecipitation with anti-human factor B polyclonal antibody, and subjected to SDS-PAGE. B(+) supernatants yielded a predominant band of 90 kDa in apparent molecular size, the appropriate position for mature human factor B (Fig. 1, lane2). A weak band present in the B(-) cell supernatant (Fig. 1, lane4) was most likely endogenous monkey factor B synthesized by the COS cells. Neither band was present when normal goat serum was used in place of anti-factor B antibody (Fig. 1, lanes1 and 3).

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 (leq1% activity for an equivalent volume of supernatant).

Analysis of the SCR Region by Site-specific Mutagenesis

SCR mutants were generated employing a strategy known as homolog-scanning mutagenesis(22) . By this method, amino acids are replaced by those of the corresponding region of a structural homolog. In principle, homolog-scanning mutagenesis can identify sequences causing functional variation among homologous proteins. In practice, it has proved helpful for the analysis of the SCRs of complement receptor 1(16, 23) . Thus, a panel of SCR mutations was generated that spanned all three factor B SCRs (Fig. 2). Most mutants feature short amino acid substitutions derived from the corresponding SCR region of human C2, which is 46% homologous to factor B in this region (24) but does not interact with C3b or factor D. In a few cases substitutions derived from a sea lamprey factor B/C2 sequence were used(25) .

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 PIGTRKV 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.

Effects of SCR Mutations on C3b Binding Capacity

An ELISA procedure was developed that detected the binding of factor B to immobilized C3b at salt and cation concentrations similar to those of the hemolytic assay. Binding of the wild type factor B was Mg-dependent (9 ± 2% binding observed without Mg), and binding capacity was constant between 25 and 75 mM NaCl (data not shown). Moreover, factor B von Willebrand factor mutations Bmut28 and Bmut29 were also examined; in each of these mutants, an amino acid residue that is homologous to one that coordinates Mg binding in the von Willebrand factor domain of CR3 (26, 27) has been replaced. Bmut28 and Bmut29 lacked both C3b binding capacity and hemolytic activity (Fig. 2).

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.

Epitope Mapping of mAb 014-III-33

The factor B mutant panel was used to map the epitope of 014-III-33, an anti-Ba mAb reported to block factor B function (Quidel). It was seen that 014-III-33 recognized all but three of the 26 SCR mutants (Fig. 3). Those three mutants defined a discrete region of 19 amino acids in length between the conserved tryptophan of SCR-1 and the first conserved cysteine of SCR-2.

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.


Effects of mAb 014-III-33 on Factor B Function

mAb 014-III-33 was examined for its effects on hemolytic capacity (Table 4, Experiment I). Our observations confirmed its inhibitory effects: Even at a 2:1 mAb:factor B molar ratio, 014-III-33 inhibited over 80% of hemolytic activity. Two factor B mutations not recognized by the mAb (Bmut7L and Bmut8) were similarly unaffected by 014-III-33 in hemolytic assays (Table 4, Experiment 2).

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).

DISCUSSION

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, leq2% 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.

FOOTNOTES

*
This work was funded by the Washington University School of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Div. of Rheumatology, Washington University School of Medicine, 660 S. Euclid, Box 8045, St. Louis, MO 63110. Tel.: 314-362-8397; Fax: 314-362-1366.

(^1)
The abbreviations used are: SCR, short consensus repeat; PBS, phosphate-buffered saline; mAb, monoclonal antibody; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PB, phosphate buffer.

ACKNOWLEDGEMENTS

We thank Dr. Rick Wetsel of St. Louis Children's Hospital and the Washington University School of Medicine for the cDNA library and factor B partial cDNA probe, Dr. Paul Higgins of CytoMed, Inc. (Cambridge, MA) for purified human C2, and Dr. John Tamerius of Quidel (San Diego, CA) for helpful discussions pertaining to mAb 14-III-33.

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