High-Affinity C3b-Binding Complement Factor B That Promote

Mutations of the Type A Domain of Complement Factor B That Promote High-Affinity C3b-Binding

This information is current as Dennis E. Hourcade, Lynne M. Mitchell and Teresa J. of September 26, 2021. Oglesby J Immunol 1999; 162:2906-2911; ; http://www.jimmunol.org/content/162/5/2906 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Mutations of the Type A Domain of Complement Factor B That Promote High-Afﬁnity C3b-Binding

Dennis E. Hourcade,1 Lynne M. Mitchell, and Teresa J. Oglesby

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes “gain of function” mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the ␤A-␣1Mg2؉ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and 2؉ C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg binding cleft, when combined with the D254G Downloaded from substitution, resulted in increased afﬁnity for C3b and iC3b, a C3b derivative. These ﬁndings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding. The Journal of Immunology, 1999, 162: 2906–2911.

he complement system consists of ϳ30 proteins that func- microscopy has indicated that Bb is a dumbbell-shaped protein

tion in innate and acquired immunity (1–4). The comple- with one lobe that becomes attached to C3b (13, 16). Several lines http://www.jimmunol.org/ T ment activation pathways respond to foreign objects and of evidence obtained with factor B and its classical pathway/lectin immune complexes by assembling the C3 convertases. These mul- pathway homologue C2 indicate that convertase stability is deter- ticomponent serine proteases cleave C3, and the resulting C3 de- mined at least in part by elements of the type A domain (17–19). rivatives covalently attach to nearby targets and direct Ag selec- Thus, by the simplest interpretation of the available evidence, it is tion, immune clearance, and cell lysis. the type A domain that is in contact with C3b in the C3 convertase. Factor B is the zymogen that carries the catalytic site of the The type A domain in the ␣-chain of CR3 (20), an integrin, is alternative pathway (AP)2 convertases (5). Assembly of the AP C3 structurally homologous to the factor B type A domain (21). A convertase begins with the association of factor B with C3b in the three-dimensional model of the CR3 type A domain has been gen- ϩ presence of Mg2 . In this context, factor B can be cleaved by erated by x-ray crystallographic analysis (22). It features a divalent by guest on September 26, 2021 factor D, resulting in the Ba and Bb fragments. Ba dissociates from cation that resides in a cleft at one end of the domain, coordinated the complex, while Bb and its associated divalent cation remain by five conserved amino acid sidechains. This region, termed a bound to C3b (6). C3bBb, the active AP C3 convertase, is capable metal ion dependent adhesion site (22), mediates binding to iC3b, of catalyzing C3 cleavage. Dissociation of the convertase is irre- a cleavage product of C3b (1). The CR3 model, and a similar versible, and the Bb fragment alone has no convertase activity. model derived from the type I domain of LFA-1 (23), could pro- Factor B is a 90-kDa single-chain glycoprotein composed of five vide a good approximation of the factor B type A region. Muta- protein domains (7). Its amino-terminal region (Ba) consists pri- tions in predicted metal-binding residues and loops of factor B marily of three short consensus repeats, also found in a number of abrogate ligand-binding activity (11, 15). This report describes complement control proteins (8). The middle region is a type A factor B “gain of function” mutants produced by substituting factor domain similar to those found in von Willebrand factor, some in- B residues near the putative Mg2ϩ and ligand-binding sites with tegrin ␣-chains, and several collagen forms (9). The carboxy ter- homologous CR3 residues. Several of these mutants show in- minus is a serine protease (SP) domain similar to that of trypsin creased hemolytic activity, convertase stability, and greater affinity (10), but controlled by novel regulatory mechanisms (5). for the factor B ligand, C3b, as well as iC3b, a C3b derivative. While all three factor B module types have been implicated in C3b binding (11–15), analysis of factor B and C3bBb by electron Materials and Methods Recombinant factor B forms Department of Medicine, Washington University School of Medicine, St. Louis, MO The transient expression of mutant and wild-type recombinant factor B 63110 forms was conducted in COS-7 cells transfected with factor B cDNA sub- Received for publication August 24, 1998. Accepted for publication December cloned into the expression vector pSG5 (Stratagene, La Jolla, CA) using 1, 1998. serum-free medium (see Ref. 11). Supernatants were dialyzed and stored in

The costs of publication of this article were defrayed in part by the payment of page phosphate buffer (PB; 11 mM Na2HPO4 and 1.8 mM NaH2PO4 (pH 7.4)) charges. This article must therefore be hereby marked advertisement in accordance supplemented with 25 mM NaCl. Factor B content was assessed by ELISA with 18 U.S.C. Section 1734 solely to indicate this fact. using immobilized mouse anti-human Ba mAb (Quidel, San Diego, CA) 1 Address correspondence and reprint requests to Dr. Dennis Hourcade, Division of for capture and goat anti-human factor B polyclonal Ab followed by rabbit Rheumatology, Washington University School of Medicine, 660 South Euclid, Box anti-goat IgG polyclonal Ab for detection (11). Mutant factor B forms were 8045, St. Louis, MO 63110. E-mail address: [email protected] initially compared with wild-type factor B by either immunoprecipitation 2 Abbreviations used in this paper: AP, alternative pathway; PB, phosphate buffer; of biosynthetically-labeled protein followed by PAGE (11) or by Western DGVB2ϩ, dextrose veronal-buffered saline containing gelatin and cations; SP, serine blot analysis of unlabeled full-length and Bb forms (24). Mutations were protease; P, properdin. introduced into the factor B/pSG5 construct using three different methods:

the transformer site-directed mutagenesis method (25) (Clontech Labora- tories, Palo Alto, CA), the QuikChange site-directed mutagenesis method (Stratagene), and the double-take double-stranded site-directed mutagenesis method (Stratagene), following the manufacturers’ instructions.

Hemolysis assays Cells were prepared by ﬁrst assembling classical pathway C3 convertases on sheep erythrocytes and then using them to coat the cell surface with C3b. Ab-sensitized sheep erythrocytes (EA cells, 5 ml, 5 ϫ 108/ml) obtained from Advanced Research Technology (San Diego, CA) were washed twice and resuspended in 5 ml of dextrose veronal-buffered saline containing gelatin and cations (DGVB2ϩ) (26), mixed with 37.5 ␮gof human C1 in 5 ml of DGVB2ϩ, and incubated for 15 min at 30°. The resulting cells (EAC1) were washed twice and resuspended in 5 ml of DGVB2ϩ, mixed with 50 ␮g of human C4 suspended in 5 ml of DGVB2ϩ, and incubated for 15 min at 30°. These cells (EAC1, 4) were washed twice and suspended in 5 ml of DGVB2ϩ, mixed with 250 ␮g of human C3 and 5 ␮g of human C2 suspended in 5 ml of DGVB2ϩ, and incubated for 30 min at 30°. The resulting cells (EAC1, 4, 2, 3) were washed and resuspended in 5 ml of 10 mM EDTA buffer (26) and incubated at 37°C for 2 h to allow dissociation of the active classical pathway convertases. The re-

sulting C3b-coated cells were washed twice in 5 ml 10 mM EDTA buffer, Downloaded from twice in 5 ml of 10 mM Mg2ϩ EGTA buffer (26), and resuspended in 10 mM Mg2ϩ EGTA buffer to a final concentration of 1 ϫ 108/ml. All purified complement proteins in this study were obtained from Advanced Research Technologies. For each determination, duplicate samples were made from 100 ␮lof prepared C3b-coated sheep erythrocytes, 50 ␮l of purified factor D (5 ng in Mg2ϩ EGTA buffer), 50 ␮l of properdin (P; 45 ng in Mg2ϩ EGTA buffer), and 50 ␮l of factor B source or standard (typically 0.1–0.5 ng) http://www.jimmunol.org/ were mixed together and incubated at 30°C for 30 min. In some cases, the FIGURE 1. Schematic diagram of Bb. A, Position of Mg2ϩ and the P addition was replaced by 50 ␮lofMg2ϩ EGTA buffer. The negative ϩ N-linked oligosaccharide are derived from Refs. 22 and 23. B, Schematic ␮ 2 ϩ control substituted 50 l of DGVB buffer for the factor B source. AP C3 diagram of the factor B residues that participate in the Mg2 coordination ␮ convertase sites were developed with 300 l of a 1/40 dilution of guinea sphere. C, Factor B type A mutants. The factor B type ␤A-␣1 loop se- pig serum (Colorado Serum, Denver, CO) in 40 mM EDTA buffer (26) at quence is aligned with the homologous region of CR3 as in Ref. 21. Extent 37°C for 60 min. Additional controls included complete cell lysis by 450 ␤ ␣ ␮l of distilled water and a negative control in which cells were mixed with of the factor B A- 1 loop, as predicted in Ref. 15, is indicated by the 450 ␮l of DGVB2ϩ buffer only. These samples were also incubated at 37°C horizontal bar. Mutations utilized in this study are noted below with their

for 60 min. All samples were then centrifuged and the OD414 of the su- respective replacement amino acids. The Ch1 mutation is from Tuckwell et

pernatants determined. Hemolytic activity levels for the factor B mutants al. (15). N-linked oligosaccharide at factor B position 260 is indicated by by guest on September 26, 2021 The three putative Mg2ϩ-coordination residues in the ␤A-␣1 loop .” ء “ were expressed as percent of the wild-type Z value, which is equivalent to an the average number of lytic sites per cell (26). are marked by “ ∧ ”. Measurement of convertase stability AP convertases were assembled on sheep erythrocytes using 0.15–0.25 ng of factor B. After the 30 min incubation of duplicate samples of C3b-coated cells with factor B, factor D, and P, 250 ␮l of 40 mM EDTA buffer was Results added to prevent the assembly of new convertases, and incubation was Factor B type A mutants that increase C3b-binding afﬁnity continued at 30°C to allow decay. At various times, 50 ␮l of 1/8 guinea pig serum diluted in 40 mM EDTA buffer was added, and samples were in- Complement proteins factor B, C2, and CR3 each feature a type A cubated at 37°C for 1 h. Hemolysis proceeded during this period. The domain that is a major ligand-binding site. To explore the basis for

remaining cells were centrifuged, the OD414 of the supernatants were ob- their ligand-specificity we began to substitute factor B type A tained, and Z values were calculated. t1/2 was calculated for each experi- amino acids with homologous residues of C2 and CR3. In the ment by linear regression analysis of log Z vs t. t was expressed as the 1/2 course of this process a hybrid protein of particular interest was average t Ϯ SD for n independent experiments. 1/2 generated. C3b binding and iC3b binding The ␤A-␣1 loop (notation of Lee et al. (22)), which carries three Mg2ϩ coordination residues (Fig. 1, A and B) (22, 23), was sub- Factor B in PB supplemented to 75 mM NaCl, 4% BSA, 0.05% Tween 20, jected to site-directed mutagenesis. Five factor B amino acids were and 10 mM MgCl2, 5 mM NiCl2, or 20 mM EDTA was incubated in duplicate C3b-coated microtiter wells at 37°C for 30–120 min, the wells replaced with CR3 residues (254-DSIGASN* to 254-GSIIPHD, washed in PB supplemented with 25 mM NaCl, 4% BSA, and 0.05% where N* indicates an N-linked oligosaccharide). The Mg2ϩ co- Tween 20, and detected by ELISA using goat anti-human factor B poly- ordination residues were not altered. The resulting mutant form, clonal Abs followed by peroxidase-conjugated rabbit anti-goat IgG poly- Bmut31 (Fig. 1C) exhibited a 2-fold to 3-fold increase in hemo- clonal Abs (11). For iC3b binding, microtiter wells were prepared as with C3b, but in the factor B incubation step NaCl was 25 mM. lytic activity over wild type (Table I). Several observations indicated that the Bmut31 substitution in- Generation of Bb creases C3b-binding affinity: 1) Bmut31 convertase was relatively Mutant or wild-type recombinant factor B (500 ng/ml) was treated with stable over a 2-h period at 30°C, while wild-type convertase de- factor D (200 ng/ml) and C3b (2000 ng/ml) in PB supplemented with 10 cayed with a half-life of 53 Ϯ 6 min (n ϭ 4) (Fig. 2). Moreover, mM MgCl2 and 25 mM NaCl for 30 min at 37°C, and the factor B cleavage Bmut31 convertases assembled in the absence of P decayed with a was confirmed by Western blot analysis (24). Control uncleaved factor B half-life of 17 Ϯ 4 min (n ϭ 3), while equivalent wild-type con- was prepared similarly except C3b was omitted from the reaction. In those vertases were not detected; 2) Bmut31 attained 30–40% maximal cases, the Western blot analysis confirmed that factor D-mediated cleavage did not occur. The Bb fraction retained Ͻ3% of the hemolytic activity of hemolytic activity without P, an agent that stabilizes AP conver- the full-length factor B fraction. tases (1), while wild-type factor B was dependent on P for activity 2908 GAIN OF FUNCTION MUTATIONS OF THE FACTOR B TYPE A DOMAIN

Table I. Hemolytic activity of factor B mutant forms

Mutant % of Wild Type (n)

␤A-␣1 loop mutations, Mg2ϩ-coordination residues conserved Bmut31 246 (3) D254G 264 (3) D254A 258 (3) D254N 148 (3) N*260D 91 (4) D254G, N*260D 285 (3) D254A, N*260D 183 (3) D254N, N*260D 41 (3) 2ϩ Mg -coordination residue mutations FIGURE 3. Hemolytic activity of recombinant factor B forms. Mutant D364A 2 (2) and wild-type factor B forms (0.5 ng) were assayed for hemolytic activity Bmut31, D364A Յ2 (2) in the presence (ϩP) and absence (ϪP) of P. Hemolytic activity is ex- D254G, D364A Յ2 (2) N*260D, D364A Յ2 (2) pressed in each case as a Z value, which is the average number of inde- D254G, N*260D, D364A Յ2 (4) pendent lytic sites per target cell (26). Factor D cleavage site mutations K233RK3AAA Յ2 (3) Downloaded from D254G, K233RK3AAA Յ3 (3) slightly increased C3b binding (Fig. 4A). The N*260D single mu- D254G, N*260D, K233RK3AAA Յ2 (3) tation was similar to wild-type factor B by both of these criterion (Figs. 3 and 4A). Combining D254G with N*260D resulted in a mutant factor B form that was very similar to Bmut31 in both (Fig. 3); 3) Bmut31 bound immobilized C3b readily while wild- hemolytic activity and C3b-binding capacity (Fig. 4A). ϩ type native factor B and recombinant factor B were relatively in- High-affinity C3b binding was dependent on Mg2 , although a 2ϩ active (Fig. 4A). wide range of Mg concentrations would suffice (Fig. 4B). C3b http://www.jimmunol.org/ The Bmut31 mutation incorporates five amino acid changes de- binding was abolished by EDTA (not shown) and by the replace- ϩ rived from human CR3:G.IPHD. Three of these residues (IPH) are ment of putative Mg2 coordination residue D364 with alanine in not conserved in murine CR3 where the homologous positions are the D254G, N*260D, D364A triple mutant (Fig. 5A), and the NNI (27), and so were not expected to be critical determinants of Bmut31, D364A double mutant (not shown). Both EDTA treat- ligand binding in CR3. On the contrary, the G residue is conserved ment (not shown) and D364A substitution (Table I) also abrogated in murine CR3 and is also seen in the type A domain of human hemolytic activity. In contrast, high-affinity C3b binding did not CR4 (28), which, like CR3, binds iC3b. The D substitution, also require an intact factor D-mediated cleavage site. When the factor seen in murine CR3, was also regarded with interest since it re- D cleavage site, K233RK3AAA, was disrupted by triple Ala sub- moves an N-linked oligosaccharide (Ref. 7, and Hourcade and stitution (K233RK3AAA), ligand binding was not impaired (Fig. by guest on September 26, 2021 Mitchell, unpublished observations) that is near the Mg2ϩ binding 5A), although hemolytic activity was abolished (Table I) and fluid cleft (22). phase C3b-dependent factor D-mediated cleavage was abrogated The D254G and N*260D substitutions were introduced sepa- (not shown). High-affinity binding was also observed in the rately and together into the factor B sequence. D254G alone pro- D254G, N*260A double mutant Fig. 5B), suggesting that C3b duced hemolytic activity similar to Bmut31 (Fig. 3), but only binding was accentuated by the loss of the glycosylated asparagine at this position rather than the gain of a specific amino acid sidechain. In general, as the residue at position 254 became smaller (D,N 3 A 3 G), the observed ligand-binding activity and hemolytic activity became greater. The D254G, N*260D double mutant bound C3b better than the D254A, N*260D double mutant that bound C3b better than the D254N, N*260D double mutant (Fig. 5B). Bb derived from the D254G, N*260D mutant did not bind C3b (Fig. 6) and was hemolytically inactive.

The Bmut31-related mutants bind iC3b The Bmut31 substitution was derived from the type A domain of CR3, a domain that binds iC3b, a cleavage product of C3b but not a factor B ligand. Given that the Bmut31 substitution greatly accentuated C3b binding, it seemed plausible that it might also affect iC3b binding. Thus, iC3b was immobilized to microtiter wells and treated with mutant and wild-type factor B forms. Ni2ϩ, which enhances convertase assembly and stability (17), was used in addition to Mg2ϩ as divalent cation. The main results of these experiments were: 1) The Bmut31 derivative, D254G, N*260D, bound iC3b in the presence of Ni2ϩ, although at lower afﬁnity than FIGURE 2. Decay of preassembled convertases. C3 convertases were C3b (Fig. 7, A and B); 2) The binding of D254G, N*260D to iC3b assembled on C3b-coated sheep erythrocytes and allowed to decay at 30°C 2ϩ for varying lengths of time before their detection (see Materials and Meth- was distinguished from C3b binding by its strong Ni -depen- dence (Fig. 7, A and B) and its salt sensitivity (Fig. 7D). In addi- ods). t1/2 values in text were derived from several experiments such as the one shown here. In the absence of P, hemolytic activity could not be de- tion, iC3b binding required both D254G and N*260D substitu- tected, even at t ϭ 0. tions, was sensitive to EDTA, and occurred when the factor D The Journal of Immunology 2909 Downloaded from http://www.jimmunol.org/

FIGURE 5. Effects of factor B mutations on C3b-afﬁnity. Factor B by guest on September 26, 2021 forms in ELISA buffer supplemented with 75 mM NaCl and 10 mM MgCl2 were incubated in C3b-coated microtiter wells for 30 min at the indicated concentrations. Wells were washed and the factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab. A, Effects of mutations of a Mg2ϩ-coordination residue (D364A) and of the factor D cleavage site (K233RK3AAA) on the C3b-binding activity of the D254G, N*260D double mutant. B, Effects of factor B ␤A-␣1 loop double muta- ␤ ␣ FIGURE 4. Effects of A- 1 loop mutations on C3b afﬁnity. A, Factor tions on C3b binding. B forms in ELISA buffer supplemented with 75 mM NaCl and 10 mM

MgCl2 were incubated in C3b-coated microtiter wells for2hattheindi- cated concentrations. Wells were washed and the factor B bound was de- binding, C3bBb assembly, and/or C3bBb stability. However, in these tected by ELISA employing goat anti-human factor B polyclonal Ab. B, cases it is difficult to rule out indirect negative effects on either Mg2ϩ- The D254G, N*260D mutant (25 ng/ml; filled squares), the Bmut31 mutant binding or C3b-binding elements in the SP domain (14). (filled triangles), and wild-type recombinant factor B (open squares) in In this report we describe factor B “gain of function” mutations ELISA buffer supplemented with 75 mM NaCl and the indicated MgCl 2 ␤ ␣ concentrations were incubated in C3b-coated microtiter wells for 2 h. altered in a type A domain A- 1 loop, a region previously im- 2ϩ Wells were washed and the factor B bound was detected by ELISA em- plicated directly in Mg binding (22, 23) and indirectly in the ploying goat anti-human factor B polyclonal Ab. The OD averaged 0.08 generation and/or maintenance of C3b-binding (11, 15). Amino 450 ϩ in the absence of any factor B form. acids were derived from CR3, without altering the Mg2 coordination residues. One mutant, Bmut31, was hemolytically more active than wild type, especially in the absence of P, an agent that stabilizes AP convertase. Bmut31 bound immobilized C3b much cleavage site was disrupted (data not shown). No iC3b-binding more effectively than wild-type factor B and produced a relatively was observed with the wild-type factor B form (Fig. 7C). stable convertase. Two amino acid substitutions together accounted for the novel Discussion properties of Bmut31: replacing D254, positioned directly between Factor B is a mosaic protein consisting of three different types of two metal-coordination residues on the ␤A-␣1 loop, with glycine protein modules: three short consensus repeats, one type A do- resulted in enhanced hemolytic activity. Combining the D254G main, and one SP domain. Activity of the SP catalytic site is mutation with mutations that remove an N-linked oligosaccharide strictly regulated by an assembly process that culminates in a located near the Mg2ϩ-binding cleft (22, 23) resulted in factor C3bBb complex, the active C3 convertase. The factor B type A D-independent high-affinity C3b binding. High-affinity C3b bind- domain is believed to mediate C3b binding in C3bBb, based on the ing required divalent cation but did not require factor D-mediated negative effects of several site-directed type A mutations on C3b cleavage. It was EDTA-sensitive and abolished by mutations that 2910 GAIN OF FUNCTION MUTATIONS OF THE FACTOR B TYPE A DOMAIN

FIGURE 6. C3b-binding properties of Bb. The full-length factor B and the Bb forms of the D254G, N*260D double mutant were prepared as described in Materials and Methods. They were mixed at the indicated concentrations in ELISA buffer supplemented with 75 mM NaCl and 10 mM MgCl and were incubated in C3b-coated microtiter wells for 30 min.

2 Downloaded from Wells were washed, and the factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab.

ϩ FIGURE 7. Effects of factor B mutations on iC3b-binding. A, The factor disrupt Mg2 binding, but not by mutations that disrupt the factor B D254G, N*260D double mutant was mixed at 125 ng/ml in ELISA D cleavage site. High-affinity binding derivatives also bound the buffer supplemented with 25 mM NaCl and either 10 mM MgCl or5mM http://www.jimmunol.org/ CR3 ligand, iC3b. Together, these findings present a compelling 2 NiCl2 and incubated in iC3b-coated microtiter wells for the indicated case for the positive role of the type A domain in C3b-binding times. Factor B bound was detected by ELISA employing goat anti-human affinity, C3bBb stability, and ligand-binding specificity. factor B polyclonal Ab. B, The factor B D254G, N*260D double mutant The assembly of the AP convertases involves critical changes in was mixed at 10 ng/ml in ELISA buffer supplemented with 25 mM NaCl the Bb region, including the formation of a C3b/Bb connection and and either 10 mM MgCl2 or 5 mM NiCl2 and incubated in C3b-coated the activation of the serine protease domain. We have observed microtiter wells for the indicated times. Factor B bound was detected by that D254G alone is sufficient for P-independent hemolytic activ- ELISA employing goat anti-human factor B polyclonal Ab. C, Wild-type ity, but removal of the oligosaccharide at position 260 is also re- recombinant factor B was mixed at 125 ng/ml in ELISA buffer supple-

mented with 25 mM NaCl and either 10 mM MgCl or 5 mM NiCl and by guest on September 26, 2021 quired for enhanced factor D-independent C3b binding. Thus, the 2 2 incubated in iC3b-coated microtiter wells for the indicated times. Factor B D254G substitution may directly alter the C3b-binding site, while bound was detected by ELISA employing goat anti-human factor B poly- removal of the oligosaccharide at N*260 may allow access of the clonal Ab. Similar results were obtained with purified wild-type factor B. modified C3b-binding site to C3b without factor D-mediated cleav- D, The factor B D254G, N*260D double mutant was mixed in ELISA age and Ba release. By this model, the transition from C3bB to buffer supplemented with 5 mM NiCl2 and the indicated NaCl concentra- C3bBb would involve at least two important events: 1) improved tions and incubated in iC3b-coated microtiter wells (125 ng/ml factor B) or access of the type A ligand-binding region to C3b by a conforma- C3b-coated microtiter wells (10 ng/ml factor B) for 30 min. Factor B bound tional shift of the N260-linked oligosaccharide, and 2) formation was detected by ELISA employing goat anti-human factor B polyclonal Ab. of a new type A/C3b interaction at the Mg2ϩ site. Both of these events would be normally driven by factor D-mediated cleavage and/or Ba release. The type A domain serves as a ligand-binding site in several affinity by allowing greater access of C3b and iC3b to the metal- integrins, and in some of those cases, amino acids that are struc- coordination sphere. turally homologous to D254 and N*260 of factor B have been The possible role of N260-linked oligosaccharide in the regu- implicated in ligand binding. In LFA-1, the M140Q and E146D lation of ligand binding may be unique to the type A domain of substitutions reduced ICAM-1 binding by 35% and 50%, respec- mammalian factor B. The N260-linked oligosaccharide has also tively (29), while in CR3, binding to neutrophil inhibitory factor been observed in murine factor B (31), although not in frog (32), was abolished by G143 M and reduced ϳ50% by D149K (30). The fish (33, 34), or lamprey (35) equivalents. Interestingly, both negative effects of the integrin mutations and the factor B Ch1 human and murine C2 (31, 36) have a potentially glycosylated mutation of Tuckwell et al. (see Fig. 1 of Ref. 15), and the positive asparagine at positions equivalent to human factor B residue effects reported here with factor B, indicate the general signifi- 324, which, by three-dimensional models, lies close to the N260 cance of these two positions to type A metal ion dependent adhe- position (22, 23). sion site ligand interactions. It has been proposed for CR3 that the As with wild-type factor B, dissociation of the high-affinity Bb divalent cation is coordinated by five type A residues and a single derivative from C3b is irreversible. The biochemical basis for this acidic sidechain of iC3b, forming a bridge between receptor and effect is not understood. Previous findings indicate that the Ba ligand (22). While it is unclear from our results whether the cation region is critically involved in the assembly of C3bBb, possibly provides a ligand contact point, it is of interest that the D254 res- contributing an essential C3b-binding site or providing a scaffold idue of factor B would be expected to be positioned adjacent to the necessary for type A/C3b interactions (11–13). Thus, loss of Ba proposed ligand sidechain (see Fig. 5 of Ref. 22). Thus, the bridge during convertase assembly could be a negative controlling factor. model could account for several of the observations reported here. Alternatively, it is also possible that upon dissociation of conver- Substitution of D254 with smaller amino acids may increase ligand tase, the ligand-binding site on Bb is itself deactivated. The Journal of Immunology 2911

In summary, this report describes “gain of function” mutations 14. Lambris, J. D., and H. J. Muller-Eberhard. 1984. Isolation and characterization of of the complement factor B type A domain that increase hemolytic a 33,000-Dalton fragment of complement factor B with catalytic and C3b binding activity. J. Biol. Chem. 259:12685. activity, convertase stability, and affinity for the natural ligand 15. Tuckwell, D. S., Y. Xu, P. Newham, M. J. Humphries, and J. E. Volanakis. 1997. C3b, as well as iC3b, the C3b derivative. Two amino acid changes Surface loops adjacent to the cation-binding site of the complement factor B von account for these new properties. D254 lies in the ␤A-␣1Mg2ϩ- Willebrand factor type A module determine C3b binding specificity. Biochem- istry 36:6605. binding loop but does not coordinate the divalent cation. Its re- 16. Smith, C. A., C. W. Vogel, and H. J. Muller-Eberhard. 1984. MHC class III placement with the smaller amino acids increases hemolytic ac- products: an electron microscope study of the C3 convertase of human comple- tivity and C3bBb stability. Removal of the oligosaccharide at ment. J. Exp. Med. 159:324. 2ϩ 17. Fishelson, Z., and H. J. Muller-Eberhard. 1982. C3 convertase of human com- position 260, near the Mg -binding cleft, when combined with plement: enhanced formation and stability of the enzyme generated with nickel D254G, results in high affinity ligand binding without factor D- instead of magnesium. J. Immunol. 129:2603. mediated cleavage. These findings offer strong evidence for the 18. Polley, M. J., and H. J. Muller-Eberhard. 1967. Enhancement of the hemolytic activity of the second component of human complement by oxidation. J. Exp. direct involvement of the type A domain in C3b binding and are Med. 126:1013. suggestive that steric effects of the D254 residue and the nearby 19. Horiuchi, T., K. J. Macon, J. A. Engler, and J. E. Volanakis. 1991. Site-directed N-linked oligosaccharide may contribute to the regulation of the mutagenesis of the region around cys-241 of complement component C2. J. Im- munol. 147:584. type A C3b-binding site. 20. Corbi, A. L., T. K. Kishimoto, S. J. Miller, and T. A. Springer. 1988. The human leukocyte adhesion glycoprotein Mac-1 (complement receptor type 3, CD11b) ␣ Acknowledgments subunit: cloning, primary structure, and relation to the integrins, von Willebrand factor and factor B. J. Biol. Chem. 263:12403. We thank Drs. John Atkinson and Evan Sadler for helpful readings of the 21. Perkins, S. J., K. F. Smith, S. C. Williams, P. I. Haris, D. Chapman, and Downloaded from manuscript and Madonna Bogacki for assistance in preparing the R. B. Sim. 1994. The secondary structure of the von Willebrand factor Type A domain in factor B of human complement by Fourier transform infrared spec- manuscript. troscopy. J. Mol. Biol. 238:104. 22. Lee, J. O., P. Rieu, M. A. Arnaout, and R. Liddington. 1995. Crystal structure of References the A domain from the a subunit of integrin CR3 (CD116/CD18). Cell 80:631. 23. Qu, A., and D. J. Leahy. 1995. Crystal structure of the I-domain from the CD11a/ 1. Muller-Eberhard, H. J. 1992. Inflammation: basic principles and clinical corre- CD18 (LFA-1, aLb2) integrin. Proc. Natl. Acad. Sci. USA 92:10277. lates. In Complement: Chemistry and Pathways. 2nd Ed. J. Gallin, I. Goldstein, 24. Hourcade, D. E., L. M. Mitchell, and T. J. Oglesby. 1998. A conserved element and R. Snyderman, eds. Raven Press, New York, p. 33. in the serine protease domain of complement Factor B. J. Biol. Chem. 273:25996. http://www.jimmunol.org/ 2. Volanakis, J. E. 1998. Overview of the complement system. In The Human Com- 25. Deng, W. P., and J. A. Nickoloff. 1992. Site-directed mutagenesis of virtually any plement System in Health and Disease. 10th Ed. J. E. Volanakis, and M. M. plasmid by eliminating a unique site. Anal. Biochem. 200:81. Frank, eds. Marcel Dekker, New York, p. 9. 3. Fearon, D. T., and R. M. Locksley. 1996. The instructive role of innate immunity 26. Whaley, K. 1985. Measurement of complement. In Methods in Complement for in the acquired immune response. Science 272:50. Clinical Immunologists. K. Whaley, ed. Churchill Livingstone, New York, p. 77. ␣ 4. Carroll, M. C. 1998. The role of complement and complement receptors in in- 27. Pytela, R. 1988. Amino acid sequence of the murine Mac-1 chain reveals duction and regulation of immunity. Annu. Rev. Immunol. 16:545. homology with the integrin family and an additional domain related to von Wil- 5. Volanakis, J. E., and G. J. Arlaud. 1998. Complement enzymes. In The Human lebrand factor. EMBO J. 7:1371. Complement System in Health and Disease. J. E. Volanakis, and M. M. Frank, 28. Corbi, A. L., J. Garcia-Aguilar, and T. A. Springer. 1990. Genomic structure of eds. Marcel Dekker, New York, p. 49. an integrin a subunit, the leukocyte p150,95 molecule. J. Biol. Chem. 265:2782. 6. Fishelson, Z., M. K. Pangburn, and H. J. Muller-Eberhard. 1983. C3 convertase 29. Huang, C., and T. Springer. 1995. A binding interface on the I domain of lym- of the alternative complement pathway: demonstration of an active, stable C3b, phocyte function-associated antigen-1 (LFA-1) required for specific interaction by guest on September 26, 2021 Bb(Ni) complex. J. Biol. Chem. 258:7411. with intercellular adhesion molecular 1 (ICAM-1). J. Biol. Chem. 270:19008. 7. Mole, J. E., J. K. Anderson, E. A. Davison, and D. E. Woods. 1984. Complete 30. Rieu, P., T. Sugimori, D. L. Griffith, and M. A. Arnaout. 1996. Solvent-accessible primary structure for the zymogen of human complement factor B. J. Biol. Chem. residues on the metal ion-dependent adhesion site face of integrin CR3 mediate 259:3407. its binding to the neutrophil inhibitory factor. J. Biol. Chem. 271:15858. 8. Kristensen, T., P. D’Eustachio, R. T. Ogata, L. P. Chung, K. B. M. Reid, and 31. Ishikawa, N., M. Nonaka, R. A. Wetsel, and H. R. Colten. 1990. Murine com- B. F. Tack. 1991. The superfamily of C3b/C4b-binding proteins. Fed. Proc. 46: plement C2 and factor B genomic and cDNA cloning reveals different mecha- 2463. nisms for multiple transcripts of C2 and B. J. Biol. Chem. 265:19040. 9. Colombatti, A., and P. Bonaldo. 1991. The superfamily of proteins with von 32. Kato, Y., L. Salter-Cid, M. F. Flajnik, M. Kasahara, C. Namikawa, M. Sasaki, Willebrand factor type A-like domains: one theme common to components of and M. Nonaka. 1994. Isolation of the Xenopus complement factor B comple- extracellular matrix, cellular adhesion, and defense mechanisms. Blood 77:2305. mentary DNA and linkage of the gene to the frog MHC. J. Immunol. 153:4546. 10. Perkins, S. J., and K. F. Smith. 1993. Identity of the putative serine-proteinase fold in proteins of the complement system with nine relevant crystal structures. 33. Kuroda, N., H. Wada, K. Naruse, A. Simada, A. Shima, M. Sasaki, and Biochem. J. 295:109. M. Nonaka. 1996. Molecular cloning and linkage analysis of the Japanese 11. Hourcade, D. E., L. M. Wagner, and T. J. Oglesby. 1995. Analysis of the short medaka fish complement Bf/C2 gene. Immunogenetics 44:459. consensus repeats of human complement factor B by site-directed mutagenesis. 34. Seeger, A., W. Mayer, and J. Klein. 1996. A complement factor B-like cDNA J. Biol. Chem. 270:19716. clone from the zebrafish (Brachydanio Rerio). Mol. Immunol. 33:511. 12. Pryzdial, E. L. G., and D. E. Isenman. 1987. Alternative complement pathway 35. Nonaka, M., M. Takahashi, and M. Sasaki. 1994. Molecular cloning of a lamprey activation fragment Ba binds to C3b. J. Biol. Chem. 262:1519. homologue of the mammalian MHC class III gene, complement factor B. J. Im- 13. Ueda, A., J. F. Kearney, K. H. Roux, and J. E. Volanakis. 1987. Probing func- munol. 152:2263. tional sites on complement protein B with monoclonal antibodies. J. Immunol. 36. Bentley, D. R. 1986. Primary structure of human complement component C2. 138:1143. Biochem. J. 239:339.