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The Journal of Immunology, 1999, 162: 2906-2911.
Copyright © 1999 by The American Association of Immunologists

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

Dennis E. Hourcade1, Lynne M. Mitchell and Teresa J. Oglesby

Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 ßA-{alpha}1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The complement system consists of ~30 proteins that function in innate and acquired immunity 1, 2, 3, 4 . The complement activation pathways respond to foreign objects and immune complexes by assembling the C3 convertases. These multicomponent serine proteases cleave C3, and the resulting C3 derivatives covalently attach to nearby targets and direct Ag selection, immune clearance, and cell lysis.

Factor B is the zymogen that carries the catalytic site of the alternative pathway (AP)2 convertases 5 . Assembly of the AP C3 convertase begins with the association of factor B with C3b in the presence of Mg2+. In this context, factor B can be cleaved by factor D, resulting in the Ba and Bb fragments. Ba dissociates from the complex, while Bb and its associated divalent cation remain bound to C3b 6 . C3bBb, the active AP C3 convertase, is capable of catalyzing C3 cleavage. Dissociation of the convertase is irreversible, and the Bb fragment alone has no convertase activity.

Factor B is a 90-kDa single-chain glycoprotein composed of five protein domains 7 . Its amino-terminal region (Ba) consists primarily of three short consensus repeats, also found in a number of complement control proteins 8 . The middle region is a type A domain similar to those found in von Willebrand factor, some integrin {alpha}-chains, and several collagen forms 9 . The carboxy terminus is a serine protease (SP) domain similar to that of trypsin 10 , but controlled by novel regulatory mechanisms 5 .

While all three factor B module types have been implicated in C3b binding 11, 12, 13, 14, 15 , analysis of factor B and C3bBb by electron microscopy has indicated that Bb is a dumbbell-shaped protein with one lobe that becomes attached to C3b 13, 16 . Several lines of evidence obtained with factor B and its classical pathway/lectin pathway homologue C2 indicate that convertase stability is determined at least in part by elements of the type A domain 17, 18, 19 . Thus, by the simplest interpretation of the available evidence, it is the type A domain that is in contact with C3b in the C3 convertase.

The type A domain in the {alpha}-chain of CR3 20 , an integrin, is structurally homologous to the factor B type A domain 21 . A three-dimensional model of the CR3 type A domain has been generated by x-ray crystallographic analysis 22 . It features a divalent cation that resides in a cleft at one end of the domain, coordinated by five conserved amino acid sidechains. This region, termed a metal ion dependent adhesion site 22 , mediates binding to iC3b, a cleavage product of C3b 1 . The CR3 model, and a similar model derived from the type I domain of LFA-1 23 , could provide a good approximation of the factor B type A region. Mutations in predicted metal-binding residues and loops of factor B abrogate ligand-binding activity 11, 15 . This report describes factor B "gain of function" mutants produced by substituting factor B residues near the putative Mg2+ and ligand-binding sites with homologous CR3 residues. Several of these mutants show increased hemolytic activity, convertase stability, and greater affinity for the factor B ligand, C3b, as well as iC3b, a C3b derivative.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant factor B forms

The transient expression of mutant and wild-type recombinant factor B forms was conducted in COS-7 cells transfected with factor B cDNA subcloned into the expression vector pSG5 (Stratagene, La Jolla, CA) using serum-free medium (see 11 . Supernatants were dialyzed and stored in phosphate buffer (PB; 11 mM Na2HPO4 and 1.8 mM NaH2PO4 (pH 7.4)) supplemented with 25 mM NaCl. Factor B content was assessed by ELISA using immobilized mouse anti-human Ba mAb (Quidel, San Diego, CA) for capture and goat anti-human factor B polyclonal Ab followed by rabbit anti-goat IgG polyclonal Ab for detection 11 . Mutant factor B forms were initially compared with wild-type factor B by either immunoprecipitation of biosynthetically-labeled protein followed by PAGE 11 or by Western blot analysis of unlabeled full-length and Bb forms 24 . Mutations were introduced into the factor B/pSG5 construct using three different methods: the transformer site-directed mutagenesis method 25 (Clontech Laboratories, 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 x 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 (DGVB2+) 26 , mixed with 37.5 µg of human C1 in 5 ml of DGVB2+, and incubated for 15 min at 30°. The resulting cells (EAC1) were washed twice and resuspended in 5 ml of DGVB2+, mixed with 50 µg of human C4 suspended in 5 ml of DGVB2+, and incubated for 15 min at 30°. These cells (EAC1, 4) were washed twice and suspended in 5 ml of DGVB2+, mixed with 250 µg of human C3 and 5 µg of human C2 suspended in 5 ml of DGVB2+, 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 resulting C3b-coated cells were washed twice in 5 ml 10 mM EDTA buffer, twice in 5 ml of 10 mM Mg2+ EGTA buffer 26 , and resuspended in 10 mM Mg2+ EGTA buffer to a final concentration of 1 x 108/ml. All purified complement proteins in this study were obtained from Advanced Research Technologies.

For each determination, duplicate samples were made from 100 µl of prepared C3b-coated sheep erythrocytes, 50 µl of purified factor D (5 ng in Mg2+ EGTA buffer), 50 µl of properdin (P; 45 ng in Mg2+ EGTA buffer), and 50 µl of factor B source or standard (typically 0.1–0.5 ng) were mixed together and incubated at 30°C for 30 min. In some cases, the P addition was replaced by 50 µl of Mg2+ EGTA buffer. The negative control substituted 50 µl of DGVB2+ buffer for the factor B source. AP C3 convertase sites were developed with 300 µl of a 1/40 dilution of guinea pig serum (Colorado Serum, Denver, CO) in 40 mM EDTA buffer 26 at 37°C for 60 min. Additional controls included complete cell lysis by 450 µl of distilled water and a negative control in which cells were mixed with 450 µl of DGVB2+ buffer only. These samples were also incubated at 37°C for 60 min. All samples were then centrifuged and the OD414 of the supernatants determined. Hemolytic activity levels for the factor B mutants were expressed as percent of the wild-type Z value, which is equivalent to the average number of lytic sites per cell 26 .

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 µl of 40 mM EDTA buffer was added to prevent the assembly of new convertases, and incubation was continued at 30°C to allow decay. At various times, 50 µl of 1/8 guinea pig serum diluted in 40 mM EDTA buffer was added, and samples were incubated at 37°C for 1 h. Hemolysis proceeded during this period. The remaining cells were centrifuged, the OD414 of the supernatants were obtained, and Z values were calculated. t1/2 was calculated for each experiment by linear regression analysis of log Z vs t. t1/2 was expressed as the average t1/2 ± SD for n independent experiments.

C3b binding and iC3b binding

Factor B in PB supplemented to 75 mM NaCl, 4% BSA, 0.05% Tween 20, 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 washed in PB supplemented with 25 mM NaCl, 4% BSA, and 0.05% Tween 20, and detected by ELISA using goat anti-human factor B polyclonal Abs followed by peroxidase-conjugated rabbit anti-goat IgG polyclonal Abs 11 . For iC3b binding, microtiter wells were prepared as with C3b, but in the factor B incubation step NaCl was 25 mM.

Generation of Bb

Mutant or wild-type recombinant factor B (500 ng/ml) was treated with factor D (200 ng/ml) and C3b (2000 ng/ml) in PB supplemented with 10 mM MgCl2 and 25 mM NaCl for 30 min at 37°C, and the factor B cleavage was confirmed by Western blot analysis 24 . Control uncleaved factor B was prepared similarly except C3b was omitted from the reaction. In those cases, the Western blot analysis confirmed that factor D-mediated cleavage did not occur. The Bb fraction retained <3% of the hemolytic activity of the full-length factor B fraction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Factor B type A mutants that increase C3b-binding affinity

Complement proteins factor B, C2, and CR3 each feature a type A domain that is a major ligand-binding site. To explore the basis for their ligand-specificity we began to substitute factor B type A amino acids with homologous residues of C2 and CR3. In the course of this process a hybrid protein of particular interest was generated.

The ßA-{alpha}1 loop (notation of Lee et al. 22), which carries three Mg2+ coordination residues (Fig. 1Go, A and B) 22, 23 , was subjected to site-directed mutagenesis. Five factor B amino acids were replaced with CR3 residues (254-DSIGASN* to 254-GSIIPHD, where N* indicates an N-linked oligosaccharide). The Mg2+ coordination residues were not altered. The resulting mutant form, Bmut31 (Fig. 1GoC) exhibited a 2-fold to 3-fold increase in hemolytic activity over wild type (Table IGo).



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  		FIGURE 1. Schematic diagram of Bb. A, Position of Mg2+ and the N-linked oligosaccharide are derived from Refs. 22 and 23. B, Schematic diagram of the factor B residues that participate in the Mg2+ coordination sphere. C, Factor B type A mutants. The factor B type ßA-{alpha}1 loop sequence is aligned with the homologous region of CR3 as in Ref. 21. Extent of the factor B ßA-{alpha}1 loop, as predicted in Ref. 15, is indicated by the horizontal bar. Mutations utilized in this study are noted below with their respective replacement amino acids. The Ch1 mutation is from Tuckwell et al. (15). N-linked oligosaccharide at factor B position 260 is indicated by an " * ". The three putative Mg2+-coordination residues in the ßA-{alpha}1 loop are marked by " {wedge} ".

 

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  		Table I. Hemolytic activity of factor B mutant forms

 
Several observations indicated that the Bmut31 substitution increases C3b-binding affinity: 1) Bmut31 convertase was relatively stable over a 2-h period at 30°C, while wild-type convertase decayed with a half-life of 53 ± 6 min (n = 4) (Fig. 2Go). Moreover, Bmut31 convertases assembled in the absence of P decayed with a half-life of 17 ± 4 min (n = 3), while equivalent wild-type convertases were not detected; 2) Bmut31 attained 30–40% maximal hemolytic activity without P, an agent that stabilizes AP convertases 1 , while wild-type factor B was dependent on P for activity (Fig. 3Go); 3) Bmut31 bound immobilized C3b readily while wild-type native factor B and recombinant factor B were relatively inactive (Fig. 4GoA).



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  		FIGURE 2. Decay of preassembled convertases. C3 convertases were assembled on C3b-coated sheep erythrocytes and allowed to decay at 30°C for varying lengths of time before their detection (see Materials and Methods). 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 detected, even at t = 0.

 


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  		FIGURE 3. Hemolytic activity of recombinant factor B forms. Mutant and wild-type factor B forms (0.5 ng) were assayed for hemolytic activity in the presence (+P) and absence (-P) of P. Hemolytic activity is expressed in each case as a Z value, which is the average number of independent lytic sites per target cell (26).

 


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  		FIGURE 4. Effects of ßA-{alpha}1 loop mutations on C3b affinity. A, Factor B forms in ELISA buffer supplemented with 75 mM NaCl and 10 mM MgCl2 were incubated in C3b-coated microtiter wells for 2 h at the indicated concentrations. Wells were washed and the factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab. B, The D254G, N*260D mutant (25 ng/ml; filled squares), the Bmut31 mutant (filled triangles), and wild-type recombinant factor B (open squares) in ELISA buffer supplemented with 75 mM NaCl and the indicated MgCl2 concentrations were incubated in C3b-coated microtiter wells for 2 h. Wells were washed and the factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab. The OD450 averaged 0.08 in the absence of any factor B form.

 
The Bmut31 mutation incorporates five amino acid changes derived from human CR3:G.IPHD. Three of these residues (IPH) are not conserved in murine CR3 where the homologous positions are NNI 27 , and so were not expected to be critical determinants of ligand binding in CR3. On the contrary, the G residue is conserved in murine CR3 and is also seen in the type A domain of human CR4 28 , which, like CR3, binds iC3b. The D substitution, also seen in murine CR3, was also regarded with interest since it removes an N-linked oligosaccharide (Ref. 7, and Hourcade and Mitchell, unpublished observations) that is near the Mg2+ binding cleft 22 .

The D254G and N*260D substitutions were introduced separately and together into the factor B sequence. D254G alone produced hemolytic activity similar to Bmut31 (Fig. 3Go), but only slightly increased C3b binding (Fig. 4GoA). The N*260D single mutation was similar to wild-type factor B by both of these criterion (Figs. 3Go and 4GoA). Combining D254G with N*260D resulted in a mutant factor B form that was very similar to Bmut31 in both hemolytic activity and C3b-binding capacity (Fig. 4GoA).

High-affinity C3b binding was dependent on Mg2+, although a wide range of Mg2+ concentrations would suffice (Fig. 4GoB). C3b binding was abolished by EDTA (not shown) and by the replacement of putative Mg2+ coordination residue D364 with alanine in the D254G, N*260D, D364A triple mutant (Fig. 5GoA), and the Bmut31, D364A double mutant (not shown). Both EDTA treatment (not shown) and D364A substitution (Table IGo) also abrogated hemolytic activity. In contrast, high-affinity C3b binding did not require an intact factor D-mediated cleavage site. When the factor D cleavage site, K233RK->AAA, was disrupted by triple Ala substitution (K233RK->AAA), ligand binding was not impaired (Fig. 5GoA), although hemolytic activity was abolished (Table IGo) and fluid phase C3b-dependent factor D-mediated cleavage was abrogated (not shown). High-affinity binding was also observed in the D254G, N*260A double mutant Fig. 5GoB), suggesting that C3b 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 -> A -> 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. 5GoB). Bb derived from the D254G, N*260D mutant did not bind C3b (Fig. 6Go) and was hemolytically inactive.



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  		FIGURE 5. Effects of factor B mutations on C3b-affinity. Factor B 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 Mg2+-coordination residue (D364A) and of the factor D cleavage site (K233RK->AAA) on the C3b-binding activity of the D254G, N*260D double mutant. B, Effects of factor B ßA-{alpha}1 loop double mutations on C3b binding.

 


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  		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 MgCl2 and were incubated in C3b-coated microtiter wells for 30 min. Wells were washed, and the factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab.

 
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. Ni2+, which enhances convertase assembly and stability 17 , was used in addition to Mg2+ as divalent cation. The main results of these experiments were: 1) The Bmut31 derivative, D254G, N*260D, bound iC3b in the presence of Ni2+, although at lower affinity than C3b (Fig. 7Go, A and B); 2) The binding of D254G, N*260D to iC3b was distinguished from C3b binding by its strong Ni2+-dependence (Fig. 7Go, A and B) and its salt sensitivity (Fig. 7GoD). In addition, iC3b binding required both D254G and N*260D substitutions, was sensitive to EDTA, and occurred when the factor D cleavage site was disrupted (data not shown). No iC3b-binding was observed with the wild-type factor B form (Fig. 7GoC).



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  		FIGURE 7. Effects of factor B mutations on iC3b-binding. A, The factor B D254G, N*260D double mutant was mixed at 125 ng/ml in ELISA buffer supplemented with 25 mM NaCl and either 10 mM MgCl2 or 5 mM NiCl2 and incubated in iC3b-coated microtiter wells for the indicated times. Factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab. B, The factor B D254G, N*260D double mutant was mixed at 10 ng/ml in ELISA buffer supplemented with 25 mM NaCl and either 10 mM MgCl2 or 5 mM NiCl2 and incubated in C3b-coated microtiter wells for the indicated times. Factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab. C, Wild-type recombinant factor B was mixed at 125 ng/ml in ELISA buffer supplemented with 25 mM NaCl and either 10 mM MgCl2 or 5 mM NiCl2 and incubated in iC3b-coated microtiter wells for the indicated times. Factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab. Similar results were obtained with purified wild-type factor B. D, The factor B D254G, N*260D double mutant was mixed in ELISA buffer supplemented with 5 mM NiCl2 and the indicated NaCl concentrations and incubated in iC3b-coated microtiter wells (125 ng/ml factor B) or C3b-coated microtiter wells (10 ng/ml factor B) for 30 min. Factor B bound was detected by ELISA employing goat anti-human factor B polyclonal Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Factor B is a mosaic protein consisting of three different types of protein modules: three short consensus repeats, one type A domain, and one SP domain. Activity of the SP catalytic site is strictly regulated by an assembly process that culminates in a C3bBb complex, the active C3 convertase. The factor B type A domain is believed to mediate C3b binding in C3bBb, based on the negative effects of several site-directed type A mutations on C3b binding, C3bBb assembly, and/or C3bBb stability. However, in these cases it is difficult to rule out indirect negative effects on either Mg2+-binding or C3b-binding elements in the SP domain 14 .

In this report we describe factor B "gain of function" mutations altered in a type A domain ßA-{alpha}1 loop, a region previously implicated directly in Mg2+ binding 22, 23 and indirectly in the generation and/or maintenance of C3b-binding 11, 15 . Amino 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 more effectively than wild-type factor B and produced a relatively stable convertase.

Two amino acid substitutions together accounted for the novel properties of Bmut31: replacing D254, positioned directly between two metal-coordination residues on the ßA-{alpha}1 loop, with glycine resulted in enhanced hemolytic activity. Combining the D254G mutation with mutations that remove an N-linked oligosaccharide located near the Mg2+-binding cleft 22, 23 resulted in factor D-independent high-affinity C3b binding. High-affinity C3b binding required divalent cation but did not require factor D-mediated cleavage. It was EDTA-sensitive and abolished by mutations that disrupt Mg2+ binding, but not by mutations that disrupt the factor D cleavage site. High-affinity binding derivatives also bound the CR3 ligand, iC3b. Together, these findings present a compelling case for the positive role of the type A domain in C3b-binding affinity, C3bBb stability, and ligand-binding specificity.

The assembly of the AP convertases involves critical changes in the Bb region, including the formation of a C3b/Bb connection and the activation of the serine protease domain. We have observed that D254G alone is sufficient for P-independent hemolytic activity, but removal of the oligosaccharide at position 260 is also required for enhanced factor D-independent C3b binding. Thus, the D254G substitution may directly alter the C3b-binding site, while removal of the oligosaccharide at N*260 may allow access of the modified C3b-binding site to C3b without factor D-mediated cleavage and Ba release. By this model, the transition from C3bB to C3bBb would involve at least two important events: 1) improved access of the type A ligand-binding region to C3b by a conformational shift of the N260-linked oligosaccharide, and 2) formation of a new type A/C3b interaction at the Mg2+ 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 integrins, and in some of those cases, amino acids that are structurally homologous to D254 and N*260 of factor B have been implicated in ligand binding. In LFA-1, the M140Q and E146D substitutions reduced ICAM-1 binding by 35% and 50%, respectively 29 , while in CR3, binding to neutrophil inhibitory factor was abolished by G143 M and reduced ~50% by D149K 30 . The negative effects of the integrin mutations and the factor B Ch1 mutation of Tuckwell et al. (see Fig. 1Go of 15 , and the positive effects reported here with factor B, indicate the general significance of these two positions to type A metal ion dependent adhesion site ligand interactions. It has been proposed for CR3 that the divalent cation is coordinated by five type A residues and a single acidic sidechain of iC3b, forming a bridge between receptor and ligand 22 . While it is unclear from our results whether the cation provides a ligand contact point, it is of interest that the D254 residue of factor B would be expected to be positioned adjacent to the proposed ligand sidechain (see Fig. 5Go of 22 . Thus, the bridge model could account for several of the observations reported here. Substitution of D254 with smaller amino acids may increase ligand affinity by allowing greater access of C3b and iC3b to the metal-coordination sphere.

The possible role of N260-linked oligosaccharide in the regulation of ligand binding may be unique to the type A domain of mammalian factor B. The N260-linked oligosaccharide has also been observed in murine factor B 31 , although not in frog 32 , fish 33, 34 , or lamprey 35 equivalents. Interestingly, both human and murine C2 31, 36 have a potentially glycosylated asparagine at positions equivalent to human factor B residue 324, which, by three-dimensional models, lies close to the N260 position 22, 23 .

As with wild-type factor B, dissociation of the high-affinity Bb derivative from C3b is irreversible. The biochemical basis for this effect is not understood. Previous findings indicate that the Ba region is critically involved in the assembly of C3bBb, possibly contributing an essential C3b-binding site or providing a scaffold necessary for type A/C3b interactions 11, 12, 13 . Thus, loss of Ba during convertase assembly could be a negative controlling factor. Alternatively, it is also possible that upon dissociation of convertase, the ligand-binding site on Bb is itself deactivated.

In summary, this report describes "gain of function" mutations of the complement factor B type A domain that increase hemolytic activity, convertase stability, and affinity for the natural ligand C3b, as well as iC3b, the C3b derivative. Two amino acid changes account for these new properties. D254 lies in the ßA-{alpha}1 Mg2+-binding loop but does not coordinate the divalent cation. Its replacement with the smaller amino acids increases hemolytic activity and C3bBb stability. Removal of the oligosaccharide at position 260, near the Mg2+-binding cleft, when combined with D254G, results in high affinity ligand binding without factor D-mediated cleavage. 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 residue and the nearby N-linked oligosaccharide may contribute to the regulation of the type A C3b-binding site.


   Acknowledgments
 
We thank Drs. John Atkinson and Evan Sadler for helpful readings of the manuscript and Madonna Bogacki for assistance in preparing the manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Dennis Hourcade, Division of Rheumatology, Washington University School of Medicine, 660 South Euclid, Box 8045, St. Louis, MO 63110. E-mail address: Back

2 Abbreviations used in this paper: AP, alternative pathway; PB, phosphate buffer; DGVB2+, dextrose veronal-buffered saline containing gelatin and cations; SP, serine protease; P, properdin. Back

Received for publication August 24, 1998. Accepted for publication December 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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