Mutations of the Type A Domain of
Complement Factor B That Promote
High-Affinity C3b-Binding
This information is current as
of June 14, 2017.
Dennis E. Hourcade, Lynne M. Mitchell and Teresa J.
Oglesby
J Immunol 1999; 162:2906-2911; ;
http://www.jimmunol.org/content/162/5/2906
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References
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
he complement system consists of ;30 proteins that function in innate and acquired immunity (1– 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 Mg21. 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 a-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 (11–15), analysis of factor B and C3bBb by electron
T
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 (17–19).
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 a-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 Mg21 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
Recombinant factor B forms
Department of Medicine, Washington University School of Medicine, St. Louis, MO
63110
Received for publication August 24, 1998. Accepted for publication December
1, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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: [email protected]
2
Abbreviations used in this paper: AP, alternative pathway; PB, phosphate buffer;
DGVB21, dextrose veronal-buffered saline containing gelatin and cations; SP, serine
protease; P, properdin.
Copyright © 1999 by The American Association of Immunologists
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 Ref. 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:
0022-1767/99/$02.00
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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 bA-a1 Mg21 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 Mg21 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. The Journal of Immunology, 1999, 162: 2906 –2911.
The Journal of Immunology
2907
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
FIGURE 1. Schematic diagram of Bb. A, Position of Mg21 and the
N-linked oligosaccharide are derived from Refs. 22 and 23. B, Schematic
diagram of the factor B residues that participate in the Mg21 coordination
sphere. C, Factor B type A mutants. The factor B type bA-a1 loop sequence is aligned with the homologous region of CR3 as in Ref. 21. Extent
of the factor B bA-a1 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 “ p ”. The three putative Mg21-coordination residues in the bA-a1 loop
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 ml 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 ml 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 6 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
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 bA-a1 loop (notation of Lee et al. (22)), which carries three
Mg21 coordination residues (Fig. 1, 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 Mg21 coordination residues were not altered. The resulting mutant form,
Bmut31 (Fig. 1C) exhibited a 2-fold to 3-fold increase in hemolytic activity over wild type (Table I).
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 6 min (n 5 4) (Fig. 2). Moreover,
Bmut31 convertases assembled in the absence of P decayed with a
half-life of 17 6 4 min (n 5 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
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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 3 108/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 (DGVB21) (26), mixed with 37.5 mg of
human C1 in 5 ml of DGVB21, and incubated for 15 min at 30°. The
resulting cells (EAC1) were washed twice and resuspended in 5 ml of
DGVB21, mixed with 50 mg of human C4 suspended in 5 ml of DGVB21,
and incubated for 15 min at 30°. These cells (EAC1, 4) were washed twice
and suspended in 5 ml of DGVB21, mixed with 250 mg of human C3 and
5 mg of human C2 suspended in 5 ml of DGVB21, 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 Mg21 EGTA buffer (26), and resuspended in 10
mM Mg21 EGTA buffer to a final concentration of 1 3 108/ml. All purified
complement proteins in this study were obtained from Advanced Research
Technologies.
For each determination, duplicate samples were made from 100 ml of
prepared C3b-coated sheep erythrocytes, 50 ml of purified factor D (5 ng
in Mg21 EGTA buffer), 50 ml of properdin (P; 45 ng in Mg21 EGTA
buffer), and 50 ml 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 ml of Mg21 EGTA buffer. The negative
control substituted 50 ml of DGVB21 buffer for the factor B source. AP C3
convertase sites were developed with 300 ml 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
ml of distilled water and a negative control in which cells were mixed with
450 ml of DGVB21 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).
2908
GAIN OF FUNCTION MUTATIONS OF THE FACTOR B TYPE A DOMAIN
Table I. Hemolytic activity of factor B mutant forms
Mutant
246 (3)
264 (3)
258 (3)
148 (3)
91 (4)
285 (3)
183 (3)
41 (3)
2 (2)
#2 (2)
#2 (2)
#2 (2)
#2 (4)
#2 (3)
#3 (3)
#2 (3)
(Fig. 3); 3) Bmut31 bound immobilized C3b readily while wildtype native factor B and recombinant factor B were relatively inactive (Fig. 4A).
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 Mg21 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. 3), but only
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 (1P) and absence (2P) 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).
slightly increased C3b binding (Fig. 4A). The N*260D single mutation 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
hemolytic activity and C3b-binding capacity (Fig. 4A).
High-affinity C3b binding was dependent on Mg21, although a
wide range of Mg21 concentrations would suffice (Fig. 4B). C3b
binding was abolished by EDTA (not shown) and by the replacement of putative Mg21 coordination residue D364 with alanine in
the D254G, N*260D, D364A triple mutant (Fig. 5A), and the
Bmut31, D364A double mutant (not shown). Both EDTA treatment (not shown) and D364A substitution (Table I) 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, K233RK3 AAA, was disrupted by triple Ala substitution (K233RK3 AAA), ligand binding was not impaired (Fig.
5A), although hemolytic activity was abolished (Table I) 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. 5B), 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 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
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 5 0.
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. Ni21, which
enhances convertase assembly and stability (17), was used in addition to Mg21 as divalent cation. The main results of these experiments were: 1) The Bmut31 derivative, D254G, N*260D,
bound iC3b in the presence of Ni21, although at lower affinity than
C3b (Fig. 7, A and B); 2) The binding of D254G, N*260D to iC3b
was distinguished from C3b binding by its strong Ni21-dependence (Fig. 7, A and B) and its salt sensitivity (Fig. 7D). In addition, iC3b binding required both D254G and N*260D substitutions, was sensitive to EDTA, and occurred when the factor D
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bA-a1 loop mutations, Mg21-coordination
residues conserved
Bmut31
D254G
D254A
D254N
N*260D
D254G, N*260D
D254A, N*260D
D254N, N*260D
Mg21-coordination residue mutations
D364A
Bmut31, D364A
D254G, D364A
N*260D, D364A
D254G, N*260D, D364A
Factor D cleavage site mutations
K233RK3AAA
D254G, K233RK3AAA
D254G, N*260D, K233RK3AAA
% of Wild Type (n)
The Journal of Immunology
cleavage site was disrupted (data not shown). No iC3b-binding
was observed with the wild-type factor B form (Fig. 7C).
Discussion
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
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 Mg21-coordination residue (D364A) and of the factor D
cleavage site (K233RK3 AAA) on the C3b-binding activity of the D254G,
N*260D double mutant. B, Effects of factor B bA-a1 loop double mutations on C3b binding.
binding, C3bBb assembly, and/or C3bBb stability. However, in these
cases it is difficult to rule out indirect negative effects on either Mg21binding 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 bA-a1 loop, a region previously implicated directly in Mg21 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 Mg21 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 bA-a1 loop, with glycine
resulted in enhanced hemolytic activity. Combining the D254G
mutation with mutations that remove an N-linked oligosaccharide
located near the Mg21-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
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FIGURE 4. Effects of bA-a1 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.
2909
2910
GAIN OF FUNCTION MUTATIONS OF THE FACTOR B TYPE A DOMAIN
disrupt Mg21 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 Mg21 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. 1 of Ref. 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. 5 of Ref. 22). Thus, the bridge
model could account for several of the observations reported here.
Substitution of D254 with smaller amino acids may increase ligand
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.
affinity by allowing greater access of C3b and iC3b to the metalcoordination 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 (11–13). 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.
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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 Journal of Immunology
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 bA-a1 Mg21binding 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 Mg21-binding cleft, when combined with
D254G, results in high affinity ligand binding without factor Dmediated 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
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We thank Drs. John Atkinson and Evan Sadler for helpful readings of the
manuscript and Madonna Bogacki for assistance in preparing the
manuscript.
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