Two Clusters of Acidic Amino Acids Near the
NH 2 Terminus of Complement Component
C4 α′-Chain Are Important for C2 Binding
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J Immunol 2000; 165:2518-2527; ;
doi: 10.4049/jimmunol.165.5.2518
http://www.jimmunol.org/content/165/5/2518
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References
Qun Pan, Roger O. Ebanks and David E. Isenman
Two Clusters of Acidic Amino Acids Near the NH2 Terminus
of Complement Component C4 ␣ⴕ-Chain Are Important for
C2 Binding1
Qun Pan, Roger O. Ebanks,2 and David E. Isenman3
I
n terms of the functional manifestations of complement activation, including its opsonic, inflammatory, and lytic roles
in immune clearance, the respective cleavages of C3 and C5
represent the most critical steps in the activation process. In the
classical pathway, the cleavage of C3 and C5 is accomplished by
convertases having the compositions C4b2a and C4bC3b2a, respectively. Because only in its C4b-bound state is C2a capable of
cleaving its physiologic protein substrates C3 and C5 (1, 2), C4b
may be thought of as a modulator of the serine protease activity of
C2a. The equivalent C3 and C5 convertases of the alternative pathway have the composition C3bBb and C3bC3bBb, respectively.
Thus, in these convertases, the serine protease moiety of factor B
(Bb) fulfills the proteolytic role and C3b fulfills the enzyme-modulatory role. Therefore, both in terms of sequence and function, C3
and C4 are homologs, as are C2 and factor B.
Human C2 is a 102-kDa serum glycoprotein showing 39% sequence identity with its functional homolog factor B (3). C2 and
factor B have similar modular structures consisting from the NH2
to COOH terminus of three short consensus repeats (SCRs)4 of the
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
Received for publication March 24, 2000. Accepted for publication June 14, 2000.
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
This work was supported by funding from the Medical Research Council of Canada
(MT-7081).
2
Current address: Institut National de la Recherche Scientifique-Institut Armand
Frappier, 531 Boulevard des Prairies, Laval, Quebec, Canada, H7V 1B7.
3
Address correspondence and reprint requests to Dr. David E. Isenman, Department
of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8. E-mail
address: [email protected]
4
Abbreviations used in this paper: SCR, short consensus repeat; vWFA, von Willebrand factor type A; CR, complement receptor; C4BP, C4 binding protein; C-EDTA,
a C3-C9 reagent; oxyC2, iodine-oxidized C2; EAC, sheep erythrocytes coated with Ab
Copyright © 2000 by The American Association of Immunologists
type found in complement regulatory proteins encoded within the
regulators of compliment activation locus, a von Willibrand factor
type A (vWFA) domain, and a serine protease domain. The genes
encoding C2 and factor B are located within the MHC class III
region (4), are ⬍500 bp apart, and have similar intron/exon structures (5–7). Cumulatively, these data suggest that a gene duplication event has led to the divergence and evolution of factor B and
C2 and, indeed, there is an example of a single protein in rainbow
trout displaying both classical and alternative pathway C3 convertase subunit activity (8).
When viewed by transmission electron microscopy, human C2
and factor B appear structurally similar (9) with each molecule
consisting of three 40-Å-diameter lobes. Presumably, the NH2terminal-derived C2b and Ba fragments, which are each comprised
of three SCR domains, would occupy a single lobe, whereas C2a
and Bb, each being comprised of a vWFA domain and a serine
protease domain, would occupy two of the lobes. For both C2 and
factor B, evidence has been presented that C4b/C3b binding sites
are located both within the C2b (2, 10, 11) and Ba activation fragments (12, 13), as well as within their respective vWFA domains
(14, 15).
The proteins C3 and C4 contain an intramolecular thioester
bond that not only controls their conformational state (16), and
thereby their ligand binding properties, but also mediates their covalent attachment to target nucleophiles on pathogen surfaces in a
proteolytic activation-dependent manner (17). Whereas mature
plasma C3 is a disulfide-linked heterodimer consisting of a 119kDa ␣-chain and a 75-kDa ␤-chain, plasma C4 is a disulfide-linked
heterotrimer made up of a 93-kDa ␣-chain, a 75-kDa ␤-chain, and
and the indicated complement components or fragments thereof; MIDAS, metal-iondependent adhesion site; rC4, recombinant C4; VBS, veronal-buffered saline; SVB,
low ionic strength VBS made isotonic with sucrose; SGVB, SVB with gelatin; KLH,
keyhole limpet hemocyanin; API, Alberta Peptide Institute.
0022-1767/00/$02.00
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Previous work has indicated a role for the NH2-terminal segment of the C3 ␣ⴕ-chain in the binding interactions of C3b with a
number of its protein ligands. In particular, we have identified two clusters of acidic residues, namely, E736 and E737 and to a
lesser extent D730 and E731, as being important in the binding of C3b to factor B and complement receptor 1 and the binding
of iC3b to complement receptor 3. Whereas human C3 and C4 have an overall sequence identity of 29%, over a segment near the
NH2 termini of their respective ␣ⴕ-chains the sequence identity is 56% (70% chemical similarity). Given the functional similarity
between the C4b-C2 and C3b-B interactions in the respective formation of the classical and alternative pathway C3 convertases,
as well as the sequence conservation of two acidic clusters, we hypothesized that residues 744EED and 749DEDD within the
NH2-terminal segment of the C4 ␣ⴕ-chain would mediate in part the binding of C2 to C4b. We tested this hypothesis using three
independent approaches. Site-directed mutagenesis experiments revealed that replacing subsets of the charged residues by their
isosteric amides within either acidic cluster resulted in molecules having reduced C2 binding activity. Moreover, a synthetic
peptide (C4 residues 740 –756) encompassing the two acidic clusters was a specific inhibitor of the binding of C2 to red cellassociated C4b. Finally, Ab raised against the above peptide was able to block the interaction between red cell-associated C4b and
fluid phase C2. Taken together, these results strongly suggest that the NH2-terminal acidic residue-rich segment of C4 ␣ⴕ-chain
contributes importantly to the interaction of C4b with C2. The Journal of Immunology, 2000, 165: 2518 –2527.
The Journal of Immunology
which has been identified in C3 as containing a binding site for
factors B, H, and CR1. Whereas human C3 and human C4 have an
overall sequence identity of ⬃29%, over a stretch encompassing
27 residues near the NH2 terminus of their respective ␣⬘-chains the
sequence identity is ⬃56% (70% if chemically similar residues are
included; see Fig. 1). This region of sequence similarity is then
immediately followed by a stretch of sequence showing little or no
similarity between C3 and C4. It can also be seen in Fig. 1 that
when comparing several representative species of C3 and C4, although the sequence identities are not always absolute, within the
two NH2-terminal-most acidic subclusters there tends to be overall
compositional conservation of acidic amino acids. Given the functional homology between the C3b-B and C4b-C2 interactions, as
well as the sequence conservation noted above, we hypothesized
that the acidic residues within the NH2-terminal segment of the C4
␣⬘-chain would mediate, at least in part, the binding of C2 to C4b.
Accordingly, in this study we have assessed the C2 binding activities of a series of human C4B isotype mutants in which residues
within the negatively charged clusters 744EED and 749DEDD were
systematically replaced by their isosteric amides. We have also
tested our hypothesis through the use of both the synthetic peptide
mimetic and anti-peptide Ab approaches. Our cumulative results
clearly demonstrate the involvement of the acidic residue-rich
NH2-terminal segment of the C4 ␣⬘-chain in mediating the interaction between C4b and C2.
Materials and Methods
Buffers
The following diethyl barbiturate (veronal)-NaCl buffers were used (31):
veronal-buffered saline (VBS), 4 mM veronal, 0.15 M NaCl, 0.15 mM
CaCl2, and 0.5 mM MgCl2, pH 7.2 (␮ ⫽ 0.15); GVB, VBS containing
0.1% gelatin; GVB-Mg2⫹, GVB containing 5 mM MgCl2; GVBE, VBS
containing gelatin and 10 mM EDTA; low ionic strength VBS made isotonic with sucrose (SVB), 4 mM veronal, 0.06 M NaCl, 0.15 mM CaCl2,
0.5 mM MgCl2, and 0.17 M sucrose, pH 7.2 (␮ ⫽ 0.06); SVB-Mg2⫹, SVB
containing 5 mM MgCl2; SVB containing 0.1% gelatin (SGVB); SGVBMg2⫹, SGVB containing 5 mM MgCl2.
Purified complement components
C1̄s (32), C2 (33), and C4 (34) were purified from fresh frozen human
plasma as described previously. A functionally pure human C1 reagent,
used in the preparation of complement component cellular intermediates,
was prepared from a euglobulin precipitation of whole human serum (35).
Guinea pig complement (Sigma, St. Louis, MO) was diluted 50-fold in
VBS containing gelatin and EDTA to obtain a C3-C9 reagent. Protein
radioiodination was performed by the lactoperoxidase procedure and
yielded typical specific activities of 106 cpm/␮g (36).
FIGURE 1. Sequence alignment of the 738 – 808 region of C4 from various species and the 727–793 region of C3 from various species. A738 is the first
residue of human C4 ␣⬘-chain, and S727 is the first residue of the human C3 ␣⬘-chain. Sequence identities between various C4 and C3 species, respectively,
are indicated by periods and gaps by hyphens. Identical residues between human C4 and human C3 are indicated by vertical lines and conservative residue
replacements by dashed vertical lines. The underlined residues denote the crucial residues for factor B, CR1, and CR3 binding to C3 as determined from
previous mutagenesis studies in this laboratory (25). The targets chosen for the current site-directed mutagenesis studies are indicated in bold letters.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
a 33-kDa ␥-chain. In both cases, proteolytic removal of a 77-residue activation peptide from the NH2-terminal of the respective
␣-chains, i.e., C4a and C3a, respectively, results in exposure and
activation of the thioester. Following thioester transacylation, or
the competing hydrolysis reaction, the resulting C3b and C4b molecules acquire ligand-binding properties that were not present in
the respective native molecules. The same conformational end
state, and therefore the same ligand-binding profile, can also be
achieved by direct scission of the thioester in the absence of proteolytic activation (16). Among the new binding sites in C4b or
thioester-cleaved forms of C4, e.g., C4(CH3NH2) or C4(H2O), are
those for C2, nascent C3b, C5, and the complement regulatory
proteins complement receptor 1 (CR1), C4 binding protein
(C4BP), membrane cofactor protein, decay accelerating factor, and
factor I (18). Likewise, C3b, and its thioester-cleaved analogs such
as C3(CH3NH2) and C3(H2O), acquire interaction sites for a similar set of complement family ligands and receptors except that
instead of C2 and C4BP, these molecules bind the respective alternative pathway homologs, factors B and H.
There have been quite a large number of studies aimed at identifying ligand interaction sites in C3 (18). There is now general
agreement that at least one of the major contacts through which
C3b binds the proteins B, H, and CR1 is located within a 42-aa
peptide segment at the NH2 terminus of the C3b ␣⬘-chain. This
conclusion has been reached using a number of distinct, but complementary, experimental approaches including the use of synthetic peptide mimetics (19 –23), the functional site-blocking effect
of an anti-peptide Ab (20), the functional profile of engineered
chimeric or segment-deleted molecules (24), and, finally, the use
of site-directed mutagenesis to identify specific residues in the segment contributing to these various interactions (25, 26). The latter
two studies have specifically implicated a number of acidic residues spanning amino acids 730 –758 (mature protein numbering of
human C3 and C4 used here and throughout), although the subset
of the most important acidic residues for each of the three interactions is only partially overlapping. Other studies have suggested
the presence of a second contact point for B, H, and CR1 within
the C3d region of the molecule (23, 27), although specific C3d
contact residues have yet to be identified.
By contrast with C3, relatively little is known about the location
of the functionally homologous binding sites in C4 for CR1, C2,
and C4BP. One mAb that blocks C4BP binding to C4b also blocks
C2 binding to this fragment (28), a finding consistent with C2 and
C4BP being antagonistic ligands. Another anti-C4 mAb that
blocks the C4b-C4BP interaction has been an epitope mapped to
an 89-aa segment located at the NH2 terminus of the C4 ␣⬘-chain
(29, 30), i.e., the analogous segment of the C4 ␣-chain to that
2519
2520
A C2 BINDING SITE IN C4
Cell culture media
The high-glucose formulation of DMEM supplemented with 2 mM L-glutamine and 100 U/ml of penicillin/streptomycin was the basal tissue culture
medium used in this study. The pH of the medium was maintained by 5%
CO2 in a humidified incubator. COS-1 cells were maintained in DMEM
supplemented with 10% heat-inactivated FCS (complete DMEM; Life
Technologies, Grand Island, NY). The growth medium for transfected
COS-1 cells was complete DMEM. FCS-free DMEM consisted of complete DMEM in which the 10% FCS was replaced with 1% Nutridoma-HU
(Boehringer Mannheim, Montreal, Quebec, Canada). In the metabolic labeling experiment described below, both the basal DMEM and the Metand Cys-free DMEM were supplemented with 4% K76-COOH-treated
FCS and 1% Nutridoma (DMEM/K76). The procedure for K76-COOH
treatment of FCS, which irreversibly inactivates bovine factor I, has been
described previously (25).
Synthetic peptides
Antibodies
Rabbit anti-sheep RBC stroma, rabbit polyclonal IgG against human C4,
rabbit polyclonal IgG against human C3c, and goat alkaline-phosphataseconjugated Ab against rabbit IgG were purchased from Sigma. Abs against
the synthetic peptide C4740 –756 were raised in rabbits by s.c. injections of
500 ␮g of the KLH-peptide conjugate, initially in CFA, and at two subsequent 2-wk intervals in IFA. The immunizations and the collection of the
rabbit antisera were done by technical staff in the Department of Comparative Medicine, Faculty of Medicine, University of Toronto, in accordance
with the guidelines of the Canadian Council on Animal Care. The rabbit
IgG was fractionated from other serum proteins by ion-exchange chromatography on a QAE Sephadex A-50 as described previously (37) and dialyzed against PBS, pH 7.3. An immunoadsorbant column was prepared by
coupling the BSA-C4-peptide conjugate to Affi-Gel 15 (Bio-Rad, Hercules,
CA) according to the manufacturer’s instructions. The affinity matrix was
equilibrated with PBS, pH 7.3, and the IgG fraction of the rabbit antiserum
was applied to the column by gravity flow. Following washing in PBS, the
bound Abs were eluted with 100 mM glycine, pH 2.5, and neutralized to
pH 8.0 by the addition of 1.0 M phosphate buffer, pH 8.0. The pooled
anti-peptide Ab fractions were dialyzed extensively against SVB-Mg2⫹
containing 0.02% sodium azide.
740 –756
ELISA for determining the specificity of the C4
anti-peptide Ab
Standard ELISAs in which BSA-C4 peptide conjugate, control BSA, or
C4(CH3NH2) were coated on the wells as capture Ag were used to assess
the specificity and titer of the preimmune and postimmune rabbit antisera.
To assess the relative binding activity of the immunoaffinity-purified antiC4740 –756 Ab toward native C4, C4b (i.e., C1̄s-treated native C4), and
C4(CH3NH2), the immunoaffinity-purified Ab was coated as capture Ab on
ELISA wells (10 ␮g/ml). Following blocking, the wells were incubated
with a 2-fold dilution series of native C4, C4b, or C4(CH3NH2), where the
initial Ag concentration was 50 ␮g/ml. Using standard ELISA procedures,
Ag capture was detected using a goat anti-human-C4 polyclonal Ab followed by alkaline phosphatase-conjugated rabbit anti-goat IgG. Control
wells contained no capture Ab, but were otherwise treated the same as
experimental wells. Relative binding activity was assessed by the horizon-
Plasmid constructions and site-directed mutagenesis
The cDNA expression plasmid pSV-C4B, coding for the wild-type human
C4B isotype under the control of an SV40 promoter, has been described
previously (38). The plasmid pUC-C4-5⬘ consists of a 2.8-kb 5⬘ SalI-EcoRI
fragment of the C4B cDNA cloned into pUC19. Site-directed mutants were
produced by the overlap extension PCR mutagenesis method (39) using the
proofreading enzyme Vent DNA polymerase (New England Biolabs, Beverly, MA) and pSV-C4B as the wild-type template. The resulting 932-bp
PCR fragment encompassed unique restriction sites for Eco47III and
EcoRI (at bases 2132 and 2788 of the C4B cDNA, respectively) and was
digested with these enzymes to generate a 657-bp mutation-containing
fragment that could be subcloned into similarly digested pUC-C4-5⬘, where
the sites for Eco47III and EcoRI are also unique (the intermediate vector
step is required because the digestion site for Eco47III is not unique in
pSV-C4B). Following confirmation of the desired mutation(s), and the absence of any undesired mutations within the 657-bp target sequence by
strand-denaturation dideoxy sequencing (T7 polymerase sequencing kit;
Amersham Pharmacia Biotech, Piscataway, NJ), pUC-C4-5⬘ was restricted
with SalI and EcoRI to produce a 2780-bp fragment encompassing the
mutation(s). The SalI/EcoRI mutation-containing fragment was then exchanged for the corresponding wild-type region in pSV-C4B.
Expression of recombinant C4 (rC4)
COS-1 cells were transiently transfected by the DEAE-dextran method
essentially as described in our earlier studies (40) using 15 ␮g of pSV-C4B
plasmid per 100-mm plate seeded 16 –20 h earlier with 106 cells. Following
the transfection, the cells were allowed to grow in 9 ml complete DMEM
for 72 h, after which time the medium was changed to FCS-free DMEM.
Supernatants containing rC4 were harvested after a further 48 –72 h of
incubation.
Metabolic labeling, biosynthetic characterization, and
immunoprecipitation
After being allowed to grow for 72 h in complete DMEM, transfected cells
were washed in PBS and then incubated for 1 h in Met- and Cys-free
DMEM (ICN Biomedicals, Costa Mesa, CA) containing 4% K76-treated
FCS and 1% Nutridoma-HU (3 ml/100-mm plate) to deplete internal stores
of methionine and cysteine. The medium was then supplemented with 250
␮Ci of [35S]methionine/[35S]cysteine (ICN Biochemicals TransLabel,
⬃1300 Ci/mmol). After 5 h of incubation, an equal volume of Met- and
Cys-sufficient DMEM/K76 was added, and the incubation was continued
overnight. To assess the biosynthetic processing and C1̄s cleavability of the
rC4, metabolically labeled supernatants were immunoprecipitated with rabbit IgG anti-human C4 in conjunction with a Staphylococcus aureus suspension (Sigma), both with and without prior treatment of the supernatants
with C1̄s (2 ␮g/ml, 1 h, 37°C). The buffers and wash procedures for the
immunoprecipitations have been described previously (41). All samples
were analyzed on 8% SDS-PAGE under reducing conditions, followed by
phosphorimage analysis using a Storm 860 scanner (Molecular Dynamics,
Sunnyvale, CA). Quantitative measurement of band intensities within a
lane was accomplished by peak profile integration using the program
IPLabGel 2.0 f (Signal Analytics, Vienna, VA).
Quantitative measurement of secreted rC4
The concentration of rC4 in dialyzed culture supernatants of transfected
cells was determined by a competitive solid-phase RIA (42) using 125Ilabeled purified human C4 as the probe and rabbit IgG anti-human C4 as
the capture Ab adsorbed to opaque polystyrene microtiter plates (Packard
Instruments, Meriden, CT). Purified human C4 was used to obtain a standard curve. Radioactivity was measured by liquid scintillation counting
directly in the plates using a TopCount instrument (Packard Instruments).
Classical pathway-dependent hemolytic assay
The hemolytic activity of rC4 in SVB-Mg2⫹-dialyzed transfection supernatants was determined by using sheep erythrocytes coated with Ab and C1
(EAC1), iodine-oxidized C2 (oxyC2), and C3-C9 reagent as described previously (35, 40). After correcting for background, the degree of specific
lysis was converted to Z units where Z ⫽ ⫺ln(1 ⫺ fractional lysis) and is
physically equal to the number of hemolytically effective molecules per
erythrocyte. Comparisons of activity were made on the basis of Z units per
amount of immunochemically determined rC4.
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The synthetic peptide EILQEEDLIDEDDIPVR, corresponding to residues
740 –756 of mature C4 (C4740 –756), was obtained from two independent
sources: Procyon Biopharma (London, Ontario, Canada) and the Alberta
Peptide Institute (API; Edmonton, Alberta, Canada). The Procyon peptide
had a C-terminal cysteine-amide residue in place of the naturally occurring
serine, and the API peptide had at its C terminus the photoactivatable
amino acid derivative ornithine-amide-benzoylbenzoate. The API peptide
was also supplied as photo-cross-linked conjugates of keyhole limpet hemocyanin (KLH) and BSA, each substituted with on average 17 molecules
of peptide per molecule of carrier protein. The KLH- and BSA-peptide
conjugates were used, respectively, for rabbit immunizations and for antipeptide Ab detection and purification (see below). The peptides SIERPD
SAPPRVGDT and EDPGKQLYNVEATSY, respectively, corresponding
to residues 455– 469 of mature C4 ␤-chain and residues 1198 –1212 of
human C3, were obtained from Chiron Mimotopes (Raleigh, NC) for previous studies and were used as control peptides. Before use, all peptides
were lyophilized from H2O several times to remove traces of volatile compounds and then dissolved in H2O to yield a concentration of about 700
␮M. The exact concentration was determined spectrophotometrically at
1%
220 nm using an E220
nm, 1 cm of 100.
tal displacement of the dose-response curves, with the concentration of Ag
being plotted on a logarithmic scale.
The Journal of Immunology
Fluid-phase C2-dependent hemolytic inhibition assay
Synthetic peptide competition assay
Varying amounts of synthetic peptide in 160 ␮l of H2O were mixed with
40 ␮l of 5⫻ SGVB-Mg2⫹, and these solutions were then incubated with 16
ng of oxyC2 for 10 min at 0°C. EAC4b cells (1.5 ⫻ 107) were then added
and the remaining steps of the assay were done exactly as described above
for the fluid-phase C2-dependent hemolytic inhibition by rC4(CH3NH2).
Anti-peptide Ab hemolytic inhibition assays
In one type of assay, various amounts of anti-peptide Ab were incubated
with EAC4b cells (1.5 ⫻ 107) in 65 ␮l SGVB-Mg2⫹ at 0°C for 60 min, at
which time 16 ng of oxyC2 were added and the assay volume was increased
to 200 ␮l with SGVB-Mg2⫹. After 10 min of incubation at 0°C, all samples
were centrifuged and the supernatants were discarded. The resulting
EAC4b2 cells were treated with C1 and C3-C9 reagents, and the degree of
lysis was determined as described above. A second assay using the antiC4740 –756 reagent was designed to restrict the effect of the Ab solely to the
C4b-C2 interaction. In this assay, EAC4b cells (1 ⫻ 108), in sufficient
amount to bind virtually all of a test quantity of C2, were incubated with
various amounts of anti-C4740 –756 for 60 min at 0°C, followed by washing
with cold GVB-Mg2⫹. The cells were then resuspended in 100 ␮l of
SGVB-Mg2⫹, and 23 ng oxyC2 were added. After incubation for 10 min at
0°C, the cells were centrifuged, and fixed amounts (95 ␮l) of the respective
supernatants were transferred to another tube. The supernatants were then
supplemented with SGVB-Mg2⫹ to give a total volume of 200 ␮l and
incubated with fresh EAC4b cells (1.5 ⫻ 107) for 10 min at 0°C. The
hemolytic activity of C2 captured on the second set of cells was then
determined by the addition of C1 and C3-C9 reagents as described above.
Table I. Nomenclature and amino acid sequences of 744 –752 segment
mutants examined in this study a
C4BWT
QED
744
EEN
744
QQD
744
QQN
749
DQDD
749
DEND
749
DQND
749
NQNN
744
a
744
EEDLIDEDD752
QEDLIDEDD752
744
EENLIDEDD752
744
QQDLIDEDD752
744
QQNLIDEDD752
744
EEDLIDQDD752
744
EEDLIDEND752
744
EEDLIDQND752
744
EEDLINQNN752
744
The residues changed are indicated in bold letters.
levels that were comparable to the wild-type control in terms of
total C4 Ag per milliliter of culture supernatant (typically ⬃1.5–2
␮g/ml at the time of harvesting). Fig. 2 shows the relative hemolytic activities of the mutants in a purely classical pathway-dependent assay. It can be seen that 744QQN, 749DEND, 749DQND, and
749
NQNN are severely compromised in this activity with the defect being ⱖ20-fold. An intermediate level of defect ranging from
8- to 3-fold is seen for mutants 744QQD, 744QED, and 749DQDD,
but by contrast mutant 744EEN shows a ⬍2-fold defect in hemolytic activity. However, the hemolytic activity defect need not necessarily arise solely from a defect at the C2 binding stage as other
impairments, such as in biosynthetic processing or cleavability by
C1̄s, could also give rise to the defect in C4 hemolytic activity.
These latter two points were addressed by performing a metabolic
labeling experiment on the various transfectants and then carrying
out an immunoprecipitation before and after treatment of the labeled supernatants with C1̄s. The results of such an experiment are
shown in Fig. 3, A and B. We have previously noted that the posttranslational proteolytic processing of even wild-type human C4 to
the mature three-chain form in COS-1 cell transfections is incomplete (40, 46), especially at the junction between ␤- and ␣-chains.
Results
The site-directed mutagenesis approach
The first approach used to test the hypothesis that the two acidic
clusters of residues at the NH2 terminus of the C4b ␣⬘-chain contribute to a binding site for C2 involved the systematic replacement
of these residues with their isosteric amides. Isosteric amide substitution was chosen in preference to alanine scanning, as this approach did not superimpose a potential steric effect on the loss of
charge mutation. The various mutant molecules constructed, and
their respective names, are indicated in Table I. The mutations
were introduced either into the first cluster having the sequence
744
EED or the second cluster having the sequence 749DEDD, but
not simultaneously into both clusters. Therefore, for simplicity of
annotation, the nomenclature of the mutants that we have used will
be of the form 744EEN, 749DEND, 749DQND, etc., where the
amide replacement residues are indicated in the context of their
neighbors.
When transiently expressed in COS-1 cells and harvested in
serum-free medium, all of the rC4 molecules were expressed at
FIGURE 2. Comparison of the hemolytic activities of recombinant
wild-type and mutant C4 molecules. The hemolytic activities of rC4B species present in dialysed culture supernatants were assessed in a purely
classical pathway-dependent hemolytic assay as described in Materials and
Methods. Comparisons of hemolytic activities were made on the basis of Z
units per amount (nanograms) of rC4, where Z ⫽ ⫺ln(1 ⫺ fractional lysis).
Results are expressed as a percentage of wild-type hemolytic activity.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Secreted rC4 was converted to rC4(CH3NH2) by treating the culture supernatants from transfected cells with 0.1 M methylamine, pH 8.0, for 6 h
at 37°C (43). The methylamine-treated supernatants were then dialyzed
extensively against SVB-Mg2⫹ and concentrated to approximately oneeighth of original volume by using either Centricon-100 concentrators
(Amicon, Beverly, MA) or Ultrafree-15 Centrifugal Filter Devices (Millipore, Bedford, MA). Following this procedure, the concentration of the
rC4(CH3NH2) was determined by a competitive RIA as described above.
oxy
C2 (44) in SGVB-Mg2⫹ in an amount sufficient to generate ⬃80% hemolysis in the absence of inhibitor protein in a C2 hemolytic assay to be
described below (typically 16 ng of C2) was incubated with variable
amounts of rC4(CH3NH2) in a total volume of 200 ␮l of SGVB-Mg2⫹ at
0°C for 5 min. EAC4b cells (1.5 ⫻ 107), prepared as described previously
(45), were added, and the tubes were further incubated at 0°C for 10 min,
at which time the cells were washed with cold GVB-Mg2⫹. The resulting
EAC4b2 cells were resuspended in 200 ␮l of GVB-Mg2⫹ containing excess C1 reagent and incubated for 10 min at 30°C. Lysis was developed by
the addition of C3-C9 reagent (1 ml) at 37°C for 45 min. After spinning
down unlysed cells, the degree of lysis was determined by measuring the
absorbance of the supernatant at 412 nm. Hemolytic data were used to
calculate the relative C2 binding ability of each rC4(CH3NH2) species by
comparing the 50% inhibition of hemolytic activity of a given molecule to
that of the wild-type molecule.
2521
2522
A C2 BINDING SITE IN C4
Thus the ␤-␣ band is a major species on the SDS-PAGE phosphorimage analysis of the metabolic labeling experiment shown.
Additionally, because the ␣-␥ junction is cleaved more efficiently
than is the ␤-␣ junction, the ␥-chain band is overrepresented relative to ␤ and ␣. To compare quantitatively the extent of processing to the mature three-chain form of C4, the only form that has
hemolytic activity (47), the gels shown in Fig. 3, A and B, were
subjected to quantitative peak profile analyses. We then determined for all of the non-C1̄s-treated lanes the ratio of the ␣-chain
pixel intensity to the total pixel intensity of all the chains in the
lane. From the bar graph representation of this analysis shown in
Fig. 3C, it can be seen that mutants 744EEN, 744QED, 744QQD, and
749
DQDD were fairly comparable to the wild type with respect to
their posttranslational proteolytic processing. By contrast, mutants
744
QQN, 749DEND, 749DQND, and 749NQNN showed defects of
2- to 3-fold in their respective processing relative to wild-type C4.
It can also be seen in Fig. 3 that whereas mutants 744EEN, 744QED,
744
QQD, and 749DQDD are cleaved by C1s to an extent comparable to that occurring in the wild-type molecule, whatever ␣-chain
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FIGURE 3. Biosynthetic processing and susceptibility to C1̄s cleavage of recombinant wildtype and mutant C4 molecules. A and B, Phosphorimage of 8% reducing SDS-PAGE of metabolically
labeled C4, immunoprecipitated with rabbit IgG antihuman C4 from transfected COS-1 cell supernatants,
before (⫺) and after (⫹) treatment with C1̄s (2 ␮g/
ml, 37°C, 1 h). A and B, Immunoprecipitations of the
744
EED and 749DEDD cluster mutants. “No DNA”
refers to the immunoprecipitation of labeled supernatants from mock-transfected COS-1 cells. “C” refers to the control immunoprecipitation of wild-type
rC4B supernatants with rabbit IgG anti-human C3c.
C, Quantitative phosphorimage analysis of gels
shown in A and B. The ratios of the ␣-chain band
pixel intensity over the total pixel intensity of all the
chains in the respective C1̄s (⫺) lanes were calculated and plotted.
is present in the 749DQND, 749NQNN, 749DEND, and 744QQN
mutants is relatively resistant to cleavage by C1̄s. It is known from
the work of Ogata and colleagues that cleavability by C1s is quite
sensitive to changes immediately downstream of the cleavage site
(48). Even in the absence of sequence changes in this region, thioester-hydrolyzed C4, which spontaneously forms in the culture supernatants, will be resistant to cleavage by C1̄s (49).
The acquisition by C4 of a conformation that is capable of interacting with C2 is achievable either by proteolytic activation
with C1̄s, which would yield a thioester-hydrolyzed fluid-phase
C4b fragment, or by direct nucleophilic scission of the thioester in
native C4 with the small nucleophile methylamine (43). To avoid
the problem of C1̄s cleavage resistance and, at the same time, to
maximize the concentration of the rC4 molecules in the supernatants capable of interacting with C2, the supernatants were reacted
with methylamine to generate the C4(CH3NH2) species having a
C4b-like conformational state. Molecules that had already undergone spontaneous thioester hydrolysis (i.e., forming the species
C4(H2O)) would also be in a C4b-like conformation. These C4b-
The Journal of Immunology
The peptide mimetic approach
Although the mutagenesis results strongly suggested that the NH2terminal ␣⬘-chain segment of C4, and, in particular, some of the
acidic residues, contributed to the binding interaction with C2,
these results on their own were not definitive because, at least for
some of the mutants analyzed, there was evidence for some undesirable effects on global conformation. To further test the involvement of the NH2-terminal ␣⬘-chain segment in C2 binding from an
independent direction, a synthetic peptide corresponding to C4 residues 740 –756 and having the sequence EILQEEDLIDEDDIPVR
was assessed for its ability to inhibit the binding of C2 to EAC4b
cells. It can be seen in Fig. 5 that this peptide was able to compete
with cell-associated C4b for the binding of C2, with the 50% inhibition point requiring ⬃0.2 mM of peptide. In contrast, a control
peptide derived from a segment of human C3d failed to show any
interaction with C2. Similar inhibitory effects on C2 binding were
obtained when this experiment was repeated with an independently
synthesized peptide of the same C4 sequence, whereas a second
control peptide, this time corresponding to human C4 ␤-chain seg-
ment 455– 469, was still without effect in the assay (data not
shown).
The blocking Ab approach
To further corroborate the involvement of the NH2-terminal C4
␣⬘-chain segment in the interaction with C2, a polyclonal rabbit Ab
was raised against the peptide corresponding to C4 residues 740 –
756. This Ab was immunoaffinity-purified and was then assessed
for its ability to block the interaction between C4b and C2. In
preliminary ELISA experiments, we confirmed that the antipeptide Ab, but not preimmune rabbit IgG, would not only recognize the peptide as a conjugate of BSA (peptide-KLH was the
immunogen) but also would react, albeit with much lower titer,
with methylamine-treated C4 that had been coated on the ELISA
plate. Moreover, when the affinity-purified anti-peptide Ab was
coated on ELISA plates it had the ability to capture C4 Ag from
solution with its reactivity against C4b being about 3-fold higher
than against either native C4 or C4(CH3NH2) (data not shown).
These latter observations suggest that a subset of the epitopes recognized by the anti-peptide polyclonal Ab are available even in the
native molecule, but removal of the C4a activation peptide either
results in their increased exposure or the liberated free amino
group forms part of an epitope.
In the experiment shown in Fig. 6, EAC4b cells, made with
excess purified C4, were preincubated with various amounts of
specific anti-peptide Ab or with control nonspecific rabbit IgG.
The cells were then incubated with a fixed quantity of C2, and then
lysis was developed upon addition of excess C1 and C-EDTA. It
can be seen that the anti-C4-peptide IgG, but not the control IgG,
inhibited C2-dependent hemolysis in a dose-dependent manner.
Although the results were strongly suggestive of a blockage of C2
binding to C4b by the anti-peptide Ig, we could not exclude the
possibility that this Ab might also interfere with the C5 convertase
subunit functionality of C4b, either by sterically masking the
transacylation target residues of nascent C3b in C4b ␣⬘-chain or by
blocking the C5 binding site in C4b. To circumvent this ambiguity,
a second hemolytic assay was used in which the effect of the antipeptide Ab would be limited to the C4b-C2 interaction step. Specifically, EAC4b cells, made with excess human C4, were preincubated with the peptide-specific or control IgG as before.
Following washing, these cells were incubated with a limiting
amount of C2, an amount that in the absence of any pretreatments
would become almost completely bound to the EAC4b cells. The
cells were pelleted and the residual C2 activity in the supernatant
was assessed using a fresh aliquot of EAC4b cells. The experiment
depicted in Fig. 7 shows that as the amount of anti-peptide Ab
added in the preincubation step with the EAC4b cells is increased,
the amount of C2 that remains uncomplexed, and thus available for
binding and inducing hemolysis in the second aliquot of EAC4b
cells, also increases. The control IgG added in an amount equivalent to the highest amount of the specific anti-peptide IgG used
showed no ability to block the interaction between C2 and the C4b
present on the initial sample of EAC4b cells. Cumulatively, these
experiments show that an Ab recognizing an epitope within the
740 –756 segment of target-bound C4 ␣⬘-chain can block the interaction between C4b and C2.
Discussion
Whereas there is a considerable body of knowledge regarding the
localization of complement ligand-binding sites in C3 (reviewed in
Ref. 18), comparable studies on C4 are much more limited. At the
outset of this investigation, the only binding sites in C4 that were
identified at the specific amino-acid level were a C5 binding site
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like molecules were then assessed for their ability to interact with
C2 in the fluid phase and thereby inhibit its detection in a subsequent C2 hemolytic assay. Thus, culture supernatants containing
various amounts of methylamine-modified recombinant wild-type
or mutant C4 in SGVB-Mg2⫹ were allowed to react in the fluid
phase with a constant amount of C2, the amount of C2 having been
predetermined to yield ⬃80% hemolysis in a C2 hemolytic assay.
Uncomplexed C2 was then captured by EAC4b cells made with an
excess of purified C4. Following washing, hemolysis of these
EAC4b2 cells was developed by the addition of C1 and subsequently
C-EDTA, a C3-C9 reagent, as a source of C3-C9. Therefore, if the
744 –752 segment mutants have impaired C2 binding capacity, their
fluid-phase C4(CH3NH2) derivatives will not bind C2 in the preincubation step and thus there will be no decrease in the amount of
C2-dependent hemolytic activity observed relative to a sham preincubation. In contrast, active C4(CH3NH2) molecules will yield a
dose-dependent inhibition curve, and partial defects will yield a
horizontal displacement from wild-type inhibition curve when the
data are presented in the form of a semilogarithmic plot. The results of one such C2 binding experiment are depicted in Fig. 4,
with the data for the 744EED and 749DEDD clusters being grouped
in A and B, respectively. Fig. 4C shows a summary in bar graph
form of the relative C2 binding capacities of the various mutant C4
molecules in which data from three independent experiments have
been averaged. These data provide strong evidence for the involvement of residues in both acidic clusters in mediating C2 binding
and generally indicate that multiple mutations result in a cumulative defect. One notes a striking similarity between the bar graph
showing the relative C2 binding activities of the mutants (Fig. 4C)
and the bar graph showing overall hemolytic activity (Fig. 2). For
the case of mutants 744EEN, 744QED, 744QQD, and 749DQDD,
which show normal processing and C1s cleavability, the correlation between these two assays suggests that the hemolytic defect
can indeed be accounted for at the C2 binding stage. For the case
of mutants 744QQN, 749DEND, 749DQND, and 749NQNN, which
have, respectively, ⬃3, ⬃6, ⬃1, and ⬍1% of wild-type C2 binding
activity, interpretation of the data is clouded by the defects in
posttranslational proteolytic processing. Nevertheless, quantitative
analysis of the metabolic labeling experiment (Fig. 3) suggests that
the extent of the C2 binding defect is much greater than the processing defect in all cases. For example, whereas the processing
defect in 749DEND is about 2-fold, the C2 binding defect is about
16-fold, thereby suggesting an important contribution by C4 residue D751 to the interaction with C2.
2523
2524
A C2 BINDING SITE IN C4
surrounding ␤-chain residue R462 (46) and a noncovalent interaction site for nascent C3b in the vicinity of ␣-chain residue S1217
(50). The sole other relevant data in the literature pertained to an
anti-C4 mAb that could block the interaction between C4b and
C4BP and that was shown to bind to an 89-aa segment spanning
residues 738 – 826 within the NH2-terminal-most ␣-chain fragment
of C4c (30). This investigation has identified two acidic clusters of
residues that are a subset of the above segment, specifically located
near its NH2 terminus, as contributing to the binding interaction
with C2. This is consistent with C4BP and C2 being antagonistic
ligands, and therefore binding to the same general area of the molecule, although not necessarily to the same key contact residues.
The two clusters of acidic residues spanning residues 744 –752
were targeted for investigation because of their general conservation with the analogous residues in C3 ␣⬘-chain and because the
C3 residues had been shown to contribute to the binding interaction between C3b and factor B (25), this interaction being the
alternative pathway analog of the C4b-C2 interaction. We believe
that the congruency of results obtained using three independent
binding site-mapping approaches, namely, site-directed mutagenesis, synthetic peptide competition, and anti-peptide Abs, strongly
supports a role for this segment of C4b in mediating the interaction
with C2. Interpretation of the site-directed mutagenesis data for a
subset of the mutants was complicated by the fact that in addition
to the loss in C2 binding, there was clear evidence for more general
conformational perturbations that affected the extent of posttranslational proteolytic processing and cleavability by C1̄s. Nevertheless, half of the mutants analyzed displayed wild-type-like processing and cleavability by C1̄s and yet also showed 2- to 8-fold
FIGURE 4. Binding of C2 to recombinant wild-type and mutant
C4(CH3NH2) molecules. The ability of nucleophile-modified C4 to bind
C2 was assessed in a fluid-phase C2-dependent hemolytic assay. Dilution
series of culture supernatants containing the various rC4(CH3NH2) molecules were incubated with oxyC2 (16 ng) at 0°C for 5 min. The hemolytic
activity of uncomplexed C2 was measured using a C2-dependent hemolytic
assay as described in Materials and Methods. A and B, Representative
experiments for the 744EED and the 749DEDD cluster mutants, respectively. Dashed lines indicate the hemolysis in the absence of rC4(CH3NH2)
inhibitor. C, Relative C2 binding ability of each rC4(CH3NH2) species by
comparing the amount giving 50% inhibition of hemolytic activity of a
given C4 mutant to that of the wild-type molecule. The results show the
means and SD of three independent experiments.
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FIGURE 5. Inhibition of C2 binding to EAC4b by synthetic peptide.
Various amounts of synthetic peptide C4740 –756 (API sample) were incubated with oxyC2 (16 ng) at 0°C for 10 min, at which time EAC4b cells
(1.5 ⫻ 107) were added and the tubes were further incubated at 0°C for 10
min. Following washing, the cells were treated with excess C1 and hemolysis was developed by the addition of C-EDTA (1 ml). The nonspecific
peptide used in this experiment corresponds to the 1198 –1212 region of
human C3d.
The Journal of Immunology
2525
FIGURE 7. Inhibition of C2 binding to EAC4b by anti-peptide Ab. Various amounts of anti-peptide Ab (anti-C4740 –756) were preincubated with
EAC4b cells (1 ⫻ 108) at 0°C for 90 min. The cells were washed with cold
GVB-Mg2⫹, followed by the addition of oxyC2 (23 ng). After incubation
for 10 min at 0°C, the cells were centrifuged and the supernatants were
collected. The hemolytic activity of residual C2 in the supernatant was
determined using fresh EAC4b cells (1.5 ⫻ 107), C1 and C-EDTA as a
source of C3-C9. “Control IgG” refers to the fraction of IgG that ran
through the immunoaffinity column containing BSA-C4740 –756 conjugate.
defects in C2 binding (i.e., 744EEN, 744QED, 744QQD, and
749
DQDD), thereby clearly indicating a role for E744, E745,
D746, and E750 in mediating the binding interaction with C2.
Furthermore, even for a mutant such as 749DEND that showed a 2to 3-fold defect in processing, the defect in C2 binding was much
greater (⬃16-fold) than the processing defect. Thus the C2 binding
defect in this case could not simply be accounted for by the lower
concentration of the fully processed three-chain form of the molecule. Although clearly not definitive for this amino acid, these
data nevertheless point toward D751 also being a participant in the
binding interaction with C2. C4 residues E750 and D751 correspond to C3 residues E736 and E737, whose mutation we have
previously shown decreases the strength of the C3b-B interaction
⬃3-fold (25). One notable difference between the charged residue
mutational analysis of the corresponding C4 and C3 segments is the
apparently greater importance of the NH2-terminal-most 744EED
acidic cluster of C4 for the C2-C4b interaction than is the case for the
corresponding C3 730DED cluster in mediating the C3b-B interaction.
Although even single mutations within the 744EED acidic cluster of
C4 have fairly pronounced effects on the C2-binding interaction, even
the double mutation of C3 residues D730 and E731 led to only about
a 20 –30% decrease in factor B binding activity (25).
The general conclusions from the site-directed mutagenesis data
regarding the participation of the two acidic clusters in forming a
C2 binding site were further supported by experiments showing
the ability of a synthetic peptide encompassing residues 740 –756
to inhibit the authentic C4b-C2 interaction in a specific and dosedependent manner and by the ability of an Ab to this peptide segment to also block the C4b-C2 interaction. Negative results using
peptide mimetics are inconclusive because the conformation of the
peptide may be very different as a free peptide than within the
context of the rest of the protein. However, a positive result such
as we have in this case can only be artifactual if the array of side
chains available in the peptide are ones that would not normally be
accessible in the whole protein. This is clearly not the present case
as the anti-peptide Ab was not only able to recognize the epitope
in the context of C4b, but also its binding to C4b blocked the
interaction with C2. Furthermore, although the steric “footprint” of
the Ab on the C4b molecule would almost certainly extend beyond
the acidic residue-rich peptide epitope, the fact that both the sitedirected mutagenesis and the synthetic peptide competition experiments did not extend much beyond the cluster of acidic residues,
and yet in each case had an effect on the C4b-C2 interaction, is
consistent with the functional site-blocking effect of the antipeptide Ab being a direct rather than an indirect one.
An anti-peptide Ab against the NH2-terminal ␣⬘-chain segment
of C3 corresponding to C3 residues 727–768 was shown to block
the C3b-B interaction (20), analogous to the effect that we observe
on the C4b-C2 interaction by the anti-C4740 –756 anti-peptide Ab.
In the case of the anti-C3727–768 Ab, the antigenic determinant
recognized only became available upon proteolytic conversion of
native C3 to C3b, iC3b, or C3c. Thus the immunodominant epitope
behaved as a classic neoepitope. In our case, there is only a 3-fold
enhancement in Ab reactivity toward C4b vs native C4. It is possible that in the case of C4, the two acidic clusters are largely
available to C2 even in the native molecule, and that it is the
formation/exposure of an independent binding site in another part
of the molecule that determines the ability of C4b and
C4(CH3NH2), but not native C4, to bind C2. Alternatively, there
may actually be no difference in the relative exposures of the NH2terminal acidic clusters in native C3 and native C4. Rather, it may
be that a more C-terminal epitope becomes exposed upon proteolytic activation of C3, an immunodominant epitope that was
present in the C3727–768 peptide immunogen, but that was not
present in the C4740 –756 peptide corresponding to only the NH2terminal-most 40% of the immunogen used in the C3 study.
As suggested above, the interaction between C2 and C4b involves multiple points of attachment. In terms of the currently
known C4b interaction sites within C2, the NH2-terminal ␣⬘-chain
segment of C4b could potentially interact with the SCR domains of
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FIGURE 6. Inhibition of C2-dependent hemolytic activity by antipeptide Ab. Varying amounts of anti-peptide Ab (anti-C4740 –756) were preincubated with EAC4b cells (1.5 ⫻ 107) at 0°C for 90 min, at which time
oxy
C2 (16 ng) was added and the incubation was continued for 10 min at
0°C. After centrifugation, the cells were treated with excess C1 and hemolysis was developed by the addition of C-EDTA (1 ml). “Control IgG”
refers to the fraction of IgG that ran through the immunoaffinity column
containing BSA-C4740 –756 conjugate.
2526
C3b ␣⬘-chain showed that binding interactions with factor H and
CR1, both of which are composed entirely of SCR motifs, are
compromised only by the loss of negative charges, despite there
being four basic residues that were mutated within the target segment (26). Loss of charge mutations at C3 residues E736 and E737
not only cause loss in CR1 binding activity, but also factor B
binding activity. Owing to the commonality of SCR motifs to CR1
and factor B, it is tempting to speculate that the defect in factor B
binding may reflect the loss of an interaction between the acidic
residues of the NH2-terminal ␣⬘-chain segment of C3b and the
SCR motif-containing Ba region of factor B. Furthermore, it has
been observed that in a series of substitution mutants in the SCR
domains of CR1 that gain of function mutants for both C3b and
C4b binding often correlated with an increase of positive charge,
or decrease of negative charge, suggesting an electropositive interface on the CR1 molecule and an electronegative interface on
the C3b or C4b molecule (55). Further evidence for an electropositive interface on an SCR-containing protein binding to C4b comes
from a recent modeling study on SCR domains of C4BP that predicted an electropositive surface at the interface between SCR
modules 1 and 2 (56). Furthermore, this work showed that mutation of these basic residues in C4BP to neutral ones led to a loss in
C4b binding activity. Given the evidence in the literature suggesting a C4BP binding site within the NH2-terminal segment of C4b
␣⬘-chain, Blom et al. (56) hypothesized that the highly acidic segment of C4b ␣⬘-chain that has been the focus of our present study
may also be responsible for the interaction with the electropositive
interface that they had identified on C4BP. However, they found
that a peptide corresponding to the C4b740 –757 segment did not
inhibit the interaction between C4b and C4BP. This peptide was
virtually identical with the one that we used and that was found to
inhibit the interaction between C4b and C2. Although C2 and
C4BP are antagonistic ligands for C4b, they need not use exactly
the same contact residues to sterically block one another. Indeed,
for the analogous pair of antagonistic alternative pathway proteins,
i.e., factors B and H, data from both site-directed mutagenesis and
peptide mimetic studies have shown that these binding sites are
separable within the NH2-terminal ␣⬘-chain segment of C3b (22,
26) with the acidic residues essential for H binding being ⬃7 aa
COOH-terminal of those mediating factor B binding.
In summary, we have demonstrated using three independent experimental approaches that the acidic residues 744EED and
749
DEDD within the NH2-terminal segment of the C4b ␣⬘-chain
contribute importantly to the binding interaction between C4b and
C2. On balance, we believe that the arguments in favor of these
acidic residues interacting with the SCR domains of C2b appear
stronger than those that favor the interaction of this region with the
vWFA domain of C2a. Clearly, however, future studies using recombinant fragments corresponding to C2b and to the vWFA domain of C2, in conjunction with the mutant C4 molecules engineered in this study, will be necessary to resolve this issue.
References
1. Müller-Eberhard, H. J., M. J. Polley, and M. A. Calcott. 1967. Formation and
functional significance of a molecular complex derived from the second and the
fourth component of human complement. J. Exp. Med. 125:359.
2. Kerr, M. A. 1980. The human complement system: assembly of the classical
pathway C3 convertase. Biochem. J. 189:173.
3. Bentley, D. R. 1986. Primary structure of human complement component C2:
homology to two unrelated protein families. Biochem. J. 239:339.
4. Carroll, M. C., R. D. Campbell, D. R. Bentley, and R. R. Porter. 1984. A molecular map of the human major histocompatibility complex class III region linking complement genes C4, C2 and factor B. Nature 307:237.
5. Campbell, R. D., and D. R. Bentley. 1985. The structure and genetics of the C2
and factor B genes. Immunol. Rev. 87:19.
6. Campbell, R. D., S. K. A. Law, K. B. M. Reid, and R. B. Sim. 1988. Structure,
organization, and regulation of the complement genes. Annu. Rev. Immunol.
6:161.
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C2b or with the vWFA domain portion of C2a. Like many complement protein-protein interactions, that between C4b and C2 is
known to be ionic strength-dependent (2). Furthermore, the C2bC4b interaction, which has been hypothesized as being important
in the assembly of the classical pathway C3 convertase (10), is not
only exquisitely sensitive to ionic strength, but also to the presence
of Mg2⫹ (2). In this latter respect, the interaction between C2b and
C4b differs from that of the equivalent alternative pathway interaction between Ba and C3b as the latter was found to be metal
ion-independent (13). The ionic strength dependence as well as the
Mg2⫹ dependence of the C2b-C4b interaction are both consistent
with the involvement on the C4 side of the interface of the acidic
residue-rich segment near the NH2 terminus of the C4b ␣⬘-chain.
One or more of the carboxylate groups might be a coordinate ligand for the metal ion, which in turn facilitates bridge formation
with the partner protein C2. Besides the putative metal coordination site within the C2b region, both C2a and Bb have a known
metal binding motif, the MIDAS (metal-ion-dependent adhesion
site) motif, which is part of their respective vWFA domains. The
three-dimensional structure of examples of vWFA domains from
the adhesion molecules ␣M␤2 (CR3) and ␣L␤2 (LFA-1) have been
determined, and five metal ion-coordinating residues have been
identified (51, 52). It has been suggested that the sixth metal ioncoordinating residue would be contributed by the ligand protein
(51). Our previous finding that mutation of the C3 ␣⬘-chain NH2terminal acidic residues E736 and E737 resulted not only in a loss
in factor B binding by C3b, but also in a loss in CR3 binding by
iC3b, led us to suggest that because of the commonality of Mg2⫹
dependence and the presence of a vWFA domain in both factor B
and CR3 that the contact of E736 and E737 might be with the
respective vWFA domains (25). Therefore, one might speculate
that the C4b NH2-terminal ␣⬘-chain acidic cluster interacts with
the vWFA domain of C2. Through homology modeling, the vWFA
domain of factor B has recently been visualized (53, 54). Furthermore, a study using the technique of surface-enhanced laser desorption-ionization affinity mass spectrometry, in conjunction with
homology modeling, has suggested that peptide segments within
the MIDAS cleft were directly involved in C3b binding (54).
Therefore, by extension, the vWFA domain of C2 should also be
involved in contacting C4b. However, electrostatic surface potential renditions of the MIDAS surface for either factor B or modeled
factor B-C2 chimeras did not show an obvious electropositive surface that might interact with the highly negatively charged 730 –
736 segment of C3 ␣⬘-chain or, by extension, the corresponding
744 –752 segment of C4 ␣⬘-chain (53, 54). Indeed, the homology
modeling revealed that the floor of the MIDAS cleft is highly
negatively charged, and the only significant electropositive patch
visualized on the surface of the factor B vWFA is remote from the
C3b-binding peptide segments identified by the affinity-based mass
spectrometry (54). Although it is still possible that the acidic residues at the amino terminus of the C3b and C4b ␣⬘-chain might
directly contribute to the coordination of the Mg2⫹ ion within the
MIDAS, it is hard to explain why in the case of C4b so many of
the isosteric amide mutations would affect the single coordinating
residue that one would expect to come from that side of the interface. Thus, based on the modeled data available at this time, it does
not seem likely that the MIDAS surfaces of factor B or C2 would
mediate multiple ionic interactions with the acidic residues located
near the NH2 termini of, respectively, the C3b and C4b ␣⬘-chains.
By contrast to the above, there are several lines of circumstantial
evidence suggestive of an ionic interaction between SCR motifcontaining proteins and the negatively charged residues located at
the NH2 termini of both C3b and C4b ␣⬘-chains. First, mutagenic
analyses of the charged residues within the 730 –768 segment of
A C2 BINDING SITE IN C4
The Journal of Immunology
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