From the Department of Immunology and
¶ Department of Biochemistry, University of Toronto,
Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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Mapping approaches employing blocking antibodies
and synthetic peptides have implicated the 727-767 segment at the
NH2 terminus of C3b Stringent regulation of the alternative complement pathway C3
convertase is essential for the prevention of C3 hypercatabolism and
for the protection of host tissue from the deleterious effects of
alternative pathway propagation. In particular, it is important to
regulate alternative pathway propagation that can employ as a nidus C3b
molecules that have become adventitiously bound to host cell membrane.
In primates, this regulation involves primarily the soluble serum
proteins factor H and factor I, as well as the membrane-resident
proteins decay-accelerating factor (CD55), membrane cofactor protein
(MCP,1 CD46), and complement
receptor 1 (CR1, CD35) (reviewed in Ref. 1). With the exception of the
serum protease factor I, the other regulatory molecules are members of
a superfamily consisting almost entirely of varying numbers of a
sequence motif alternatively referred to as a short consensus repeat
(SCR) or complement control protein (CCP) module. Although this motif
is also found in many non-complement proteins, the genes encoding the
complement regulatory molecules are clustered on the long arm of
chromosome 1 at what has been termed the RCA locus, for
regulators of complement activation. Structural analyses of single and
paired CCP modules have directly demonstrated that they each form
independently folded compact globular domains consisting of two
interacting layers of anti-parallel CCP-containing proteins that bind to C3b exert their regulatory effect
via two mechanisms as follows: decay-accelerating activity and factor
I-cofactor activity. Specifically, the binding of H, decay-accelerating
factor, and CR1 to C3bBb, the alternative pathway C3 convertase, leads
to an accelerated rate of unidirectional dissociation of the serine
protease Bb from the C3b modulatory subunit of the convertase. I
cofactor activity refers to the accessory role of the SCR-containing
protein in the I-mediated cleavage of C3b COOH terminus to residues
1281 and 1298 (mature C3 numbering), yielding the major fragmentation
product iC3b and a minor fragment termed C3f. Since the iC3b fragment
can no longer associate with factor B, this permanently inactivates the
C3 molecule with respect to being a nidus for alternative pathway C3
convertase formation. However, this factor I-mediated cleavage can only
take place when the C3b is complexed with one of three cofactors, these
being the soluble protein factor H and the host membrane-associated proteins CR1 and MCP. Although there is some suggestive evidence in the
literature that binding of H to C3b causes a conformational change in
the latter (5), thereby increasing the affinity for factor I, there is
also recent evidence that factor I can simultaneously bind to both H
and C3b, thereby stabilizing the intrinsically weak C3b-I interaction
sufficiently to permit cleavage (6). Whether factor I similarly binds
directly to cofactors CR1 or MCP is as yet unknown. At physiological
ionic strength, only CR1 can efficiently act as an I cofactor for the
further cleavage of iC3b COOH-terminal to residue 932, yielding
fragments C3c and C3dg (7).
Domain deletion and domain exchange studies have succeeded in
identifying CCP/SCR regions within the complement regulatory proteins
that are required for their C3b-binding and I cofactor activity
(8-13). Usually 3-4 SCR domains are required for functional activity.
For example, in the case of factor H, SCRs 1-4 are minimally required
for the expression of its I cofactor activity (8, 9). Factor H has also
been shown to possess C3b-binding sites involving SCRs 6-10 and
16-20, although these sites do not contribute to the I cofactor
activity of the molecule (9). The two C3b-binding sites of human CR1
are contributed by SCRs 8-11 and 15-18, respectively, and these
entities are each capable of mediating full I cofactor activity (10). A
recent study on CR1 has shown that the first three SCR domains in each
case account for most of the functional activity (11) and critical
residues within these functionally important domains have also been
identified (11, 14, 15).
In contrast to the situation in the complement regulatory molecules
where the repeating sequence motifs suggested an obvious experimental
approach for functional site-mapping studies, the absence of any
identifiable sequence motifs within C3 precludes the domain deletion or
domain exchange approach. Nevertheless, several independent groups have
used a variety of other approaches to putatively identify segments
within C3 that mediate association with its membrane-bound receptors
and soluble protein ligands. The approaches have included functional
site blocking antibodies against C3, together with the identification
of the polypeptide segments to which they bind (16, 17) and the
combined use of proteolytic fragments and synthetic peptides derived
from C3 as functional mimetics of the intact physiologic fragments (18, 19). Additionally, once one has a candidate site, xenogeneic sequence
comparisons, together with knowledge about whether a particular
non-human species of C3b can or cannot interact with the human ligands,
can provide additional evidence for the involvement of a particular C3
segment in a binding interaction (20). Lambris and co-workers (21) have
in some cases used this information to construct and assess the ligand
binding activities of C3 molecules in which a segment of human C3 has
either been deleted or replaced with the homologous segment of the
non-human ligand-binding xenogeneic species in order to verify a
proposed binding site. Ultimately, one can employ site-directed
mutagenesis to test further the validity of proposed binding sites, to
identify important residues, and in some cases to establish the
chemical nature of the side chains that are required for a particular
binding reaction (22, 23).
All of the above described approaches have implicated the
NH2-terminal segment of C3 Fishelson (19) had analyzed a series of overlapping hexameric and
heptameric peptides spanning the 727-767 region for their ability to
bind factor B and factor H. The results suggested that the segment
730DEDIIAEENI contributed to factor B binding, whereas the
segment 744EFPESWLWNVE contributed to the binding of factor
H. Site-directed mutagenesis work on intact C3 examined the role of the
carboxylate side chains within the 730-739 segment and identified
those of glutamic acid residues 736 and 737 as being important not only for the interaction of C3b with factor B but also for the ability of
target-bound C3b and iC3b to bind, respectively, to CR1 and CR3 on
phagocytes (22). In keeping with the predictions of the Fishelson work,
these mutations did not alter the interaction with human factor H. In
the present work, we have extended our previous mutagenesis study on
human C3 to cover the remainder of the charged residues in the 727-768
segment, initially with the aim of identifying residues crucial for the
interaction with factor H. Since like factor H, CR1 and MCP also act as
cofactors for the factor I-mediated cleavages of C3, the same series of mutants were also examined for their ability to interact with soluble
forms of CR1 (sCR1) and MCP (sMCP). We have identified Glu-744 and
Glu-747 within the predicted "Fishelson" segment as residues whose
carboxylate side chains contribute to the interaction with factor H. We
have also found that the charged residue contacts with CR1, although
partially overlapping with factor H, extend over a much larger portion
of the segment. In contrast, none of the mutations examined had any
effect on the interaction with MCP.
Purified Complement Components and Antibodies--
C
Rabbit polyclonal IgG against human C3c (Sigma), goat antiserum against
human C3 (Quidel, San Diego), and alkaline phosphatase-conjugated antibody against goat IgG (Jackson ImmunoResearch, West Grove, PA) were
purchased from the indicated suppliers. Mouse monoclonal IgG1 4C2 against human C3d was a gift from Dr. V. Koistinen
(Helsinki, Finland). A mouse monoclonal IgG1 recognizing a determinant
in human C3c was purified on protein A-agarose (35) from the culture supernatants of the hybridoma cell line F63-3A2, originally provided by
Dominion Biologicals Ltd. (Truro, Nova Scotia, Canada).
Cell Culture--
All cell lines were maintained at 37 °C
with 5% CO2 in a humidified tissue culture incubator.
COS-1 cells were maintained in Dulbecco's modified Eagle's medium
(DMEM, Life Technologies, Inc.) supplemented with 10% fetal calf serum
(FCS), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (complete DMEM). Murine myeloma J558L cells that
were stably transfected with pKG5-En-C3, a vector encoding wild-type
human C3 and harboring a neomycin resistance gene, were maintained in
complete DMEM containing 0.6 mg/ml active G418-sulfate (Geneticin, Life
Technologies, Inc.).
When rC3-containing culture supernatants were to be harvested for use
in the cofactor-dependent factor I cleavage assay (see below), it was necessary to use FCS in which the bovine factor I had
been irreversibly inactivated with K76-monocarboxylic acid (K76-COOH,
obtained from Dr. W. Miyazaki, Otsuka Pharmaceuticals, Tokushima,
Japan). Our modification of the procedure for K76-COOH treatment of FCS
has been described previously (22). One day before culture supernatants
were to be harvested, the cells were washed in Hanks' buffered salt
solution (Life Technologies, Inc.) and then incubated in DMEM/K76, this
medium being complete DMEM in which the 10% FCS is replaced with 4%
K76-COOH-treated FCS and supplemented with 1% Nutridoma-HU (Boehringer
Mannheim, Montreal, Quebec, Canada).
Site-directed Mutagenesis--
The construction of a full-length
cDNA expression plasmid for human C3, pSV-C3, has been described
previously (36). The same cDNA was also inserted as a
HindIII fragment into pBluescript in both orientations, and
the resulting plasmids were designated pBST-C3A (C3 coding strand is
top strand of plasmid) and pBST-C3B (C3 coding strand is bottom strand
of plasmid), respectively. pBST-C3B was used as a wild-type template
and as an intermediate subcloning vector for the production of
site-directed mutants by the overlap extension polymerase chain
reaction method (37) using the proofreading enzyme Vent DNA polymerase
(New England Biolabs, Beverly, MA). The resulting 612-bp polymerase
chain reaction fragment encompassed unique restriction sites for
SphI and PmlI (at bases 2149 and 2459 of C3
cDNA, respectively), and after restricting with these enzymes, we
purified a 312-bp mutation-containing fragment which could be subcloned
into similarly digested pBST-C3B, where the sites for SphII
and PmlI are also unique (SphI is not unique in
pSV-C3, thus necessitating the intermediate cloning step). Following
confirmation of the desired mutation(s), and the absence of any
undesired mutations within the 312-bp target sequence by strand
denaturation dideoxy DNA sequencing (T7 polymerase sequencing reagents,
Amersham Pharmacia Biotech), a 1300-bp ApaI-SalI
fragment encompassing the mutation(s) is exchanged for the
corresponding wild-type segment in pSV-C3.
Expression of Recombinant C3--
The DEAE-dextran method (38,
39) was used in the transfection of COS-1 cells with the wild-type and
mutant pSV-C3 expression vectors using 5 µg of plasmid per 35-mm
plate seeded 18 h earlier with 1 × 105 cells.
Following the transfection, the cells were allowed to grow in complete
DMEM for 48 h, after which time the medium was changed to
DMEM/K76. The cells were cultured overnight in this medium prior to
metabolic labeling as described below. For hemolytic assays and
ELISA-based experiments, the culture supernatants were harvested
48 h after the change of the medium and then dialyzed extensively
against VBS.
Metabolic Labeling, Biosynthetic Characterization, and
Immunoprecipitation--
The transfected cells were depleted of
endogenous methionine and cysteine by incubating for 1 h in
methionine- and cysteine-free DMEM (ICN Biochemicals, Costa Mesa, CA)
containing 4% K76-treated FCS and 1% Nutridoma (1 ml per 35-mm
plate). The cells were then labeled with 200 µCi of
Tran35S-label (ICN Biochemicals) for 6 h at which time
an equal volume of methionine- and cysteine-sufficient DMEM/K76 was
added, and the incubation was continued overnight. To assess the
biosynthetic processing and C3 convertase cleavability of the
recombinant molecules, metabolically labeled supernatants were
immunoprecipitated with anti-C3c and analyzed by SDS-PAGE (9% gel)
autofluorography, both with and without prior treatment of the
supernatants with C4boxy2a. The procedures for the
generation of the C4boxy2a and for the immunoprecipitation
and wash conditions have been described previously (40, 41).
Quantitative densitometry of band intensities on pre-flashed film was
performed by scanning the film on an Epson-ES-1000C color scanner and
quantifying band intensities using the program NIH Image version
1.58.
Quantitative Measurement of Secreted Recombinant C3--
The
amount of recombinant C3 secreted by the transfectants, both as
unlabeled and 35S-labeled proteins, was determined by a
competitive solid-phase radioimmunoassay (42) using
125I-labeled purified human C3 as the probe and rabbit
polyclonal IgG anti-human C3c as the capture antibody. A standard curve
was obtained using known amounts of purified human C3 as the
competitor. The assay was performed in opaque polystyrene microtiter
plates (Packard Instruments, Meridan, CT), and bound radioactivity was measured by liquid scintillation counting directly in the plates using
a TopCount instrument (Packard Instruments).
Classical Pathway-dependent Hemolytic
Assays--
Antibody and C3-convertase bearing sheep erythrocytes,
EAC4boxy2a (1.5 × 107), prepared by
standard methods (34), were incubated for 1 h at 37 °C with 500 µl of VBS-dialyzed culture supernatants containing varying amounts of
rC3, as determined by RIA. The resulting EAC423b cells were washed in
GVBE (VBS containing 0.1% gelatin and 10 mM EDTA) and then
incubated with human C5 (1 µg) and guinea pig C6-9 reagent (1/50
dilution in GVBE, 1 ml) at 37 °C for approximately 30 min until a
suitable degree of lysis developed in the tubes containing a comparable
concentration range of purified human C3. After spinning down unlysed
cells, the degree of lysis in each tube was determined by measuring the
absorbance of the supernatant at 412 nm. After correcting for
background, the degree of specific lysis was converted to
"Z" units where Z = ( ELISA--
For the purpose of determining the conformational
dependence of the reactivity of a pair of C3-specific monoclonal
antibodies, polystyrene microtiter plates were first coated (2 h,
37 °C) with 200 µl of varying dilutions of purified human C3 or
C3(H2O) in PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) that had either been treated or not
treated with SDS (10% v/v). A starting concentration of 30 µg/ml was
used for coating, and a 3-fold dilution series in PBS was established.
Following blocking with Blotto/Tween (5% skim milk in PBS, 0.05%
Tween 20) and three washes in PBS/Tween, mouse monoclonal antibodies
against either a C3c epitope or a C3d epitope were then added (2 µg/ml in Blotto/Tween; 200 µl/well). A polyclonal rabbit anti-human
C3c IgG (10 µg/ml in Blotto/Tween, 200 µl/well) was used as a
positive control for the presence of C3/C3(H2O) on the
plates. These antibodies were allowed to adhere for 2 h at
37 °C. Following washing, plate-bound mouse or rabbit IgG were
respectively detected with alkaline phosphatase-conjugated donkey
anti-mouse IgG or alkaline phosphatase-conjugated goat anti-rabbit IgG,
in conjunction with the alkaline phosphatase substrate
p-nitrophenyl phosphate (Harlow and Lane (42)).
When the two monoclonals were used as conformational probes of the
recombinant C3, a capture ELISA protocol was employed. Briefly
polystyrene microtiter plates were coated with one of three
antibodies as follows: rabbit polyclonal IgG anti-human C3c, mouse
monoclonal IgG anti-human C3c, and mouse monoclonal IgG anti-human C3d
(2 µg/ml in 0.01 M NaHCO3, pH 9.8; 200 µl/well). After blocking with Blotto/Tween, the wells were incubated
with equal concentrations (100 ng/ml) of the recombinant C3 molecules for 2 h at 37 °C (200 µl/well). The primary sandwiching
antibody, goat anti-human C3, was then added followed by the detecting
antibody of alkaline phosphatase-conjugated rabbit anti-goat IgG,
2 h at 37 °C for each incubation. From the blocking stage
onwards, the wells were washed with PBS/Tween between each incubation.
Hydrolysis of p-nitrophenyl phosphate was measured
colorimetrically after approximately 30 min at room temperature.
Negative controls included not adding capture antibody, C3, or primary antibody.
Cleavage of 35S-Labeled C3(H2O) by Factor
I and Cofactors H, sCR1, and sMCP--
35S-Labeled
C3-containing supernatants were collected and then treated for 4 h
at 37 °C with an equal volume of 4 M KBr in order to
deliberately hydrolyze the thioester and convert native C3 to the
C3b-like C3(H2O) molecule. The supernatants were then
dialyzed extensively against VBS at 4 °C. Equal concentrations of
35S-labeled C3(H2O) (100 ng/ml), as determined
by RIA, were treated for 2 h at 37 °C with a single amount of
purified human factor I and with variable amounts of one of the three
soluble human I cofactors examined in this study, i.e. H,
sCR1, and sMCP (exact quantities added are given in various figure
legends). A control sample in each case had factor I added without any
cofactor. Following the incubation, the samples were immunoprecipitated
using rabbit polyclonal IgG against C3c in conjunction with formalized
Staphylococcus aureus (Sigma) as described previously (41).
These samples were then treated with SDS-PAGE sample buffer under
reducing conditions, separated on 9% SDS-PAGE, and visualized by
autofluorography (Kodak XAR-5 film) using 1 M sodium
salicylate as the gel-impregnating fluor. A variant of this assay done
with factor H as the I cofactor involved adding to the
35S-labeled C3(H2O)-containing supernatants
fresh fetal calf serum (10% final concentration) as a source of
heterologous (i.e. bovine) factors H and I. In all cofactor
assays, quantification of band intensities was performed by
densitometric analysis as described above.
General Characterization and Conformational Integrity of the Mutant
C3 Proteins--
The observation that the interactions between RCA
proteins and their ligands are strengthened under conditions of low
ionic strength (44-46) suggests an important contribution of ionic
forces to the binding event. Consequently, we have focused our
mutagenic alanine scan on the charged residues of the 742-767 target
region. Additionally, in the absence of structural data that would
identify surface-exposed residues, and thus guide the mutagenic
analysis, the charged residue to alanine scan approach has been
experimentally validated as being non-disruptive to the conformation of
the protein, while at the same time being able to identify segments of
a molecule contributing to a binding interface (47, 48). Fig.
1 lists the mutant
proteins2 examined in this
study for their interaction with the I cofactors H, CR1, and MCP. In
addition to the new mutants engineered within the 742-767 target
region, we have also re-examined three mutant C3 molecules that were
engineered for our previous study (22) and that span the
NH2-terminal-most charged cluster of C3
The classical pathway-dependent hemolytic activities of the
various mutant molecules were also examined. As indicated in Fig. 1,
all of the mutants show some level of defect, with hemolytic activities
ranging from approximately 50 to 70% of wild-type activity. The level
of hemolytic activity generally correlates with the extent to which the
molecule is cleaved by a limiting concentration of fluid-phase
classical pathway C3 convertase (Fig. 1). A corollary to this
observation is that events downstream of C3 convertase cleavage, such
as thioester-mediated transacylation efficiency and C5 convertase
subunit activity, are essentially normal in the various mutant
molecules. Collectively, these data suggest that the mutations have not
introduced conformational alterations that extend beyond the target
segment. The partial defect observed on cleavability by the classical
pathway C3 convertase in virtually all of the mutants is in keeping
with a previous study (50) showing that susceptibility of mouse C3 to
cleavage by the alternative pathway C3 convertase was quite sensitive
to sequence alterations in the segment immediately downstream of the
cleavage site.
Optimization of Cofactor-dependent Factor I Cleavage
Assay--
The respective abilities of the various
rC3(H2O) molecules to interact with the human
SCR-containing regulatory molecules factor H, soluble CR1 (sCR1), or
soluble membrane cofactor protein (sMCP) were assessed by their
cofactor binding-dependent susceptibility to cleavage by
human factor I. The rC3(H2O) molecules were present as
metabolically labeled entities in the KBr-treated COS-1 cell transfection supernatants. The fetal bovine serum employed in the
culture medium had been treated with K76-COOH in order to inactivate
bovine factor I and thereby reduce the background cleavage of
rC3(H2O) by bovine H and I (22). In the case of the
interactions with factor H and sMCP, rC3(H2O) is cleaved by
factor I to the iC3b-like species iC3(H2O). Upon SDS-PAGE
analysis of the immunoprecipitated material, this cleavage is detected
by the conversion of
At least for the case of factor H and CR1 binding to C3b, there is
evidence in the literature that in addition to a binding site within
the NH2-terminal Interaction of Factor H with the Series of Mutant
C3(H2O) Molecules--
Shown in Fig.
3A is the H
cofactor-dependent cleavage assay for wild-type C3 and for
the 9 charged residue alanine-scan mutants constructed within the
742-767 target region. Visual inspection of the autoradiogram reveals
that mutants E744A and E747A show a significant impairment in
H-dependent cleavage by factor I that is apparent at both
100 and 200 ng/ml of the cofactor. At least an equal degree of
impairment was also observed in E744A/E747A double mutant. However,
mutations at any of the other 7 charged residues in the 742-767 target
segment were without effect in this assay. In order to determine
whether the negatively charged side chains of Glu-744 and Glu-747 were
required, isosteric amide substitutions were engineered, and the
functional activities of these molecules were determined. It can be
seen in Fig. 3A that the E744Q and E747Q mutants showed the
same degree of impairment as did the equivalent alanine substituents,
thereby implying that the negative charge per se is
important for mediating binding of factor H.
Although the three mutants within the 727-737 segment (see Fig. 1) had
previously been examined for their interaction with human factor H in a
cofactor-dependent cleavage assay and were reported to be
unimpaired in this binding interaction (22), it is now clear that the
assay had been carried out using saturating concentrations of factor H. When reexamined under the current subsaturating assay conditions, only
the tetra-mutant D730N/E731Q/E736Q/E737Q showed a degree of impairment
that was similar to that seen with either E744Q or E747Q alone.
In order to assess the reproducibility of the assay, and to determine
whether the effects of the mutations at Glu-744 and Glu-747 were
cumulative, this assay, as well as two other independent replicates,
were subjected to densitometric analyses. For each lane the intensity
of the
As a second approach to assessing subtle defects in H binding activity
produced by the various mutations, a version of the cofactor-dependent cleavage assay was employed in which
10% non-K76-COOH-treated fetal calf serum was used as a source of
heterologous factors H and I. We reasoned that the interaction between
the human C3(H2O) derivatives and the bovine H would be
less strong than with its autologous counterpart and therefore that
subtle defects or cumulative effects that were difficult to detect when
using the autologous components may be observed using the heterologous
components. The results of this experiment are shown in Fig.
4, and they essentially mirror those
obtained using the autologous components under non-saturating conditions. In this case, however, the cumulative effect of having residues Glu-744 and Glu-747 both mutated to alanine is more readily apparent.
Interaction of sCR1 with the Series of Mutant C3(H2O)
Molecules--
The results from a representative
CR1-dependent I-cleavage experiment are shown in Fig.
5A, and 5B depicts
results from replicate experiments as a bar graph. Our
previous work had shown that isosteric amide substitutions at Glu-736
and Glu-737, but not at Asp-730 and Glu-731, had resulted in a marked
loss in the ability of the mutant red cell-bound C3b to mediate rosette
formation with CR1 on neutrophils (22). The present analysis of these
mutants in the CR1-dependent I-cleavage assay therefore
provided the opportunity to validate this assay as a surrogate CR1
binding assay. In accordance with the CR1-dependent rosette
assay results, mutant proteins containing the E736Q/E737Q substitutions
show markedly impaired conversion of C3(H2O) to
iC3(H2O) and no evidence of the third cleavage converting
In examining the 742-767 series of mutants in this assay, it became
apparent that the mutants showing impaired cleavage fell into two
categories: those affecting the first I-mediated cleavage at residue
1281 and those affecting only the third cleavage at residue 932. For
example, whereas mutant E747A showed significantly impaired
CR1-dependent conversion of C3(H2O) to
iC3(H2O), and no evidence of the third cleavage at the high
concentration of sCR1, mutant E744A showed no impairment in
C3(H2O) to iC3(H2O) conversion but nevertheless
was resistant to the third cleavage. An impairment in
C3(H2O) to iC3(H2O) conversion was also seen with the E754A/D755A mutant. Although there was no impairment in this
conversion for neighboring K757A/E758A mutant, there was impairment of
the third cleavage. Combination mutants showed the phenotype of the
individual mutations, and at least within this range of sCR1
concentrations, there was no obvious indication of a cumulative effect.
Finally, as was the case for the interaction with factor H, isosteric
amide substitutions at residues 744 and 747 showed the same degree of
impairment as did their alanine-substituent counterparts. Thus for
these two residues at least, one can conclude that the negative charge
per se is important to the binding with CR1. In contrast,
mutation to alanine of the positively charged residues Arg-742,
Lys-761, and Lys-767 were each without effect on interaction between
rC3(H2O) and CR1.
Interaction of sMCP with the Series of Mutant C3(H2O)
Molecules--
Like factor H and CR1, MCP is also a cofactor for the
factor I-mediated cleavage of C3b to iC3b (or C3(H2O) to
iC3(H2O)), and furthermore since no binding site for MCP
has been delineated, the complete series of 727-767 segment mutants
were analyzed in a factor I cleavage assay in which a soluble form of
MCP acted as the I cofactor. The results of a representative experiment are shown in Fig. 6A and as a
bar graph from replicate experiments in Fig. 6B.
In contrast to what was observed with both factors H and sCR1, when
sMCP was used as the I cofactor, there appears to be no impairment in
the interaction of this molecule with any of the mutant
rC3(H2O) molecules examined.
As summarized in Table I, the
observations in this study have identified critical residues within the
hydrophilic 42 amino acid segment at the NH2 terminus of
C3b '-chain as contributing to the
interactions with factor B, factor H, and CR1. Our previous mutagenesis
study on the NH2-terminal acidic cluster of this segment
identified residues Glu-736 and Glu-737 as contributing to the binding
of C3b to factor B and CR1 but not factor H. We have now extended the
charged residue mutagenic scan to cover the remainder of the segment
(738-767) and have assessed the ability of the C3b-like
C3(H2O) form of the mutant molecules to interact with
factor H, CR1, and membrane cofactor protein (MCP) using a
cofactor-dependent factor I cleavage assay as a surrogate
binding assay. We have found that the negatively charged side chains of
Glu-744 and Glu-747 are important for the interaction between
C3(H2O) and factor H, a result in general agreement with an
earlier synthetic peptide study (Fishelson, Z. (1991) Mol.
Immunol. 28, 545-552) which implicated residues within the
744-754 segment in H binding. The interactions of the mutants with
soluble CR1 (sCR1) revealed two classes of residues. The first are
residues required for sCR1 to be an I cofactor for the first two
cleavages of
-chain. These are all acidic residues and include the
Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The
second class affects only the ability of sCR1 to be a cofactor for the
third factor I cleavage and include Glu-744 and the Lys-757/Glu-758
pairing. The dominance of acidic residues in the loss-of-function
mutants is striking and suggests that H and CR1 contribute basic
residues to the interface. Additionally, although there is partial
overlap, the contacts required for CR1 binding appear to extend over a
wider portion of the 727-767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction
between soluble MCP and C3(H2O), indicating that despite
its functional homology to H and CR1, MCP differs in its mode of
binding to C3b/C3(H2O).
INTRODUCTION
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Abstract
Introduction
References
-sheet (2-4). It has been
speculated that the protruding loops, some of which are of variable
length, are good candidates for mediating interaction with ligand.
'-chain (residues 727-767)
as contributing at least one contact site to the interaction of C3b
with factor B, factor H, and CR1. In particular, an anti-peptide
antibody directed against this segment recognizes a neoepitope in C3b
and can block the interaction with factor B, factor H, and CR1 (24). Furthermore, the 727-767 peptide can compete with factor H and CR1 for
binding to C3b (24, 25), and in one study (24), but not the other (25),
was capable of inhibiting the interaction between C3b and factor B. Finally, a recombinant molecule in which the 727-767 segment was
deleted lost the ability to interact with all three proteins (21).
EXPERIMENTAL PROCEDURES
s
(26), C2 (27), C3 and C5 (28), C4 (29), factor B (30), factor H (31),
and factor I (32) were purified from human plasma as described
previously. C3(H2O) was generated from native C3 by
incubation with KBr, 2 M final concentration, at 37 °C
for 4 h, followed by extensive dialysis against veronal-buffered saline (VBS, 4 mM veronal, pH 7.2, 0.15 M NaCl,
0.15 mM CaCl2, 0.5 mM
MgCl2). A functionally pure human C1 reagent was obtained by euglobulin precipitation of serum (33), and a guinea pig C6-9
reagent was prepared from guinea pig serum (Life Technologies, Inc.,
Oakville, Ontario, Canada) as described (34). Soluble CR1 (sCR1) and
soluble MCP (sMCP) were generously provided by Dr. Richard Smith of
SmithKline Beecham (Harlow, UK) and Dr. Grace Yeh of Cytomed Inc.
(Cambridge, MA), respectively.
ln(1
fractional lysis)) and corresponds to the number of hemolytically
effective molecules of C3 per target cell (43). Comparisons were made on the basis of Z units per amount of C3 added within the
linear portion of the dose-response curve.
RESULTS
'-chain. When
transiently expressed in COS-1 cells, all of the mutant molecules were
secreted at levels comparable to wild type. Furthermore, as deduced
from metabolic labeling experiments, the ratio of mature 2 chain C3 to
pro-C3 present in the secretions was in each case also comparable to
the ratio seen in the wild-type protein (see for example zero cofactor
lanes of Figs. 3-6). Both of these pieces of data point toward the
general conformational integrity of the various mutant molecules having
been maintained in a native-like state. This was further assessed by
measuring the binding of the various rC3(H2O) molecules
(i.e. thioester-hydrolyzed C3 produced by KBr treatment) to
two monoclonal antibodies, each of which recognized an epitope in
native C3 or C3(H2O), but which in preliminary experiments
(detailed under "Experimental Procedures") showed greatly
diminished binding to the equivalent SDS-denatured molecules. One of
these monoclonals (F63-3A2) recognizes an as yet uncharacterized epitope in C3c. The other monoclonal (4C2) is C3d-specific and has
previously been shown to be a blocking antibody for the interactions of
C3b with factor H, CR1, and factor B (49). When we compared the amount
of wild-type or mutant rC3(H2O) antigen captured by the
respective monoclonal antibodies to the amount captured in each case by
a polyclonal anti-C3c, we found that for each of the two monoclonal
antibodies this ratio was invariant for all of the rC3(H2O)
molecules tested. We conclude that all of the mutant
rC3(H2O) molecules engineered for this study exhibited wild-type reactivity with respect to the conformational epitopes recognized by these two monoclonal antibodies.
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Fig. 1.
Mutant C3 molecules assessed in this study,
their classical pathway-dependent hemolytic activities, and
their cleavability by the fluid-phase classical pathway C3 convertase
C4b2a. The procedures for measurement of classical pathway
hemolytic activity and for the quantitation of cleavage by C4b2a are
described under "Experimental Procedures." The first three mutant
molecules of the list were produced in an earlier study (22). As a
point of reference, the Fishelson (19) peptides that bound factor B and
factor H, respectively, are indicated above the wild-type
target sequence with the thickness of the bar
representing the strength of binding that was observed.
-chain into two major fragments,
-75, that
co-migrates with the
-chain and
-40. By using sCR1 as the
cofactor, in addition to the above products, one can get a further
cleavage of
-75 COOH-terminal to residue 932, thereby producing an
NH2-terminal 37-kDa fragment (
-37) and a COOH-terminal
38-kDa fragment corresponding to C3dg. Since in our experiments
immunoprecipitation was with an anti-C3c reagent, only the
-37
fragment would be visualized on SDS-PAGE in cases where the factor I
cleavage COOH-terminal to residue 932 had occurred.
'-chain segment, there is at a minimum another binding site located in the C3d region of the molecule (24,
49). Thus a mutation that affects only the NH2-terminal segment might only partially impair the binding and might even be
missed in our cofactor-dependent cleavage assay if
saturating concentrations of the cofactor were used. To establish a
concentration range over which H was subsaturating in the assay,
metabolically labeled wild-type C3(H2O) was treated with a
constant amount of factor I and a range of H concentrations. It can be
seen in Fig. 2 that the extent of
-chain conversion to
-75 and
-40 was dependent upon the
concentration of H up to 100 ng/ml, after which the assay was saturated
with respect to cofactor. Also shown are the
concentration-dependent cleavage profiles using sCR1 and sMCP
as the I cofactors. For sMCP the assay is subsaturating with respect to
cofactor up to 400 ng/ml. For sCR1, by 200 ng/ml there is essentially
quantitative conversion of
-chain into
-75 and
-40. However,
the further cleavage of
-75 into the 38-kDa C3dg fragment and the
NH2-terminal 37-kDa fragment only became apparent at the
400 ng/ml concentration of sCR1. Since it was desirable to
simultaneously assay all of the mutants, there was a practical need to
restrict the number of samples to be handled. Consequently, in addition
to the zero cofactor control, each rC3(H2O) molecule was
analyzed at two cofactor concentrations, one of these concentrations
giving an intermediate level of cleavage of wild-type
rC3(H2O) and one giving near total cleavage of this
molecule. These concentrations corresponded to 100 and 200 ng/ml for
each of factor H and sMCP. For sCR1, the two concentrations chosen were
100 and 400 ng/ml, the latter being chosen in order to see whether the
mutations had an effect on the cleavage of
-75 to C3dg and
-37.
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Fig. 2.
Cofactor concentration dependence of the
factor I-mediated cleavage assay using wild-type recombinant
C3(H2O). Culture supernatants from stably transfected
J588L cells containing wild-type human 35S-labeled
C3(H2O), diluted to 100 ng/ml, were treated with increasing
concentrations of human cofactor in the indicated amounts plus 500 ng/ml of human factor I. Digestion products were immunoisolated using
rabbit anti-human C3c and resolved on a 9% SDS-PAGE gel under reducing
conditions, followed by autofluorography.
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Fig. 3.
Factor I-mediated cleavage of recombinant
C3(H2O) molecules using H as the I cofactor.
A, culture supernatants from COS-1 cell transfections
containing 100 ng/ml of wild-type or mutant 35S-labeled
rC3(H2O) (as determined by RIA) were digested for 2 h
at 37 °C with the concentrations of factor H (ng/ml) indicated
above each lane and 500 ng/ml factor I. Digestion products
were immunoisolated using rabbit anti-human C3c and resolved on 9%
SDS-PAGE under reducing conditions followed by autofluorography.
B, autofluorographs from three independent experiments
were analyzed using densitometry to quantify the major bands. The ratio
of the -chain band pixel intensity over the combined signals from
the
-chain,
-75,
-40, and
-chains (total) was calculated
and plotted as a bar graph as a function of cofactor
concentration. Error bars represent the S.D. of the mean.
The order of the bars (left to right)
is the same as the order in the legend (top to
bottom).
-chain was expressed as a fraction of the summed intensities
arising from
-chain,
-75,
-chain, and
-40. The means and
standard deviations of fractional
-chain intensity for each
recombinant at the two factor H concentrations are shown in bar
graph form in Fig. 3B. The bar graph presentation of
the data, especially for the 200 ng/ml factor H concentration, readily
identifies molecules having substitutions at either or both of Glu-744
or Glu-747 as being impaired with respect to factor H binding. There is
also some suggestion that the effects on H binding of the individual
mutations at residues 744 and 747 are cumulative, although not strictly additive.
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Fig. 4.
Susceptibility of wild-type and mutant
C3(H2O) to cleavage by bovine factors H and I. Equal
concentrations of 35S-labeled rC3(H2O) (100 ng/ml), harvested from COS-1 cell transfections cultured in the
presence of K-76COOH-treated FCS, were incubated with 10% untreated
FCS as a source of bovine factors H and I for 1 h at 37 °C
(lanes denoted +). Control samples were incubated without
addition of the untreated FCS (lanes denoted ). These
treatments were followed by immunoprecipitation with anti-C3c and
analysis on 9% SDS-PAGE autofluorography (reducing conditions).
-75 to C3dg and
-37. Similarly, as was the case for the rosette
assay, mutation of residues 730 and 731 on their own resulted in
unimpaired activity in the CR1-dependent I-cleavage assay.
These results therefore confirm that the cofactor-dependent I-cleavage assay faithfully reports on the relative strength of the
binding interaction between the SCR-containing I cofactor and the
rC3(H2O) molecule.
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Fig. 5.
Factor I-mediated cleavage of recombinant
C3(H2O) molecules using sCR1 as the I cofactor.
A, culture supernatants from COS-1 cell transfections
containing 100 ng/ml of wild-type or mutant 35S-labeled
rC3(H2O) (as determined by RIA) were digested for 2 h
at 37 °C with the concentrations of sCR1 (ng/ml) indicated
above each lane and 500 ng/ml factor I. Digestion products
were immunoisolated using rabbit anti-human C3c and resolved on 9%
SDS-PAGE under reducing conditions followed by autofluorography.
B, autofluorographs from three independent experiments
were analyzed using densitometry to quantify the major bands. The ratio
of the -chain band pixel intensity over the combined signals from
the
-chain,
-75,
-40, and
-chains (total) was calculated
and plotted as a bar graph as a function of cofactor
concentration. Error bars represent the S.D. of the mean.
Note,
-37 represents the NH2-terminal half of
-75
that is produced as result of the third factor I-mediated cleavage of
C3(H2O). The COOH-terminal half corresponds to the similar
mass C3dg fragment; however, this fragment is not immunoprecipitated by
the anti-C3c antibody. Therefore in cases where this cleavage occurs,
the denominator of the ratio will be somewhat underestimated. The order
of the bars (left to right) is the
same as the order in the legend (top to
bottom).
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Fig. 6.
Factor I-mediated cleavage of recombinant
C3(H2O) molecules using sMCP as the cofactor.
A, culture supernatants from COS-1 cell transfections
containing 100 ng/ml of wild-type or mutant 35S-labeled
rC3(H2O) (as determined by RIA) were digested for 2 h
at 37 °C with the concentrations of sMCP (ng/ml) indicated
above each lane and 500 ng/ml factor I. Digestion products
were immunoisolated using rabbit anti-human C3c and resolved on 9%
SDS-PAGE under reducing conditions followed by autofluorography.
B, autofluorographs from three independent experiments
were analyzed using densitometry to quantify the major bands. The ratio
of the -chain band pixel intensity over the combined signals from
the
-chain,
-75,
-40, and
-chains (total) was calculated
and plotted as a bar graph as a function of cofactor
concentration. Error bars represent the S.D. of the mean.
The order of the bars (left to right)
is the same as the order in the legend (top to
bottom).
DISCUSSION
'-chain that contribute to the binding interaction with factor H
and CR1. In contrast, we were unable to show any defect in the
association of MCP with any of mutant proteins examined. With respect
to a number of criteria including level of expression, biosynthetic
processing, reaction with two conformationally sensitive monoclonal
antibodies and hemolytic activity, the mutant molecules examined can be
considered to be native-like with respect to their conformational
state. Although the extent of hemolytic activity, which largely
reflected the extent to which the C3 convertase cleavage site was
affected, ranged from 73 to 46% of wild-type levels, it is important
to note that there was no correlation between the extent of the
hemolytic defect and whether or not an I cofactor binding site was
affected.
Summary of cofactor binding activities of mutant C3 molecules examined
in this study
Although the factor H and CR1 sites of interaction within the 727-767
segment partially overlap, the charged residue contacts required by CR1
extend over a wider portion of the segment than is the case for factor
H. In particular, whereas the negatively charged side chains of Glu-744
and Glu-747 appear to be most important for the interaction with factor
H, for CR1 there were 3 clusters of charged residues, namely
Glu-736/Glu-737, Glu-747, and Glu-754/Asp-755 that had approximately
equal effects on the CR1-dependent cleavage of
C3(H2O) to iC3(H2O) upon replacement of the
negative charge by a neutral residue. Additional contacts with Glu-744
and the Lys-757/Glu-758 pair appeared to be required for CR1 to act as a cofactor for the third factor I-mediated cleavage. This additional requirement is reminiscent of previous results using factor H as the I
cofactor for the third cleavage. Whereas H is not a cofactor for this
cleavage at physiologic ionic strength, it becomes one at low ionic
strength (7), suggesting a need for additional contacts facilitated by
relatively weak ionic bonds in order to alter either the conformation
of iC3b/iC3(H2O) or more likely to position the factor I
appropriately to enable the third cleavage of C3 -chain. The recent
observation that factors H and I directly interact with one another (6)
would be consistent with the latter possibility.
The observation that residues Glu-744 and Glu-747 are crucial for the binding of factor H to C3(H2O) is fully consistent with the data of the Fishelson (19) overlapping hexa/heptapeptide study which suggested that C3 residues 744-754 contribute to the binding of factor H. The only other mutant in which the interaction with H appeared to be compromised was the tetra mutant D730N/E731Q/E736Q/E737Q. Since the magnitude of impairment is the same as that seen with either E744Q or E747Q alone, it would suggest that either the contributions of the Asp-730/Glu-731 and Glu-736/Glu-737 charged pairs on their own to the binding of factor H were too small to be detectable in our assay or, more likely, that there is sufficient local conformational distortion caused by the loss of four negative charges over a space of seven amino acids to have had an effect on the nearby segment which now by two independent approaches has been shown to make a measurable contribution to H binding.
We believe it noteworthy that all of the mutant proteins for which the I cofactor assay suggested a diminished interaction with either factor H or CR1 had lost at least one negative charge, whereas none of the single positive charge substitutions, representing one-third of the non-overlapping mutations examined, showed any defect. The only ambiguity involves the K757A/E758A mutant which showed resistance to only the third factor I cleavage obtainable with CR1 as the cofactor. It is thus possible that Lys-757 is not involved at all in the interaction with CR1 or that at most it contributes only to the additional cofactor binding site required to mediate the third factor I cleavage. The dominance of negatively charged residues as putative contacts on the C3 side of the interactions with CR1 and factor H, together with the documented ionic strength dependence of these interactions (44, 45), strongly suggests that ionic bonds to positively charged residues on the cofactor side of the interaction form an essential component of the binding interface. Indeed, with respect to the extensively studied C3b- and C4b-binding sites within human CR1, Krych et al. (11) noted that all loss-of-function mutations either resulted from the loss of a positive charge or the addition of a negative charge. These observations are therefore in keeping with the hypothesis that the ionic interface is formed between positively charged residues of CR1 and negatively charged residues contributed by C3b and C4b. Similar point mutagenesis studies of factor H SCR domains have yet to be done. Based upon other known structures of protein-protein interfaces, it is likely that there is also a hydrophobic component to the interactions of C3b with CR1 and factor H (51-53). Indeed within the peptide segment suggested by the Fishelson study (19) to contain a binding site for factor H, there is a fairly hydrophobic stretch of residues, 749WLWNV, some of which may contribute a hydrophobic patch to the binding interfaces for factor H and CR1.
Lambris et al. (21) have examined the contribution of the
NH2-terminal '-chain segment of human C3 to the
interactions with factor H, factor B, sCR1, and sMCP, all of human
origin, by either deleting most of the segment or by replacing it with the homologous segments from Xenopus and trout C3 and from
the C3-related molecule cobra venom factor (CVF). As summarized in Fig.
7, these molecules differ in their
abilities to bind to the human ligands (20), although it does not
necessarily follow that the NH2-terminal
'-chain segment
on its own dictates the inter-species compatibilities. The recombinant
molecule in which the 727-767 segment was deleted lost the ability to
interact with human H, B, and sCR1 but not sMCP (21). Notwithstanding
the conformational integrity caveat about the interpretation of
loss-of-function data from a molecule containing a 36-residue deletion,
there is general agreement between the major conclusions reached from
the deletion study and those from our point mutation study. There are,
however, some inconsistencies with respect to the results obtained with
the homologous replacement mutants as the only deleterious effects seen
were on the third factor I-mediated cleavage with sCR1 as the cofactor.
In all other respects, the chimeric molecules displayed wild-type
behavior. As a point of reference, a minimum gap alignment of the
relevant peptide segments from the various species, along with a
denotation of point mutants determined in our study to affect the
interaction with human H and sCR1, is shown in Fig. 7. One can easily
rationalize the lack of effect of the interaction with factor H of the
Hu/Xe chimera, since residues Glu-744 and Glu-747 are conserved in
Xenopus C3. However, in the case of the Hu/Tr and Hu/CVF
chimeras, one of the two crucial acidic residues is replaced by either
its isosteric amide (Gln-744 in Tr C3) or a positively charged residue
(Lys-747 in CVF), and based on our current findings, these changes
would have been expected to decrease the extent of cleavage in the
H-cofactor assay. One possible technical reason why this was not seen
is that the assay performed by Lambris and colleagues (21) may not have
been performed under limiting conditions of cofactor. We would have
also expected compromised CR1-mediated cleavages in the case of the
Hu/Tr and Hu/CVF chimeras that were not limited solely to the third
factor I cleavage site because of the presence of Lys at residue 747 in
the Hu/CVF chimera and the replacement of Glu-754/Asp-755 by a neutral
TN pairing in the Hu/Tr chimera. However, in the case of the latter
change, the presence of an ED pairing at residues 752 and 753 may
compensate for the loss of negative charge at residues 754 and 755. Similarly, it is perhaps not surprising that substitutions of the human
Glu-736/Glu-737 pairing in the various chimeric molecules were without
effect in the CR1-dependent conversion of
C3(H2O) to iC3(H2O) because in each case a
triplet composed of two negative side chains and one neutral side chain (EEN in human C3) is replaced by a similarly composed triplet (Xe and
CVF have DSD and Tr has SED). At least with respect to factor B binding
activity, we have previously shown that the human EEN triplet can be
replaced by the CVF-like DSD triplet without effect (22).
|
There is now a congruence of evidence from synthetic peptide, blocking
antibody, and protein engineering approaches that the acidic
residue-rich segment at the amino terminus of C3 '-chain contributes
a ligand-binding site for factor H, CR1, and factor B (19, 21, 22, 24, 25, 54, and the present work). The protein engineering approach has
also implicated this region in contributing a binding site for CR3
(22). Nevertheless, there is also considerable evidence that this is
not the only binding site in the C3 molecule for these ligands.
Blocking monoclonal antibody studies (49), as well as binding and
inhibition studies with proteolytic fragments (24, 55, 56) and
synthetic peptides (55) all implicate the C3d fragment as contributing
to these binding interactions. Given the functional homology among the
three I cofactors, together with the fact that CR1, factor H, and MCP
are competitive ligands of C3b (46), one might expect that they might
share a common, or at least partially overlapping, binding site in C3d.
The three-dimensional structure of human C3d has recently been solved
(57) and can now serve as a platform for a structure-guided mutagenic
analysis that will hopefully further define the C3d-resident
interactions sites for the various protein ligands of C3.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Grace Yeh of CytoMed, Inc., and Dr. Richard Smith of SmithKline Beecham for providing us with sMCP and sCR1, respectively.
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FOOTNOTES |
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* This work was supported by Grant MT-7081 from the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322.
To whom correspondence should be addressed: Dept. of
Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-2703; Fax: 416-978-8548; E-mail:
d.isenman{at}utoronto.ca.
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ABBREVIATIONS |
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The abbreviations used are: MCP, membrane cofactor protein; CCP, complement control protein; CR, complement receptor; CVF, cobra venom factor; DMEM, Dulbecco's modified Eagle's medium; DMEM/K76, DMEM containing K76-COOH-treated fetal calf serum; EAC, sheep erythrocytes coated with antibody and the indicated complement components or fragments thereof; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; PBS, phosphate-buffered saline; rC3, recombinant C3; RCA, regulators of complement activation; RIA, radioimmunoassay; SCR, short consensus repeat; sCR1, soluble CR1; sMCP, soluble MCP; VBS, veronal-buffered saline; PAGE, polyacrylamide gel electrophoresis; bp, base pair; Hu, human; Tr, trout; Xe, Xenopus.
2 In Fig. 1 and throughout the text, the standard convention for naming single and multiple mutations is employed. However, in order to be able to annotate subsequent figures within the restricted space available, it was necessary to adopt a non-standard but nevertheless unambiguous nomenclature system.
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REFERENCES |
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