From the Laboratoire d'Enzymologie
Moléculaire, ¶ Laboratoire de Spectrométrie de Masse
des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel
(CEA-CNRS), 41 Avenue des Martyrs, 38027 Grenoble Cedex 1, France
and the
University of Missouri School of Biological Sciences,
Kansas City, Missouri 64110-2499
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ABSTRACT |
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The Ca2+-dependent
interaction between complement serine proteases C1r and C1s is mediated
by their C11 (1) is the complex
modular protease that triggers the classical pathway of complement in
response to the formation of antigen-antibody complexes and to
infection by various microorganisms. The human C1 complex comprises
three types of proteins (C1q, C1r, and C1s) that form two distinct
entities: the recognition subunit C1q and the catalytic subunit
C1s-C1r-C1r-C1s, a Ca2+-dependent tetrameric
assembly of the two homologous but distinct serine proteases C1r and
C1s. Binding of C1 to the target microorganism is mediated by C1q and
triggers autocatalytic activation of C1r into C A particular feature of C1r and C1s is that they exert their catalytic
activities within the Ca2+-dependent
C1s-C1r-C1r-C1s complex. The non-covalent C1r-C1r homodimer forms the
core of the tetramer, with its catalytic regions in the center, and
each of its distal Previous studies performed on C1s have suggested that the structural
determinants required for Ca2+ binding and interaction with
C1r are contributed by both the N-terminal CUB and EGF modules (12,
13), an hypothesis that is supported by recent functional studies on
the recombinant CUB-EGF module pair (14). In the case of C1r, a
deletion mutant lacking the first CUB module was shown to lose the
ability to bind C1s in the presence of Ca2+ (15).
Conversely, the isolated EGF module of C1r, which exhibits the
consensus sequence pattern characteristic of the particular subset of
EGF modules involved in Ca2+ binding (16), retains the
ability to bind Ca2+ ions. However, compared with the whole
C1r Materials--
Diisopropyl phosphorofluoridate and trypsin from
bovine pancreas (treated with
1-chloro-4-phenyl-3-(L-tosylamido)-butan-2-one) were from
Sigma. Peptide:N-glycosidase F was purified from cultures of
Flavobacterium meningosepticum according to the method of
Tarentino et al. (19), modified as described by Aude
et al. (20). Polyclonal anti-C1r antiserum was raised in
rabbits according to standard procedures. Restriction enzymes were from
Boehringer Mannheim. VentR polymerase was from New England
Biolabs. The pHC1r3 plasmid containing full-length C1r cDNA (21)
was kindly provided by Dr Agnès Journet (Commissariat à
l'Energie Atomique, Grenoble, France). Antibiotics and molecular
biology reagents were from Appligene Oncor. Oligonucleotides were
obtained from Life Technologies, Inc. (Cergy-Pontoise, France).
Proteins--
Activated C1r and C1s were purified from human
plasma as described previously (22). Their C1r Cell Lines and Cell Culture Conditions--
The Spodoptera
frugiperda insect cells (Ready-Plaque Sf9 cells from
Novagen) were routinely grown and maintained in serum-free Sf900
II SFM medium (Life Technologies, Inc.) supplemented with 50 IU/ml
penicillin and 50 mg/ml streptomycin (Life Technologies, Inc.). The
Trichoplusia ni (High FiveTM) insect cells
(provided by Dr. Jadwiga Chroboczek, Institut de Biologie Structurale,
Grenoble) were maintained in TC100 medium (Life Technologies, Inc.)
containing 10% fetal calf serum (Dominique Dutscher SA, Brumath,
France) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin.
Pichia pastoris GS115 (his4) yeast cells
(Invitrogen) were propagated in buffered minimal glycerol complex
medium (BMGY) containing 100 mM potassium phosphate, pH
6.0, 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base
(without amino acids), 0.00004% biotin and 1% glycerol. Expression of
recombinant protein was induced in methanol complex medium (BMMY, same
composition as BMGY, with 0.5% methanol instead of glycerol). For
selecting P. pastoris transformants, MD plates containing
1% dextrose, 1.34% yeast nitrogen base, and 0.00004% biotin were
used. Mut+/Muts selection was performed on MM
plates (same composition as MD with 0.5% methanol instead of dextrose).
Cloning and Expression of the C1r CUB-EGF Fragment in a
Baculovirus/Insect Cell System--
A DNA fragment encoding the C1r
signal peptide plus the N-terminal CUB-EGF module pair (amino acids
1-175 of the mature C1r protein) was amplified by PCR using
VentR polymerase and pHC1r3 as a template, according to
established procedures. The sequences of the sense
(5'-CGGAATTCATGTGGCTCTTGTAC-3') and antisense
(5'-GGGGTACCCTACTCAGCCTGGCAGGA-3') primers
introduced an EcoRI site (underlined) at the 5' end of the
PCR product and a stop codon (boldface) followed by a KpnI site (underlined) at the 3' end. The amplified DNA was purified using
the Geneclean kit (Bio 101) and subcloned into the pCR-Script Amp SK(+)
intermediate vector (Stratagene) according to the manufacturer's instructions. The fragment was excised by digestion with
EcoRI and KpnI and cloned into the
EcoRI/KpnI sites of the pFastBac1 baculovirus
transfer vector (Life Technologies, Inc.). The resulting construct was
characterized by restriction mapping and checked by double-stranded DNA
sequencing (Genome Express, Grenoble, France).
The recombinant baculovirus was generated using the
Bac-to-BacTM system (Life Technologies, Inc.). The bacmid
DNA was purified using the Qiagen midiprep purification system (Qiagen
S.A., Courtaboeuf, France) and used to transfect Sf9 insect
cells with Cellfectin (Life Technologies, Inc.) in Sf900 II SFM
medium as described by the manufacturer. Recombinant virus particles
were collected 4 days later, titered by virus plaque assay, and
amplified as described by King and Possee (25).
High Five cells (1.75 × 107 cells/175-cm2
tissue culture flask) were infected with the recombinant virus at a
multiplicity of infection of 2 in Sf900 II SFM medium for
84 h at 28 °C. The supernatant was collected by centrifugation,
and diisopropyl phosphorofluoridate was added to a final concentration
of 1 mM.
Cloning and Expression of the C1r CUB Fragment in P. pastoris--
A DNA fragment encoding the CUB module of C1r (amino
acids 1-124 of the mature protein) was amplified by PCR using the
VentR polymerase and pHC1r3 as a template. The sequence of
the sense primer (5'-GGTACGTATCCATTCCCATCCCTC-3')
introduced an SnaBI restriction site (underlined) at the 5'
end of the PCR product and allowed in-frame cloning with the
The colony expressing the recombinant CUB fragment was grown overnight
in 10 ml of BMGY. This culture was inoculated into 150 ml of BMGY and
grown at 30 °C in a shaking incubator until the
A600 reached a value of 6.0. The cells were
collected by centrifugation at 1500 × g, resuspended
in 600 ml of BMMY, and cultured for 30 h, and pure methanol was
added to a final concentration of 0.5% after 24 h of induction.
The supernatant was separated from the cells by centrifugation, and
diisopropyl phosphorofluoridate added to a final concentration of 1 mM.
Purification of the Recombinant CUB-EGF and CUB
Fragments--
The culture supernatant containing the CUB-EGF fragment
was dialyzed against 50 mM NaCl, 1 mM
CaCl2, 50 mM triethanolamine hydrochloride, pH
8.5, and loaded at 1.5 ml/min onto a Q-Sepharose-Fast Flow column
(Amersham Pharmacia Biotech) (2.8 × 14 cm) equilibrated in the
same buffer. Elution was carried out by applying a 1.2-liter linear
gradient from 50 to 500 mM NaCl in the same buffer, and fractions containing the recombinant fragments were identified by
Western blot analysis.
The CUB-containing culture supernatant was dialyzed against 25 mM MES, pH 6.0, and loaded at 1 ml/min onto a
carboxymethylcellulose CM52 column (2.8 × 14 cm) (Whatman)
equilibrated in the same buffer. Elution was carried out by applying a
1-liter linear gradient from 0 to 150 mM NaCl in the same
buffer. Fractions containing the recombinant fragment were identified
by Western blot analysis.
Fractions containing the CUB-EGF or CUB fragments were pooled and
dialyzed against 1.0 M ammonium sulfate, 1 mM
CaCl2, 0.1 M triethanolamine hydrochloride, pH
7.4. Further purification of both fragments was achieved by high
pressure hydrophobic interaction chromatography on a TSK-phenyl 5PW
column (7.5 × 75 mm) (Beckman). Elution was carried out by
decreasing the ammonium sulfate concentration from 1.0 M to
0 in 20 min at a flow rate of 1 ml/min. The purified fragments were
dialyzed against 145 mM NaCl, 50 mM
triethanolamine hydrochloride, pH 7.4, and concentrated if necessary to
0.1 mg/ml by ultrafiltration on Microsep microconcentrators (molecular
weight cut-off = 3,000) (Filtron).
Chemical Characterization of Recombinant
Proteins--
N-terminal sequence analyses were performed after
SDS-PAGE and electrotransfer, using an Applied Biosystems model 477 A
protein sequencer as described previously (26). Mass spectrometry
analysis of the recombinant proteins was performed using the
matrix-assisted laser desorption ionization technique on a Voyager
Elite XL instrument (PerSeptive Biosystems, Cambridge, MA), under
conditions described previously (27).
Enzymic Deglycosylation of the Recombinant Proteins--
Fifty
µl of 10-fold concentrated culture supernatants containing the
CUB-EGF or CUB fragments were incubated with 0.5 µg of peptide:N-glycosidase F for 4 h at 25 °C.
Deglycosylation of the recombinant fragments was monitored by SDS-PAGE
and Western blot analysis of the samples. The purified CUB-EGF fragment
(0.1 mg/ml) in 145 mM NaCl, 50 mM
triethanolamine hydrochloride, pH 7.4, was incubated in the presence of
20% (w/w) peptide:N-glycosidase F for 20 h at
25 °C. Protein deglycosylation was monitored by SDS-PAGE analysis of
the samples.
Polyacrylamide Gel Electrophoresis and
Immunoblotting--
SDS-PAGE analysis was performed as described
previously (8). Western blot analysis and immunodetection of the
recombinant proteins were performed as described (28), using rabbit
polyclonal anti-C1r antiserum (1:400 dilution).
High Pressure Gel Permeation--
The C1r CUB-EGF and Real Time Surface Plasmon Resonance Measurements and Data
Evaluation--
Real time analysis of the
Ca2+-dependent interaction between C1r or its
fragments and C1s or C1s
Sensorgrams were analyzed by non-linear least squares curve fitting
using the BIAevaluation 2.1 Software (Amersham Pharmacia Biotech). A
single-site binding model was used for kinetic analysis of all
interactions. The equation Rt = R0 exp(
Whenever this was possible, i.e. when the binding reaction
reached or approached equilibrium, the association constant
(KA(eq)) was also determined from
equilibrium levels of the analyte binding to the surface
(Req). The equation
Req/C = KA(eq) × Rmax Fluorescence Measurements--
Intrinsic protein fluorescence
was measured at 20 °C using a SLM-Aminco Bowman Series 2 Luminescence Spectrometer at a photomultiplier voltage of 900 V with a
10-mm path length cell. The excitation wavelength was 275 nm, and the
band path was 4 nm. Emission spectra were recorded at a scanning rate
of 1 nm/s in the 285-355 nm range using a band path of 4 nm. All
measurements were corrected for buffer (145 mM NaCl, 50 mM triethanolamine hydrochloride, pH 7.4, containing 0.1 mM EGTA or 0.9 mM CaCl2) background emission.
Expression of the Recombinant C1r Fragments--
The modular
structures of human C1r and of the various fragments used in the
present study are depicted in Fig. 1. The
recombinant baculovirus for expression of the CUB-EGF fragment of C1r
was generated as described under "Experimental Procedures" and used to infect Sf9 and High Five insect cells for various periods at 28 °C. Secretion of the recombinant protein into the culture medium was monitored by SDS-PAGE and Western blot analysis, as illustrated in
Fig. 2. The anti-C1r antibody labeled a
band of apparent molecular mass 20 kDa in both cell culture
supernatants, a major, broader band of about 22 kDa in the case of High
Five cells (Fig. 2B), and two minor bands of 21 and 22 kDa
in the case of Sf9 cells (Fig. 2A). All bands became
detectable after 24 h of infection, and their intensity reached a
maximum at 96 h. Incubation of the 96-h Sf9 culture medium
with peptide:N-glycosidase F led to the disappearance of the
21- and 22-kDa bands, with a concomitant slight increase in the
intensity of the 20-kDa band (data not shown), indicating that the
latter represented an unglycosylated form of the CUB-EGF fragment. The
presence of unglycosylated material in the supernatant was clearly not
the result of the release of intracellular material from lysed infected
cells, as the relative amounts of the glycosylated and unglycosylated
forms was kept constant throughout the course of infection. Analysis of
protein production in the cell pellets showed the presence of
equivalent amounts of both forms for Sf9 and High Five insect
cells. The amount of unglycosylated fragment present in the 96-h
culture supernatant was estimated to be 1 µg/ml for both Sf9
and High Five cells. In contrast, the level of expression of the
glycosylated CUB-EGF fragment was about 5 times higher in High Five
cells (about 2.5 µg/ml) than in Sf9 cells. High Five cells
were therefore selected for subsequent production of the recombinant
CUB-EGF fragment.
Expression of the CUB fragment of C1r was carried out in the GS115
strain of the methylotrophic yeast P. pastoris using the pPIC9K expression vector, as described under "Experimental
Procedures." SDS-PAGE and Western blot analysis of the culture medium
and cell pellet revealed the presence of a single immunoreactive
protein of about 14 kDa that was totally secreted. Treatment of the
supernatant with peptide:N-glycosidase F induced no shift in
the electrophoretic mobility of the recombinant fragment, indicating
that it was likely unglycosylated. The amount of secreted CUB fragment
was estimated to be approximately 1.5 µg/ml culture medium.
Purification of the Recombinant C1r Fragments--
The culture
supernatant containing both the glycosylated and unglycosylated CUB-EGF
species was initially fractionated by anion-exchange chromatography in
the presence of 1 mM CaCl2. This step did not
allow separation of the two species, although the unglycosylated
fragment eluted slightly later than its glycosylated counterpart during
the ascending salt gradient. Final purification of the two species was
achieved by hydrophobic interaction chromatography. The glycosylated
and unglycosylated fragments eluted in that order after the end of the
descending ammonium sulfate gradient, indicating a slightly more
hydrophobic character of the unglycosylated fragment. The amounts of
purified proteins obtained were typically about 1.3 and 0.5 µg/ml
culture for the glycosylated and unglycosylated CUB-EGF species,
respectively. Both were very sensitive to aggregation and could not be
concentrated over 0.1 mg/ml.
The recombinant CUB fragment was purified by fractionation of the
culture supernatant by cation-exchange chromatography, followed by the
same hydrophobic interaction chromatography step as above. The CUB
fragment also was hydrophobic, as it eluted between the glycosylated
and unglycosylated CUB-EGF species and was prone to aggregation upon
concentration above 0.1 mg/ml. The amount of purified fragment obtained
was typically 0.5 µg/ml culture.
The recombinant fragments were essentially pure as judged from SDS-PAGE
analysis (Fig. 3). Each yielded a single
band, which was more diffuse in the case of the glycosylated CUB-EGF
fragment, with apparent molecular masses of 21.5 (glycosylated
CUB-EGF), 20 (unglycosylated CUB-EGF), and 14 kDa (CUB), consistent
with expected values. Incubation of the purified glycosylated CUB-EGF fragment with 7% (w/w) peptide:N-glycosidase F for 1 h
at 25 °C, i.e. under conditions known to remove both
N-linked oligosaccharides at positions 108 and 204 of C1r Chemical Characterization of the Recombinant C1r
Fragments--
Edman degradation of both purified CUB-EGF species
yielded a single sequence
Ser-Ile-Pro-Ile-Pro-Gln-Lys-Leu-Phe-Gly ... , corresponding to the
N-terminal end of C1r. The CUB fragment also yielded a single sequence
Tyr-Val-Ser-Ile-Pro-Ile-Pro-Gln-Lys-Leu ... , corresponding to the above sequence preceded by the two residues
Tyr-Val expected to be added at the N terminus, due to in-frame cloning
with the signal sequence of the yeast
Analysis by matrix-assisted laser desorption ionization mass
spectrometry of the various recombinant CUB-EGF species is summarized in Table I. Both the unglycosylated and
the enzymatically deglycosylated fragment exhibited a major peak
(approximately 70% of the total material) with a mass consistent with
the sequence of the N-terminal Ser1-Glu175 C1r
fragment (calculated mass = 19,790 Da). A minor peak with an
average extra mass of 164 ± 10 Da was consistently observed in
both cases. This difference probably accounts for covalent attachment
of a diisopropyl phosphate group (164 Da), considering that diisopropyl
phosphorofluoridate was present at all steps of the purification
procedure and that nonspecific labeling of the C1r interaction region
with radioactive diisopropyl phosphorofluoridate has been previously
reported (30). Analysis of the glycosylated CUB-EGF species yielded a
wide, heterogeneous peak comprising four major components (Table I),
with deduced mass values for the non-polypeptide component of the
fragment of 1,539 ± 10, 1,694 ± 10, 1,880 ± 10, and
2,016 ± 10 Da. The first three values are compatible with the
presence of an heterogeneous high mannose oligosaccharide comprising 2 N-acetylglucosamine and 7, 8, or 9 mannose residues
(calculated masses 1,542, 1,704, and 1,866 Da, respectively), whereas
additional binding of a diisopropyl phosphate group would account for
the highest value 2,016 ± 10 Da. Mass spectrometry analysis of
the CUB fragment yielded a single peak with a mass of 14,243 ± 7 Da, consistent with a sequence comprising residues Tyr-Val
followed by the N-terminal segment Ser1-Val124
of C1r (calculated mass = 14,243 Da).
In summary, mass spectrometry analyses clearly showed that the
recombinant CUB fragment was unglycosylated, whereas the CUB-EGF fragment was only partially glycosylated, with a heterogeneous high
mannose oligosaccharide attached to Asn108 within the CUB module.
Size-exclusion Chromatography--
The ability of the recombinant
CUB-EGF fragment to mediate Ca2+-dependent
interaction with the
Size-exclusion chromatography of the isolated glycosylated CUB-EGF pair
in the presence of CaCl2 (0.2 or 2.5 mM) or
EDTA indicated a 0.9-min Ca2+-induced shift of its elution
position (from 16.4 to 17.3 min). A similar but less important shift
(0.3 min) was observed in the case of C1r Effect of Ca2+ on Protein Intrinsic
Fluorescence--
With a view to detect potential conformational
modifications induced upon Ca2+ binding to the interaction
region of C1r, the intrinsic fluorescence spectra of different
fragments from this region were recorded in the presence of either EGTA
or CaCl2. As the Surface Plasmon Resonance Analysis of the Interaction between C1r
or Its Fragments and C1s or C1s
We next studied the Ca2+ dependence of the binding of C1r
or its fragments to immobilized C1s. Sensorgrams were recorded at different Ca2+ concentrations, and resonance units at
equilibrium (Req) were determined for each
analyte (C1r, C1r
Further experiments were aimed at determining the kinetic parameters of
the C1r/C1s interaction. This was achieved by recording sensorgrams at
varying protein concentrations with a fixed, saturating CaCl2 concentration of 250 µM. Fig.
7A shows the association and dissociation curves from a representative series of experiments performed with five C1r concentrations (13-103 nM). The
association phase was analyzed by nonlinear least squares fitting as
described under "Experimental Procedures" to yield
ks values at each concentration. A plot of
ks versus C1r concentration produced a
straight line (Fig. 7B) with a slope corresponding to the
association rate constant kon (Table
II). The dissociation phase (415-675 s)
was also analyzed by nonlinear least squares curve fitting as described
under "Experimental Procedures" to yield the dissociation rate
constant koff (Table II). The apparent equilibrium dissociation constant KD determined from the (koff/kon) ratio was
10 nM. This experiment was repeated on two different chips,
and the resulting mean KD value was 10.9 ± 0.9 nM (Table II). As Ca2+-dependent
binding of C1r to C1s is known to involve the C1s interaction region, a
similar experiment was performed using the
Binding of C1r
The fact that C1r Baculovirus/insect cells systems have been used previously to
express human complement proteases C1r (31) and C1s (32) and more
recently to produce C1r deletion mutants (15) and modular fragments
from the catalytic and interaction regions of C1s (14, 28).
Baculovirus-mediated expression was used in the present study to
produce the N-terminal CUB-EGF module pair of human C1r, and
unexpectedly, this fragment was secreted under both glycosylated and
unglycosylated forms. The coexistence of both forms in the culture
medium as well as in the cell lysate indicates that glycosylation is
not an absolute prerequisite for secretion of the CUB-EGF fragment by
insect cells, in contrast to what has been observed for expression of
human decorin (33). It should also be mentioned that a similar expression pattern, with a mixture of glycosylated and unglycosylated species, was obtained when the signal peptide of human C1r was replaced
by that of honey bee
melittin.2 Indeed,
inefficient glycosylation of the CUB-EGF fragment does not appear to be
inherent in the baculovirus/insect cells expression system, as the
recombinant protein secreted by the P. pastoris yeast was
totally unglycosylated (34), as was the recombinant CUB fragment
produced in the present study. A likely explanation is therefore that
the observed inefficiency of the glycosylation at Asn108
mainly results from a lack of accessibility of this particular site,
both in the isolated CUB fragment and in the CUB-EGF pair. However, the
fact that the unglycosylated CUB-EGF fragment had the correct
N-terminal sequence and that its functional properties were
indistinguishable from those of its glycosylated counterpart indicates
that the lack of carbohydrate at Asn108 in the CUB module
does not significantly affect the folding of the fragment. In the same
way, the presence of an oligosaccharide chain had no significant effect
on the solubility of the CUB-EGF fragment. In this respect, it should
be mentioned that the solubility limit of the CUB-EGF pair (about 0.1 mg/ml) was about 6 times lower than that of the larger tryptic fragment
C1r Surface plasmon resonance spectroscopy has been used successfully to
study various Ca2+-dependent interactions, such
as the binding of recoverin to phospholipids (35), of calpain to
calpastatin (36, 37), and of tissue factor to coagulation factor VIIa
(38-40). This technique was used in the present work to study
Ca2+-dependent binding of C1r and its fragments
to C1s. The data obtained with intact C1r and C1s indicate that the
proteins associate in the presence of Ca2+ with high
affinity, with a KD ranging from 10.9 (immobilized C1s) to 29.7 nM (immobilized C1r), close to the value (32 nM) determined previously by tracer sedimentation
equilibrium (41). Based on the KD values determined
in the present study, and on the physiological serum concentrations of
C1r and C1s (about 0.2 and 0.4 µM, respectively (2)), it
may be anticipated that virtually all of the C1r and C1s molecules are
associated within the Ca2+-dependent
C1s-C1r-C1r-C1s tetrameric complex in normal sera.
Both size-exclusion chromatography and surface plasmon resonance
spectroscopy studies show that, as previously found for the larger
proteolytic fragment C1r It appears clear that although the soluble CUB-EGF module pair does
bind immobilized C1s in the presence of Ca2+, its binding
affinity shows an important decrease compared with intact C1r, mainly
because of an increased tendency of the CUB-EGF·C1s complex to
dissociate. A possible explanation is that the CUB-EGF pair lacks
accessory ligand(s) that would provide further stabilization of the
interaction within intact C1r. As C1r Analysis of the interaction between soluble C1s Our study of the Ca2+ dependence of the interaction between
C1r or its fragments and immobilized C1s shows that half-maximal binding occurs at Ca2+ concentrations ranging from 5 µM in the case of C1r to 10-16 µM for the
various fragments. These values are very close to one another and are
in good agreement with the KD values (32-38
µM) determined previously by equilibrium dialysis for
Ca2+ binding by fragments C1r The size-exclusion chromatography and fluorescence measurements
performed in this work both strongly support the hypothesis that
binding of Ca2+ to the CUB-EGF fragment induces a more
compact conformation of this module pair, a process that is also
observed in the C1r regions, encompassing the major part of their N-terminal
CUB-EGF-CUB (where EGF is epidermal growth factor) module array. In
order to define the boundaries of the C1r domain(s) responsible for
Ca2+ binding and Ca2+-dependent
interaction with C1s and to assess the contribution of individual
modules to these functions, the CUB, EGF, and CUB-EGF fragments were
expressed in eucaryotic systems or synthesized chemically. Gel
filtration studies, as well as measurements of intrinsic Tyr
fluorescence, provided evidence that the CUB-EGF pair adopts a more
compact conformation in the presence of Ca2+.
Ca2+-dependent interaction of intact C1r with
C1s was studied using surface plasmon resonance spectroscopy, yielding
KD values of 10.9-29.7 nM. The C1r
CUB-EGF pair bound immobilized C1s with a higher KD
(1.5-1.8 µM), which decreased to 31.4 nM when CUB-EGF was used as the immobilized ligand and C1s was free. Half-maximal binding was obtained at comparable Ca2+
concentrations ranging from 5 µM with intact C1r to
10-16 µM for C1r
and CUB-EGF. The isolated CUB and
EGF fragments or a CUB + EGF mixture did not bind C1s. These data
demonstrate that the C1r CUB-EGF module pair (residues 1-175) is the
minimal segment required for high affinity Ca2+ binding and
Ca2+-dependent interaction with C1s and
indicate that Ca2+ binding induces a more compact folding
of the CUB-EGF pair.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
r, which in turn
converts C1s into C
s, the protease responsible for enzymic
activity of C
(2-4). Both steps of the C1 activation process
occur through cleavage of a single peptide bond in the proenzymes,
generating active proteases comprising two disulfide-linked chains. C1r
and C1s are modular proteins exhibiting homologous overall structures
comprising, from the N terminus, two CUB modules (5) surrounding a
single EGF-like module, two CCP modules (6), a connecting segment
homologous to the activation peptide in chymotrypsinogen, and a serine
protease domain. In the same way, each monomeric protease is thought to be organized in two functional regions, a C-terminal catalytic region
and an N-terminal (
) interaction region.
regions is connected in a Ca2+-dependent fashion to the homologous region
of a C1s molecule. N-terminal fragments corresponding to the
regions of C1r and C1s, each comprising the first CUB module, the
EGF-like module, and a small N-terminal portion of the following second
CUB module, have been obtained by limited proteolysis with trypsin in
the presence of Ca2+ (7, 8). Each of these fragments
contains a single high affinity Ca2+-binding site and
retains the ability to mediate Ca2+-dependent
heterologous (C1r/C1s) interaction (8). The
regions of C1r and C1s
both exhibit similar low temperature transitions, with midpoints of
26-31 °C, which are shifted upward at high ionic strength or in the
presence of Ca2+ ions (7, 9). The
regions of both C1r
and C1s also likely participate in the interaction between the
C1s-C1r-C1r-C1s tetramer and C1q and therefore represent key elements
of the architecture of the C1 complex (10, 11). In addition to their
structural role, various studies suggest that the
regions of C1r
may control autoactivation of the catalytic regions of the protease
(11).
fragment, its affinity for Ca2+ is decreased about
300-fold (17, 18), strongly suggesting that Ca2+ binding by
C1r involves residues located outside the EGF module. The objective of
the present study was to measure the ability of the N-terminal CUB-EGF
module pair of C1r to bind Ca2+ ions and to mediate
Ca2+-dependent interaction with C1s, as well as
to assess the contribution of the individual CUB and EGF modules to
these functions. For this purpose, the CUB module and CUB-EGF pair were
expressed in eucaryotic systems, and the EGF module was synthesized
chemically. Comparative analysis of the physicochemical and functional
properties of these recombinant molecules indicates that the CUB-EGF
module pair binds Ca2+ with high affinity and mediates
Ca2+-dependent interaction with C1s and
supports the hypothesis that Ca2+ induces a more compact
conformation of this domain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and C1s
fragments
were obtained by limited proteolysis with trypsin and purified as
described previously (8). The EGF-like module from C1r was synthesized chemically and characterized as described (17). The concentrations of
purified proteins were determined using the following absorption coefficients (A1 cm1% at 280 nm) and molecular weights as follows: dimeric C1r, 12.4 and 172,600 (8); C1s, 14.5 and 79,800 (8, 23); C1r
, 7.2 and 27,600 (8); and
C1s
, 10.0 and 24,200 (8). The absorption coefficients
(A1 cm1% at 280 nm) used for
the C1r EGF-like module (7.0), the recombinant fragments CUB (6.4),
aglycosylated CUB-EGF (6.7), and glycosylated CUB-EGF (6.2) were
calculated from the number of Trp, Tyr, and disulfides by the method of
Edelhoch (24), using molecular weights of 5,970 (17), 14,243, 19,790, and 21,500, respectively, as determined by mass spectrometry analysis
(see "Results").
factor
yeast secretion signal. The antisense primer
(5'-GGCCTAGGCTACACAGCTTGGTAGTAGGC-3') introduced
a stop codon (boldface) followed by an AvrII restriction site (underlined) at the 3' end of the PCR product. The purified amplified DNA fragment was subcloned into the pCR-Script Amp SK(+) intermediate vector (Stratagene), excised with SnaBI and
AvrII, and ligated into the corresponding restriction sites
of the pPIC9K yeast expression vector (Invitrogen). The final construct
was submitted to double-stranded DNA sequencing. Ten µg of
recombinant plasmid DNA were linearized with SacI and used
for transformation of P. pastoris GS115 cells by
electroporation as described in the user's manual of the
Pichia Expression kit (Invitrogen). Histidine-independent transformants were selected on MD plates, replicated on MM plates, and
allowed to grow for 3 days at 30 °C. Pure methanol (100 µl) was
supplied every 24 h inside the lid of the plates. The colonies were then screened for CUB expression by overlaying the plates with a
0.2-µm nitrocellulose disc (Schleicher & Schuell) overnight at
30 °C and probing it with polyclonal anti-C1r antibodies. Screening for Mut+/Muts phenotypes indicated that the
clone yielding the strongest CUB immunoreactive signal was
Mut+. An optimal methanol induction time of 30 h for
liquid medium expression was determined for this clone as described in
the Pichia Expression kit manual (Invitrogen).
fragments (190 pmoles each), either alone or in equimolar mixture with
C1s
, were analyzed by high pressure gel permeation on a TSK G3000
SWG column (7.5 × 600 mm) (Toso Haas) equilibrated in 145 mM NaCl, 50 mM triethanolamine hydrochloride, pH 7.4, containing EDTA (2.5 mM) or CaCl2 (2.5 or 0.2 mM), and run at 1 ml/min. Sample volumes ranged from
42 to 56 µl. Proteins were detected from their absorbance at 280 nm.
was performed at 25 °C using an upgraded
BIAcoreTM instrument (BIAcore AB). The running buffer for
protein immobilization was 145 mM NaCl, 5 mM
EDTA, 10 mM HEPES, pH 7.4. Protein ligands were diluted to
10-20 µg/ml either in 10 mM formate, pH 3.0 (C
r, C
s, and C1s
), or in 10 mM acetate, 10 mM NaCl, pH 4.8 (CUB-EGF and unglycosylated CUB-EGF), and
coupled to the carboxymethylated dextran surface of a CM5 sensor chip
(BIAcore AB) using the amine coupling chemistry (BIAcore AB amine
coupling kit) according to the manufacturer's instructions. Binding of
C1r and its fragments was measured over 530 and 3100 resonance units
(RU) of immobilized C1s (or 150 and 600 RU of C1s
), at a flow rate
of 10 µl/min in 145 mM NaCl, 0.25 mM
CaCl2, 50 mM triethanolamine hydrochloride, pH
7.4. Binding of C1s was measured under the same conditions as above,
over 1000 RU of immobilized C1r, 150 and 450 RU of CUB-EGF, or 350 RU
of unglycosylated CUB-EGF. Binding of C1s
was measured over 3200 RU
of immobilized C1r. Equivalent volumes of each protein sample were
injected over a surface with immobilized bovine serum albumin (instead
of C1s or C1r) or ovalbumin (instead of C1s
or CUB-EGF) to serve as
blank sensorgrams for subtraction of bulk refractive index background.
Regeneration of the surfaces was achieved by injection of 10 µl of 20 mM EDTA. The effect of Ca2+ concentration on
the interaction between C1r or its fragments and C1s was studied in 145 mM NaCl, 50 mM triethanolamine hydrochloride, pH 7.4, containing 1 mM EGTA and varying amounts of
CaCl2 calculated to give the desired free calcium
concentrations as described (29).
koff
(t
t0)) was used for the
dissociation phase, where Rt is the amount of ligand
(in RU) remaining bound at time t, and t0 is the beginning of the dissociation phase.
The final dissociation rate constant koff was
calculated from the mean of the values obtained from a series of
injections. To analyze the association phase, the equation
Rt = Req(1
exp(
ks(t
t0))) was employed, where
Req is the amount of bound ligand (in RU) at
equilibrium, t0 is the starting time of
injection, and ks = kon × C + koff, where C is the
concentration of analyte injected over the sensor chip surface. The
association rate constant kon was determined
from the slope of a plot of ks versus
C, based on a series of at least five analyte
concentrations. The apparent equilibrium dissociation constant
(KD(kin)) was calculated from the ratio
of these two kinetic constants
(koff/kon).
KA(eq) × Req was
used, where Rmax is the maximal binding capacity
of the immobilized ligand, and the association constant KA(eq) was determined from the slope of
a Scatchard plot of Req/C
versus Req. The dissociation constant
derived from KA(eq) is referred to in
the text as KD(eq).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparative representation of the modular
structures of human C1r and of the various fragments used in this
study. The nomenclature and symbols used for protein modules are
those defined by Bork and Bairoch (1). Ser Pr, serine
protease domain. The arrow indicates the
Arg208-Val209 bond cleaved by trypsin (6). The
only disulfide bridge shown is that connecting the activation peptide
to the serine protease domain. , oligosaccharides linked to
Asn108 and Asn204.
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Fig. 2.
Time course of the secretion of the
recombinant CUB-EGF fragment from infected Sf9 and High Five
insect cells. Sf9 (A) and High Five
(B) cells were infected with the recombinant baculovirus at
a multiplicity of infection of 2. Culture supernatants (A,
250 µl; B, 125 µl) were collected at various times,
concentrated 10 times, and submitted to SDS-PAGE and Western blot
analysis. Lanes 1-4, supernatants collected at 96, 72, 48, and 24 h, respectively; lane 5, 96-h supernatant of
mock- infected cells. All samples were analyzed under non-reducing
conditions. Molecular masses of standard proteins (expressed in kDa)
are shown on the left side of the blots.
(8), did not yield significant deglycosylation. Incubation of the
fragment with 20% (w/w) enzyme for 20 h at 25 °C led to the
appearance of a 20-kDa deglycosylated species, but deglycosylation
remained partial (~50%) under these conditions. No sign of
proteolytic degradation of the recombinant proteins was detected by
SDS-PAGE analysis upon storage of the recombinant proteins for several
weeks at 0 °C.
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Fig. 3.
SDS-PAGE analysis of the recombinant
proteins. Lane 1, standard proteins (molecular masses
expressed in kDa); lane 2, CUB-EGF fragment, glycosylated (5 µg); lane 3, CUB-EGF fragment, unglycosylated (5 µg);
lane 4, CUB fragment (7.5 µg). All samples were analyzed
under reducing conditions.
-factor (see "Experimental
Procedures").
Mass spectrometry analysis of the recombinant CUB-EGF fragments
fragment of C1s was first investigated using
high pressure size-exclusion chromatography, as described previously
for the C1r
fragment (8). When mixtures containing equimolar amounts
of C1s
and either C1r
or the glycosylated CUB-EGF fragment were
applied to the gel filtration column in the presence of 2.5 mM CaCl2, the peaks corresponding to C1r
or
CUB-EGF (retention times 15.2 and 17.3 min, respectively) disappeared to yield peaks with respective retention times of 14.1 and 14.3 min.
The observed shifts and the relative intensity of the resulting peaks
were consistent with the formation of a (CUB-EGF)-C1s
heterodimer, analogous to the previously observed C1r
-C1s
complex (8). Identical results were obtained when the CaCl2
concentration was decreased to 0.2 mM. Formation of such a
heterodimer was also observed with the unglycosylated CUB-EGF fragment,
although no significant peak was eluted when the isolated fragment was
injected on the column, indicating that most of the unglycosylated
species was likely adsorbed to the matrix, as previously observed with deglycosylated C1r
(8).
. This observation showed
that the CUB-EGF and C1r
fragments behaved in a similar way with
respect to Ca2+ binding and suggested that the CUB-EGF pair
adopts a more compact conformation upon Ca2+ binding. No
evidence for complex formation between the isolated CUB and EGF
fragments was obtained when an equimolar mixture of both fragments was
applied to the column in the presence of 2.5 mM
CaCl2.
region of C1r contains no tryptophan
residue, the intrinsic fluorescence is contributed only by the 8, 3, 10, and 15 tyrosine residues present in the CUB, EGF, CUB-EGF, and
C1r
fragments, respectively. As depicted in Fig.
4, maximum emission occurred at 306 nm
for C1r
and at about 301 nm for the other fragments, and no
significant shift in the wavelength was observed in the presence of
Ca2+. In contrast, Ca2+ induced a small
(7-10%) but highly reproducible decrease in the fluorescence
intensity of C1r
(Fig. 4A) and both the glycosylated and
unglycosylated CUB-EGF fragments (Fig. 4B). The
isolated CUB and EGF modules, and an equimolar mixture of these
fragments, exhibited no detectable fluorescence change in the presence
of calcium (Fig. 4C). These experiments were consistent with
a Ca2+-induced quenching of intrinsic tyrosine fluorescence
within the CUB-EGF module pair, suggesting shielding of one or more
tyrosine residues, an effect that could not be reproduced from a
mixture of the individual CUB and EGF modules.
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Fig. 4.
Intrinsic fluorescence emission spectra of
various fragments from the C1r region.
Effect of calcium. Fluorescence spectra of C1r fragments were recorded
in 145 mM NaCl, 50 mM triethanolamine
hydrochloride, pH 7.4, in the presence of 0.1 mM EGTA (---)
and after addition of CaCl2 to a final concentration of 0.9 mM (···). A, C1r
fragment (2.6 µM); B, recombinant CUB-EGF
fragment, unglycosylated (2.7 µM; curve
1), and glycosylated (1.8 µM; curve 2);
C, CUB fragment (3.5 µM; curve 1),
equimolar mixture of CUB and EGF fragments (2.5 µM each;
curve 2), EGF fragment (5 µM; curve
3).
--
The ability of C1r and its
fragments to bind C1s in the presence of Ca2+ was studied
using surface plasmon resonance spectroscopy. As shown in Fig.
5A, both C1r and C1r
bound
to immobilized C1s in the presence of 1 mM
CaCl2, as shown by the increase in the resonance units
during the association phase of the sensorgrams. However, when the
running buffer was substituted for the analyte, the proteins exhibited
very different dissociation phases; only very slow dissociation was
observed in the case of C1r, whereas almost complete dissociation of
C1r
occurred over the same period. As expected in view of the
Ca2+ requirement of the C1r/C1s interaction, bound C1r
could be eluted at the end of the dissociation phase by a pulse
injection of EDTA, and no binding of the C1r
fragment was observed
when the running buffer contained 2 mM EDTA instead of
CaCl2. Binding of the glycosylated and unglycosylated C1r
CUB-EGF fragments to C1s was then tested in the same way, and the
resulting sensorgrams were quite comparable to that observed for
C1r
, as shown in Fig. 5B. In contrast, no binding to C1s
was observed in the case of the isolated CUB or EGF modules (Fig.
5B) or for an equimolar mixture of these fragments (not
shown). In the same way, the CUB + EGF mixture was unable to compete
with either C1r or the CUB-EGF fragment for binding to C1s, even at
molar ratios of 32:1 and 7:1, respectively. In contrast, prior
incubation of the C1r CUB-EGF and
fragments with the
fragment
of C1s (1:1 molar ratio) almost abolished their ability to bind to
immobilized C1s. These results clearly indicated that the C1r CUB-EGF
module pair mediates specific, Ca2+-dependent
interaction with the C1s
region, in agreement with gel filtration
experiments.
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Fig. 5.
Analysis by surface plasmon resonance
spectroscopy of the interaction between C r or various C1r
fragments and immobilized C
s. C
s was immobilized on
the sensor chip as described under "Experimental Procedures." Fifty
µl of each analyte were injected in the running buffer containing 1 mM CaCl2, at a flow rate of 10 µl/min. The
plots are as follows from top to bottom:
A, C
r (100 nM) and C1r
fragment (1 µM); B, recombinant CUB-EGF, glycosylated (1.6 µM), recombinant CUB-EGF, unglycosylated (1 µM), recombinant CUB (2.8 µM), chemically
synthesized EGF (3 µM). The sensor chip was regenerated
after each experiment by a 10-µl injection of 20 mM EDTA.
The specific binding signal shown was obtained by subtracting the
background signal as described under "Experimental
Procedures."
, glycosylated, and unglycosylated CUB-EGF) by
fitting the data as described under "Experimental Procedures."
Binding of the four proteins to C1s increased with Ca2+
concentration to reach a plateau, at Ca2+ concentrations
ranging from 100 µM for C1r to 300 µM for
the fragments. Increasing the Ca2+ concentration to 10 mM resulted in reduced binding, possibly due to
Ca2+-induced aggregation of C1r and its fragments (8). No
binding of the fragments was observed at Ca2+
concentrations below 100 nM, but residual C1r binding was
still observed in the absence of Ca2+. In order to
determine the Ca2+ concentration yielding half-maximal
binding (EC50), Req values were
normalized for each fragment to the maximal value
(Req(max)) obtained at 300 µM
CaCl2 (Fig. 6). Half-maximal
Ca2+-dependent binding to C1s was found to
occur at similar CaCl2 concentrations, namely 5 µM for C1r, and 10, 16, and 14 µM for C1r
, the glycosylated and unglycosylated CUB-EGF fragments,
respectively.
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Fig. 6.
Ca2+ dependence of the
interaction between C r or its fragments and C
s.
Sensorgrams were recorded in the running buffer containing 1 mM EGTA and varying amounts of CaCl2 to give
free Ca2+ concentrations ranging from 10 nM to
1 mM, as described under "Experimental Procedures." One
hundred µl of 90 nM C
r (
), 1 µM
C1r
(
), 1 µM CUB-EGF, glycosylated (
), and 750 nM CUB-EGF, unglycosylated (
), were injected over
immobilized C
s at a flow rate of 10 µl/min.
Req values were determined from the association
phases of the sensorgrams as described under "Experimental
Procedures." These values were normalized for each analyte to the
maximal value obtained at 300 µM CaCl2 and
plotted as a function of the free Ca2+ concentration.
fragment of C1s
immobilized on the sensor chip. As shown in Table II, comparable kon values were obtained for both immobilized
ligands, whereas the koff increased about 2-fold
in the case of C1s
, resulting in a 2-fold increase in the apparent
KD value.
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Fig. 7.
Kinetics of C r binding to immobilized
C
s. A, representative sensorgrams (after
background subtraction) illustrating the binding of C
r
(bottom to top curves: 13, 25, 38, 77, and 103 nM) to immobilized C
s (530 RU) in the running buffer
containing 250 µM CaCl2; B,
concentration dependence of ks values determined by
nonlinear regression analysis of the association data from
A. The slope of the curve obtained by linear transformation
gives the kon value.
Kinetic and dissociation constants for the interaction between C1r or
its fragments and C1s or C1s
, and of the glycosylated and unglycosylated C1r
CUB-EGF fragments to C1s, was then analyzed by recording sensorgrams at
various protein concentrations in the presence of 250 µM
CaCl2. A representative experiment with five concentrations
of the glycosylated CUB-EGF fragment (225 nM to 1.2 µM) is shown in Fig.
8A. Compared with C1r, both
the association and dissociation phases for the three fragments were
fitted less satisfactorily using monoexponential equations. However,
this method allowed us to estimate kon and koff values, from which KD
values ranging from 1.2 to 1.6 µM were derived (Table
II). As the binding of the fragments approached equilibrium, the
affinity of the interactions was also estimated by determining the
equilibrium dissociation constant from a Scatchard plot of
Req/C versus
Req, as described under "Experimental
Procedures." A representative plot for the glycosylated CUB-EGF
fragment is shown in Fig. 8B. KD values,
calculated from two independent experiments for each analyte, were
2.2 ± 1.0, 1.8 ± 0.6, and 1.5 ± 0.1 µM
for C1r
, the glycosylated and unglycosylated CUB-EGF pair,
respectively (Table II). These values were fully consistent with those
estimated using monoexponential equations (Table II) and
comparable for the three C1r fragments but strikingly differed from the
KD value determined for intact C1r by a factor of
150-200. Interaction of the above three fragments with immobilized
C1s
was also observed, and the shape of the binding curves was
similar to that obtained with immobilized C1s.
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Fig. 8.
Kinetic analysis of the binding of CUB-EGF to
immobilized C s. A, representative sensorgrams
(after background subtraction) illustrating binding of the glycosylated
CUB-EGF fragments (bottom to top curves: 225, 400, 575, 750, and 1,200 nM) to immobilized C
s
(3,100 RU) in the running buffer containing 250 µM
CaCl2; B, Scatchard plot analysis of the data.
Sensor responses at equilibrium (Req) were
determined for each protein concentration from A, and
Req/C values were plotted as a
function of Req. The slope of the curve obtained
by linear transformation yields the association constant
KA.
and CUB-EGF showed a decreased affinity for C1s
suggested that these fragments exhibited a decreased stability compared
with intact C1r. To test this hypothesis, the CUB-EGF fragment itself
was immobilized, whereas C1s was used as the soluble analyte. Under
this configuration, C1s was found to bind readily to the C1r CUB-EGF
fragment (Fig. 9A), and
analysis of the data obtained at varying C1s concentrations by plotting
Req/C versus Req (Fig. 9B) yielded a mean
KD of 31.4 ± 4.2 nM, in agreement
with the value estimated from monoexponential equations (Table
II). Slightly higher KD values (48.5-56
nM) were determined for C1s when the unglycosylated CUB-EGF
species was immobilized (Table II). Control experiments using C1s as
the analyte and intact C1r as the immobilized ligand allowed us to
determine a KD of 29.7 nM, close to the
value obtained above for the interaction between C1s and the
immobilized CUB-EGF fragment (Table II). Further experiments were aimed
at measuring the ability of the soluble fragment C1s
to bind to
immobilized C1r. Under this configuration, the KD of
the interaction was estimated at 133-157 nM, indicating a
5-fold decrease of the affinity of C1s
compared with intact C1s
(Table II), much smaller than the factor of 150-200 determined in the
case of the C1r CUB-EGF fragment. Taken together, these data suggested
that the large decrease of the affinity of CUB-EGF in solution was
mainly due to a decreased stability and that covalent attachment of the
fragment to the surface of the sensor chip stabilized it in a
conformation appropriate for optimal binding affinity.
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Fig. 9.
Kinetic analysis of the binding of C s
to the immobilized CUB-EGF fragment. A, representative
sensorgrams (after background subtraction) illustrating binding of
C
s (bottom to top curves: 34.8, 46.5, 69.7, 93, and 139 nM) to the immobilized CUB-EGF fragment
(450 RU) in the running buffer containing 250 µM
CaCl2. B, Scatchard plot analysis of the data,
performed as described in the legend to Fig. 8.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Also, complete enzymatic deglycosylation of C1r
could be
achieved (8), whereas the recombinant CUB-EGF fragment could only be
partially deglycosylated using large amounts of
peptide:N-glycosidase F. The above differences suggest that
the C-terminal part of C1r
, corresponding to the N-terminal end of
the second CUB module (residues 176-208) and including another
oligosaccharide linked to Asn204, has a stabilizing effect
on the CUB-EGF module pair. Removal of the C-terminal region of C1r
could unmask hydrophobic areas and thereby decrease the solubility of
the CUB-EGF fragment. This may possibly also reduce the accessibility
to peptide:N-glycosidase F of the Asn108-linked
oligosaccharide in the first CUB module.
(8), the CUB-EGF module pair itself
essentially retains the ability of intact C1r to bind to the
region
of C1s in the presence of Ca2+ ions. However, the latter
technique clearly shows that the affinity of both fragments C1r
and
CUB-EGF for immobilized C1s is 150-200 times lower than that of intact
C1r. It is clear from the koff values of the
interactions that most of the difference arises from the dissociation
phase, which proceeds very slowly in the case of C1r
(koff = 3.7 × 10
4
s
1) and about 30 times faster in the case of the
fragments (koff = 0.9-1.4 × 10
2 s
1) (see Table II). It may be argued
that this difference arises from the fact that intact C1r is a dimer,
whereas both its
and CUB-EGF fragments are monomers. However, the
possibility that a single C1r-C1r dimer could simultaneously bind two
C1s molecules appears unlikely because of the low density of
immobilized C1s used, and considering that our data of the interaction
between C1r and C1s satisfactorily fitted a single-site binding model when either protein was immobilized. Nevertheless, it appears likely
that the koff value for the C1r/C1s interaction
was slightly underestimated when C1r was used as the soluble analyte,
considering that a 3-fold increase was observed when C1r was
immobilized (see Table II). In any case, as discussed above, the
KD values determined in the present study for the
interaction between intact C1r and C1s are similar to that obtained
previously by a different method (41), and a value in the nanomolar
range is fully consistent with formation of a tight C1s-C1r-C1r-C1s
tetramer in serum.
shows a similar decreased
affinity for immobilized C1s, such ligands could be located within the
second CUB module, in the region C-terminal to the cleavage site by
trypsin (see Fig. 1). Alternatively, the missing part of the second CUB
module may not be directly involved in the interaction per
se but could simply stabilize the structure of the preceding
CUB-EGF pair and hence tighten the interaction. As discussed above,
C1r
itself shows a significantly increased solubility compared with
CUB-EGF, suggesting a stabilizing effect of the N-terminal segment of
the second CUB module, but further stabilization of the module pair may
require the remainder of the second CUB module. Indeed, our binding
studies using CUB-EGF as the immobilized ligand clearly show that,
under these conditions, this fragment recovers a binding affinity for
C1s that is very close to that of the intact C1r molecule. A likely
hypothesis is therefore that the isolated CUB-EGF fragment of C1r
contains all of the ligands required for efficient binding to C1s but
exhibits a decreased stability due to the lack of contacts normally
occurring with the remainder of the protein.
and immobilized C1r
shows that C1s
also exhibits a decreased affinity compared with
intact C1s. However, this decrease is 30-40 times less than that
observed in the case of C1r CUB-EGF and is contributed by both a
decrease in kon and an increase in
koff (see Table II). In this respect, it should
be pointed out that the C1s CUB-EGF fragment was found to be soluble at
a concentration of 10 mg/ml (14), i.e. a value that is 100 times higher than the estimated solubility limit of C1r CUB-EGF (see
"Results"). In view of this large difference, it appears likely
that the postulated instability of the C1r CUB-EGF fragment in solution
thought to be responsible for its decreased binding affinity is related
to its poor solubility and to its observed tendency to form aggregates
at high concentration. This observation appears consistent with the
above hypothesis that the CUB-EGF moiety of C1r makes contacts with
other parts of the protein.
and C1s
(8). Thus,
whereas the C1r EGF module alone binds Ca2+ very poorly
(KD = 10 mM) (12, 18), both C1r
and CUB-EGF bind Ca2+ with an affinity comparable to that of
intact C1r. It may be concluded therefore that the C1r CUB-EGF module
pair, alone, contains all of the ligands that participate in
Ca2+ binding and that the observed decreased affinity of
C1r
and CUB-EGF for C1s is not a consequence of a decreased affinity
for Ca2+ ions.
fragment. This hypothesis is fully consistent
with previous data indicating that Ca2+ ions stabilize the
C1r
fragment, shifting upwards the midpoint of its melting
transition by more than 20 °C (7). Formation of a compact
Ca2+-dependent CUB-EGF assembly is also
reminiscent of data obtained with the N-terminal Gla-EGF-EGF and
Gla-EGF modular fragments from blood coagulation factors IX (42) and X
(43), respectively. In the former case, size-exclusion chromatography
and spectroscopic measurements indicated a more compact conformation of
the Gla-EGF-EGF fragment in the presence of Ca2+, and a
Ca2+-dependent complex between the isolated Gla
module and EGF-EGF pair could be reconstituted from a mixture of these
fragments (42). This ability to assemble spontaneously in the presence of Ca2+ is not shared by the isolated C1r CUB and EGF
modules, as judged by both size-exclusion chromatography and
fluorescence spectroscopy. In the same way, a CUB + EGF mixture was
unable to mediate Ca2+-dependent C1s binding or
even to compete for C1s binding. In the case of factor X, small angle
x-ray scattering studies showed a contraction of the Gla-EGF module
pair upon addition of Ca2+, and NMR spectroscopy indicated
that the two modules move toward each other using the
Ca2+-binding site as a hinge (43). The authors proposed
that a general property of EGF modules exhibiting the consensus
sequence for Ca2+ binding may be the ability to induce an
intermodule orientation compatible with biological activity, an
hypothesis that is supported by recent studies on coagulation factor
VII suggesting that the Ca2+-binding site in the N-terminal
EGF module may stabilize the orientation of the Gla module relative to
the EGF module in order to facilitate interaction with tissue factor
(39). Our present data are consistent with the occurrence of a similar
mechanism in the N-terminal interaction region of C1r, as schematized
in the model presented in Fig. 10. In
this model, Ca2+ binding through ligands in the EGF module
of C1r (18) would allow the CUB and EGF modules to move toward each
other, inducing formation of a more compact conformation with
concomitant shielding of one or more tyrosine residues. This
Ca2+-dependent CUB-EGF assembly would provide
the appropriate conformation as well as the ligands required for
interaction with the homologous CUB-EGF pair of C1s.
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Fig. 10.
Schematic model of Ca2+-induced
conformational changes in the N-terminal CUB-EGF module pair of C1r and
of its subsequent assembly with the CUB-EGF module pair of C1s.
Binding of one Ca2+ atom through ligands in the EGF module
of C1r (38) allows formation of a
Ca2+-dependent, compact CUB-EGF assembly
exhibiting the appropriate conformation for interaction with the
homologous CUB-EGF pair of C1s. The latter is hypothesized to undergo a
conformational change similar to that occurring in C1r. The C1r/C1s
interaction model is adapted from Ref. 12 and features ionic bonds
between complementary charges in the CUB modules of C1r and C1s. As
discussed in the text, involvement of accessory ligands from regions
outside the CUB-EGF pair of C1r cannot be excluded.
Detailed knowledge of the three-dimensional structure of the CUB-EGF
module pair of C1r is now required to test this model at the atomic
level. Among other unsolved and intriguing questions are the structure
of the CUB module and the nature of its contribution to
Ca2+ binding, as this module may either stabilize the
Ca2+-binding site of the EGF module in the appropriate
conformation or possibly provide an additional ligand for the
Ca2+ ion. Expression of the CUB module and CUB-EGF pair was
initially undertaken in part with a view to solve their structure by
NMR spectroscopy, but the low solubility of both fragments clearly precludes such a study. Given the possible stabilizing effect of the
neighboring second CUB module on the CUB-EGF pair, expression of a
larger fragment encompassing the CUB-EGF-CUB array is being considered.
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ACKNOWLEDGEMENTS |
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We thank Dr. Agnès Journet for providing the human C1r cDNA, Dr. Jean Gagnon for determining N-terminal sequences, Dr. Anna Mitraki for assistance with the fluorescence measurements, and Myriam Ben Khalifa for assistance with the BIAcoreTM experiments. The help provided by Dr. Joan Stader during the initial stages of this work is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by the Commissariat à l'Energie Atomique, the Centre National de la Recherche Scientifique, the European Union Biotechnology Programme Contract BIO 4 CT 960662, and National Institutes of Health Grant RO1-AI 19478 (to A. F. E.). This is publication number 568 from the Institut de Biologie Structurale Jean-Pierre Ebel. A preliminary report of this study was presented at the XVIIth International Complement Workshop in Rhodes, Greece, October 11-16, 1998.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.
§ To whom correspondence should be addressed. Tel.: 33 4 76 88 95 79; Fax: 33 4 76 88 54 94; E-mail: thielens{at}ibs.ibs.fr.
2 N. M. Thielens, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
the nomenclature of
complement proteins is that recommended by the World Health
Organization, activated components are indicated by an overbar,
e.g. Cr;
the nomenclature of protein modules is that
defined by Bork and Bairoch (1), CCP module, complement control protein
module;
CUB module, module found in complement subcomponents C1r/C1s,
Uegf, and bone morphogenetic protein-1;
EGF, epidermal growth factor;
Gla module,
-carboxyglutamic acid containing module;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
RU, resonance unit(s);
Req, resonance unit(s) at
equilibrium;
MES, 2-(N-morpholino)ethanesulfonic acid.
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REFERENCES |
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