From the Laboratoire d'Enzymologie
Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel
(CEA-CNRS), 41 avenue des Martyrs,
38027 Grenoble Cedex 1, France and the ¶ University of
Missouri-Kansas City School of Biological Sciences,
Kansas City, Missouri 64110-2499
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ABSTRACT |
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C1s is the modular serine protease responsible
for cleavage of C4 and C2, the protein substrates of the first
component of complement. Its catalytic region (-B) comprises two
complement control protein (CCP) modules, a short activation peptide
(ap), and a serine protease domain (SP). A baculovirus-mediated
expression system was used to produce recombinant truncated fragments
from this region, deleted either from the first CCP module
(CCP2-ap-SP) or from both CCP modules (ap-SP). The
aglycosylated fragment CCP2-ap-SPag was also
expressed by using tunicamycin. The fragments were produced at yields
of 0.6-3 mg/liter of culture, isolated, and characterized chemically
and then tested functionally by comparison with intact C1s and its
proteolytic
-B fragment. All recombinant fragments were expressed in
a proenzyme form and cleaved by C
r to generate active enzymes
expressing esterolytic activity and reactivity toward C1 inhibitor
comparable to those of intact C
s. Likewise, the activated
fragments
-B, CCP2-ap-SP, and ap-SP retained C
s ability to cleave C2 in the fluid phase. In contrast, whereas fragment
-B cleaved C4 as efficiently as C
s, the C4-cleaving activity
of CCP2-ap-SP was greatly reduced (about 70-fold) and that
of ap-SP was abolished. It is concluded that C4 cleavage involves
substrate recognition sites located in both CCP modules of C
s,
whereas C2 cleavage is affected mainly by the serine protease domain.
Evidence is also provided that the carbohydrate moiety linked to the
second CCP module of C
s has no significant effect on catalytic
activity.
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INTRODUCTION |
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The first component of complement comprises two homologous, yet
distinct modular serine proteases
C1r1 and C1s assembled in the
form of a Ca2+-dependent tetramer
C1s-C1r-C1r-C1s. C1r is responsible for intrinsic C1 activation, a
two-step process first involving autolytic C1r activation and then
Cr-mediated cleavage of proenzyme C1s. The active enzyme
C
s is a highly specific protease with trypsin-like specificity
that mediates limited proteolysis of C4 and C2, the two protein
substrates of C
, thereby triggering activation of the classical
pathway of complement in response to infection by various
microorganisms (2-5). C4 cleavage occurs in the fluid phase and
generates fragment C4b, which has the transient ability to bind
covalently to the surface of the target. Cleavage of the second
substrate C2, in contrast, takes place after prior formation of a
C4b-C2 complex and yields C4b-C2a, the protease responsible for
cleavage of complement protein C3 (5). This double proteolytic activity
of C
is controlled by C1 inhibitor, a member of the serine
protease inhibitor (serpin) family, which reacts stoichiometrically with both C
r and C
s to form covalent protease-inhibitor
complexes, resulting in disruption of the
C
s-C
r-C
r-C
s tetramer (2).
Human C1s is synthesized as a 673-residue single chain zymogen which,
upon activation by Cr, is cleaved between Arg422 and
Ile423 to yield two disulfide-linked polypeptides, the
N-terminal A chain comprising a series of five protein modules and the
serine protease B domain (6-8). Limited proteolysis of C
s with
plasmin yields a C-terminal fragment
-B, comprising two contiguous
complement control protein (CCP) modules, a 15-residue intermediary
segment homologous to the activation peptide in chymotrypsinogen, and the serine protease domain (9). The CCP modules (originally known as
short consensus repeats) are structural motifs of about 60 residues
homologous to those mostly found in various complement regulatory
proteins known to interact with components C3b and/or C4b (10). The
C-terminal CCP module of C1s bears a heterogeneous, complex-type
N-linked oligosaccharide with both biantennary and triantennary species (11). Based on chemical cross-linking and three-dimensional homology modeling, it was shown recently that this
module closely interacts with the serine protease domain on the side
opposite to both the active site and the
Arg422-Ile423 bond cleaved upon activation
(12).
From a functional point of view, the fact that the proteolytic fragment
-B retains a functional active site (9) and studies based on the use
of monoclonal antibodies indicating that the
segment may be
involved in C4 binding (13, 14) together suggest that
-B may
represent the catalytic region of C
s. However, no precise
information is available yet on the respective role of the serine
protease domain and CCP modules in the various aspects of C
s
catalytic activity. The present study provides identification of the
domains of C1s involved in its activation by C
r, proteolytic activity toward C4 and C2, and reactivity toward C1 inhibitor, based on
baculovirus-mediated expression of truncated recombinant fragments from
the
-B region.
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EXPERIMENTAL PROCEDURES |
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Materials--
Diisopropylphosphorofluoridate, neuraminidase
(type X), and the synthetic substrate
N-benzoyl-L-arginine ethyl
ester were from Sigma. Human plasmin was obtained from Chromogenix AB
(Mölndal, Sweden). Polyclonal anti-C
s antiserum was raised
in rabbits according to standard procedures. Restriction enzymes were
from Boehringer Mannheim. VentR polymerase was from New
England Biolabs. The pHC1s46 vector encoding human C1s (6) was kindly
provided by Dr. Mario Tosi (Laboratoire d'Immunogénétique,
Institut Pasteur, Paris). The pVT-Bac vector was kindly provided by Dr.
Thierry Vernet (Institut de Biologie Structurale, Grenoble).
Antibiotics and molecular biology reagents were from Appligen
Oncor.
Proteins--
Proenzyme C1s and activated C1r and C1s were
purified from human plasma as described previously (15, 16). The
Cs
-B fragment was obtained by limited proteolysis with
plasmin (17) and purified by ion-exchange chromatography on a Mono Q HR
5/5 column (Pharmacia Biotech Inc.) as described previously (12). The
two glycoforms of the C
s
-B fragment, containing either a
biantennary or a triantennary oligosaccharide, were isolated by
affinity chromatography on concanavalin A-Sepharose (Pharmacia) as
described previously (11). The desialylated C
s
-B fragment was obtained by treatment with neuraminidase (10 units/mg protein) for
5 h at 25 °C. Complement proteins C2, C4, and C1 inhibitor were
isolated from human plasma by means of published procedures (18-20).
For the determination of protein concentrations the following absorption coefficients (A (1%, 1 cm) at 280 nm) and
molecular weights were used: C1r, 12.4 and 86,300 (21); C1s, 14.5 and 79,800 (21, 11); C
s
-B, 18.3 and 47,520 (22, 11); C2, 10.0 and 100,000 (18); C4, 8.2 and 205,000 (23, 24); C1 inhibitor, 3.86 and
105,000 (25). The absorption coefficients (A (1%, 1 cm) at
280 nm) used for the recombinant fragments ap-SP (17.0) and
CCP2-ap-SP (16.4) were calculated from their amino acid
composition by the method of Edelhoch (26), and their molecular weights
were determined by mass spectrometry analysis (see "Results").
Construction of C1s Fragments CCP2-ap-SP and
ap-SP-containing Expression Plasmids--
The following primers were
obtained from Eurogentec (Seraing, Belgium) and used to amplify the
desired human C1s cDNA sequences in a polymerase chain reaction
using VentR polymerase, according to established
procedures: GAAGATCTTGACTGTGGCATTCCT (sense for CCP2-ap-SP); GGAATTCTTAGTCCTCACGGGGGGT
(antisense for CCP2-ap-SP and ap-SP); and
GAAGATCTTCCAGTCTGTGGAGTC (sense for ap-SP). The underlined sequences represent bases introduced either at the 5 end of
the C1s cDNA to create a BglII site for in-frame cloning into the pNT-Bac expression vector or at the 3
end to create an
EcoRI site. The pNT-Bac vector was constructed from the
baculovirus expression vectors pVT-Bac (27) and pFast
Bac1TM (Life Technologies Inc.). Briefly, the segment
comprised between the EcoRV and KpnI sites was
excised from plasmid pVT-Bac and introduced between the
SnaBI and KpnI sites in pFast Bac1. The resulting
vector contains the polyhedrin promoter and the multiple cloning site
region of pVT-Bac (Fig. 1). It also
encodes the melittin signal peptide previously used to enhance
secretion of the papain precursor (27) and is therefore adapted to the
production in a secreted form of fragments derived from the C-terminal
end of proteins. The amplified DNAs were designated
CCP2-ap-SP, encoding residues 343-673 in mature human C1s,
and ap-SP, encoding residues 408-673. These were purified and cloned
into the pCR-Script Amp SK(+) intermediate vector (Stratagene)
according to the manufacturer's instruction. Both fragments were
excised with BglII and EcoRI and introduced into
the BamHI and EcoRI sites of the pNT-Bac vector by means of the T4 DNA ligase to create the plasmids
pNT-Bac/CCP2-ap-SP and pNT-Bac/ap-SP. Clones containing the
insert were identified by restriction enzyme analysis and checked for
the absence of mutations introduced by polymerase chain reaction by
double-stranded DNA sequencing using the
T7SequencingTM kit (Pharmacia Biotech
Inc.).
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Cells and Viruses-- The insect cells, Spodoptera frugiperda (Sf21), were provided by Dr. Jadwiga Chroboczek (Institut de Biologie Structurale, Grenoble). The cells were maintained in TC100 medium (Life Technologies, Inc.) containing 5% fetal calf serum (JRH Biosciences) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin (Sigma). Recombinant baculoviruses were generated by using the Bac-to-BacTM system (Life Technologies, Inc.) based on site-specific transposition of an expression cassette carried by the pNT-Bac-based recombinant plasmids into a baculovirus shuttle vector (bacmid) propagated in Escherichia coli (28). Transposition of the pNT-Bac/CCP2-ap-SP and pNT-Bac/ap-SP plasmids into DH10Bac E. coli cells and selection of recombinant bacteria were performed as recommended by the manufacturer (Life Technologies, Inc.). The recombinant bacmids were purified using the Qiagen midiprep DNA purification system (Qiagen S.A., Courtaboeuf, France) and transfected into Sf21 cells with cellfectin in Sf900 II SFM medium (Life Technologies, Inc.) as recommended by the manufacturer. The transfection supernatant was decanted 4 days later, centrifuged, and stored at 4 °C until use. The recombinant viruses, designated rCCP2-ap-SP and rap-SP, were titered by virus plaque assay and amplified as described by King and Possee (29).
Protein Production and Purification--
Sf21 cells
(5 × 105 cells/ml in spinner flasks or 2 × 107 cells/175-cm2 tissue culture flask) were
infected with the recombinant viruses at a multiplicity of infection of
2-3 in Sf900 II SFM medium containing 50 IU/ml penicillin and
50 µg/ml streptomycin. Tunicamycin (5 µg/ml) was added for the
production of the aglycosylated form of the CCP2-ap-SP
fragment. After 96 h at 28 °C, the supernatant was collected by
centrifugation, and diisopropylphosphorofluoridate was added to a final
concentration of 2 mM. The culture supernatant was dialyzed
against 5 mM EDTA, 20 mM sodium phosphate, pH
8.6, and loaded at 2 ml/min onto a Mono Q HR 10/10 column (Pharmacia) equilibrated in the same buffer containing 1 mM
diisopropylphosphorofluoridate. Elution was carried out with a linear
NaCl gradient from 0 to 350 mM in 30 min. Fractions
containing the recombinant proteins were identified by Western blot
analysis and dialyzed against 1.5 M ammonium sulfate, 0.1 M sodium phosphate, pH 7.4. Further purification was
achieved by high pressure hydrophobic interaction chromatography on a
TSK-Phenyl 5PW column (Beckman) equilibrated in the same buffer
containing 1 mM diisopropylphosphorofluoridate. Elution was
carried out by decreasing the ammonium sulfate concentration from 1.5 M to 0 in 30 min at a flow rate of 1 ml/min. Both
recombinant fragments were dialyzed against 50 mM
triethanolamine HCl, 145 mM NaCl, pH 7.4, concentrated to
0.2-0.5 mg/ml by ultrafiltration on MicrosepTM
microconcentrators (molecular weight cut-off = 10,000) (Filtron), and stored at 20 °C.
Chemical Characterization of the Recombinant Proteins-- N-terminal sequence analyses were performed either on soluble proteins or after SDS-PAGE analysis and electrotransfer, using an Applied Biosystems model 477A protein sequencer as described previously (12). The matrix-assisted laser desorption ionization (MALDI) technique was used for mass spectrometry analysis of the recombinant proteins, using a Voyager Elite XL instrument (PerSeptive Biosystems, Cambridge, MA), under conditions described previously (30).
Functional Tests--
Activation of proenzyme C1s and the
recombinant fragments CCP2-ap-SP and ap-SP was tested by
incubation of the proteins (6.5 µM) in the presence of
active Cr (C
r/protein molar ratio = 0.045) for
various periods at 37 °C, in 50 mM triethanolamine HCl,
145 mM NaCl, pH 7.4. In the case of C1s and
CCP2-ap-SP, activation was monitored by SDS-PAGE analysis
under reducing conditions and subsequent quantification by gel scanning
of the two-chain structure characteristic of the active state of the
proteins (see Fig. 2). Activation of the
smaller fragment ap-SP was measured by its ability to cleave the
synthetic substrate
N
-benzoyl-L-arginine ethyl
ester.
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Other Methods--
SDS-PAGE analysis was performed as described
previously (12). Western blot analysis of the recombinant proteins
after SDS-PAGE was performed by electrotransfer to a nitrocellulose
membrane and blocking of unoccupied sites with 5% non-fat dry milk in
10 mM Tris-HCl, 200 mM NaCl, pH 7.2. The
immobilized proteins were visualized by using a rabbit polyclonal
anti-Cs antiserum, followed by a goat anti-rabbit immunoglobulin
fraction conjugated to alkaline phosphatase (Sigma) and staining
according to the manufacturer's instructions.
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RESULTS |
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Expression and Purification of Recombinant Proteins-- The modular structures of human C1s and of the various truncated fragments used in the present study are depicted in Fig. 2. Construction of the recombinant baculoviruses, transposition of the pNT-Bac/CCP2-ap-SP and pNT-Bac/ap-SP plasmids into DH10Bac E. coli cells, and transfection of the corresponding recombinant bacmids into Sf21 cells were performed as described under "Experimental Procedures." The virus titer of the transfection supernatants after amplification was about 107 plaque forming units/ml in both cases. Infection of Sf21 cells by the supernatants was conducted for various periods at 28 °C, and the amount of recombinant proteins secreted into the culture medium was monitored by SDS-PAGE and Western blot analysis. Under normal culture conditions, protein bands with the expected apparent molecular weights (see below) became detectable at 48 h, their intensity increasing progressively to reach a plateau at 96 h. In contrast, secretion of the CCP2-ap-SP fragment in the presence of tunicamycin reached a maximum at 72 h and decreased considerably after 96 h. Analysis of protein production in the cell pellets showed kinetic profiles similar to those observed for the supernatants. Under optimal production conditions, the amounts of recombinant protein secreted into the culture medium were estimated to be approximately 0.6 µg/ml for ap-SP, 3 µg/ml for CCP2-ap-SP, and 1.5 µg/ml for the aglycosylated fragment CCP2-ap-SP (CCP2-ap-SPag) produced in the presence of tunicamycin.
The recombinant fragments were initially purified by fractionation of the culture supernatants by ion-exchange chromatography on a Mono Q column (Pharmacia). Both fragment CCP2-ap-SP and CCP2-ap-SPag yielded visible peaks at 280 nm eluting during the ascending salt gradient, slightly later than the C
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Chemical Characterization of Recombinant Proteins--
Edman
degradation of the purified fragments CCP2-ap-SP and
CCP2-ap-SPag yielded a single sequence
Asp-Leu-Asp-(Cys)-Gly-Ile-Pro-Glu-Ser-Ile ... in both
cases, corresponding to the segment
Asp343-Ile350 of human C1s preceded by the two
residues Asp-Leu expected to be added at the N terminus, due to the
introduction of bases at the 5 end of the cDNA to create a
BglII site (see "Experimental Procedures"). In the same
way, fragment ap-SP yielded a single sequence
Asp-Leu-Pro-Val-(Cys)-Gly-Val-Pro-Arg-Glu ... derived
from the segment Pro408-Glu415 of C1s,
corresponding to the N-terminal end of the activation peptide.
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Functional Characterization of Recombinant Proteins--
As judged
from SDS-PAGE analysis under reducing conditions, fragments
CCP2-ap-SP, CCP2-ap-SPag, and ap-SP
retained a monochain structure upon prolonged incubation at 37 °C,
indicating that the preparations were free of contaminating proteases.
To test their activation by Cr, i.e. their ability to
be split at the Arg422-Ile423 bond cleaved upon
activation of intact C1s, each fragment was incubated for various
periods at 37 °C in the presence of C
r. SDS-PAGE analysis
under reducing conditions indicated that all three fragments were
cleaved by C
r: (i) in the case of CCP2-ap-SP and
CCP2-ap-SPag, a two-chain structure became
clearly visible, as shown by the appearance of bands corresponding to
the serine protease domain and to the CCP2-ap moieties;
(ii) the apparent molecular weight of ap-SP was slightly decreased,
suggesting removal of the short N-terminal activation peptide. Edman
degradation of the C
r-treated fragments gave evidence for a
second sequence 423Ile-Ile-Gly-Gly-Ser-Asp-Ala ... , in addition to the single N-terminal sequence yielded upon analysis of
the monochain fragments (see above), confirming C
r-mediated
cleavage of the 422Arg-Ile423 bond in all
cases. A comparative kinetic analysis of the activation of proenzyme
C1s and the recombinant fragments CCP2-ap-SP and ap-SP by
C
r showed that all three proteins were activated in a comparable
manner (Fig. 6). It was concluded,
therefore, that the structural determinants involved in the cleavage of
proenzyme C1s by C
r are contained solely in the C-terminal part
of the protein, comprising the activation peptide and the serine
protease domain.
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DISCUSSION |
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The baculovirus/insect cells expression system has been used
successfully to produce a number of intact multidomain proteins, including human complement proteases C1r and C1s (35, 36). In the
present study, the recently developed Bac-to-BacTM system
(28) was used to express recombinant modular fragments from the
catalytic region of human C1s. Although the secretion yields (0.6-3.0
mg/liter cell culture) were low compared with some of those reported
for intact proteins, they were sufficient to produce the purified
fragments for chemical and functional characterization. In this
respect, the significantly lower level of secretion of fragment ap-SP
is probably related, at least in part, to a decreased stability and/or
solubility compared with the larger fragment CCP2-ap-SP.
Indeed, we have provided evidence that the second CCP module of
Cs is closely associated with the serine protease domain (12).
Removal of this module may therefore be expected to unmask hydrophobic
areas of the serine protease domain and thereby to decrease its
solubility. In contrast, the observed slight decrease in the secretion
yield of the aglycosylated fragment CCP2-ap-SPag
is probably not linked to a reduced stability of the protein in
solution due to the lack of the oligosaccharide chain but rather is an
indirect consequence of the deleterious effect of tunicamycin on insect
cells, which is known to affect secretion of some proteins (37). As
shown by Edman degradation and mass spectrometry analyses, the
recombinant C1s fragments produced in this study were homogeneous at
the polypeptide level and had the expected N-terminal sequences and
amino acid compositions. In contrast, the analyses performed on the
glycosylated fragment CCP2-ap-SP are consistent with the
occurrence at Asn391 of heterogeneous high mannose type
oligosaccharides containing a varying number of mannose residues
ranging from 4 to 8, depending on the preparation, and even within a
given preparation (see Fig. 5). Such a heterogeneity of the
carbohydrate moiety of the glycoproteins expressed in
baculovirus/insect cells systems could be a major disadvantage if these
are produced for crystallographic purposes.
Several lines of evidence indicate that the carbohydrate borne by the
second CCP module of Cs is not significantly involved in the
functional activities of this protease. Thus, the two glycoforms and
the asialylated form of fragment C
s
-B all display comparable proteolytic activities toward C2 and C4. Moreover, the aglycosylated form of fragment CCP2-ap-SP exhibits proteolytic kinetic
constants similar to those of its glycosylated counterpart. It is very
likely therefore that the two naturally occurring glycoforms of human C
s display identical catalytic properties in vivo.
The fact that complete removal of the carbohydrate from fragment
CCP2-ap-SP does not impair its functional activity also
suggests that this does not significantly affect the folding of the
protein. Thus, despite the location of Asn391 in the
vicinity of the CCP module/serine protease domain interface (12), the
oligosaccharide chain itself is probably not directly involved in the
interaction between the two domains. This observation lends further
support to the above-mentioned hypothesis that the decreased secretion
of fragment CCP2-ap-SPag results from an
indirect effect of tunicamycin on insect cells.
All of the recombinant fragments were produced and remained stable in a
proenzyme form, indicating that, like the parent C1s molecule, none of
them undergoes self-activation. In contrast, both fragments
CCP2-ap-SP and ap-SP were readily activated in solution by
Cr, with an efficiency comparable to intact C1s. The shorter C1s
fragment ap-SP therefore contains the structural information required
for C
r-mediated cleavage. Indeed, this appears consistent with
the fact that C1s cleavage by C
r normally does not occur in the
fluid phase, but inside the C1 complex, in which the activation site of
the former protease and the active site of the latter are expected to
be pre-positioned with respect to each other (4). It is likely,
therefore, that C1s recognition by C
r mainly involves a
restricted number of residues on either side of the C1s
Arg422-Ile423 bond cleaved upon activation. In
any case, it is clear that efficient cleavage does not require
accessory binding sites outside the serine protease domain of C1s. It
is noteworthy that C
r-mediated cleavage of fragments
CCP2-ap-SP and ap-SP leads to the development of
esterolytic activity in both cases, which again implies that the latter
is self-sufficient for the formation of a fully active site, in
agreement with current knowledge on active site formation in other
serine proteases (38).
Our observation that the short Cs fragment ap-SP retains the
ability to form a stable complex with C1 inhibitor indicates that this
contains the structural elements required for reaction with C1
inhibitor. It should be mentioned, however, that the reaction of
C
s with C1 inhibitor is a complex and still not fully understood process. Like other members of the serpin family, C1 inhibitor acts as
a pseudosubstrate: C
s cleaves C1 inhibitor at the
Arg444-Thr445 "reactive center," then the
cleavage products do not dissociate, but a tight, SDS-stable
enzyme-inhibitor complex is formed (25). It has been recently proposed
that formation of this stable complex involves a secondary interaction
site on C1 inhibitor (39). Obviously, a precise kinetic analysis of the
reaction of C1 inhibitor with native C
s and its ap-SP fragment
would be necessary to check whether the latter behaves exactly as the
parent protein at the different steps of the reaction. Nevertheless,
our observation that C
s and its fragment ap-SP yield comparable
amounts of stable complexes with C1 inhibitor suggests that the
proposed secondary interaction site on C1 inhibitor binds to the serine
protease domain of C
s.
With regard to C2 cleaving activity, all Cs fragments were found
to cleave this substrate with an efficiency comparable with that of the
intact protease. The fact that the shorter fragment ap-SP exhibits a
slightly decreased kcat value can be
rationalized by the following observations: (i) as discussed above,
removal of the second CCP module may be expected to uncover hydrophobic patches of the serine protease domain and possibly to induce local denaturation resulting in partial loss of substrate binding site(s) at
the surface of the domain; (ii) due to its lower solubility, the
uncertainty on concentration measurements and hence on
kcat was probably higher in the case of fragment
ap-SP. Indeed, the observation that ap-SP exhibits a
Km value for C2 comparable with that of the other
fragments tends to favor the latter explanation. Therefore, our
conclusion is that C2 recognition and cleavage by C
s only
involve structural motifs located within the ap-SP fragment and
probably confined to the serine protease domain. In any case, our data
provide no indication of a significant involvement of the remainder of
the C
s molecule, particularly the CCP modules.
In contrast, our data clearly show that, while fragment -B exhibits
a C4 cleaving activity comparable with that of intact C
s, this
activity is greatly reduced upon removal of the first CCP module and
abolished when both CCP modules are deleted. Therefore, we conclude
that both of these modules contribute accessory recognition sites for
C4, which likely mediate binding and positioning of the substrate in
such a way that the scissile bond fits into the C
s active site,
thereby allowing efficient cleavage of the protein. This hypothesis is
consistent with the observation that fragment CCP2-ap-SP
shows both an increased Km for C4 and a decreased kcat value (see Table II). Thus, removal of the
first CCP module would result in partial loss of the C4 binding site,
resulting in improper positioning of the protein, and thereby in
decreased cleavage efficiency. Our finding that both CCP modules of
C
s participate in C4 binding is in full agreement with previous
studies (13, 14) indicating that monoclonal antibodies directed to the
segment of the protease inhibit its ability to cleave C4. From a
more general point of view, it should be emphasized that all complement
regulatory proteins known to bind the C4b fragment of C4 (including
complement receptor type 1 (CR1), membrane cofactor protein, decay
accelerating factor, and C4b-binding protein) are composed mainly or
entirely of CCP modules (10). In the case of CR1, it has been shown by
deletion experiments and site-directed mutagenesis that both the first
and second CCP modules are required for C4b binding (40). By analogy,
the most likely hypothesis is therefore that the two CCP modules of
C
s bind to the C4b moiety of C4. This does not preclude,
however, that other binding sites may be involved in the C
s-C4
interaction. In this respect, it should be mentioned that plasmin
degradation of the serine protease domain of C
s was reported to
result in the loss of C4 binding ability (14). In the same way, it has
been suggested that C
s may interact with the C4a moiety of C4
through acidic residues close to the active site (41). It may be
hypothesized, therefore, that efficient positioning of C4 with respect
to C
s requires two types of interactions: (i) between the C4b
moiety and the CCP modules and (ii) between the C4a moiety and the
serine protease domain.
It is not surprising that C4 and C2 recognition by Cs involves
distinct structural requirements, since these protein substrates are
quite different both in terms of size and modular organization. Also,
it is well established that, in vivo, C4 cleavage occurs in
the fluid phase, whereas C2 is cleaved within the C2-C4b complex that
forms after covalent attachment of C4b in the vicinity of the C
complex (5). It may be expected, therefore, that appropriate "presentation" of C2 with respect to the C
s active site is
effected, at least in part, by C4b. In this respect, it is noteworthy
that initial binding of C2 to C4b has recently been shown to involve the CCP modules of C2 (42). Thus, the C4b moiety of native C4 would
bind first to the CCP modules of C
s, then the C4b fragment released upon C
s cleavage would acquire preferential affinity for the homologous modules of C2.
The present study provides the first precise identification of the
domains of Cs involved in the various aspects of its catalytic
activity, and this study also shows that its very restricted specificity and its ability to discriminate its two protein substrates arise for a large part from the occurrence of accessory substrate binding sites located outside the serine protease domain. Further studies are in progress to identify the residues of the CCP modules of
C
s that participate in C4 binding.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mario Tosi for providing the human C1s cDNA; Dr. Thierry Vernet for providing the pVT-Bac expression vector; Dr. Jean Gagnon for determining N-terminal sequences; and Dr. Michel Jaquinod for performing mass spectrometry analyses. The help provided by Dr. Thierry Vernet and Dr. Jadviga Chroboczek 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 (CEA), the Centre National de la Recherche Scientifique, and National Institutes of Health Grant RO1-AI 19478 (to A. F. E.). This is publication number 493 from the Institut de Biologie Structurale Jean-Pierre Ebel.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: University of Missouri-Kansas City School of Biological Sciences, 5100 Rockhill Rd., Kansas City, MO 64110-2499.
To whom correspondence should be addressed. Tel.: (33) 4 76 88 49 81; Fax: (33) 4 76 54 94; E-mail: arlaud{at}ibs.ibs.fr.
1
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. Cs; the nomenclature of protein modules is that
defined by Bork and Bairoch (1); CCP2-ap-SP, ap-SP,
recombinant fragments from the C1s catalytic region lacking the
N-terminal CCP module and both CCP modules, respectively;
CCP2-ap-SPag, aglycosylated fragment
CCP2-ap-SP; CCP, complement control protein; MALDI,
matrix-assisted laser desorption ionization; PAGE, polyacrylamide gel
electrophoresis.
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